CN102850397B - Multi-target antitumor compounds and preparation method and application thereof - Google Patents

Multi-target antitumor compounds and preparation method and application thereof Download PDF

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CN102850397B
CN102850397B CN201110177810.0A CN201110177810A CN102850397B CN 102850397 B CN102850397 B CN 102850397B CN 201110177810 A CN201110177810 A CN 201110177810A CN 102850397 B CN102850397 B CN 102850397B
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quinazoline
chloroethyl
bis
phosphoric acid
chloro
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CN102850397A (en
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李润涛
葛泽梅
林松文
李日东
孙崎
王欣
程铁明
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Peking University
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Abstract

The invention provides multi-target antitumor compounds shown as general formula I or pharmaceutical salts thereof, especially relates to EGFR, HER-2 and DNA multi-target antitumor compounds, and also provides preparation method thereof and application thereof in antitumor drug. Researches show that the inventive chemicals have remarkable antitumor activity, and the antitumor activity of some chemicals is superior to the positive control lapatinib.

Description

Mutiple Targets antineoplastic compound and its preparation method and application
Technical field
The present invention relates to a class Mutiple Targets antineoplastic compound, particularly relate to EGFR, HER-2 and DNA Mutiple Targets antineoplastic compound, also relates to and its preparation method and application, belongs to medicinal chemistry art.
Background technology
The biological procedures of the complexity that the generation of tumour and development are multifactor effects, polygene participates in, the methods for the treatment of of the single target spot of single medicine often also exists the problem that resistance appears greatly and easily in unsatisfactory curative effect, toxic side effect.At present, the treated with combined medication of multiple different mechanism of action is often adopted in clinical treatment, to overcome above-mentioned problem.According to this clinical experience, scientist has has designed and synthesized many small-molecule drugs with multiple target effect mechanism, and pharmacological evaluation proves, this thinking is feasible.
EGFR inhibitor is the antitumor drug gone on the market in recent years, and result for the treatment of is remarkable, but in clinical application, has been found that to undergo mutation in the T790M position of acceptor, cause the appearance of resistance; And a lot of cell expresses EGFR and HER-2 acceptor simultaneously, the result for the treatment of of EGFR inhibitor is obviously reduced.This just requires that design can act on EGFR and HER-2 simultaneously, and to the effective new inhibitor of saltant type acceptor.
Endoxan is a kind of application DNA alkylating agent series antineoplastic medicament for many years, but serious toxic side effect limits application clinically.For addressing this problem, scientists devises numerous open loop cyclophosphamide derivatives, Cyclophosphamide quaternary ammonium salt derivant and open loop Cyclophosphamide quaternary ammonium salt derivant, the toxicity that the pharmacological results shows these compounds has had obvious reduction, but anti-tumor activity is also not as endoxan.
Although had scientist EGFR inhibitor and DNA alkylating agent to be combined devise a series of pair of target drug molecule, the anti-tumor activity of this compounds is general, and can not act on HER-2 acceptor.Based on the thinking of multiple target effect, and in conjunction with this experimental group for many years about the Research foundation of Cyclophosphamide quaternary ammonium salt derivant, we have proposed new mentality of designing: be combined by suitable connecting arm with Glyciphosphoramide by the precursor structure of EGFR/HER-2 inhibitor, EGFR and HER-2 acceptor can be acted on to find, the Mutiple Targets antitumor drug of the high-efficiency low-toxicity of alkanisation can be produced again DNA.
Summary of the invention
The present invention is based on the thinking of Mutiple Targets medicinal design, antitumor drug EGFR/HER-2 inhibitor merged mutually with the structure of alkylating agent endoxan, designs, synthesized a class EGFR, HER-2 and DNA Mutiple Targets antineoplastic compound.
The invention provides the class Mutiple Targets antineoplastic compound as shown in general formula I
Wherein, R 1be selected from hydrogen, lower alkoxy; Preferably from hydrogen, methoxy or ethoxy.
R 2for being selected from substituted or unsubstituted phenyl, the aryl of described replacement refers to that phenyl is replaced by one or more halogen, lower alkoxy or alkoxy aryl; Preferably from 3-bromophenyl, 3-chloro-4-fluorophenyl or the chloro-4-of 3-(3-fluorine benzyloxy) phenyl.
X is CH 2or CO;
Y is O or NH;
N is the integer being selected from 1-10, or when n is 0, X and Y does not exist.
Defined in the present invention:
Described " lower alkoxy " can be the alkoxyl group of carbonatoms at 1-6 or the alkoxyl group of replacement, such as, methoxyl group, oxyethyl group, isopropoxy, positive propoxy, n-butoxy, isobutoxy, sec-butoxy, tert.-butoxy, n-pentyloxy, isopentyloxy, positive hexyloxy, different hexyloxy etc., when for replace alkoxyl group time, substituting group can be such as halogen or substituted-phenyl etc.
Described " alkoxy aryl " can be the benzyl oxygen base of such as benzyl oxygen base or rudimentary replacement, and described rudimentary substituting group can be provided at the alkyl, alkoxyl group etc. of one or more halogens, amino or the 1-3 carbon on phenyl ring.
Described " halogen " is chlorine, fluorine or bromine.
Further, the present invention also provides the preparation method of compound of Formula I.
Wherein, when X is CH 2, Y to be O or n be 0 and X and Y does not exist time, R 1, R 2with n as aforementioned formula I define time compound synthetic route as shown in Figure 1:
(1) synthesis of key intermediate 4,6-dihydroxyl quinazoline and 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride
5-hydroxyl-2-benzaminic acid and the cyclization of methane amide high temperature obtain 4,6-dihydroxyl quinazoline;
Or, vanillic acid obtains 3 through methylating, 4-dimethoxy p-methyl, 2-nitro-4 is obtained through nitration reaction, 5-dimethoxy p-methyl, carry out demethylating reaction again and hydrolysis of ester group is obtained by reacting 2-nitro-4-methoxyl group-5-hydroxy-benzoic acid, be prepared into 2-nitro-4-methoxyl group-5-methyl hydroxybenzoate again, 2-amino-4-methoxyl-5-methyl hydroxybenzoate is obtained again through catalytic hydrogen reduction nitro, at 150 DEG C, 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride is obtained by reacting again with ammonium formiate and methane amide;
7 R 1the synthetic method of 4,6-dihydroxyl quinazolines replaced and the synthetic method of 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride similar;
(2) synthesis of formula I
7 R 1the 6-OH highly selective acylation of 4, the 6-dihydroxyl quinazolines replaced, obtains 7 R 16-acetoxyl group-4-the hydroxyquinazoline replaced, then become 7 R with reacting with sulfur oxychloride 16-acetoxyl group-4-the chloro-quinazoline replaced, not purified directly and substituted aniline be obtained by reacting 7 R 1the 4-R replaced 2substituted benzene amino-6-acetoxyl group quinazoline, then in ammonia hydroxide/methanol system, deacetylation obtains 7 R 1the 4-R replaced 2substituted benzene amino-6-hydroxyquinazoline; 7 R 1the 4-R replaced 2substituted benzene amino-6-hydroxyquinazoline directly reacts with dichlor-phosphoryl mustargen, or elder generation and halohydrin react, and products therefrom reacts with dichlor-phosphoryl mustargen again, namely obtains formula I.
Wherein, when X be CO, Y is NH, R 1, R 2with n as aforementioned formula I define time compound synthetic route as shown in Figure 2, specific as follows:
(1) synthesis of the carboxylic acid of benzoyloxy replacement
The glycol of 1.5 equivalents and the Benzoyl chloride of 1.0 equivalents react, and obtain the alcohol that benzoyloxy replaces, then through Jone ' s reagent oxidation, obtain the carboxylic acid that benzoyloxy replaces, then at SOCl 2/ CHCl 3middle backflow obtains the acyl chlorides that benzoyloxy replaces, and is directly used in next step reaction;
(2) synthesis of formula I
4 R 1the 2-benzaminic acid replaced and methane amide at high temperature cyclization obtain 7 R 14, the 6-dihydroxyl quinazolines replaced, further nitration reaction obtains 7 R 16-nitro-4-the hydroxyquinazoline replaced, then change 7 R into sulfur oxychloride 16-nitro-4-the chloro-quinazoline replaced, not purified directly and substituted aniline be obtained by reacting 7 R 1the 4-R replaced 2substituted benzene amino-6-nitro-quinazoline, obtains 7 R with glass putty-hydrochloric acid reduction nitro further 1the 4-R replaced 2substituted benzene amino-6-amido quinazoline; The acyl chloride reaction replaced with above-mentioned benzoyloxy again, products therefrom, without separation, directly removes benzoyl and obtains 4-R in sodium hydroxide/methanol/water system 2the amido quinazoline that substituted benzene amino-6-hydroxyl replaces, then under sodium hydride effect, be obtained by reacting formula I with dichlor-phosphoryl mustargen.
Synthetic method of the present invention also comprises makes pharmaceutical salts with corresponding acid-respons further by the general formula obtained (I) product, such as tosilate, mesylate, hydrochloride or maleate etc.
Invention further provides the pharmaceutical composition of described Mutiple Targets antineoplastic compound, described compound or pharmaceutically acceptable salt thereof wherein containing treatment significant quantity, and one or more pharmaceutically acceptable carriers, can by the mixture of compound itself or its pharmaceutical salts and pharmaceutically acceptable vehicle, thinner etc. with the form oral administration of tablet, capsule, granule, powder or syrup or with the form non-oral administration of injection.This pharmaceutical composition preferably containing weight ratio be the Mutiple Targets antineoplastic compound of the present invention of 0.1%-99.5% or its pharmaceutical salts as activeconstituents, be more preferably the activeconstituents of 0.5%-99.5% containing weight ratio.
Above-mentioned preparation is prepared by conventional pharmaceutical method.The example of available medicinal adjuvant comprises vehicle, and (such as carbohydrate derivative is as lactose, sucrose, glucose, mannitol and Sorbitol Powder, starch derivative is as W-Gum, potato starch, dextrin and carboxymethyl starch, derivatived cellulose is as crystalline cellulose, hydroxypropylcellulose, carboxymethyl cellulose, calcium carboxymethylcellulose, Xylo-Mucine, gum arabic, dextran, silicate derivative is as Neusilin US2, phosphate derivative is as calcium phosphate, carbonate derivative is as calcium carbonate, sulfate-derivatives is as calcium sulfate etc.), tackiness agent (such as gelatin, polyvinylpyrrolidone and polyoxyethylene glycol), (such as derivatived cellulose is as Xylo-Mucine for disintegrating agent, polyvinylpyrrolidone), lubricant (such as talcum, calcium stearate, Magnesium Stearate, spermaceti, boric acid, Sodium Benzoate, leucine), stablizer (methyl p-hydroxybenzoate, propylparaben etc.), correctives (such as conventional sweeting agent, acidic flavoring agent and spices etc.), thinner and injection liquid solvent (such as water, ethanol and glycerine etc.).
Present invention also offers the application of described compound or pharmaceutically acceptable salt thereof at anti-tumor aspect, research shows it and has significant anti-tumor activity, the restraining effect of some of them compound to HCT116, MDA-MB-468 and SK-BR-3 tri-kinds of tumor cell line growths is better than positive control medicine lapatinibditosylate.Primary action Mechanism Study result shows: this compounds both had inhibit activities to EGFR/HER-2, has again DNA alkanisation, belongs to irreversible inhibition agent.
Accompanying drawing explanation
Fig. 1 is the synthetic route 1 of compound of Formula I of the present invention.
Fig. 2 is the synthetic route 2 of compound of Formula I of the present invention.
Fig. 3 is EGFR phosphorylation assays (p-EGFR: the EGFR of phosphorylation) the result figure of the compounds of this invention EMB-3.
Fig. 4 is damaging action (a.p < 0.05, versus control) the result figure of the compounds of this invention EMB-3 to DNA.
Fig. 5 be the compounds of this invention EMB-3 cell cycle affect result figure.
Contraction table
Embodiment
The preparation of embodiment 1:4-(3-bromobenzene amido) quinazoline phenol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-1)
The preparation of 1a:4,6-dihydroxyl quinazoline
5-hydroxyl-2-benzaminic acid (21.44g, 140mmol) is added in 22mL methane amide, at 150 DEG C, reacts 0.5h, after cooling, add 200mL water, stir 1h, separate out a large amount of white solid, suction filtration, washing, dry, obtain 14.40g, productive rate 63%.mp＞300℃。
1H NMR(300MHz，DMSO-d 6)：δ7.25(s，1H)，7.40(s，1H)，7.52(s，1H)，7.90(s，1H)，10.07(s，1H)，12.03(s，1H).
The preparation of 1b:6-acetoxyl group-4-hydroxyl-quinazoline
4,6-dihydroxyl quinazoline (23.55g, 145.24mmol) and 27mL pyridine are added in 220mL diacetyl oxide, at 100 DEG C, react 2h, after cooling, pour in trash ice, separate out a large amount of solid, suction filtration, dry, obtain white solid 22.40g, productive rate 76%.mp＞300℃。
1H NMR(300MHz，DMSO-d 6)：δ2.32(s，3H)，7.60(dd，J＝2.7，8.7Hz，1H)，7.73(d，J＝8.7Hz，1H)，7.83(d，J＝2.7Hz，1H)，8.11(s，1H)，12.35(s，1H).
The preparation of 1c:4-(3-bromoanilino)-6-acetoxyl group quinazoline
Be added in 10mL thionyl chloride by 6-acetoxyl group-4-hydroxyl-quinazoline (0.62g, 3mmol) and DMF (0.1mL), 6h is answered in backflow.Pressure reducing and steaming sulfur oxychloride, then with chloroform (10mL × 3) revolve steam take away residual sulfur oxychloride, obtain yellow solid.Add 20mL Virahol, 3-bromaniline (0.62g, 3.6mmol) and triethylamine (0.36g, 3.6mmol), stirred at ambient temperature 6h, then the 3h that refluxes makes to react completely, cooling, suction filtration, wash with Virahol, water and ether successively, dry, obtain faint yellow solid, 0.74g, productive rate 69%.mp 238-239℃
1H NMR(300MHz，DMSO-d 6)：δ2.40(s，3H)，7.44-7.54(m，2H)，7.78(d，J＝7.5Hz，1H)，7.93-8.09(m，3H)，8.67(s，1H)，8.99(s，1H)，11.39(s，1H).
The preparation of 1d:4-(3-bromoanilino)-6-hydroxyquinazoline
Be added in 30mL methyl alcohol by 4-(3-bromoanilino)-6-acetoxyl group quinazoline (1.40g, 3.91mmol) and strong aqua 8mL, room temperature reaction 2h, boils off solvent, add 30mL water, stir 10min, suction filtration, washing, dry, obtain 1.16g, productive rate 94%.mp＞300℃。
1H NMR(300MHz，DMSO-d 6)：δ7.28-7.37(m，1H)，7.46(d，J＝9.0Hz，1H)，7.71(d，J＝9.0Hz，1H)，7.80(s，1H)，8.27(s，1H)，8.52(s，1H)，9.60(s，1H)，10.11(s，1H).
The preparation of 1e:4-(3-bromobenzene amido) quinazoline phenol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
4-(3-bromoanilino)-6-hydroxyquinazoline (0.32g, 1mmol) is placed in 100mL there-necked flask, N 2protection; add 40mL anhydrous tetrahydro furan; n-Butyl Lithium (0.63mL is dripped at 0 DEG C; the hexane solution of 1.6M); after reaction 1h; this reaction solution be added drop-wise to tetrahydrofuran (THF) (10mL) solution of the dichlor-phosphoryl mustargen (0.31g, 1.2mmol) be placed at 0 DEG C and react 3h, more logical ammonia 30min.25mL saturated aqueous common salt is added toward reaction system, adjust PH to neutral, separatory, water layer, again with extraction into ethyl acetate (25mL × 2), merges organic phase, anhydrous sodium sulfate drying, filter, concentrated, dodge post (E → E: MeOH=25: 1) separation and obtain white solid, 0.22g, productive rate 42%.mp 200-202℃。
1H NMR(400MHz，DMSO-d 6)：δ3.41-3.46(m，4H)，3.66-3.71(m，4H)，7.32-7.40(m，2H)，7.85-7.91(m，3H)，8.21(s，1H)，8.27(s，1H)，8.64(s，1H)，
13C NMR(100MHz，DMSO-d 6)：δ42.44，48.92(d，J P-C＝4.0Hz)，113.28，115.59，120.87，121.33，124.35，126.25，127.86，129.44，130.49，140.83，146.83，149.02(d，J P-C＝7.0Hz)，153.62，157.25.
31P NMR(122MHz，DMSO-d 6)：δ15.59.
Anal.Cald for C 18H 19BrCl 2N 5O 2P：C，41.64；H，3.69；N，13.49；Found：C，41.70；H，3.83；N，13.53.
The preparation of embodiment 2:2-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-2)
The preparation of 2a:4-(3-bromoanilino)-6-(2-hydroxyl-oxethyl) quinazoline
By 4-(3-bromoanilino)-6-hydroxyquinazoline (0.32g, 1.00mmol), ethylene chlorhydrin (0.10g, 1.25mmol), salt of wormwood (0.28g, 2.00mmol) with potassiumiodide (0.017g, 0.10mmol) be added in 10mL acetonitrile, backflow 12h, pressure reducing and steaming solvent, add 20mL water, with extraction into ethyl acetate (30mL × 2), merge organic phase, saturated common salt washing (30mL × 2), anhydrous sodium sulfate drying, filter, concentrated, dodge post (P: E=1: 2 → 1: 5) separation and obtain faint yellow solid 0.22g, productive rate 60%.mp 213-215℃。
1H NMR(300MHz，DMSO-d 6)：δ3.57(br s，1H)，3.85(t，J＝4.8Hz，2H)，4.21(t，J＝4.8Hz，2H)，7.30-7.43(m，2H)，7.54(dd，J＝2.4，9.0Hz，1H)，7.79(d，J＝9.0Hz，1H)，7.92-7.94(m，2H)，8.21(s，1H)，8.57(s，1H).
13C NMR(75MHz，DMSO-d 6)：δ59.52，70.41，102.80，115.74，120.81，121.36，124.25，124.86，126.10，129.62，130.56，141.05，145.17，152.22，156.72，157.08.
Anal.Calcd for C 16H 14BrN 3O 2：C，53.35；H，3.92；N，11.67.Found：C，53.36；H，3.95；N，11.69.
The preparation of 2b:2-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(3-bromoanilino)-6-(2-hydroxyl-oxethyl) quinazoline.Productive rate 37%.mp 143-144℃。
1H NMR(400MHz，CDCl 3)：δ3.07(br s，2H)，3.38-3.64(m，8H)，4.20-4.47(m，4H)，7.22-7.25(m，2H)，7.35(d，J＝2.0，8.8Hz，1H)，7.79(d，J＝9.2Hz，1H)，7.98-8.01(m，2H)，8.25(s，1H)，8.70(s，1H)，9.08(br s，1H)
13C NMR(100MHz，DMSO-d 6)：δ42.54，48.93(d，J P-C＝4.0Hz)，62.87(d，J P-C＝4.0Hz)，68.05(d，J P-C＝7.0Hz)，102.98，115.67，120.73，121.34，124.20，124.71，126.08，129.55，130.49，140.98，145.10，152.26，156.55，156.75
31P NMR(122MHz，DMSO-d 6)：δ18.68.
Anal.Cald for C 20H 23BrCl 2N 5O 3P：C，42.65；H，4.12；N，12.43；Found：C，42.67；H，4.16；N，12.45.
The preparation of embodiment 3:3-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-3)
The preparation of 3a:4-(3-bromoanilino)-6-(3-hydroxy propyloxy group) quinazoline
Preparation method is with embodiment 2a, and wherein the ethylene chlorhydrin of 2a replaces with 3-propylene chlorohydrin.Productive rate 60%.mp 184-186℃。
1H NMR(300MHz，DMSO-d 6)：δ1.94-2.02(m，2H)，3.62-3.68(m，2H)，4.24(t，J＝6.3Hz，2H)，4.68(t，J＝5.1Hz，1H)，7.30-7.40(m，2H)，7.52(dd，J＝2.4，9.0Hz，1H)，7.75(d，J＝9.0Hz，1H)，7.93-7.94(m，1H)，8.20(t，J＝1.8Hz 1H)，8.57(s，1H)，9.70(s，1H).
13C NMR(75MHz，DMSO-d 6)：δ32.13，57.31，65.63，102.81，115.77，120.80，121.36，124.26，124.73，126.05，129.56，130.52，141.08，145.12，152.16，156.71，157.12.
Anal.Calcd for C 17H 16BrN 3O 2：C，54.56；H，4.31；N，11.23.Found：C，54.63；H，4.37；N，11.41.
The preparation of 3b:3-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(3-bromoanilino)-6-(3-hydroxy propyloxy group) quinazoline.Productive rate 30.71%.mp 158-159℃。
1H NMR(400MHz，DMSO-d 6)：δ2.12-2.18(m，2H)，3.26-3.33(m，4H)，3.65(t，J＝7.2Hz，4H)，4.03-4.08(m，2H)，4.28(t，J＝5.6Hz，2H)，7.32(d，J＝7.6Hz，1H)，7.38(t，J＝8.0Hz，1H)，7.55(d，J＝8.8Hz，1H)，7.77(d，J＝9.2Hz，1H)，7.93(d，J＝8.0Hz，1H)，7.96(s，1H)，8.21(s，1H)，8.56(s，1H)，
13C NMR(100MHz，DMSO-d 6)：δ29.85(d，J P-C＝7.0Hz)，42.56，48.86(d，J P-C＝4.0Hz)，61.28(d，J P-C＝5.0Hz)，65.05，103.10，115.76，120.80，121.32，124.25，124.67，126.07，129.61，130.52，141.04，145.18，152.22，156.75，156.87
31P NMR(122MHz，DMSO-d 6)：δ18.12.
Anal.Cald for C 21H 25BrCl 2N 5O 3P：C，43.70；H，4.37；N，12.13；Found：C，43.51；H，4.65；N，11.84.
The preparation of embodiment 4:4-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-4)
The preparation of 4a:4-(3-bromoanilino)-6-(4-hydroxybutoxy) quinazoline
Preparation method is with embodiment 2a, and wherein the ethylene chlorhydrin of 2a replaces with 4-butylene-chlorohydrin.Productive rate 68%.mp 202-204℃。
1H NMR(300MHz，DMSO-d 6)：δ1.60-1.69(m，2H)，1.82-1.91(m，2H)，3.48-3.53(m，2H)，4.18(t，J＝6.0Hz，2H)，4.50(t，J＝4.8Hz，1H)，7.29-7.40(m，2H)，7.52(d，J＝9.0Hz，1H)，7.75(d，J＝9.0Hz，1H)，7.91-7.94(m，2H)，8.20(s，1H)，8.56(s，1H)，9.65(s，1H).
13C NMR(100MHz，DMSO-d 6)：δ25.50，29.05，60.42，68.43，102.92，115.76，120.79，121.32，124.24，124.62，126.04，129.56，130.50，141.04，145.07，152.13，156.69，157.06
Anal.Calcd for C 18H 18BrN 3O 2：C，55.68；H，4.67；N，10.82.Found：C，55.68；H，4.77；N，10.84.
The preparation of 4b:4-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(3-bromoanilino)-6-(4-hydroxybutoxy) quinazoline.Productive rate 36%.mp 70-71℃
1H NMR(400MHz，CDCl 3)：δ1.80-1.93(m，4H)，3.13(s，2H)，3.39-3.45(m，4H)，3.59(t，J＝6.4Hz，4H)，3.97-4.12(m，4H)，7.18-7.22(m，2H)，7.33(dd，J＝2.0，4.8Hz，1H)，7.72-7.77(m，2H)，7.84-7.85(m，1H)，8.03(s，1H)，8.64(s，1H)，9.40(br s，1H)
13C NMR(100MHz，CDCl 3)：δ24.79，26.70(d，J P-C＝7.0Hz)，42.26，48.73(d，J P-C＝5.0Hz)，64.69(d，J P-C＝4.0Hz)，67.63，102.72，116.05，120.88，121.94，124.53，125.03，126.62，129.10，129.81，140.49，144.85，152.36，156.84，157.28
31P NMR(122MHz，DMSO-d 6)：δ17.98.
Anal.Cald for C 22H 27BrCl 2N 5O 3P：C，44.69；H，4.60；N，11.84；Found：C，44.63；H，4.72；N，11.68.
The preparation of embodiment 5:3-(6-(4-(3-bromobenzene amido)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-5)
5a:3,4-dimethoxy p-methyl
By vanillic acid (3.36g, 20mmol), methyl iodide (5mL, 100mmol) and salt of wormwood (6.91g, 50mmol) are added in 50mL acetone, backflow 9h, suction filtration, acetone is washed, and obtains white solid after mother liquor concentrations to dodge post (P: E=8: 1) separation, 2.65g, productive rate 67%.mp 56-57 DEG C.
1H NMR(400MHz，CDCl 3)：δ3.90(s，3H)，3.93(s，3H)，3.94(s，3H)，6.89(d，J＝8.4Hz，1H)，7.55(d，J＝2.0Hz，1H)，7.69(dd，J＝2.0，8.4Hz，1H).
5b:2-nitro-4,5-dimethoxy p-methyl
3,4-dimethoxy p-methyl (0.98g, 5mmol) is dissolved in CHCl 3(25mL) in, drip the nitration mixture (1mL: 1mL) of concentrated nitric acid and the vitriol oil at 0 DEG C, then naturally rise to room temperature and react 1h, then to wash (25mL), water layer is with CHCl 3(25mL) extract; Merge organic phase, saturated common salt is washed to neutrality, anhydrous sodium sulfate drying, filters, concentrated, and separate out solid, suction filtration obtains faint yellow solid, 0.87g; 0.17g is obtained with ethyl acetate-light petrol recrystallization after mother liquor concentrations.Amount to 1.04g, productive rate 86%.mp144-145℃。
1H NMR(400MHz，CDCl 3)：δ3.92(s，3H)，3.98(s，3H)，3.99(s，3H)，7.09(s，1H)，7.46(s，1H).
5c:2-nitro-4-methoxyl group-5-hydroxy-benzoic acid
By (5mL) soluble in water for NaOH (1.22g), add 2-nitro-4,5-dimethoxy p-methyl (0.96g), backflow 17h.Cooling, adjust PH to 1 ~ 2 to make a large amount of yellow solid of precipitation with concentrated hydrochloric acid, suction filtration, is washed to neutrality, dries, obtains yellow solid 0.74g, productive rate 87%.mp 182-184℃。
1H NMR(400MHz，CDCl 3)：δ4.03(s，3H)，6.14(br s，1H)，7.27(s，1H)，7.48(s，1H).
5d:2-nitro-4-methoxyl group-5-methyl hydroxybenzoate
At 0 DEG C, 1mL sulfur oxychloride is added drop-wise in 50mL methyl alcohol, after stirring 5min, add 2-nitro-4-methoxyl group-5-hydroxy-benzoic acid (0.64g, 3mmol), 12h is reacted at 50 DEG C, solvent evaporated, obtains faint yellow solid 0.50g, productive rate 73% to dodge post (P: E=3: 2) separation.mp 150-151℃。
1H NMR(400MHz，DMSO-d 6)：δ3.80(s，3H)，3.91(s，3H)，7.07(s，1H)，7.62(s，1H)，10.94(br s，1H).
5e:2-amino-4-methoxyl-5-methyl hydroxybenzoate
By 2-nitro-4-methoxyl group-5-methyl hydroxybenzoate (0.70g, 3.08mmol) be added in 10mL ethyl acetate with 0.07g palladium carbon (10%), catalytic hydrogenation 12h under 4atm, suction filtration, concentrated, dodge post (P: E=3: 1 → 2: 1) separation and obtain white solid 0.57g, productive rate 94%.mp 158-159℃。
1H NMR(400MHz，DMSO-d 6)：δ3.71(s，3H)，3.74(s，3H)，6.24(br s，2H)，6.31(s，1H)，7.08(s，1H)，8.30(s，1H).
5f:4,6-dihydroxyl-7-methoxyquinazoline hydrochloride
By 2-amino-4-methoxyl-5-methyl hydroxybenzoate (2.0g, 10mmol) be added in 8mL methane amide with ammonium formiate (0.95g, 15mmol), at 150 DEG C, react 4h, cooling, add 50mL water, stir 30min, suction filtration, washing, dry, obtain faint yellow solid 1.50g, productive rate 77%.mp 296-298℃。
1H NMR(300MHz，DMSO-d 6)：δ3.90(s，3H)，7.10(s，1H)，7.38(s，1H)，7.91(s，1H)，9.82(br s，1H)，11.95(br s，1H).
5g:6-acetoxyl group-7-methoxyl group-4-hydroxyl-quinazoline
Preparation method is with embodiment 1b, and wherein 4, the 6-dihydroxyl quinazolines of 1b replace with 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride.Productive rate 87%.mp 290-292℃。
1H NMR(400MHz，DMSO-d 6)：δ2.30(s，3H)，3.92(s，3H)，7.28(s，1H)，7.75(s，1H)，8.08(s，1H)，12.21(br s，1H).
5h:4-(3-bromoanilino)-6-acetoxyl group-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1c, and wherein the 6-acetoxyl group-4-hydroxyquinazoline of 1c replaces with 6-acetoxyl group-7-methoxyl group-4-hydroxyquinazoline.Productive rate 48%.mp 180-182℃。
1H NMR(400MHz，DMSO-d 6)：δ2.39(s，3H)，4.01(s，3H)，7.42-7.48(m，3H)，7.77(d，J＝8.0Hz，1H)，8.07(s，1H)，8.66-8.69(m，1H)，8.94(s，1H)，11.19(br s，1H).
5i:4-(3-bromoanilino)-6-hydroxyl-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1d, and wherein 4-(3-the bromoanilino)-6-acetoxyl group quinazoline of 1d replaces with 4-(3-bromoanilino)-6-acetoxyl group-7-methoxyquinazoline hydrochloride.Productive rate 79%.mp 280-282℃。
1H NMR(400MHz，DMSO-d 6)：δ3.98(s，3H)，7.22-7.34(m，3H)，7.81(s，1H)，7.90(d，J＝7.6Hz，1H)，8.25(s，1H)，8.50(s，1H)，9.44(s，1H)，9.50(br s，1H).
5j:4-(3-bromoanilino)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 2a, and wherein the ethylene chlorhydrin of 2a replaces with 3-propylene chlorohydrin, and 4-(3-bromoanilino)-6-hydroxyquinazoline replaces with 4-(3-bromoanilino)-6-hydroxyl-7-methoxyquinazoline hydrochloride.Productive rate 78%.mp251-253℃。
1H NMR(400MHz，DMSO-d 6)：δ1.96-2.02(m，2H)，3.61-3.66(m，2H)，3.95(s，3H)，4.23(t，J＝6.4Hz，2H)，4.61(t，J＝4.8Hz，1H)，7.21-7.37(m，3H)，7.85(s，1H)，7.88(d，J＝8.4Hz，1H)，8.15(s，1H)，8.52(s，1H)，9.53(br s，1H)。
13C NMR(75MHz，DMSO-d 6)：δ32.10，55.94，57.37，66.05，102.38，107.21，109.03，120.63，121.33，124.08，125.71，130.45，141.32，147.00，148.59，152.66，154.58，156.00。
Anal.Calcd for C 18H 18BrN 3O 3：C，53.48；H，4.49；N，10.39.Found：C，53.31；H，4.62；N，10.09.
5k:3-(6-(4-(3-bromobenzene amido)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(3-bromoanilino)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride.Productive rate 30%.
1H NMR(400MHz，CDCl 3)：δ2.20-2.33(m，2H)，3.09(d，J＝4.8Hz，2H)，3.42-3.49(m，4H)，3.62(t，J＝6.0Hz，4H)，3.96(s，3H)，4.17-4.37(m，4H)，7.18-7.21(m，3H)，7.89-7.92(m，2H)，8.04(s，1H)，8.63(s，1H)，9.27(s，1H).
13C NMR(100MHz，CDCl 3)：δ29.46(d，J P-C＝5.0Hz)，42.39，48.76(d，J P-C＝5.0Hz)，56.03，62.93(d，J P-C＝6.0Hz)，67.00，105.14，107.54，109.70，120.40，122.04，124.58，126.18，129.93，141.07，147.60，148.08，153.65，155.20，156.83.
31P NMR(122MHz，CDCl 3)：δ16.07.
Anal.Calcd for C 22H 27BrCl 2N 5O 4P：C，43.51；H，4.48；N，11.53.Found：C，43.24；H，4.34；N，11.62.
The preparation of embodiment 6:3-(6-(4-(the chloro-4-fluoroanilino of 3-)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-6)
The preparation of 6a:4-(the chloro-4-fluoroanilino of 3-)-6-acetoxyl group-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1c, and wherein the 6-acetoxyl group-4-hydroxyquinazoline of 1c replaces with 6-acetoxyl group-7-methoxyl group-4-hydroxyquinazoline, and 3-bromaniline replaces with the chloro-4-fluoroaniline of 3-.Productive rate 78%.mp184-186℃。
1H NMR(400MHz，DMSO-d 6)：δ2.39(s，3H)，4.01(s，3H)，7.46(s，1H)，7.54(t，J＝8.8Hz，1H)，7.71-7.75(m，1H)，8.07(dd，J＝2.4，6.8Hz，1H)，8.62(s，1H)，8.91(s，1H)，8.94(s，1H)，11.12(br s，1H).
The preparation of 6b:4-(the chloro-4-fluoroanilino of 3-)-6-hydroxyl-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1d, and wherein 4-(3-the bromoanilino)-6-acetoxyl group quinazoline of 1d replaces with 4-(the chloro-4-fluoroanilino of 3-)-6-acetoxyl group-7-methoxyquinazoline hydrochloride.Productive rate 85%.mp 285-288℃。
1H NMR(400MHz，DMSO-d 6)：δ3.98(s，3H)，7.22(s，1H)，7.41(t，J＝9.2Hz，1H)，7.78(s，1H)，7.82-7.86(m，1H)，8.21(dd，J＝2.4，6.8Hz，1H)，8.48(s，1H)，9.46(s，1H)，9.67(s，1H).
6c:4-(the chloro-4-fluoroanilino of 3-)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 2a, and wherein the ethylene chlorhydrin of 2a replaces with 3-propylene chlorohydrin, and 4-(3-bromoanilino)-6-hydroxyquinazoline replaces with 4-(the chloro-4-fluoroanilino of 3-)-6-hydroxyl-7-methoxyquinazoline hydrochloride.Productive rate 91%.mp 280-282℃。
1H NMR(400MHz，DMSO-d 6)：δ1.95-2.03(m，2H)，3.61-3.65(m，2H)，3.94(s，3H)，4.23(t，J＝6.0Hz，2H)，4.62(t，J＝4.8Hz，1H)，7.21(s，1H)，7.44(t，J＝8.8Hz，1H)，7.78-7.83(m，2H)，8.12(dd，J＝2.4，6.8Hz，1H)，8.50(s，1H)，9.56(s，1H).
13C NMR(75MHz，DMSO-d 6)：δ32.07，55.96，57.34，66.05，102.39，107.23，108.86，116.61( 2J F-C＝21Hz)，118.91( 2J F-C＝17.9Hz)，122.34( 3J F-C＝6.8Hz)，123.50，136.84，146.92，148.58，152.66，153.27( 1J F-C＝240Hz)，154.59，156.05.
Anal.Calcd for C 18H 17ClFN 3O 3：C，57.22；H，4.54；N，11.12.Found：C，56.97；H，4.70；N，10.95.
6d:3-(6-(4-(the chloro-4-fluoroanilino of 3-)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(the chloro-4-fluoroanilino of 3-)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride.Productive rate 46%.mp 150-152℃。
1H NMR(400MHz，CDCl 3)：δ2.19-2.29(m，2H)，3.19(d，J＝4.4Hz，2H)，3.39-3.50(m，4H)，3.61(t，J＝6.0Hz，4H)，3.93(s，3H)，4.16-4.36(m，4H)，7.09(t，J＝8.8Hz，1H)，7.15(s，1H)，7.72-7.76(m，1H)，7.84(s，1H)，7.96(dd，J＝2.4，6.4Hz，1H)，8.59(s，1H)，9.03(br s，1H).
13C NMR(100MHz，CDCl 3)：δ29.45(d，J P-C＝4.0Hz)，42.27，48.65(d，J P-C＝5.0Hz)，56.00，62.89(d，J P-C＝5.0Hz)，66.98，105.10，107.43，109.47，116.12( 2J F-C＝22Hz)，120.30( 2J F-C＝19Hz)，121.84( 3J F-C＝6Hz)，124.02，136.26( 3J F-C＝3Hz)，147.42，148.08，153.57，154.24( 1J F-C＝244Hz)，155.19，156.86.
31P NMR(122MHz，CDCl 3)：δ15.79.
Anal.Calcd for C 22H 26C1 3FN 5O 4P：C，45.49；H，4.51；N，12.06.Found：C，45.72；H，4.64；N，11.89.
The preparation of embodiment 7:3-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMA-7)
7a:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-acetoxyl group-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1c, and wherein the 6-acetoxyl group-4-hydroxyquinazoline of 1c replaces with 6-acetoxyl group-7-methoxyl group-4-hydroxyquinazoline, and 3-bromaniline replaces with the chloro-4-of 3-(3-fluorine benzyloxy) aniline.Productive rate is 52%.mp 194-196℃
1H NMR(400MHz，DMSO-d 6)：δ2.37(s，3H)，3.95(s，3H)，5.25(s，2H)，7.17-7.21(m，1H)，7.25-7.34(m，4H)，7.45-7.51(m，1H)，7.72(dd，J＝2.8，5.2Hz，1H)，8.04(d，J＝2.8Hz，1H)，8.28(s，1H)，8.56(s，1H).
7b:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-hydroxyl-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 1d, and wherein 4-(3-the bromoanilino)-6-acetoxyl group quinazoline of 1d replaces with 4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-acetoxyl group-7-methoxyquinazoline hydrochloride.Productive rate 86%.mp239-241 DEG C
1H NMR(400MHz，DMSO-d 6)：δ3.97(s，3H)，5.24(s，2H)，7.16-7.25(m，3H)，7.30-7.34(m，2H)，7.45-7.50(m，1H)，7.73(dd，J＝2.4，8.8Hz，1H)，7.76(s，1H)，8.04(d，J＝2.4Hz，1H)，8.43(s，1H)
7c:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride
Preparation method is with embodiment 2a, wherein the ethylene chlorhydrin of 2a replaces with 3-propylene chlorohydrin, and 4-(3-bromoanilino)-6-hydroxyquinazoline replaces with 4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-hydroxyl-7-methoxyquinazoline hydrochloride.Productive rate 83%.mp 212-213℃
1H NMR(400MHz，DMSO-d 6)：δ1.97-2.03(m，2H)，3.65(t，J＝6.0Hz，2H)，3.94(s，3H)，4.22(t，J＝6.4Hz，2H)，5.25(s，2H)，7.18-7.35(m，5H)，7.45-7.51(m，1H)，7.72(dd，J＝2.4，8.8Hz，1H)，7.81(s，1H)，7.97(d，J＝2.4Hz，1H)，8.45(s，1H).
13C NMR(100MHz，DMSO-d 6)：δ32.08，55.91，57.36，66.03，69.54，102.48，107.19，108.80，114.14( 2J F-C＝22Hz)，114.43，114.82( 2J F-C＝21Hz)，121.19，122.19，123.45( 4J F-C＝3Hz)，124.02，130.68( 3J F-C＝8Hz)，133.55，139.79( 3J F-C＝7Hz)，146.73，148.45，149.51，152.84，154.46，156.24，162.33( 1J F-C＝242Hz).
Anal.Calcd for C 25H 23ClFN 3O 4：C，62.05；H，4.79；N，8.68.Found：C，61.79；H，4.77；N，8.65.
7d:3-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 1e, and wherein 4-(3-the bromoanilino)-6-hydroxyquinazoline of 1e replaces with 4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-(3-hydroxy propyloxy group)-7-methoxyquinazoline hydrochloride.Productive rate 29%.mp137-138℃。
1H NMR(400MHz，CDCl 3)：δ2.19-2.25(m，2H)，3.20(d，J＝4.8Hz，2H)，3.38-3.45(m，4H)，3.57(t，J＝6.4Hz，4H)，3.91(s，3H)，4.13-4.33(m，4H)，6.88(d，J＝8.8Hz，1H)，6.98-7.02(m，1H)，7.14-7.22(m，3H)，7.31-7.36(m，1H)，7.65(d，J＝2.4，8.8Hz，1H)，7.79(s，1H)，7.85(d，J＝2.8Hz，1H)，8.57(s，1H)，9.21(s，1H).
13C NMR(100MHz，CDCl 3)：δ29.46(d，J P-C＝5.0Hz)，42.32，48.74(d，J P-C＝5.0Hz)，55.96，62.84(d，J P-C＝6.0Hz)，66.77，70.35，104.77，107.40，109.45，113.90( 2J F-C＝22Hz)，114.24，114.75( 2J F-C＝21Hz)，121.87，122.41( 4J F-C＝2Hz)，122.90，124.57，130.06( 3J F-C＝8Hz)，133.67，139.22( 3J F-C＝8Hz)，147.27，147.96，150.20，153.71，154.99，157.00，162.91( 1J F-C＝245Hz).
31P NMR(122MHz，CDCl 3)：δ15.75.
Anal.Calcd for C 29H 32Cl 3FN 5O 5P：C，50.71；H，4.70；N，10.20.Found：C，50.56；H，4.88；N，9.97.
The preparation of embodiment 8:2-oxo-2-(6-(4-(3-bromobenzene amido) quinazoline) is amino) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMB-1)
8a:2-benzoyloxy ethanol
Ethylene glycol (4.65g, 75mmol) is dissolved in CHCl 3(50mL), in, under stirring, Na is added 2cO 3(5.30g, 50mmol), then drip Benzoyl chloride (7.03g, 50mmol), then reflux 10h.Cooling, add 100mL water, separatory after jolting, water layer is with CHCl 3extraction (30mL × 2); Merge organic phase, with saturated common salt washing (50mL × 2), anhydrous sodium sulfate drying, filters, concentrated, and dodge post (silica gel, P: E=10: 1 → 2: 1) and be separated to obtain colorless oil 6.30g, productive rate is 76%.
1H NMR(300MHz，CDCl 3)：δ2.13(t，J＝5.4Hz，1H)，3.94-3.99(m，2H)，4.47(t，J＝4.5Hz，1H)，7.43-7.61(m，3H)，8.05-8.08(m，2H).
8b:2-benzoxy guanidine-acetic acid
The 16.9mL vitriol oil is dissolved in 200mL water, under ice-water bath, adds CrO 3(14.30g, 143mmol), is stirred to dissolve, and then slowly drips the acetone soln (80mL) of 2-benzoyloxy ethanol (6.30g, 37.91mmol).After dropwising, rise to room temperature reaction 2h, then add 100mL water, with extraction into ethyl acetate (50mL × 3); Merge organic phase, saturated common salt is washed to neutrality, then alkalizes to pH=7 ~ 8 with NaOH solution, with water extraction (70mL × 2); Combining water layer, is acidified to pH=3 ~ 4, with extraction into ethyl acetate (50mL × 3); Combined ethyl acetate layer, is washed to neutrality with saturated common salt, anhydrous sodium sulfate drying, filters, concentrated, and dodge post (silica gel, P: E: HOAc=4mL: 1mL: 2drops) separation and obtain white solid, 2.40g, productive rate is 35%.mp 111-112℃。
1H NMR(300MHz，CDCl 3)：δ4.91(s，2H)，7.43-7.63(m，3H)，8.07-8.11(m，2H)，10.15(br s，1H)。
8c:4-hydroxyquinazoline
Be added to by anthranilic acid (6.86g, 50mmol) in 10mL methane amide, react 6h at 150 DEG C, cooling, adds 100mL water, stirs 1h, separates out a large amount of white solid, suction filtration, and washing is dried, obtained 3.93g, productive rate 53%.mp 214-216 DEG C.
1H NMR(400MHz，DMSO-d 6)：δ7.52(t，J＝7.6Hz，1H)，7.67(d，J＝8.0Hz，1H)，7.82(t，J＝7.6Hz，1H)，8.09(s，1H)，8.12(d，J＝8.0Hz，1H)，12.22(br s，1H).
8d:4-hydroxyl-6-nitro-quinazoline
By 4-hydroxyquinazoline (3.93g at 0 DEG C, 26.89mmol) be added in the nitration mixture that the 8mL vitriol oil and 8mL concentrated nitric acid be made into, add under stirring, then at 90-95 DEG C, react 1h, cooling, pour in frozen water, separate out a large amount of yellow solid, suction filtration, washing, dry, obtain 4.41g, productive rate 85%.mp 275-277℃。
1H NMR(400MHz，DMSO-d 6)：δ7.87(d，J＝8.8Hz，1H)，8.32(s，1H)，8.55(dd，J＝2.4，8.8Hz，1H)，8.81(d，J＝2.4Hz，1H)，12.75(br s，1H).
8e:4-(3-bromoanilino)-6-nitro-quinazoline
4-hydroxyl-6-nitro-quinazoline (4.4g, 23mmol) is added to SOCl 2(30mL), in, add 0.1mL DMF, be back to clarification, pressure reducing and steaming SOCl 2, add i-PrOH (140mL), m-bromoaniline (3.96g, 23mol) and triethylamine (2.33g, 23mmol), backflow 6h.Cooling, separate out yellow solid, suction filtration, successively with Virahol, water and ether are washed, and dry, obtain yellow solid 5.88g, productive rate is 74%.mp 268-269℃。
1H NMR(300MHz，DMSO-d 6)：δ7.36-7.43(m，2H)，7.90-7.97(m，2H)，8.20(s，1H)，8.57(dd，J＝1.2，9.0Hz，1H)，8.78(s，1H)，9.65(s，1H)，10.48(s，1H)
8f:4-(3-bromoanilino)-6-amido quinazoline
Glass putty (5.07g, 42.75mmol) is added in 22mL concentrated hydrochloric acid, under stirring, adds 4-(3-bromoanilino)-6-nitro-quinazoline (2.95g, 8.55mmol), reaction is spent the night, and adds 200mL water and 200mL ethyl acetate, regulate PH to 8-9 with sodium hydroxide under vigorous stirring, water layer, again with 100mL extraction into ethyl acetate, merges organic phase, saturated common salt is washed, anhydrous sodium sulfate drying, filters, concentrated, obtain product 2.52g, productive rate 94%.mp 263-265℃。
1H NMR(300MHz，DMSO-d 6)：δ5.64(br s，2H)，7.22-7.35(m，4H)，7.56(d，J＝9.0Hz，1H)，7.90(d，J＝8.4Hz，1H)，8.24(t，J＝1.8Hz，1H)，8.39(s，1H)，9.46(br s，1H).
8g:4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline
2-benzoxy guanidine-acetic acid (0.91g, 5mmol) is dissolved in 15mL chloroform, at 0 DEG C, drips SOCl 2, then the 4h that refluxes (2mL).Pressure reducing and steaming CHCl 3and SOCl 2, add THF (10mL) wiring solution-forming, be added drop-wise to 4-(3-bromoanilino)-6-amido quinazoline (1.58g, 5mmol) and Na 2cO 3in the THF suspension (50mL) of (0.53g, 5mmol), room temperature reaction 12h.Boil off THF, add methyl alcohol (50mL), water (10mL) and NaOH (0.80g, 20mmol), the system of being stirred to becomes settled solution; Evaporated under reduced pressure, adds 50mL water, with extraction into ethyl acetate (30mL × 2), and saturated common salt washing (30mL × 2), anhydrous Na 2sO 4drying, filters, concentrated, and dodge post (silica gel, P: E=1: 6 → E) and be separated to obtain yellow solid, 1.75g, productive rate is 93.78%.mp 232-234℃。
1H NMR(300MHz，DMSO-d 6)：δ4.09(d，J＝5.7Hz，2H)，5.86(t，J＝5.7Hz，1H)，7.28-7.38(m，2H)，7.80(d，J＝9.0Hz，1H)，7.88(d，J＝7.8Hz，1H)，8.08(dd，J＝1.8，9.0Hz，1H)，8.18(d，J＝1.8Hz，1H)，8.59(s，1H)，8.71(s，1H)，9.85(s，1H)，9.93(s，1H)
13C NMR(75MHz，DMSO-d 6)：δ61.88，112.46，115.41，120.83，121.37，124.28，126.11，127.60，128.49，130.54，136.22，141.10，146.77，153.33，157.31，171.29
Anal.Cald for C 16H 13BrN 4O 2：C，51.49；H，3.51；N，15.01；Found：C，51.44；H，3.60；N，14.75.
8h:2-oxo-2-(6-(4-(3-bromobenzene amido) quinazoline) is amino) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline (0.38g, 1mmol) is placed in 100mL there-necked flask, N 2protection; add 40mL anhydrous tetrahydro furan; NaH (0.048g is added at 0 DEG C; 1.2mmol; the mineral oil dispersion system of 60%), after reacting 1h at 0 DEG C, this reaction solution is added drop-wise to the tetrahydrofuran solution (10mL) of the dichlor-phosphoryl mustargen (0.31g, 1.2mmol) be placed at 0 DEG C; 3h is reacted at 0 DEG C, more logical ammonia 30min.Add 50mL saturated aqueous common salt toward reaction system, adjust PH to neutral, separatory, water layer, again with extraction into ethyl acetate (50mL × 2), merges organic phase, anhydrous sodium sulfate drying, filters, concentrated, dodges post (E → E: CH 3oH=15: 1 → 8: 1) separation obtains faint yellow blister solid, 0.17g, productive rate 18%.
1H NMR(300MHz，CDCl 3)：δ2.52(d，J＝5.4Hz，2H)，3.54-3.61(m，4H)，3.72(t，J＝5.7Hz，4H)，4.72-4.93(m，2H)，7.11-7.21(m，2H)，7.56-7.61(m，3H)，7.85(s，1H)，7.96(br s，1H)，8.27(s，1H)，8.59(s，1H)，9.32(br s，1H).
13C NMR(100MHz，DMSO-d 6)：δ42.32，48.13(d，J P-C＝4.0Hz)，64.61(d，J P-C＝3.0Hz)，112.76，115.46，120.88，121.31，124.38，126.08，127.43，128.44，130.33，135.83，141.01，146.70，153.31，157.30，166.18
31P NMR(122MHz，DMSO-d 6)：δ10.94.
HRMS(ESI+)m/z calcd for C 20H 22BrCl 2N 6O 3P(M+H) +，574.00514，found574.00532
The preparation of embodiment 9:3-oxo-3-(6-(4-(3-bromobenzene amido) quinazoline) is amino) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMB-2)
9a:3-benzoyloxy propyl alcohol
Preparation method is with embodiment 8a, and wherein the ethylene glycol of 8a replaces with 1,3-PD.Productive rate is 72%.
1H NMR(300MHz，CDCl 3)：δ1.94(br s，1H)，1.98-2.06(m，2H)，3.78(t，J＝6.0Hz，2H)，4.50(t，J＝6.0Hz，2H)，7.42-7.47(m，2H)，7.55-7.60(m，1H)，8.03-8.06(m，2H)。
9b:3-benzoyloxy propionic acid
Preparation method is with embodiment 8b, and wherein the 2-benzoyloxy ethanol of 8b replaces with 3-benzoyloxy propyl alcohol.Productive rate is 68%.mp 66-68℃。
1H NMR(300MHz，CDCl 3)：δ2.83(t，J＝6.3Hz，2H)，4.58(t，J＝6.3Hz，2H)，7.40-7.59(m，3H)，8.00-8.07(m，2H)，9.73(br s，1H)。
9c:4-(3-bromoanilino)-6-(3-hydroxyl propionamido) quinazoline
Preparation method is with embodiment 8g, and wherein the 2-benzoxy guanidine-acetic acid of 8g replaces with 3-benzoyloxy propionic acid.Productive rate is 51%.mp 216-218℃。
1H NMR(300MHz，DMSO-d 6)：δ2.56(t，J＝6.3Hz，2H)，3.74-3.80(m，2H)，4.75(t，J＝5.1Hz，1H)，7.27-7.37(m，2H)，7.77-7.89(m，3H)，8.17(s，1H)，8.57(s，1H)，8.73(d，J＝1.8Hz，1H)，9.93(s，1H)，10.26(s，1H).
13C NMR(75MHz，CD 3OD)：δ40.96，59.20，112.56，116.86，122.26，123.12，126.44，128.21，128.30，128.85，131.26，138.36，141.83，147.40，154.53，159.48，172.86
Anal.Cald for C 17H 15BrN 4O 2：C，52.73；H，3.90；N，14.47；Found：C，52.48；H，4.03；N，14.28.
9d:3-oxo-3-(6-(4-(3-bromobenzene amido) quinazoline) is amino) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 8h, and wherein 4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline of 8h replaces with 4-(3-bromoanilino)-6-(3-hydroxyl propionamido) quinazoline.Productive rate 33%.mp 148-150℃
1H NMR(400MHz，DMSO-d 6)：δ2.52(d，J＝5.4Hz，2H)，3.14-3.44(m，10H)，4.48-4.66(m，2H)，7.30-7.38(m，2H)，7.75(dd，J＝2.0，8.8Hz，1H)，7.88(s，1H)，7.90(s，1H)，8.19(t，J＝2.0Hz，1H)，8.51(s，1H)，8.69(s，1H)
13C NMR(100MHz，DMSO-d 6)：δ34.12，41.99，48.23(d，J P-C＝4.0Hz)，62.22(d，J P-C＝5.0Hz)，115.25，121.05，121.35，124.26，124.54，126.52，128.94，130.60，132.57，134.87，140.65，149.40，155.19，157.56，170.91(d，J P-C＝5.0Hz)
31P NMR(122MHz，DMSO-d 6)：δ7.33.
HRMS(ESI+)m/z calcd for C 21H 24BrCl 2N 6O 3P(M+H) +，588.02079，found588.02092.
The preparation of embodiment 10:4-oxo-4-(6-(4-(3-bromobenzene amido) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMB-3)
10a:4-benzoyloxy butanols
Preparation method is with embodiment 8a, and wherein the ethylene glycol of 8a replaces with BDO.Productive rate is 82%.
1H NMR(300MHz，CDCl 3)：δ1.69-1.78(m，2H)，1.83-1.92(m，2H)，3.73(t，J＝6.3Hz，2H)，4.37(t，J＝6.3Hz，2H)，7.41-7.46(m，2H)，7.53-7.58(m，1H)，8.04-8.06(m，2H)。
10b:4-benzoyloxybutanoic acid
Preparation method is with embodiment 8b, and wherein the 2-benzoyloxy ethanol of 8b replaces with 4-benzoyloxy butanols.Productive rate is 35%.
1H NMR(300MHz，CDCl 3)：δ2.08-2.71(m，2H)，2.55(t，J＝6.0Hz，2H)，4.39(t，J＝6.3Hz，2H)，7.44(t，J＝7.5Hz，1H)，8.02(dd，J＝1.5，7.5Hz，2H)，10.79(brs，1H)。
10c:4-(3-bromoanilino)-6-(4-maloyl group is amino) quinazoline
Preparation method is with embodiment 8g, and wherein the 2-benzoxy guanidine-acetic acid of 8g replaces with 4-benzoyloxybutanoic acid.Productive rate is 50%.mp 175-176℃
1H NMR(300MHz，DMSO-d 6)：δ1.77-1.86(m，2H)，2.47(t，J＝7.5Hz，2H)，3.47-3.53(m，2H)，4.59(t，J＝4.8Hz，1H)，7.28-7.38(m，2H)，7.77-7.88(m，3H)，8.18(s，1H)，8.59(s，1H)，8.76(s，1H)，9.92(s，1H)，10.28(s，1H).
13C NMR(100MHz，DMSO-d 6)：δ28.38，33.08，60.23，111.65，115.50，120.89，121.27，124.34，125.99，127.21，128.45，130.41，137.16，141.14，146.53，153.04，157.29，171.81
Anal.Cald for C 18H 17BrN 4O 2：C，53.88；H，4.27；N，13.96；Found：C，53.58；H，4.35；N，13.68.
10d:4-oxo-4-(6-(4-(3-bromobenzene amido) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 8h, and wherein 4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline of 8h replaces with 4-(3-bromoanilino)-6-(4-maloyl group is amino) quinazoline.Productive rate 44%.
1H NMR(300MHz，CDCl 3，)：δ2.01-2.07(m，2H)，2.52(d，J＝5.4Hz，2H)，3.32-3.46(m，6H)，3.59(t，J＝8.4Hz，4H)，4.01-4.11(m，2H)，7.14-7.22(m，2H)，7.62-7.67(m，3H)，7.97(s，1H)，8.50-8.61(m，3H)，9.95(s，1H)
13C NMR(100MHz，DMSO-d 6)：δ26.41(d，J P-C＝5.0Hz)，33.07，42.55(d，J P-C＝6.0Hz)，48.81，64.57(d，J P-C＝5.0Hz)，111.08，115.40，120.67，122.11，124.86，126.86，128.24，129.93，136.69，140.14，146.21，153.32，157.38，171.98
31P NMR(122MHz，DMSO-d 6)：δ18.08.
Anal.Cald for C 22H 26BrCl 2N 6O 3P：C，43.73；H，4.34；N，13.91；Found：C，43.81；H，4.55；N，14.07.
The preparation of embodiment 11:4-oxo-4-(6-(4-(chloro-4 fluoroanilino of 3-) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMB-4)
11a:4-(the chloro-4-fluoroanilino of 3-)-6-nitro-quinazoline
The same 8e of preparation method, wherein the 3-bromaniline of 8e replaces with the chloro-4-fluoroaniline of 3-.Productive rate 65%.mp278-280℃。
1H NMR(400MHz，DMSO-d 6)：δ7.50(t，J＝9.2Hz，1H)，7.84(d，J＝8.4Hz，1H)，7.97(d，J＝8.8Hz，1H)，8.17(d，J＝4.8Hz，1H)，8.59(dd，J＝2.4，9.2Hz，1H)，8.79(s，1H)，9.64(s，1H)，10.56(br s，1H).
11b:4-(the chloro-4-fluoroanilino of 3-)-6-amido quinazoline
Preparation method is with embodiment 8f, and wherein 4-(3-the bromoanilino)-6-nitro-quinazoline of 8f replaces with 4-(the chloro-4-fluoroanilino of 3-)-6-nitro-quinazoline.Productive rate 92%.mp 253-255℃。
1H NMR(400MHz，DMSO-d 6)：δ5.63(s，2H)，7.26(dd，J＝2.0，8.8Hz，1H)，7.13(d，J＝2.0Hz，1H)，7.41(t，J＝8.4Hz，1H)，7.55(d，J＝9.2Hz，1H)，7.81-7.85(m，1H)，8.21(dd，J＝2.4，6.8Hz，1H)，8.37(s，1H)，9.48(s，1H).
11c:4-(the chloro-4-fluoroanilino of 3-)-6-(4-maloyl group is amino) quinazoline
Preparation method is with embodiment 8g, and wherein the 2-benzoxy guanidine-acetic acid of 8g replaces with 4-benzoyloxybutanoic acid, and 4-(3-bromoanilino)-6-amido quinazoline replaces with 4-(the chloro-4-fluoroanilino of 3-)-6-amido quinazoline.Productive rate is 79%.mp 208-209℃
1H NMR(400MHz，DMSO-d 6)：δ1.77-1.84(m，2H)，2.46(t，J＝7.6Hz，2H)，3.48(t，J＝6.4Hz，2H)，7.44(t，J＝8.4Hz，1H)，7.77-7.86(m，3H)，8.13(dd，J＝2.4，6.8Hz，1H)，8.55(s，1H)，8.70(d，J＝1.6Hz，1H)，10.29(s，1H).
13C NMR(75MHz，DMSO-d 6)：δ28.38，33.08，60.22，111.44，115.37，116.61( 2J F-C＝21.0Hz)，118.91( 2J F-C＝18.6Hz)，122.70( 3J F-C＝6.8Hz)，123.83，127.18，128.49，136.68，137.08，146.47，153.05，153.48( 1J F-C＝241Hz)，157.34，171.74
Anal.Cald for C 18H 16ClFN 4O 2：C，57.68；H，4.30；N，14.95；Found：C，57.48；H，4.30；N，14.90.
11d:4-oxo-4-(6-(4-(3 chloro-4 fluoroanilino) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 8h, and wherein 4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline of 8h replaces with 4-(3 chloro-4 fluoroanilino)-6-(4-maloyl group is amino) quinazoline.Productive rate 32%.
1H NMR(400MHz，CDCl 3)：δ2.00-2.06(m，2H)，2.46-2.58(m，2H)，3.40-3.63(m，10H)，3.99-4.09(m，2H)，7.05(t，J＝8.8Hz，1H)，7.52-7.67(m，3H)，7.83(dd，J＝2.4，6.4Hz，1H)，8.52(s，1H)，8.54(s，1H)，8.73(s，1H)，10.03(s，1H)
13C NMR(100MHz，CDCl 3)：δ26.56(d，J P-C＝5.0Hz)，33.02，42.26，48.74(d，J P-C＝5.0Hz)，64.50(d，J P-C＝5.0Hz)，111.03，115.27，116.25(d， 2J F-C＝22.0Hz)，120.52(d， 2J F-C＝18.0Hz)，121.99( 3J F-C＝7.0Hz)，124.25，126.77，128.45，135.32，136.52，146.44，153.36，154.65( 1J F-C＝232Hz)，157.39，171.98
31P NMR(122MHz，CDCl 3)：δ18.00.
Anal.Cald for C 22H 25C 13FN 6O 3P：C，45.73；H，4.36；N，14.54；Found：C，45.79；H，4.66；N，14.43.
The preparation of embodiment 12:4-oxo-4-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester (EMB-5)
12a:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-nitro-quinazoline
The same 8e of preparation method, wherein the 3-bromaniline of 8e replaces with the chloro-4-of 3-(3-fluorine benzyloxy) aniline.Productive rate 97%.mp 279-281℃
1H NMR(400MHz，DMSO-d 6)：δ5.30(s，2H)，7.18-7.22(m，1H)，7.31-7.36(m，3H)，7.46-7.51(m，1H)，7.71(d，J＝8.8Hz，1H)，7.98-8.04(m，2H)，8.68(t，J＝6.8Hz，1H)，8.88(d，J＝10.0Hz，1H)，9.71(d，J＝6.0Hz，1H)，11.19(br s，1H).
12b:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-amido quinazoline
Preparation method is with embodiment 8f, and wherein 4-(3-the bromoanilino)-6-nitro-quinazoline of 8f replaces with 4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-nitro-quinazoline.Productive rate 97%.mp 250-252℃
1H NMR(400MHz，DMSO-d 6)：δ5.28(s，2H)，7.17-7.21(m，1H)，7.28-7.39(m，5H)，7.45-7.51(m，1H)，7.59(d，J＝9.2Hz，1H)，7.65(dd，J＝2.0，8.8Hz，1H)，7.94(d，J＝2.4Hz，1H)，8.52(s，1H).
12c:4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-(4-maloyl group is amino) quinazoline
Preparation method is with embodiment 8g, wherein the 2-benzoxy guanidine-acetic acid of 8g replaces with 4-benzoyloxybutanoic acid, and 4-(3-bromoanilino)-6-amido quinazoline replaces with 4-(the chloro-4-of 3-(3-fluorine benzyloxy) phenylamino)-6-amido quinazoline.Productive rate is 51%.mp 209-211℃
1H NMR(400MHz，DMSO-d 6)：δ1.77-1.84(m，2H)，2.46(t，J＝7.2Hz，2H)，3.49(t，J＝7.2Hz，2H)，5.26(s，2H)，7.17-7.35(m，4H)，7.45-7.51(m，1H)，7.69-7.85(m，3H)，7.98(d，J＝2.0Hz，1H)，8.51(s，1H)，8.68(s，1H).
13C NMR(75MHz，DMSO-d 6)：δ28.41，33.09，60.24，69.52，111.57，114.15( 2J F-C＝26.0Hz)，114.34，114.83( 2J F-C＝20.4Hz)，115.36，121.15，122.47，123.45，124.26，127.05，128.41，130.70( 3J F-C＝8.0Hz)，133.41，136.93，139.80( 3J F-C＝6.8Hz)，146.40，149.69，153.27，157.45，162.35( 1J F-C＝241Hz)，171.70
Anal.Cald for C 25H 22ClFN 4O 3：C，62.44；H，4.61；N，11.65；Found：C，62.17；H，4.68；N，11.38.
12d:4-oxo-4-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester
Preparation method is with embodiment 8h, and wherein 4-(3-bromoanilino)-6-(2-hydroxyacetamido) quinazoline of 8h replaces with 4-(3 chloro-4-(3-fluorine benzyloxy) anilino)-6-(4-maloyl group is amino) quinazoline.Productive rate 26%.
1H NMR(400MHz，CDCl 3)：δ1.95-2.01(m，2H)，2.48(d，J＝4.8Hz，2H)，3.36-3.43(m，4H)，3.52-3.60(m，6H)，3.97-4.06(m，2H)，5.04(s，2H)，6.83(d，J＝9.2Hz，1H)，6.97-7.01(m，1H)，7.16-7.20(m，2H)，7.29-7.35(m，1H)，7.46(dd，J＝1.2，8.4Hz，1H)，7.57(d，J＝8.8Hz，1H)，7.64(d，J＝8.8Hz，1H)，7.73(d，J＝2.0Hz，1H)，8.52(s，2H)，8.67(br s，1H)，10.01(br s，1H)
13C NMR(100MHz，CDCl 3)：δ26.44(d，J P-C＝6.0Hz)，32.97，42.27，48.76(d，J P-C＝5.0Hz)，64.46(d，J P-C＝5.0Hz)，70.22，111.09，113.86( 2J F-C＝22.0Hz)，113.97，114.77( 2J F-C＝21.0Hz)，115.25，122.10，122.40( 4J F-C＝2.0Hz)，122.88，124.81，126.65，128.21，130.08( 3J F-C＝8.0Hz)，132.62，136.46，139.06( 3J F-C＝7.0Hz)，146.22，150.65，153.58，157.57，162.88( 1J F-C＝245Hz)，171.89.
31P NMR(122MHz，CDCl 3)：δ18.10.
HRMS(ESI+)m/z calcd for C 29H 32Cl 3FN 6O 4P(M+H) +，683.12668，found683.12562(M+H) +.
Anti-tumor activity test
The anti-tumor activity of experimental example 1 the compounds of this invention
On the impact of HCT116, MDA-MB-468 and SK-BR-3 cell proliferation
Vitro culture HCT116, MDA-MB-468 and SK-BR-3 cell.Growth of Cells is to logarithmic growth after date, and collecting cell, centrifugal 5 minutes of 1000rpm, abandons supernatant, and appropriate substratum suspends, adjustment cell concn to 3.5 × 10 4individual/mL.By cell suspension inoculation in 96 porocyte culture plates, every hole 100 μ L, placement cell culture incubator (37 DEG C, 5%CO 2) in cultivate after 24h, the every hole of medication group adds the medicine 100 μ L of cell culture medium, and three multiple holes established by often kind of medicine, and negative control group is for containing 0.5%DMSO substratum.After cultivating 72h in incubator, every hole adds the MTT 20 μ L of 5mg/mL, places 3h for 37 DEG C.Every hole adds 150 μ L DMSO, and 37 DEG C of shaking table vibration 5min, survey absorbancy (OD) in 492nm/620nm.Prism Graphpad statistical software is used to calculate IC 50value.
The Cyto toxic experiment showed of EMA-and EMB-series compound
Select SK-BR-3 (HER-2 high expression level), MDA-MB-468 (EGFR high expression level), HCT116 (all low expression of EGFR and HER-2) three kinds of tumor models, evaluate the anti-tumor activity of EMA-and EMB-series compound.Activity Results is listed in table 1.
Table 1. compound is to the IC of three tumor cell lines 50value
The data presentation of table 1, the connecting arm between Glyciphosphoramide and quinazoline ring is good with 3 ~ 4 carbon chain length; To SK-BR-3 (HER-2 high expression level), MDA-MB-468 (EGFR high expression level), HCT116 (all low expression of EGFR and HER-2) three kinds of tumor models, compd E MA-3,4,5,6,7 and compd E MB-3, the anti-tumor activity of 4,5 is better than reference substance lapatinibditosylate.
Experimental example 2 the compounds of this invention EMB-3 Study on mechanism
1, experimental technique:
Z '-LYTE Tyr 4 kinase assays test kit (Invitrogen, US) and Tyr-4 peptide substrates is used to carry out enzyme Binding experiment.
A, enzyme concn optimization
Detecting the response situation under different enzyme concn, choosing best enzyme concn and testing.The optimization of ATP concentration and incubation time is with the optimization of enzyme concn.
(1) as shown in the figure, to different concns, use multichannel pipettor from 96 orifice plate A1-12 holes difference transferase 45 μ L enzyme solution to 368 orifice plates afterwards EGFR doubling dilution in 96 orifice plates;
(2) enzyme reaction: add 5 μ L 1 × kinase buffer liquid in the 96 capable every holes of orifice plate M-N, every hole adds 5 μ L phosphopeptide solution in N is capable, adds 5 μ L peptide/ATP mixtures in the capable every hole of A-M, reacts 30min at 25 DEG C after jolting mixing.
(3) excision reaction: add 5 μ L in the capable each hole of A-N and excise liquid, reacts 30min at 25 DEG C after jolting mixing.
(4) termination reaction and fluoroscopic examination: add 5 μ l stop buffers in each hole of reaction, jolts mixing 30s.(Ex.400nm, Em.445nm is detected by microplate reader; Ex.400nm, Em.520nm) fluorescence intensity.
(5) optimize enzyme concn, ATP concentration and reaction times, make phosphorylation level be that 20-40% carries out subsequent experimental.
B, detection compound are to the inhibit activities of EGFR phosphorylation
(1) according to above-mentioned optimal conditions, preparation 1.33 × kinase buffer liquid, 4 × drug solution, kinases/peptide mixed solution, phosphopeptide solution, ATP solution, excision liquid;
(2) in 368 orifice plates, add each solution by figure below mode, carry out enzyme reaction, excision reaction and termination reaction.
(3) (Ex.400nm, Em.445nm is detected by microplate reader; Ex.400nm, Em.520nm) fluorescence intensity.
(4) according to following formulae discovery Emission Ratio and enzyme percent phosphorylation.
(5) according to the suppression per-cent of following formulae discovery compound to enzyme phosphorylation.
(6) with GraphPad Prism 5 software analysis medicine to the IC of EGFR phosphorylation 50value.
C, cell cycle analysis
(1) cell grow to 70-80% closely sealed time, the DMEM nutrient solution synchronization cell changed into containing 0.5%FBS spends the night.Afterwards with the DMEM incubated cell 24h containing different pharmaceutical concentration, with 0.125% pancreatin-0.02%EDTA digestion, collecting cell;
(2) eccentric cell, after washing cell 1 time, adds 70% ethanol and spends the night at-20 DEG C of fixed cells with the PBS of precooling;
(3) discard ethanolic soln, wash cell 1 time with the PBS of precooling, the PBS solution 100 μ l lucifuge added containing 100 μ g/mL RNAse and 50 μ g/mL propidium iodides (PI) hatches 30min;
(4) make single cell suspension with 0.5mL PBS diluting cells, use flow cytometer (BDFACSCanto immediately tM, USA) detect;
(5) cell cycle result adopts ModfitLT 3.0 software (Verity Software House, USA) to analyze.
D, comet electrophoresis are tested
(1) Pharmaceutical formulations: with reference to EMB-3 to the IC of three kinds of cytosiies 50, unified selection 25 μMs is as the criterion, use respectively 25,75,250 μMs (be denoted as 1 ×, 3 ×, 10 ×) irritation cell.In triplicate parallel, and establish a blank control wells, be denoted as nc.
(2) cell kind plate, waits to grow to 80 ~ 90% for subsequent use.Add stimulating drug, cultivate 2h for 37 DEG C.
(3) PBS of cell precooling washes once, collected by centrifugation, is 1 × 10 by resuspended its density that makes of PBS 6individual/ml.
(4) glue is spread: following each concentration agarose gel is all prepared with PBS
The preparation of the 1st layer of gel: by the frosting of slide glass upwards, 40 DEG C of preheatings, are laid on slide glass by the 0.5% normal melting point agarose (NMA) of 100 μ L of preheating 45 DEG C, cover clean cover glass, NMA are solidified being placed in 10min at 4 DEG C.
The preparation of the 2nd layer of gel: by 10 μ L cells (about 10 4individual) and the 0.7% low melting-point agarose LMA of 75 μ L mix.Then, throw off cover glass gently, rapidly celliferous LMA dripped on the first layer agarose; Cover another clean cover glass immediately, at putting 4 DEG C, 10min makes the 2nd layer of LMA solidify.
The preparation of the 3rd layer of gel: after the 2nd layer of LMA solidifies, at room temperature carefully remove cover glass, drips the 0.7% low melting-point agarose LMA of 75 μ L of preheating 37 DEG C, as above solidifies 30min at covered 4 DEG C.
(5) cover glass is removed in lysis, and slide is placed in plate, pours the Lysis Buffer of precooling into, 4 DEG C of cracking 1 ~ 2h, takes out slide glass PBS rinsing.
(6) DNA alkaline hydrolysis revolves and slide glass is placed in Horizontal electrophoresis tank.Pour the alkaline electrophoresis buffer of new preparation into, about covered slide glass glue face about 0.25cm, room temperature places 20 ~ 60min, to make DNA uncoiling and generation alkali volatility section in the basic conditions, makes DNA chain rupture be easy to migration in the electric field.
(7) Single-cell electrophoresis is at voltage 25V, electrophoresis 20 ~ 30min.
(8) after neutralization and dyeing electrophoresis, slide glass is placed in plate.Add 0.4mmol/L Tris-HCl (pH7.5) damping fluid, submerged by slide glass, 4 DEG C neutralize three times, each 10min, discards Tris-HCl damping fluid, and every slide glass adds 20 μ L SYBR Green (1: 10,000), covered, lucifuge dyeing 10min.
(9) observe, take pictures and analyze
Analysis software:
1) image analysis software: Komet 5.5
2) result statistical software: Prism5.0 & Excel
2, experimental result:
(1) EFGR Receptor Binding Assay
Inhibited to EFGR for confirming this compounds, select compd E MB-3 to be probe, carry out Receptor Binding Assay.As shown in table 2, EMB-3 is to the IC of EGFR 50value is 0.112 μM, although higher than lapatinibditosylate (IC 50, but still can illustrate that EMB-3 has stronger EGFR restraining effect=0.018).
Table 2.EMB-3 and lapatinibditosylate are to the IC of EGFR 50value
Compound EGFR restraining effect (IC 50/μM)
EMB-3 0.112±0.019
lapatinib 0.018±0.009
(2) to the restraining effect of EGFR phosphorylation
For confirming the restraining effect of EMB-3 to EFGR further, investigate the impact of EMB-3 on EGFR phosphorylation.Meanwhile, consider that Glyciphosphoramide group has alkanisation to DNA, infer that the amino-acid residue of this group to receptors bind pocket also has alkanisation, thus irreversible inhibition EGFR/HER-2 acceptor; Whether therefore, devise elution experiments, be irreversible inhibition agent to investigate compound.
As shown in Figure 3, after EMB-3 and MDA-MB-468 cell hatches 1h altogether, to the EGFR phosphorylation that Urogastron (EGF) is induced, there is obvious restraining effect, and present dose-dependently.In elution experiments, EMB-3 and MDA-MB-468 cell is hatched 1h altogether, wash-out is to remove unconjugated EMB-3, and after hatching 6h, then with Urogastron induction EGFR phosphorylation, result display EGFR phosphorylation is still subject to obvious suppression.This shows the EMB-3 killer cell by suppression EGFR really, and belongs to irreversible inhibition agent.
(3) DNA damage experiment (comet)
For verifying the damaging action of compound to DNA further, comet being carried out to EMB-3, with tail of a comet length for index, having measured the EMB-3 of different concns to the damaging action of DNA.
As shown in Figure 4, to HCT116 cell and MDA-MB-468 cell, when EMB-3 concentration is 250 μMs, there is obvious DNA damage effect (p < 0.05versus control); And to SKBR-3 cell, when concentration is 75 μMs, namely demonstrate obvious DNA damage effect (p < 0.05versus control).This illustrates that compd E MB-3 has DNA damage effect really.
(4) cell cycle experiment
Be also tested for the impact of compd E MB-3 cell cycle, to study the Anticancer Effect and Mechanism of compound further.
The impact of table 3.EMB-3 cell cycle
G1(％) S(％) G2/M(％)
Control group 57.9±2.8 22.6±2.7 19.0±5.0
1μM 64.1±3.5 19.6±4.8 16.3±8.3
2μM 67.7±2.4 17.1±4.0 15.2±6.3
4μM 66.9±1.3 17.1±3.4 16.0±4.7
8μM 72.1±3.2 14.5±1.2 13.5±4.4
As shown in table 3 and Fig. 5, EMB-3 makes most cells rest on the G1 phase, and presents dose-dependently.Consider that EGFR inhibitor Gefitinib makes cell mainly stay in the G1 phase, DNA alkylating agent endoxan makes cell mainly stay in the G2/M phase, and this shows at low concentrations, the effect of EMB-3 mainly through suppressing EGFR/HER-2 acceptor to play killing tumor cell.

Claims (6)

1. the class Mutiple Targets antineoplastic compound as shown in general formula I or its pharmaceutical salts
Wherein, R 1be selected from hydrogen, methoxy or ethoxy; R 2be selected from 3-bromophenyl, 3-chloro-4-fluorophenyl or the chloro-4-of 3-(3-fluorine benzyloxy) phenyl; X is CH 2, Y is O or X be CO, Y is NH; N is selected from 1-3 or n when being 0, X and Y does not exist.
2. compound according to claim 1 is:
4-(3-bromobenzene amido) quinazoline phenol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
2-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
3-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
4-(6-(4-(3-bromobenzene amido) quinazoline) oxygen) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
3-(6-(4-(3-bromobenzene amido)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
3-(6-(4-(the chloro-4-fluoroanilino of 3-)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
3-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino)-7-methoxyquinazoline hydrochloride) oxygen) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
2-oxo-2-(6-(4-(3-bromobenzene amido) quinazoline) is amino) ethanol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
3-oxo-3-(6-(4-(3-bromobenzene amido) quinazoline) is amino) propyl alcohol N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
4-oxo-4-(6-(4-(3-bromobenzene amido) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
4-oxo-4-(6-(4-(the chloro-4-fluoroanilino of 3-) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester;
4-oxo-4-(6-(4-(the chloro-4-of 3-(3-fluorine benzyloxy) anilino) quinazoline) is amino) butanols N, N-bis-(2-chloroethyl) phosphoric acid diamines ester.
3. prepare the method for compound described in claim 1 or 2, comprise the steps:
Wherein, when X is CH 2, Y to be O or n be 0 and X and Y does not exist time, R 1, R 2with n as aforementioned formula I define time the synthetic route of compound as follows:
(1) synthesis of key intermediate 4,6-dihydroxyl quinazoline and 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride
5-hydroxyl-2-benzaminic acid and the cyclization of methane amide high temperature obtain 4,6-dihydroxyl quinazoline;
Or vanillic acid is through being obtained by reacting 3,4-dimethoxy p-methyl with methylating reagent; Then nitratedly 2-nitro-4,5-dimethoxy p-methyl is obtained; Demethylation, hydrolysis of ester group obtains 2-nitro-4-methoxyl group-5-hydroxy-benzoic acid simultaneously; 2-amino-4-methoxyl-5-methyl hydroxybenzoate is obtained again through esterification, amino reduction; Finally, at 150 DEG C, there is cyclization with ammonium formiate and methane amide and obtain 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride;
7 R 1the synthetic method of 4,6-dihydroxyl quinazolines replaced and the synthetic method of 4,6-dihydroxyl-7-methoxyquinazoline hydrochloride similar;
(2) synthesis of formula I
7 R 1the 6-OH highly selective acylation of 4, the 6-dihydroxyl quinazolines replaced, obtains 7 R 16-acetoxyl group-4-the hydroxyquinazoline replaced; Then 4-OH chlorination, not purified directly and substituted aniline be obtained by reacting 7-R 1-4-R 2-6-acetoxyl group quinazoline; In ammonia hydroxide/methanol system, deacetylation obtains 7-R 1-4-R 2-6-hydroxyquinazoline; 7-R 1-4-R 2-6-hydroxyquinazoline directly reacts with dichlor-phosphoryl mustargen, or elder generation and halohydrin react, and products therefrom reacts with dichlor-phosphoryl mustargen again, namely obtains formula I;
Wherein, when X be CO, Y is NH, R 1, R 2with n as aforementioned formula I define time the synthetic route of compound specific as follows:
(1) synthesis of the ω-hydroxy-acid chloride of benzoyl protection
The glycol of 1.5 equivalents and the Benzoyl chloride of 1.0 equivalents react, and obtain the glycol of single benzoyl protection, through Jone ' s reagent oxidation, obtain the 'omega '-hydroxy carboxylic acid of benzoyl protection, then at SOCl 2/ CHCl 3middle backflow obtains the ω-hydroxy-acid chloride of benzoyl protection;
(2) synthesis of formula I
4-R 1-2-the benzaminic acid replaced and methane amide at high temperature cyclization obtain 7-R 1-4-the hydroxyquinazoline replaced, further nitration reaction obtains 7-R 1-6-nitro-4-the hydroxyquinazoline replaced, then change 7-R into sulfur oxychloride process 1-6-nitro-4-the chloro-quinazoline replaced, not purified directly and substituted aniline be obtained by reacting 7-R 1-the 4-R replaced 2substituted benzene amino-6-nitro-quinazoline, obtains 7-R with glass putty-hydrochloric acid reduction nitro further 1-the 4-R replaced 2substituted benzene amino-6-amido quinazoline; 7-R 1-4-R 2ω-hydroxy-acid chloride that-6-amido quinazoline is protected with the benzoyl of above-mentioned preparation again reacts, and products therefrom, without separation, directly removes benzoyl and obtains 7-R in sodium hydroxide/methanol/water system 1-4-R 2-6-(ω-alcohol amide base) quinazoline; Finally, then under sodium hydride effect, 7-R 1-4-R 2-6-(ω-alcohol amide base) quinazoline and dichlor-phosphoryl mustargen are obtained by reacting formula I;
4. compound or pharmaceutically acceptable salt thereof is preparing the application in antitumor drug as claimed in claim 1 or 2.
5. a pharmaceutical composition, compound or pharmaceutically acceptable salt thereof described in the claim 1 or 2 wherein containing treatment significant quantity, and one or more pharmaceutically acceptable carriers.
6. pharmaceutical composition is preparing the application in antitumor drug as claimed in claim 5.
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