CN113717174A

CN113717174A - Tumor targeting drug resistant O6-thienylmethylguanine-indoloquinone-chloroethylnitrosourea combined molecule and preparation method thereof

Info

Publication number: CN113717174A
Application number: CN202110951185.4A
Authority: CN
Inventors: 赵丽娇; 王娇娇; 李多; 孙国辉; 张娜; 钟儒刚
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-11-30
Anticipated expiration: 2041-08-18
Also published as: CN113717174B

Abstract

Tumor-targeting drug-resistant O⁶A-thienylmethylguanine-indoloquinone-chloroethylnitrosourea combined molecule and a preparation method thereof, relating to the field of pharmacy. The compound of the invention takes indoloquinone as hypoxia activation pharmacophore, under the condition of tumor hypoxia, indoloquinone is reduced to generate imine substances with cytotoxicity; decomposing CENUs pharmacophore to generate chloroethyl carbonium ions, resulting in cross-linking between DNA strands; simultaneously, releasing AGT inhibitor O⁶-TMG derivatives exerting AGT inhibitory action. Therefore, the compound of the invention not only can exert dual anti-tumor effects, but also has the anti-drug resistance and tumor hypoxia targetingHas high-efficiency and low-toxicity anticancer activity.

Description

Tumor targeting drug resistant O6-thienylmethylguanine-indoloquinone-chloroethylnitrosourea combined molecule and preparation method thereof

Technical Field

The invention relates to the field of pharmacy, in particular toAnd a drug-resistant tumor hypoxia targeting O⁶-thienylmethylguanine-indoloquinone-chloroethylnitrosourea combined molecule, and preparation method and anti-tumor application thereof.

Background

The Chloroethylnitrosourea (CENUs) chemotherapeutic drugs are mainly used for clinically treating malignant tumors such as brain tumor, glioma and melanoma. Mainly through inducing DNA to form dG-dC interstrand cross-linking, the process of DNA replication and transcription can not be carried out because the double-strand of DNA can not be normally opened, thereby causing cell apoptosis and playing the role of anti-tumor. The two main disadvantages of the medicine are that the medicine is easy to generate drug resistance and has toxic and side effects on normal tissues in the treatment process. O is⁶The DNA repair mediated by-alkylguanine-DNA Alkyltransferase (AGT) reduces the anti-tumor effect and is the main cause of resistance of tumor cells to CENUs. Therefore, the combination therapy of the AGT inhibitor and the CENUs is an effective method for improving the treatment effect clinically. Such as O⁶-benzylguanine (O)⁶-BG) is a potent AGT inhibitor, AGT being able to convert O⁶The benzyl group on the-BG is transferred to the active site of self cysteine to cause the loss of enzyme activity, thereby increasing the sensitivity of cancer cells to CENUs and obviously improving the treatment effect of the CENUs on tumors. However, the toxicity of the combination strategy to normal cells is greatly increased while tumor cells are acted, namely, the tumor targeting is lacked. Therefore, how to achieve combined targeting of the AGT inhibitor and the CENUs to the tumor site is a problem to be solved urgently.

By Hypoxia Activated Prodrug (HAP) is meant a drug that is itself non-toxic or less toxic under normoxic conditions, whereas under hypoxic conditions the drug can be activated to produce cytotoxic compounds, thereby killing the cell. The hypoxic microenvironment is an important characteristic that most tumor tissues are different from normal tissues, and the hypoxic condition can enable HAP to be specifically activated in the tumor tissues to play a hypoxia targeting antitumor role, so the HAP is an effective strategy for reducing toxic and side effects of anticancer drugs and realizing drug tumor targeting. The invention takes indoloquinone as a hypoxia activating group and O⁶-thienylmethylguanine (O)⁶-TMG) is an AGT inhibitor pharmacophore with 1- (2-chloroethyl) -3-alkaneThe combination of the 1-nitrosourea molecules designs and synthesizes the anti-drug resistance combined CENUs HAP of a target tumor. Indoloquinone in molecular structure as hypoxia activating group to react with O⁶-TMG pharmacophore is tethered at its C-3 position, while the nitrosourea pharmacophore is tethered at its N-1 position. The drug molecules can be specifically activated in a tumor hypoxia microenvironment through reduction reaction, and the released nitrosourea can cause the DNA inter-strand cross-linking of tumor cells; released O⁶-TMG derivatives are capable of inhibiting AGT mediated drug resistance; the released indoloquinone is converted into electrophilic imine substances to act with biological macromolecules, and DNA alkylation is promoted. The three pharmacophores have synergistic effect, and can play higher antitumor activity while realizing the tumor hypoxia targeting.

Disclosure of Invention

The invention aims to provide O with drug resistance and tumor hypoxia targeting⁶-TMG-indoloquinone-chloroethylnitrosourea combined molecule and preparation method and application thereof. The compound of the invention takes indoloquinone as hypoxia activation pharmacophore, under the condition of tumor hypoxia, indoloquinone is reduced to generate imine substances with cytotoxicity; decomposing CENUs pharmacophore to generate chloroethyl carbonium ions, resulting in cross-linking between DNA strands; simultaneously, releasing AGT inhibitor O⁶-TMG derivatives exerting AGT inhibitory action. Therefore, the compound of the invention not only can exert dual anti-tumor effects, but also has the anti-drug resistance and tumor hypoxia targeting property, and has high-efficiency and low-toxicity anti-cancer activity.

The structural formula of the drug-resistant tumor hypoxia targeting chloroethylnitrosourea combined molecule is shown as (I):

R₁is OCH₃，OCH₂CH₃，NH₂，NCH₂CH₂One of (1);

R₂is H, CH₃，CH₂CH₃，CH₂OH，CH₂CH₂OH，CH₂OHOCONH₂，CH₂CH₂OCONH₂One of (1);

R₃is H, CH₃，CH₂CH₃，CH₂CH₂CH₃，NH₂，NH(CH₃)，N(CH₃)₂，NCH₂CH₂，CHCHCH₂One of OH;

R₄is H, CH₂CH₂NH₂One of (1);

n is an integer of 2 to 6.

Preferably, R₁Is OCH₃And NCH₂CH₂，R₂Is H, R₃Is CH₃，CH₂CH₃，CH₂OHOCONH₂，R₄When the compound is H, the antitumor activity and the hypoxia selectivity of the compound are higher.

Most preferably, when R₁Are respectively OCH₃，R₃Is CH₃I.e., having the following structural formula, the antitumor activity and hypoxia selectivity of the compound are optimal.

The second purpose of the invention is to provide a pharmaceutical composition, which comprises a therapeutically effective amount of the compound with the structure of the formula (I) or pharmaceutically acceptable salt, and at least one pharmaceutical carrier.

The pharmaceutically acceptable salt is one or more of hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfite, phosphate, acetate, propionate, butyrate, lactate, methanesulfonate, p-toluenesulfonate, maleate, benzoate, succinate, citrate, fumarate, taurate, gluconate and glycinate.

The drug carrier is a substance commonly used in the field, such as a stabilizer, a dispersant, an emulsifier, a disintegrant, a lubricant, a water-retaining agent, a diluent and the like. The pharmaceutical composition can be prepared into pharmaceutically common dosage forms such as tablets, injections, capsules and the like by adopting the conventional means in the field.

A third object of the present invention is to provide a process for the preparation of the compound or pharmaceutically acceptable salt of the present invention, wherein the reaction scheme of the process at least comprises:

experiments prove that any one of the compounds or pharmaceutically acceptable salts or pharmaceutical compositions thereof can be specifically reduced and released in a tumor hypoxia area to obtain an active AGT inhibitor, so that the sensitivity of tumor cells in the hypoxia area to chemotherapeutic drugs is improved in a targeted manner, and the anti-tumor effect of chloroethyl nitrosourea drugs is further improved.

A third object of the invention is to protect the process for the preparation of the compounds or pharmaceutically acceptable salts of the invention. The reaction process is as follows:

(1) the carboxylic ester group in the compound a is reduced to obtain a compound b;

(2) amino group of Compound b is (Boc)₂Protecting with O anhydride to obtain a compound c;

(3) reacting the compound c with 1- (2-amino-9H-purine-6-yl) -1-methylpyrrolidine-1-chloride to obtain a compound d;

(4) removing amino protection from the compound d under an acidic condition to obtain a compound e;

(5) protecting hydroxyl in the compound f by tetrahydropyrane ether to obtain a compound g;

(6) reacting the compound g with dihalogenated alkane to obtain a compound h;

(7) reacting the compound h with potassium phthalimide to obtain a compound i;

(8) carrying out dehydroxylation protection reaction on the compound i to obtain a compound j;

(9) carrying out Mitsunobu reaction on the compound j and the compound e to generate a compound k;

(10) performing hydrazinolysis reaction on the compound k to obtain a compound l;

(11) reacting the compound l with 2-chloroethyl isocyanate to obtain a compound m;

(12) and reacting the compound m with nitrosonium tetrafluoroborate to obtain a compound n.

Preferably, the method comprises the following steps:

(1) compound a and LiAlH₄According to the molar ratio of 1 (0.2-2) in N₂Reacting at 0-10 ℃ under protection to obtain a compound b;

(2) compound b, (Boc)₂Adding O anhydride and 4-dimethylamino pyridine according to the molar ratio of 1 (1-3) to 0.1-1, and reacting at 0-4 ℃ to obtain a compound c;

(3) the compound c, 1- (2-amino-9H-purin-6-yl) -1-methylpyrrolidine-1-chloride, potassium tert-butoxide and dimethylaminopyridine are fed according to the molar ratio of 1 (0.5-2) to (2-4) to (0.1-0.5) in N₂Reacting at 25 ℃ under protection to obtain a compound d;

(4) treating the compound d with trifluoroacetic acid or 50% trifluoroacetic acid (trifluoroacetic acid: dichloromethane (v/v ═ 1:1)) at 25 ℃ to obtain a compound e;

(5) feeding a compound f, tetrahydropyrane ether and p-toluenesulfonic acid according to a molar ratio of 1 (1-2) to (0.005-0.01), and reacting at 25 ℃ to obtain a compound g;

(6) reacting the compound g with dihalogenated hydrocarbon according to the molar ratio of 1 (1-8) at 40-70 ℃ under the catalysis of alkali to obtain a compound h;

(7) reacting the compound h with potassium phthalimide according to the molar ratio of 1 (1-6) at 60-80 ℃ under the catalysis of a phase transfer catalyst to obtain a compound i;

(8) reacting the compound i with pyridine p-toluenesulfonate according to the molar ratio of 1 (1-4) at 25-40 ℃ to obtain a compound j;

(9) compound j, compound e, triphenyl phosphine (PPh)₃) Feeding diethyl azodicarboxylate (DEAD) according to the molar ratio of 1 (1-2) to 1-1.5 (1-1.5), firstly, adding compound i, compound e and PPh₃Dissolving in solvent, stirring for 1h, adding DEAD dropwise at 0-4 deg.C, and reacting at 25-40 deg.C to obtain compound k;

(10) reacting the compound k with hydrazine hydrate according to the molar ratio of 1 (2-6) at the temperature of 30-50 ℃ to obtain a compound l;

(11) reacting the compound l with 2-chloroethyl isocyanate according to the molar ratio of 1 (1-8) at 0-10 ℃ to obtain a compound m;

(12) the compound m and nitrosonium tetrafluoroborate react at 0-10 ℃ according to the molar ratio of 1 (1-8) to obtain a compound n.

Further preferred is:

the specific operation of the step (1) is that LiAlH is added under the ice bath condition₄Dissolving in anhydrous solvent, stirring for 5-20min, adding dropwise reactant solution a, and adding N₂Reacting for 1-2h at 0-10 ℃ under protection to obtain a compound b. Wherein, the compound a and LiAlH₄The molar ratio of (A) to (B) is preferably 1 (0.5-1); dissolving LiAlH₄Preferably Tetrahydrofuran (THF), diethyl ether, ethylene glycol or dimethyl ether; the solvent for dissolving the compound a is preferably anhydrous THF or anhydrous diethyl ether. The step (1) further comprises a step of purifying the compound b, specifically, after the reaction is finished, quenching the compound b by using water, a 10% sodium hydroxide solution and water (v/v/v ═ 1:2:3) under the condition of ice-bath stirring, and carrying out reduced pressure rotary evaporation on the reaction solution at 50 ℃; and then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is petroleum ether and ethyl acetate, gradient elution is carried out according to the ratio of the petroleum ether to the ethyl acetate (v/v) of 1:2-1:4, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 35 ℃ to obtain the purified compound b.

The specific operation of the step (2) is that the compound b, (Boc)₂Dissolving O anhydride and 4-dimethylamino pyridine in a solvent, and reacting for 6-12h at 0-4 ℃ to obtain a compound c. Wherein, compound b, (Boc)₂The mol ratio of the O anhydride to the 4-dimethylamino pyridine is preferably 1 (1-1.5) to (0.1-0.5); the reaction solvent used is preferably water or dichloromethane. The step (2) further comprises a step of purifying the compound c, specifically, dichloromethane and water (v: v ═ 1:1) are added into the reaction solution for extraction, the organic phase is collected, the organic phase is washed by saturated sodium chloride aqueous solution, anhydrous sodium sulfate is dried overnight, and the mixture is concentrated by rotary evaporation at 35 ℃ under reduced pressure; then separating and purifying the concentrated solution by column chromatography, preferably using silica gel as stationary phase and dichloromethane as mobile phaseMethanol is eluted at the same degree with dichloromethane/methanol (v/v) of 20:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 35 ℃ to obtain the purified compound c.

The specific operation of the step (3) is that the compound c is dissolved in organic solvent, 1- (2-amino-9H-purine-6-yl) -1-methyl pyrrolidine-1-chloride, potassium tert-butoxide and dimethyl aminopyridine are added in N₂Reacting for 4-8h at 25 ℃ under protection to obtain a compound d. Wherein, the mol ratio of the compound c, 1- (2-amino-9H-purin-6-yl) -1-methylpyrrolidine-1-chloride, potassium tert-butoxide and dimethylaminopyridine is preferably 1 (1-2) to (2-4) to (0.1-0.5); the organic solvent used to dissolve compound c is preferably N, N-Dimethylformamide (DMF) or acetonitrile. Step (3) further comprises a step of purifying the compound d, specifically, quenching the compound d with an acidic aqueous solution, preferably a glacial acetic acid/water (v: v ═ 1:10) solution, adding an ethyl acetate/saturated ammonium chloride (v: v ═ 1:1) solution into the reaction solution, extracting, drying over night with anhydrous sodium sulfate, and concentrating by rotary evaporation at 45 ℃ under reduced pressure; and then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is dichloromethane and methanol, gradient elution is carried out according to the ratio of dichloromethane/methanol (v/v) of 50:1-10:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 35 ℃ to obtain a purified compound d.

The specific operation of the step (4) is that the compound d is treated by trifluoroacetic acid and dichloromethane (v/v ═ 1:1) at 25 ℃ for 1-2h, and then neutralized by saturated aqueous solution of sodium bicarbonate to obtain the compound e. And (4) further comprising a step of purifying the compound e, which is to add dichloromethane and water (v: v ═ 1:1) into the reaction solution for extraction, collect the organic phase, wash the organic phase with saturated aqueous sodium chloride solution, dry the organic phase over night with anhydrous sodium sulfate, and carry out rotary evaporation and concentration at 35 ℃ under reduced pressure to obtain the purified compound e.

The specific operation of the step (5) is that the compound f is dissolved in anhydrous dichloromethane, and then the tetrahydropyrane ether and the p-toluenesulfonic acid are added to react for 2 to 8 hours at the temperature of 25 ℃ to obtain a compound g. The molar ratio of the compound f, tetrahydropyranyl ether, and p-toluenesulfonic acid is preferably 1 (1-2): 0.005. The step (5) further includes a step of purifying the compound g by adding dichloromethane and water (v: v ═ 1:1) to the reaction solution, extracting, washing with a saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, and concentrating by rotary evaporation at 35 ℃ under reduced pressure. And then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is dichloromethane and methanol, gradient elution is carried out by taking dichloromethane/methanol (v/v) as the proportion of 100:1-50:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 35 ℃ to obtain the purified compound g.

Specifically, the dihaloalkane in the step (6) is preferably dibromoalkane or dichloroalkane; more preferably a dibromoalkane. Dissolving the compound g in an organic solvent, adding inorganic base serving as a catalyst into the organic solvent, dropwise adding dibromoalkane into the organic solvent, and reacting for 48 to 72 hours at the temperature of between 40 and 70 ℃ to obtain a compound h. Wherein, the mol ratio of the compound g, the inorganic base and the dibromoalkane is preferably 1 (1-6) to 1-6; the inorganic base is preferably anhydrous potassium carbonate; the organic solvent for dissolving the compound g is preferably acetone or DMF; the reaction temperature is preferably 50 to 60 ℃. And (6) purifying the compound h, specifically filtering the reaction solution, collecting the filtrate, and concentrating the reaction solution by rotary evaporation at 50 ℃ under reduced pressure. And then separating and purifying the concentrated solution by adopting a column chromatography method, preferably selecting a stationary phase of the column chromatography as silica gel and a mobile phase as petroleum ether and ethyl acetate, carrying out isocratic elution according to the ratio of the petroleum ether to the ethyl acetate (v/v) of 1:2, and carrying out reduced pressure rotary evaporation and concentration on the eluent at 35 ℃ to obtain a purified compound h.

The specific operation of the step (7) is that the compound h is dissolved in an organic solvent, and added with phthalimide and a phase transfer catalyst to react for 6 to 8h at the temperature of between 60 and 80 ℃ to obtain the compound i. Wherein, the mol ratio of the compound h, the phthalimide and the phase transfer catalyst is preferably 1 (2-4) to (0.1-0.6); the organic solvent used for dissolving the compound h is preferably acetonitrile or DMF; the phase transfer catalyst is preferably tetrabutylammonium iodide; the reaction temperature is preferably 60 to 70 ℃. The step (7) further includes a step of purifying the compound i by adding dichloromethane and water (v: v ═ 1:1) to the reaction solution, extracting, washing with a saturated aqueous sodium chloride solution, drying over anhydrous sodium sulfate overnight, and removing the solvent by rotary evaporation under reduced pressure at 35 ℃. And then, carrying out separation and purification by adopting a column chromatography, preferably, using silica gel as a stationary phase and dichloromethane and water as mobile phases of the column chromatography, carrying out isocratic elution according to a ratio of dichloromethane/water (v/v) of 50:1, and carrying out reduced pressure rotary evaporation and concentration on eluent at 35 ℃ to obtain the purified compound i.

The specific operation of the step (8) is that the compound i is dissolved in an organic solvent, pyridine p-toluenesulfonate is added, and reflux reaction is carried out for 4 to 10 hours at the temperature of between 25 and 40 ℃ to obtain a compound j. Wherein, the mol ratio of the compound i to the pyridine p-toluene sulfonate is preferably 1 (2-4); the organic solvent used for dissolving the compound i is preferably methanol or ethanol; the reaction temperature is preferably 25 ℃. The step (8) further includes a step of purifying the compound g by adding ethyl acetate and saturated ammonium chloride (v: v ═ 1:1) to the reaction solution, extracting, drying over anhydrous sodium sulfate overnight, and concentrating by rotary evaporation under reduced pressure at 45 ℃. And then, separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is ethyl acetate and n-hexane, gradient elution is carried out according to the proportion of ethyl acetate/n-hexane (v/v) of 5:1-2:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 45 ℃ to obtain the purified compound j.

The specific operation of the step (9) is that the compound j, the compound e and the PPh are mixed₃Dissolving in organic solvent, stirring at 0-4 deg.C for 1h, adding DEAD dropwise, and reacting at 25-40 deg.C for 5-10h to obtain compound k. Wherein, the compound j, the compound e and the PPh₃The molar ratio of DEAD is preferably 1 (1-2) to 1.5; the organic solvent used for dissolving the compounds j and e is preferably anhydrous THF or a THF/acetonitrile (v/v) ═ 1/1 mixture; the reaction temperature is preferably 25 ℃. And (9) further comprising a step of purifying the compound k, specifically, the obtained reaction solution is subjected to reduced pressure rotary evaporation and concentration at 40 ℃, the concentrated solution is poured into petroleum ether and stirred, a byproduct of the tri-phenoxy phosphorus is separated out, and the filtrate is filtered and collected and is subjected to reduced pressure rotary evaporation and concentration at 30 ℃. And then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is ethyl acetate and petroleum ether, gradient elution is carried out according to the proportion of ethyl acetate/petroleum ether (v/v) of 1:1-5:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 45 ℃ to obtain the purified compound k.

The specific operation of the step (10) is that a compound k and hydrazine hydrate are dissolved in an organic solvent and react for 4 to 6 hours at the temperature of between 30 and 50 ℃ to obtain a compound l; wherein the molar ratio of the compound k to the hydrazine hydrate is preferably 1 (2-4); the reaction solvent is preferably acetonitrile or DMF; the reaction temperature is preferably 30 to 40 ℃. The step (10) further includes a step of purifying the compound i by adding dichloromethane and water (v: v ═ 1:1) to the reaction solution, extracting, washing with a saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, and concentrating by rotary evaporation at 35 ℃ under reduced pressure. And then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is dichloromethane/methanol, gradient elution is carried out according to the ratio of dichloromethane/methanol (v/v) of 30:1-5:1, and the eluent is subjected to reduced pressure rotary evaporation and concentration at 35 ℃ to obtain the purified compound l.

And (11) specifically, dissolving the compound l in dichloromethane, dropwise adding a dichloromethane solution of 2-chloroethyl isocyanate in an ice bath, and reacting at 0-10 ℃ for 2-6h to obtain a compound m. Wherein, the mol ratio of the compound l to the 2-chloroethyl isocyanate is preferably 1 (2-4). Then, the solvent was removed by rotary evaporation under reduced pressure at 35 ℃ to obtain a compound m.

The specific operation of the step (12) is that the compound m is dissolved in an organic solvent, nitrosonium tetrafluoroborate and glacial acetic acid are added under the ice bath condition, and the reaction is carried out for 4 to 6 hours at the temperature of between 0 and 10 ℃ to obtain the compound n. Wherein, the mol ratio of the compound m, the nitrosonium tetrafluoroborate and the glacial acetic acid is preferably 1 (4-8) to 0.1-0.5; the organic solvent is preferably acetonitrile or acetone. The step (12) further includes a step of purifying the compound n by adding dichloromethane and water (v: v ═ 1:1) to the reaction solution, extracting, washing with a saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, and concentrating by rotary evaporation at 35 ℃ under reduced pressure. And then separating and purifying the concentrated solution by adopting a column chromatography method, preferably, the stationary phase of the column chromatography is silica gel, the mobile phase of the column chromatography is dichloromethane and methanol, gradient elution is carried out according to the ratio of dichloromethane/methanol (v/v) of 50:1-5:1, and the purified compound n is obtained after vacuum drying at 30 ℃.

The compound n in the invention is a compound with a structure shown in a formula (I).

The fourth purpose of the invention is to protect the application of the compound or the pharmaceutically acceptable salt and the composition thereof in preparing the antitumor drugs.

Preferably, the tumor is one or more of brain tumor, glioma, myeloma, malignant melanoma, malignant lymphoma, liver cancer, breast cancer, gastric cancer, colon cancer, prostate tumor, leukemia and lymph cancer;

more preferably one or more of brain tumor, malignant melanoma, colon cancer, and lymph cancer cell. The compound or pharmaceutically acceptable salt in the general formula (I) is combined chloroethyl nitrosourea with drug resistance and tumor targeting, and the compound or pharmaceutically acceptable salt in the general formula (I) is subjected to an in-vitro anti-tumor screening test. The results show that the compound in the general formula (I) has obvious inhibition effects on the cell activities of a plurality of tumor cell lines such as DU145 prostate cancer cells, MCF-7 human breast cancer cells, human glioma cells SF763, SF126 and the like under the hypoxia condition, and the inhibition effects on the tumors are not obvious when the compound in the general formula (I) is proved under the aerobic condition. Therefore, the compounds in the general formula (I) have good targeting property and capability of killing tumor cells, and can be used as targeted tumor chemotherapy drugs. On one hand, active chloroethyl carbonium ions are generated through decomposition in vivo, so that DNA generates inter-strand cross-linking, apoptosis is induced, and an anti-tumor effect is exerted; on the other hand, indoloquinone unit has low oxygen reduction activity, can selectively act on tumor hypoxia region, and releases O with AGT inhibitory activity⁶The TMG derivative pharmacophore can reduce the drug resistance of tumor area to chloroethyl nitrosourea medicine in target direction, and the indoloquinone reduction product also has antitumor activity and forms synergistic antitumor effect with chloroethyl nitrosourea medicine pharmacophore. The compound can selectively act on tumor cells in a hypoxic region, reduces the toxic and side effects of combined medication, improves the sensitivity of the tumor cells to the chemotherapeutic drugs, and has the advantages of tumor targeting, drug resistance and high anticancer activity compared with the existing nitrosourea anticancer drugs.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The medicines involved in the following examples are not specifically described, and are commercially available; the operations involved, unless otherwise specified, are those conventional in the art.

The structural formulae of compound 1 and compound 2 referred to in the following examples are as follows:

the following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of 3- (2- (3- (((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -5-methoxy-2-methyl-4, 7-dioxo-2, 3,4, 7-tetrahydro-1H-indol-1-yl) ethyl) -1- (2-chloroethyl) -1-nitrosourea (Compound 1)

1) Synthesis of (4- (aminomethyl) thiophen-2-yl) methanol

Weighing 4- (aminomethyl) thiophene-2-carboxylic acid ethyl ester (1.85g,10mmol) and dissolving in 10mL of anhydrous THF; weighing LiAlH₄(0.2g,5.2mmol) in a 100mL round-bottom flask in N₂Adding 10mL of anhydrous THF under the protection and ice bath conditions, and stirring for 10 min; 4- (aminomethyl) thiophene-2-carboxylic acid ethyl ester solution is dripped to react for 2 hours at the temperature of 0-10 ℃; after the reaction was completed, the reaction was quenched with water, 10% sodium hydroxide solution, and water (v/v/v ═ 1:2:3 (water: 0.2mL)) with stirring in an ice bath. Concentrating the reaction solution by rotary evaporation at 50 ℃ under reduced pressure, separating and purifying the concentrated solution by silica gel column chromatography, wherein an eluant is petroleum ether and ethyl acetate, gradient elution is adopted, the volume ratio of the petroleum ether to the ethyl acetate is 1:2-1:4, and the eluant is subjected to rotary evaporation at 35 ℃ under reduced pressure to obtain white solid (4- (aminomethyl) thiophen-2-yl) methanol (0.72g,5mmol) with the yield of 50%.

IR (KBr pellet) v/cm^-1:3667.2(O-H),3577.1(-NH₂),2967.5(-CH₂),1583.1(C＝C),1075.9(C-N),1027.4(C-O),627.8(C-S)；

¹H NMR(400MHz,CDCl₃)δ:4.32(s,2H,CH₂-N),4.68(s,2H,CH₂-O),5.12(s,1H,O-H),6.5(s,1H,C₄H₂S),7.95(s,1H,C₄H₂S),8.43(s,2H,-NH₂)；

ESI-MS:m/z 144(M+H)⁺。

2) Synthesis of tert-butyl ((5- (hydroxymethyl) thiophen-3-yl) methyl) carbamate

The resulting (4- (aminomethyl) thiophen-2-yl) methanol (1.72g,12mmol), (Boc) was weighed₂O (3.1g,14mmol) and 4-dimethylaminopyridine (0.24g,2mmol) are put into a 50mL round-bottom flask, 20mL of water is added, and the reaction is carried out for 8h at the temperature of 0-4 ℃; after the reaction is finished, continuing extracting dichloromethane and water (v: v ═ 1:1), collecting an organic phase, washing the organic phase by using a saturated sodium chloride aqueous solution, drying the organic phase over night, carrying out reduced pressure rotary evaporation concentration at 35 ℃, separating and purifying concentrated solution by using silica gel column chromatography, wherein the eluent is dichloromethane and methanol, isocratic elution is adopted, the volume ratio of dichloromethane to methanol is 20:1, and the eluent is reduced pressure rotary evaporation at 35 ℃ to obtain white solid tert-butyl ((5- (hydroxymethyl) thiophen-3-yl) methyl) carbamate (2.62g,10.8mmol) with the yield of 90%.

IR (KBr pellet) v/cm^-1:3415.7(N-H),3389.7(O-H),2981.5(-CH₃)2907.9(-CH₂),1742.9(C＝O),1216.0(C-O-C),1069.0(C-N),1043.4(C-O),627.8(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.39(s,9H,-CH₃),4.22(s,2H,CH₂-N),4.81(s,2H,CH₂-O),5.96(s,1H,OH),6.59(s,1H,C₄H₂S),7.85(s,1H,C₄H₂S),8.08(s,1H,NH)；

ESI-MS:m/z 244(M+H)⁺。

3) Synthesis of tert-butyl ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methylcarbamate

The resulting tert-butyl ((5- (hydroxymethyl) thiophen-3-yl) methyl) carbamate (0.46g,1.9mmol) was weighed into a 50mL round-bottomed flask, 15mL of DMF was added, 1- (2-amino-9H-purin-6-yl) -1-methylpyrrolidine-1-chloride (0.51g,2mmol), potassium tert-butoxide (0.43g,3.8mmol), dimethylaminopyridine (0.51g,0.2mmol) were added, and the mixture was stirred under N₂Reacting for 5 hours at 25 ℃ under protection; after the reaction was completed, a 1mL glacial acetic acid solution (glacial acetic acid/water (v/v ═ v) was added1:10)), followed by extraction with ethyl acetate and saturated ammonium chloride (v: v ═ 1:1), the organic phase was collected, dried over anhydrous sodium sulfate overnight, and concentrated by rotary evaporation at 45 ℃ under reduced pressure. Separating and purifying the concentrated solution by silica gel column chromatography, wherein the eluent is dichloromethane and methanol, gradient elution is adopted, the volume ratio of dichloromethane/methanol is 50:1-10:1, and the eluent is subjected to rotary evaporation at 35 ℃ under reduced pressure to obtain white solid tert-butyl ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl carbamate (0.3g,0.8mmol), and the yield is 40%.

IR (KBr pellet) v/cm^-1:3601.7(-NH₂),3454.0(N-H),2961.5(-CH₃),2835.0(-CH₂),1742.9(C＝O),1572.3(C＝N),1188.1(C-O-C),1051.4(C-N),627.8(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.49(s,9H,-CH₃),4.29(s,2H,CH₂-N),5.09(s,2H,CH₂-O),6.24(s,2H,-NH₂),6.53(s,1H,C₄H₂S),7.49(s,1H,C₄H₂S),8.18(s,1H,C-H),11.64(s,1H,N-H)；

ESI-MS:m/z 377(M+H)⁺。

4) Synthesis of 6- ((4- (aminomethyl) thiophen-2-yl) methoxy) -9H-purin-2-amine

The resulting tert-butyl ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methylcarbamate (1.9g,5mmol) was weighed into a 50mL round-bottomed flask, 20mL of 50% trifluoroacetic acid (trifluoroacetic acid: dichloromethane (v/v ═ 1:1)) was added, and the reaction was carried out at 25 ℃ for 2H; after the reaction was completed, the reaction solution was neutralized with a saturated sodium bicarbonate solution to pH 8, followed by extraction with dichloromethane and water (v: v ═ 1:1), and the organic phase was collected, washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate overnight, and the solvent was evaporated under reduced pressure at 35 ℃ to give 6- ((4- (aminomethyl) thiophen-2-yl) methoxy) -9H-purin-2-amine (1.1g,4mmol) as a white solid in 80% yield.

IR (KBr pellet) v/cm^-1:3614.8(-NH₂),3421.9(N-H),2947.4(-CH₂),2851.7(-CH₂),1572.3(C＝N),1303.5(C-O-C),1121.8(C-N),631.1(C-S)；

¹H NMR(400MHz,CDCl₃)δ:4.37(s,2H,CH₂-N),5.49(s,2H,CH₂-O),6.42(s,2H,-NH₂),6.59(s,1H,C₄H₂S),7.89(s,1H,C₄H₂S),8.71(s,2H,-NH₂),8.84(s,1H,CH),13.12(s,1H,NH)；

ESI-MS:m/z 277(M+H)⁺。

5) Synthesis of 5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone

Weighing 3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.56g,2.5mmol) in a 50mL round-bottom flask, adding 25mL anhydrous dichloromethane, adding tetrahydropyrane ether (0.1g,5mmol) and p-toluenesulfonic acid (0.86mg,0.005mmol), and reacting at 25 ℃ for 4H; after completion of the reaction, the reaction mixture was extracted with dichloromethane and water (v: v ═ 1:1), and the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate overnight, and concentrated to remove the solvent by rotary evaporation under reduced pressure at 35 ℃. Separating and purifying the concentrated solution by silica gel column chromatography, wherein an eluent is dichloromethane and methanol, gradient elution is adopted, the volume ratio of dichloromethane to methanol is 100:1-50:1, and the eluent is subjected to decompression rotary evaporation at 35 ℃ to obtain white solid 5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (0.52g,1.7mmol), and the yield is 70%.

IR (KBr pellet) v/cm^-1:3431.6(N-H),2937.4(-CH₃),2868.8(-CH₂),1678.8(C＝O),1551.3(C＝C),1309.4(C-O-C),1163.3(C-N),697.3(N-H)；

¹H NMR(400MHz,CDCl₃)δ:1.52-1.87(m,6H,-(CH₂)₃-),2.28(s,3H,-CH₃),3.68(s,3H,-OCH₃),4.59(t,2H,CH₂-O),4.96(s,1H,CH₂-O),5.49(s,2H,CH₂-O⁶),5.45(s,1H,NH),8.54(s,1H,NH)；

ESI-MS:m/z 306(M+H)⁺。

6) Synthesis of 1- (2-bromoethyl) -5-methoxy-2-methyl-3- (((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone

Weighing the obtained 5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indolone (1.2g,4mmol) and anhydrous potassium carbonate (1.66g,12mmol) in a 100mL round-bottom flask, adding 50mL acetone, dropwise adding 1.4mL (16mmol)1, 2-dibromoethane, reacting at 50 ℃ for 72H, filtering the reaction solution, collecting the filtrate, performing reduced pressure rotary evaporation at 50 ℃ to remove the solvent, purifying by silica gel column chromatography, wherein the eluent is petroleum ether and ethyl acetate, performing isocratic elution, the volume ratio of the petroleum ether to the ethyl acetate is 1:2, and performing reduced pressure rotary evaporation at 45 ℃ to obtain a white solid 1- (2-bromoethyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) - 1H-indoloquinone (1g,2.4mmol), 61% yield.

IR (KBr pellet) v/cm^-1:2987.1(-CH₃),2801.1(-CH₂),1689.5(C＝O),1632.2(C＝C),1321.3(C-O-C),1093.4(C-N),679.4(C-Br)；

¹H NMR(400MHz,CDCl₃)δ:1.53-1.58(m,6H,-(CH₂)₃-),2.23(s,3H,-CH₃),3.68(s,3H,-OCH₃),3.74(t,2H,CH₂-O),3.89(t,2H,-CH₂),4.29(t,2H,-CH₂),4.58(s,1H,CH₂-O),4.58(s,1H,CH-O),5.23(s,1H,-NH)；

ESI-MS:m/z 412(M+H)⁺。

7) Synthesis of 1- (2- (1, 3-dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-3- ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone

Dissolving the obtained 1- (2-bromoethyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (1.6g,4mmol) in 15mL of DMF, adding potassium phthalimide (1.67g,9mmol) and tetrabutylammonium iodide (0.13g,0.4mmol), heating to 70 ℃, stirring for 8H, extracting with dichloromethane and water (v: v ═ 1:1) after the reaction is finished, washing the organic phase with saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, concentrating the organic phase under reduced pressure at 35 ℃, separating and purifying the concentrated solution by silica gel column chromatography, wherein the volume ratio of the eluent to dichloromethane and methanol is 50:1, concentrating under reduced pressure at 35 ℃ to obtain a white solid 1- (2- (1), 3-Dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-3- ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (1.6g,3.4mmol), 85% yield.

IR (KBr pellet) v/cm^-1:2955.5(-CH₃),2801.1(-CH₂),1753.6(C＝O),1621.9(C＝C),1321.3(C-O-C),1079.1(C-N),749(C-H)；

¹H NMR(400MHz,CDCl₃)δ:1.53-1.58(m,6H,-(CH₂)₃-),2.13(s,3H,-CH₃),3.73(s,3H,-OCH₃),3.84(t,2H,CH₂-O),4.12(t,2H,-CH₂),4.42(s,2H,CH₂-O⁶),4.75(t,2H,-CH₂),4.98(s,1H,CH-O),5.23(s,1H,C-H),7.23-8.15(m,4H,-C₈O₂NH₄)；

ESI-MS:m/z 479(M+H)⁺。

8) Synthesis of 1- (2- (1, 3-dioxoisoquinolin-2-yl) ethyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone

Dissolving the obtained 1- (2- (1, 3-dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (1.9g,4mmol) in 20mL of ethanol, adding pyridine p-toluenesulfonate (0.25g,10mmol), reacting at room temperature for 10H, extracting with ethyl acetate and saturated ammonium chloride (v: v ═ 1:1) after the reaction is finished, drying over night anhydrous sodium sulfate, concentrating by reduced pressure distillation at 45 ℃, separating and purifying the concentrated solution by silica gel column chromatography, eluting with ethyl acetate and n-hexane in a volume ratio of 5:1-2:1, performing gradient elution, and carrying out reduced pressure distillation on the eluent at 45 ℃ to obtain a light yellow solid 1- (2- (1), 3-Dioxoisoquinolin-2-yl) ethyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (1.4g,3.6mmol), yield 90%.

IR (KBr pellet) v/cm^-1:3462.7(O-H),2965.6(-CH₃),2868.4(-CH₂),1708.3(C＝O),1561.9(C＝C),1291.7(C-O-C),1013.6(C-N)；

¹H NMR(400MHz,CDCl₃)δ:2.15(s,3H,-CH₃),3.84(s,3H,-OCH₃),3.90(t,2H,-CH₂),4.77(t,2H,-CH₂),4.92(s,2H,-CH₂),5.26(s,1H,CH),5.76(s,1H,OH),7.23-8.15(m,4H,C₆H₄)；

ESI-MS:m/z 395(M+H)⁺。

9) Synthesis of 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (2- (1, 3-dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-1H-indoloquinone

The resulting 1- (2- (1, 3-dioxoisoquinolin-2-yl) ethyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.8g,2mmol) and compound e (0.84g,3mmol), PPh₃(0.78g,3mmol) dissolved in 20mL THF, stirred at 0-4 deg.C for 1h, added EDAD (0.5mL,3mmol) dropwise, and reacted at 25 deg.C for 6 h; after the reaction is finished, carrying out reduced pressure rotary evaporation and concentration on the solvent in the obtained reaction solution at 40 ℃, pouring the concentrated solution into petroleum ether and stirring, separating out a byproduct, namely tri-phenoxy phosphorus, filtering and collecting filtrate, and then carrying out reduced pressure rotary evaporation at 30 ℃ to remove the solvent; separating and purifying with silica gel column chromatography, eluting with ethyl acetate and petroleum ether at volume ratio of 1:1-5:1, and performing gradient elution. The eluate was rotary-evaporated under reduced pressure at 45 ℃ to give 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (2- (1, 3-dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.6g,0.9mmol) as a pale yellow solid in 45% yield.

IR (KBr pellet) v/cm^-1:3654.8(-NH₂),3321.9(N-H),2983.1(-CH₃),2861.7(-CH₂),1742.7(C＝O),1572.3(C＝N),1211.1(C-O-C),1104.5(C-N),629.1(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.92(s,1H,N-H)，2.11(s,3H,-CH₃),3.54(s,3H,-OCH₃),3.66(s,2H,-CH₂-N),3.75(s,2H,-CH₂-N),3.93(t,2H,-CH₂),4.72(t,2H,-CH₂),5.22(s,1H,CH),6.33(s,2H,-NH₂),6.59(s,1H,-C₄SH₂),6.84(s,1H,-C₄SH₂),7.42-7.85(m,4H,-C₈O₂NH₄),8.65(s,1H,CH(C₄N₄H₂)),11.3(s,1H,NH(C₄N₄H₂))；

ESI-MS:m/z 653(M+H)⁺。

10) Synthesis of 3- ((((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -1- (2-aminoethyl) -5-methoxy-2-methyl-1H-indoloquinone

The obtained 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (2- (1, 3-dioxoisoindol-2-yl) ethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.65g,1mmol) and hydrazine hydrate (1.9g,4mmol) were put in a 100mL round-bottomed flask, 15mL of dmf was added, the mixture was heated to 33 ℃ for reaction for 5 hours, after the reaction was completed, dichloromethane and water (v: v ═ 1:1) were extracted, the saturated aqueous sodium chloride solution was washed, anhydrous sodium sulfate was dried overnight, and the mixture was concentrated by rotary evaporation at 35 ℃ under reduced pressure. Separating and purifying the concentrated solution by silica gel column chromatography, wherein the mobile phase comprises dichloromethane and methanol, the dichloromethane/methanol (v/v) is subjected to gradient elution according to the ratio of 30:1-5:1, and the eluent is subjected to rotary evaporation at 35 ℃ under reduced pressure to obtain light yellow solid 3- (((((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -1- (2-aminoethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.26g,0.5mmol) with the yield of 50%.

IR (KBr pellet) v/cm^-1:3619.5(-NH₂),3425.5(N-H),2969.6(-CH₃),2857.5(-CH₂),1778.1(C＝O),1535.7(C＝N),1342.0(C-O-C),1105.1(C-N),795.4(N-H),626.2(C-S)；

¹H NMR(400MHz,CDCl₃)δ:2.01(s,1H,N-H),2.14(s,3H,-CH₃),2.93(t,2H,-CH₂),3.57(s,3H,-OCH₃),3.71(s,2H,-CH₂-N),3.79(s,2H,-CH₂-N),4.62(t,2H,-CH₂),5.02(s,2H,-NH₂-CH₂),5.26(s,1H,CH),6.33(s,2H,-NH₂),6.59(s,1H,-C₄SH₂),6.89(s,1H,-C₄SH₂),8.67(s,1H,CH(C₄N₄H₂)),11.34(s,1H,N-H-(C₄N₄H₂))；

ESI-MS:m/z 523(M+H)⁺。

11) Synthesis of 1- (2- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -5-methoxy-2-methyl-4, 7-dioxy-4, 7-dihydro-1H-indol-1-yl) ethyl) -3- (2-chloroethyl) urea

Dissolving the obtained 3- ((((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophene-3-yl) methyl) amino) methyl) -1- (2-aminoethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.26g,0.5mmol) in 20mL of dichloromethane, dropwise adding a 2-chloroethyl isocyanate solution (7mL,1.1mmol, dissolving dichloromethane) under ice bath conditions, reacting at 0-10 ℃ for 4H, and after the reaction is finished, carrying out reduced pressure rotary evaporation at 35 ℃ to obtain a brown solid 1- (2- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophene-3-yl) methyl) amino) -5-methoxy-2-methyl-4 7-dioxy-4, 7-dihydro-1H-indol-1-yl) ethyl) -3- (2-chloroethyl) urea (0.18g,0.28mmol), 55% yield.

IR (KBr pellet) v/cm^-1:3652.7(-NH₂),3409.8(N-H),1754.7(C＝O),1505.6(C＝N),1486.5(C＝N),1332.1(C-O-C),1107.9(C-N),795.4(N-H),772.8(C-Cl),631.8(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.78(s,1H,N-H),2.14(s,3H,-CH₃),3.43(t,2H,-CH₂),3.52(s,3H,-OCH₃),3.57(t,2H,-CH₂),3.67(t,2H,-CH₂),3.81(s,2H,-CH₂-N),4.23(s,2H,-CH₂-N),4.62(t,2H,-CH₂),5.24(s,1H,CH),6.03(s,2H,N-H),6.33(s,2H,-NH₂),6.59(s,1H,-C₄SH₂),6.75(s,1H,-C₄SH₂),8.67(s,1H,CH(C₄N₄H₂)),11.42(s,1H,N-H)；

ESI-MS:m/z 628(M+H)⁺。

12) Synthesis of 3- (2- (3- ((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -5-methoxy-2-methyl-4, 7-dioxo-2, 3,4, 7-tetrahydro-1H-indol-1-yl) ethyl) -1- (2-chloroethyl) -1-nitrosourea (Compound 1)

The resulting 1- (2- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -5-methoxy-2-methyl-4, 7-dioxy-4, 7-dihydro-1H-indol-1-yl) ethyl) -3- (2-chloroethyl) urea (0.31g,0.5mmol) was dissolved in 8mL of acetonitrile, a glacial acetic acid acetonitrile solution (3mL,0.2mmol) was added, nitrosonium tetrafluoroborate (0.48g,4mmol) was added under ice bath conditions, the reaction was continued for 4H, after completion of the reaction, extraction was carried out with dichloromethane and water (v: v ═ 1:1), the organic phase was washed with a saturated aqueous sodium chloride solution, drying with anhydrous sodium sulfate overnight, removing solvent by rotary evaporation at 35 deg.C under reduced pressure, separating and purifying with silica gel column chromatography, eluting with dichloromethane and methanol at volume ratio of dichloromethane/methanol of 50:1-5:1, and vacuum drying the eluate at 30 deg.C to obtain brown solid compound 1(0.18g,0.28mmol) with yield of 55%.

IR (KBr pellet) v/cm^-1:3592.5(-NH₂),3305.1(N-H),2982.1(-CH₂),2854.3(-CH₂),1724.8(C＝O),1574.5(N＝O),1332.1(C-O-C),1099.5(C-N),785.6(N-H),775.3(C-Cl),619.5(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.87(s,1H,N-H),2.16(s,3H,-CH₃),2.21(s,3H,-OCH₃),3.17(t,2H,-CH₂),3.44(t,4H,-CH₂),3.57(t,2H,-CH₂),3.84(s,2H,-CH₂-N),4.08(s,2H,-CH₂-N),5.47(s,2H,CH₂),6.03(s,1H,-NH),6.23(s,2H,-NH₂),6.47(s,1H,-C₄SH₂),6.73(s,1H,-C₄SH₂),8.57(s,1H,CH(C₄N₄H₂)),12.92s,1H,N-H)；

ESI-MS:m/z 659(M+H)⁺。

Example 2: synthesis of 3- (3- (3- ((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -5-methoxy-2-methyl-4, 7-dioxo-4, 7-dihydro-1H-indol-1-yl) propyl) -1- (2-chloroethyl) -1-nitrosourea (Compound 2)

1) Synthesis of (4- (aminomethyl) thiophen-2-yl) methanol

Weighing 4- (aminomethyl) thiophene-2-carboxylic acid ethyl ester (2.22g,12mmol) and dissolving in 10mL of anhydrous ether; weighing LiAlH₄(0.25g,6.5mmol) in a 100mL round-bottom flask in N₂Adding 10mL of anhydrous ether under the protection and ice bath conditions, and stirring for 10 min; 4- (aminomethyl) thiophene-2-carboxylic acid ethyl ester solution is dripped to react for 2 hours at the temperature of 0-10 ℃; after the reaction was completed, the reaction was quenched with water, 10% sodium hydroxide solution, and water (v/v/v ═ 1:2:3 (water: 0.25mL)) with stirring in an ice bath. Concentrating the reaction solution by rotary evaporation at 50 deg.C under reduced pressure, separating and purifying the concentrated solution by silica gel column chromatography, eluting with petroleum ether and ethyl acetate at a volume ratio of petroleum ether/ethyl acetate of 1:2-1:4, and eluting the eluate at 35 deg.C under reduced pressureRotovap gave (4- (aminomethyl) thiophen-2-yl) methanol (1.13g,8mmol) as a white solid in 66% yield.

IR (KBr pellet) v/cm^-1:3659.1(-NH₂),3523.1(O-H),2985.9(-CH₂),1525.7(C＝C),1058.0(C-N),1074.8(C-O),667.3(C-S)；

¹H NMR(400MHz,CDCl₃)δ:4.29(s,2H,CH₂-N),4.73(s,2H,CH₂-O),5.33(s,1H,O-H),6.56(s,1H,C₄H₂S),8.37(s,1H,C₄H₂S),8.49(s,2H,-NH₂)；

ESI-MS:m/z 144(M+H)⁺。

The resulting (4- (aminomethyl) thiophen-2-yl) methanol (1.72g,12mmol), (Boc) was weighed₂O (4.0g,18mmol) and 4-dimethylaminopyridine (0.24g,2mmol) are put into a 50mL round-bottom flask, 20mL of water is added, and the mixture is reacted for 10 hours at the temperature of 0-4 ℃; after the reaction is finished, continuing extracting dichloromethane and water (v: v ═ 1:1), collecting an organic phase, washing the organic phase by using a saturated sodium chloride aqueous solution, drying the organic phase over night, carrying out reduced pressure rotary evaporation concentration at 35 ℃, separating and purifying concentrated solution by using silica gel column chromatography, wherein an eluent is dichloromethane and methanol, isocratic elution is adopted, the volume ratio of dichloromethane to methanol is 20:1, and the eluent is reduced pressure rotary evaporation at 35 ℃ to obtain white solid tert-butyl ((5- (hydroxymethyl) thiophen-3-yl) methyl) carbamate (2.68g,11mmol) with the yield of 92%.

IR (KBr pellet) v/cm^-1:3486.2(N-H),3368.2(O-H),2968.9(-CH₃)2859.3(-CH₂),1739.1(C＝O),1196.7(C-O-C),1043.5(C-N),1016.8(C-O),644.4(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.41(s,9H,-CH₃),4.42(s,2H,CH₂-N),4.88(s,2H,CH₂-O),6.04(s,1H,OH),6.64(s,1H,C₄H₂S),7.93(s,1H,C₄H₂S),8.69(s,1H,NH)；

ESI-MS:m/z 244(M+H)⁺。

The resulting tert-butyl ((5- (hydroxymethyl) thiophen-3-yl) methyl) carbamate (0.61g,2.5mmol) was weighed into a 50mL round-bottomed flask, 15mL DMF was added, 1- (2-amino-9H-purin-6-yl) -1-methylpyrrolidine-1-chloride (1.02g,4mmol), potassium tert-butoxide (0.57g,5mmol), dimethylaminopyridine (0.76g,0.3mmol) were added, and the mixture was stirred under N₂Reacting for 8 hours at 25 ℃ under protection; after the reaction was complete, the reaction was quenched with 1.5mL of glacial acetic acid solution (glacial acetic acid/water (v/v ═ 1:10)), then extracted with ethyl acetate and saturated ammonium chloride (v: v ═ 1:1), the organic phase was collected, dried over anhydrous sodium sulfate overnight, and concentrated by rotary evaporation at 45 ℃. Separating and purifying the concentrated solution by silica gel column chromatography, wherein the eluent is dichloromethane and methanol, gradient elution is adopted, the volume ratio of dichloromethane/methanol is 50:1-10:1, and the eluent is subjected to rotary evaporation at 35 ℃ under reduced pressure to obtain white solid tert-butyl ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl carbamate (0.56g,1.5mmol), and the yield is 60%.

IR (KBr pellet) v/cm^-1:3635.4(-NH₂),3407.5(N-H),2948.9(-CH₃),2826.8(-CH₂),1733.5(C＝O),1516.8(C＝N),1048.4(C-O-C),1038.2(C-N),648.5(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.67(s,9H,-CH₃),4.46(s,2H,CH₂-N),5.22(s,2H,CH₂-O),6.53(s,2H,-NH₂),6.82(s,1H,C₄H₂S),7.85(s,1H,C₄H₂S),8.34(s,1H,C-H),11.84(s,1H,N-H)；

ESI-MS:m/z 377(M+H)⁺。

4) Synthesis of 6- ((4- (aminomethyl) thiophen-2-yl) methoxy) -9H-purin-2-amine

The resulting tert-butyl ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methylcarbamate (3.8g,10mmol) was weighed into a 50mL round-bottomed flask, 30mL of 50% trifluoroacetic acid (trifluoroacetic acid: dichloromethane (v/v ═ 1:1)) was added, and the reaction was carried out at 25 ℃ for 6 hours; after the reaction was completed, the reaction solution was neutralized with a saturated sodium bicarbonate solution to pH 8, followed by extraction with dichloromethane and saturated water (v: v ═ 1:1), the organic phase was collected, washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate overnight, and the solvent was evaporated under reduced pressure at 35 ℃ to give 6- ((4- (aminomethyl) thiophen-2-yl) methoxy) -9H-purin-2-amine (2.21g,8mmol) as a white solid in 80% yield.

ESI-MS:m/z 277(M+H)⁺。

Weighing 3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.9g,4mmol) into a 50mL round-bottom flask, adding 25mL anhydrous dichloromethane, adding tetrahydropyrane ether (0.1g,5mmol) and p-toluenesulfonic acid (0.86mg,0.005mmol), and reacting at 25 ℃ for 8H; after completion of the reaction, the reaction mixture was extracted with dichloromethane and water (v: v ═ 1:1), and the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate overnight, and concentrated to remove the solvent by rotary evaporation under reduced pressure at 35 ℃. Separating and purifying the concentrated solution by silica gel column chromatography, wherein the eluent is dichloromethane and methanol, gradient elution is adopted, the volume ratio of dichloromethane to methanol is 100:1-50:1, and the eluent is subjected to decompression rotary evaporation at 35 ℃ to obtain white solid 5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (0.98g,3.2mmol), and the yield is 80%.

IR (KBr pellet) v/cm^-1:3396.8(N-H),2917.9(-CH₃),2879.2(-CH₂),1709.9(C＝O),1637.2(C＝C),1366.5(C-O-C),1186.3(C-N)；

¹H NMR(400MHz,CDCl₃)δ:1.75-1.85(m,6H,-(CH₂)₃-),2.18(s,3H,-CH₃),3.87(s,3H,-OCH₃),4.69(t,2H,CH₂-O),5.02(s,1H,CH₂-O),5.66(s,2H,CH₂-O⁶),5.85(s,1H,NH),8.77(s,1H,NH)；

ESI-MS:m/z 306(M+H)⁺。

6) Synthesis of 1- (2-bromopropyl) -5-methoxy-2-methyl-3- (((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone

Weighing the obtained 5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indolone (1.8g,6mmol) and anhydrous potassium carbonate (2.5g,18mmol) in a 100mL round-bottom flask, adding 50mL acetone, dropwise adding 1.2mL,12mmol 1, 3-dibromopropane, reacting at 60 ℃ for 72H, filtering the reaction solution, collecting the filtrate, performing reduced pressure rotary evaporation at 50 ℃ to remove the solvent, purifying by silica gel column chromatography, wherein the eluent is petroleum ether and ethyl acetate, performing isocratic elution, the volume ratio of the petroleum ether to the ethyl acetate is 1:2, and performing reduced pressure rotary evaporation at 45 ℃ to obtain a white solid, namely 1- (2-bromopropyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (1.40g,3.3mmol), yield 55%.

IR (KBr pellet) v/cm^-1:2909.1(-CH₃),2867.4(-CH₂),1752.3(C＝O),1608.4(C＝C),1321.0(C-O-C),1076.3(C-N),683.1(C-Br)；

¹H NMR(400MHz,CDCl₃)δ:1.53-1.58(m,6H,-(CH₂)₃-),2.11(s,3H,-CH₃),2.36(m,2H,-CH₂),3.49(t,2H,-CH₂),3.69(t,2H,-CH₂-O),3.77(s,3H,-OCH₃),4.09(t,2H,-CH₂),4.58(s,2H,CH₂-O),4.98(s,1H,CH-O),5.23(s,1H,-NH)；

ESI-MS:m/z 426(M+H)⁺。

7) Synthesis of 1- (3- (1, 3-dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-3- ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone

Dissolving the obtained 1- (2-bromopropyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (2.6g,6mmol) in 20mL of DMF, adding potassium phthalimide (3.42g,18mmol) and tetrabutylammonium iodide (0.2g,0.6mmol), heating to 70 ℃, stirring for 8H, extracting with dichloromethane and water (v: v ═ 1:1) after the reaction is finished, washing the organic phase with saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, concentrating the organic phase by reduced pressure distillation at 35 ℃, separating and purifying the concentrated solution by silica gel column chromatography, wherein the volume ratio of dichloromethane to methanol is 30:1, carrying out reduced pressure distillation at 35 ℃ to obtain a white solid 1- (3- (1), 3-Dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-3- ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (2.1g,4.2mmol), yield 70%.

IR (KBr pellet) v/cm^-1:2947.3(-CH₃),2864.5(-CH₂),1786.1(C＝O),1594.0(C＝C),1307.6(C-O-C),1097.3(C-N)；

¹H NMR(400MHz,CDCl₃)δ:1.45-1.68(m,6H,-(CH₂)₃-),2.16(s,3H,-CH₃),2.68(m,2H,-CH₂),3.56(s,3H,-OCH₃),3.62(t,2H,-CH₂-O),4.24(t,2H,-CH₂-N),4.46(t,2H,-CH₂),4.75(s,2H,CH₂-O),5.03(s,1H,CH-O),5.45(s,1H,-NH),7.72-7.83(m,4H,C₆H₄)；

ESI-MS:m/z 493(M+H)⁺。

8) Synthesis of 1- (3- (1, 3-dioxoisoquinolin-2-yl) propyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone

Dissolving the obtained 1- (3- (1, 3-dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-3- (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-indoloquinone (2.96g,6mmol) in 20mL of ethanol, adding pyridine p-toluenesulfonate (0.3g,12mmol), reacting at room temperature for 8H, extracting with ethyl acetate and saturated ammonium chloride (v: v ═ 1:1) after the reaction is finished, drying over night anhydrous sodium sulfate, concentrating by reduced pressure distillation at 45 ℃, separating and purifying the concentrated solution by silica gel column chromatography, eluting with ethyl acetate and n-hexane in a volume ratio of 5:1-2:1, performing gradient elution, and carrying out reduced pressure distillation on the eluent at 45 ℃ to obtain a light yellow solid 1- (3- (1), 3-Dioxoisoquinolin-2-yl) propyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (1.96g,4.8mmol), yield 80%.

IR (KBr pellet) v/cm^-1:3402.6(O-H),2967.4(-CH₃),2875.8(-CH₂),1746.8(C＝O),1532.5(C＝C),1209.8(C-O-C),1056.2(C-N)；

¹H NMR(400MHz,CDCl₃)δ:2.15(s,3H,-CH₃),2.65(m,2H,-CH₂),3.56(s,1H,OH),3.84(s,3H,-OCH₃),4.30(t,2H,-CH₂),4.77(t,2H,-CH₂),4.92(s,2H,-CH₂),5.26(s,1H,CH),7.23-8.15(m,4H,-C₆H₄)；

ESI-MS:m/z 409(M+H)⁺。

9) Synthesis of 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (3- (1, 3-dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-1H-indoloquinone

The resulting 1- (3- (1, 3-dioxoisoquinolin-2-yl) propyl) -3- (hydroxymethyl) -5-methoxy-2-methyl-1H-indoloquinone (0.66g,1.6mmol) and compound e (0.7g,2.5mmol), PPh₃(0.62g,2.4mmol) in 25mL THF, stirring at 0-4 deg.C for 1h, adding DEAD (0.4mL,2.4mmol) dropwise, reacting at 25 deg.C for 8 h; after the reaction is finished, carrying out reduced pressure rotary evaporation and concentration on the solvent in the obtained reaction solution at 40 ℃, pouring the concentrated solution into petroleum ether and stirring, separating out a byproduct, namely tri-phenoxy phosphorus, filtering and collecting filtrate, and then carrying out reduced pressure rotary evaporation at 30 ℃ to remove the solvent; separating and purifying with silica gel column chromatography, eluting with ethyl acetate and petroleum ether at volume ratio of 1:1-5:1, and performing gradient elution. The eluate was rotary-evaporated under reduced pressure at 45 ℃ to give 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (3- (1, 3-dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-1H-indoloquinone (0.56g,0.8mmol) as a pale yellow solid in 50% yield.

IR (KBr pellet) v/cm^-1:3636.3(-NH₂),3321.5(N-H),2956.7(-CH₃),2871.3(-CH₂),1772.6(C＝O),1547.6(C＝N),1211.1(C-O-C),1044.9(C-N),630.5(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.85(s,1H,N-H)，2.16(s,3H,-CH₃),2.84(m,2H,-CH2),3.47(s,3H,-OCH₃),3.64(s,2H,-CH₂-N),3.79(s,2H,-CH₂-N),4.43(t,2H,-CH₂),4.72(t,2H,-CH₂),5.26(s,1H,CH),6.29(s,2H,-NH₂),6.59(s,1H,-C₄SH₂),7.18(s,1H,-C₄SH₂),7.42-7.85(m,4H,-C₈O₂NH₄),8.75(s,1H,CH(C₄N₄H₂)),10.8(s,1H,NH(C₄N₄H₂))；

ESI-MS:m/z 667(M+H)⁺。

10) Synthesis of 3- ((((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -1- (3-aminopropyl) -5-methoxy-2-methyl-1H-indoloquinone)

The obtained 3- (((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -1- (3- (1, 3-dioxoisoindol-2-yl) propyl) -5-methoxy-2-methyl-1H-indoloquinone (1.33g,2mmol) and hydrazine hydrate (3.8g,8mmol) were put in a 100mL round-bottomed flask, 25mL of acetonitrile was added thereto, the mixture was heated to 35 ℃ for reaction for 5 hours, and after completion of the reaction, dichloromethane and water (v: v ═ 1:1) were extracted, washed with a saturated aqueous sodium chloride solution, dried overnight with anhydrous sodium sulfate, and concentrated by rotary evaporation at 35 ℃. Then separating and purifying the concentrated solution by silica gel column chromatography, wherein the mobile phase comprises dichloromethane and methanol, the ratio of dichloromethane/methanol (v/v) is 30:1-5:1, gradient elution is carried out, and the eluent is subjected to decompression and rotary evaporation at 35 ℃ to obtain light yellow solid 3- (((((((5- (((2-amino-9H-purine-6-group) oxy) methyl) thiophene-3-group) methyl) amino) methyl) -1- (3-aminopropyl) -5-methoxy-2-methyl-1H-indoloquinone (0.85g,1.2mmol), and the yield is 60%.

IR (KBr pellet) v/cm^-1:3603.4(-NH₂),3445.8(N-H),29478.7(-CH₃),2847.8(-CH₂),1726.8(C＝O),1564.7(C＝N),1323.4(C-O-C),1113.6(C-N),783.1(N-H),631.4(C-S)；

¹H NMR(400MHz,CDCl₃)δ:2.03(s,1H,N-H),2.14(s,3H,-CH₃),4.56(t,2H,-CH₂),2.93(m,2H,-CH₂),3.63(s,3H,-OCH₃),3.74(s,2H,-CH₂-N),3.83(s,2H,-CH₂-N),4.25(t,2H,-CH₂),5.11(s,2H,-NH₂-CH₂),5.43(s,1H,CH),6.19(s,2H,-NH₂),6.38(s,1H,-C₄SH₂),7.19(s,1H,-C₄SH₂),8.86(s,1H,CH(C₄N₄H₂)),12.04(s,1H,N-H-(C₄N₄H₂))；

ESI-MS:m/z 537(M+H)⁺。

11) Synthesis of 1- (3- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -5-methoxy-2-methyl-4, 7-dioxy-4, 7-dihydro-1H-indol-1-yl) propyl) -3- (2-chloroethyl) urea

Dissolving the obtained 3- (((((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophene-3-yl) methyl) amino) methyl) -1- (3-aminopropyl) -5-methoxy-2-methyl-1H-indolone (0.43g,0.8mmol) in 20mL of dichloromethane, dropwise adding a 2-chloroethyl isocyanate solution (11.5mL,1.8mmol and dissolved dichloromethane) under ice bath conditions, reacting at 0-10 ℃ for 4H, and after the reaction is finished, carrying out reduced pressure rotary evaporation at 35 ℃ to obtain a brown solid 1- (3- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophene-3-yl) methyl) amino) -5-methoxy-2-methyl- 4, 7-dioxy-4, 7-dihydro-1H-indol-1-yl) propyl) -3- (2-chloroethyl) urea (0.21g,0.32mmol), yield 40%.

IR (KBr pellet) v/cm^-1:3639.5(-NH₂),3399.6(N-H),1738.4(C＝O),1484.9(C＝N),1431.9(C＝N),1308.2(C-O-C),1120.4(C-N),767.6(N-H),782.6(C-Cl),626.9(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.92(s,1H,N-H),2.23(s,3H,-CH₃),2.72(m,2H,-CH₂),3.46(s,3H,-OCH₃),3.57(t,2H,-CH₂),3.61(s,2H,-CH₂-N),3.81(s,2H,-CH₂-N),4.14(t,2H,-CH₂),4.37(t,2H,-CH₂),4.71(t,2H,-CH₂),5.31(s,1H,CH),6.03(s,2H,N-H),6.87(s,2H,-NH₂),6.45(s,1H,-C₄SH₂),7.35(s,1H,-C₄SH₂),8.45(s,1H,CH(C₄N₄H₂)),12.57(s,1H,N-H)；

ESI-MS:m/z 642(M+H)⁺。

12) Synthesis of 3- (2- (3- ((((5- (((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) methyl) -5-methoxy-2-methyl-4, 7-dioxo-2, 3,4, 7-tetrahydro-1H-indol-1-yl) ethyl) -1- (2-chloroethyl) -1-nitrosourea (Compound 2)

The resulting 1- (3- (3- ((5- ((2-amino-9H-purin-6-yl) oxy) methyl) thiophen-3-yl) methyl) amino) -5-methoxy-2-methyl-4, 7-dioxy-4, 7-dihydro-1H-indol-1-yl) propyl) -3- (2-chloroethyl) urea (0.27g,0.4mmol) was dissolved in 8mL of acetonitrile, a glacial acetic acid acetonitrile solution (3mL,0.2mmol) was added, nitrosonium tetrafluoroborate (0.38g,3.2mmol) was added under ice bath conditions, the reaction was continued for 4H, after completion of the reaction, extraction was carried out with dichloromethane and water (v: v ═ 1:1), the organic phase was washed with a saturated aqueous sodium chloride solution, drying over night with anhydrous sodium sulfate, distilling at 35 deg.C under reduced pressure to remove solvent, separating and purifying with silica gel column chromatography, eluting with dichloromethane and methanol, and gradient eluting with dichloromethane/methanol volume of 50:1-5:1, vacuum drying the eluate at 30 deg.C to obtain brown solid compound 2(0.14g,0.2mmol) with 50% yield.

IR (KBr pellet) v/cm^-1:3614.6(-NH₂),3416.7(N-H),2962.5(-CH₂),2816.8(-CH₂),1729.0(C＝O),1479.4(N＝O),1316.8(C-O-C),1065.9(C-N),769.5(C-Cl),637.8(C-S)；

¹H NMR(400MHz,CDCl₃)δ:1.99(s,1H,N-H),2.15(s,3H,-CH₃),2.48(m,2H,-CH₂),3.06(t,2H,-CH₂),3.47(t,4H,-CH₂),3.77(s,3H,-OCH₃),3.89(s,2H,-CH₂-N),4.18(s,2H,-CH₂-N),4.57(t,2H,-CH₂),5.36(s,2H,CH₂),6.13(s,1H,-NH),6.33(s,2H,-NH₂),6.38(s,1H,-C₄SH₂),7.76(s,1H,-C₄SH₂),8.63(s,1H,CH(C₄N₄H₂)),11.63(s,1H,N-H)；

ESI-MS:m/z 671(M+H)⁺。

The novel chloroethyl nitrosourea compound prepared by the invention has the following evaluation on the anti-tumor activity:

experimental example 1 evaluation of antitumor Activity

1. Experimental materials and instruments

Test Compounds Compound 1, Compound 2, free carmustine (B) prepared in the above preparation examplesCNU) and BCNU + O⁶-TMG；

Cell line: human glioma cells SF763, SF767, SF126, A549, human breast cancer cell MCF-7, human prostate cancer cell DU 145;

2. experimental methods

The six tumor cells were inoculated into a 96-well plate at 1000/well and 5% CO at 37 deg.C₂After 24h of culture, BCNU (positive control group 1), BCNU + O were administered at concentrations of 20. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 500. mu.M, 800. mu.M, 1000. mu.M and 2000. mu.M⁶TMG (positive control group 2), compound 1, compound 2 total 4 drug treatment groups, 6 duplicate wells per group, and control groups were set. The CCK-8 solution was allowed to react for 4 hours. Treating the above components under aerobic and low-oxygen conditions for 48 h; then, 10. mu.L of CCK-8 solution was added to each well; finally, the absorbance value at 450nm was determined. The cell activity was calculated according to the following formula and the median inhibition IC was calculated by regression analysis₅₀。

Tumor cell survival rate (%) ═ a_{Drug treatment group}–A_{Blank group})/(A_{Control group}–A_{Blank group})×100％

A_{Drug treatment group}Absorbance values for wells with media, tumor cells, drug solution and CCK-8 solution;

A_{blank group}Absorbance values for wells with media and CCK-8 solution, but no tumor cells and drug;

A_{control group}Absorbance values for wells with media, tumor cells, CCK-8 solution, but no drug solution.

3. The experimental results are shown in Table 1

TABLE 1 half inhibition rate (IC) of tumor cells₅₀，μM)

Table 1 the results show that:

IC of Compounds 1 and 2 on 6 tumor cells in normoxic Environment₅₀The values were similar to those of the positive control group 1, indicating that the tumor cell inhibitory activities of compounds 1 and 2 were not much different from those of the BCNU group under normoxic conditions. IC of Compounds 1 and 2 on 6 tumor cells₅₀The value is different from that of the positive control group 2, and the tumor cell inhibition activity of the compounds 1 and 2 is lower than that of the positive control group 2 for 5 tumor cells (SF763, SF767, A549, MCF-7 and DU145) with high AGT expression, which indicates that O⁶TMG derivatives in combination with BCNU, help to reduce cellular resistance; in contrast, compounds 1 and 2 do not release AGT inhibitors under normoxic conditions, and cannot effectively inhibit AGT-mediated drug resistance, so that the inhibition rate of the compounds on tumor cells is low.

IC of Compounds 1 and 2 on 5 AGT-highly expressed tumor cells in hypoxic Environment₅₀The value was significantly lower than that of the positive control group 1. This indicates that compounds 1 and 2 are reduced in a low oxygen environment, releasing O⁶TMG derivatives act as AGT inhibitors, blocking AGT mediated tumor cell resistance, making cancer cells more sensitive to alkylation by the chloroethylnitrosourea pharmacophore; meanwhile, the reduction product of indoloquinone released by the medicament under the hypoxia condition further causes DNA damage, so that the antitumor activity of the compounds 1 and 2 is obviously improved. The inhibitory activity of compounds 1 and 2 was also higher for AGT-low expressing SF126 cells than control 1 due to the dual tumor cytotoxic effect of indoloquinone reduction product with chloroethylnitrosourea.

IC comparing Compounds 1 and 2 in normoxic and hypoxic environments₅₀Values, it can be seen that the tumor cell inhibitory activity of compounds 1 and 2 is significantly improved in the hypoxic environment compared to that of compounds 1 and 2 in the normoxic environment, indicating that compounds 1 and 2 have ideal hypoxic selectivity. Therefore, the compounds 1 and 2 can selectively act on tumor cells in a hypoxic microenvironment to avoid damage to normal cells under an normoxic condition, so that the aim of targeting on the tumor cells is fulfilled.

The experimental result shows that the compound provided by the invention has higher tumor inhibition activity than the prior chloroethylnitrosourea under the low oxygen condition; meanwhile, the compounds can specifically act on tumor cells and can be used for tumor targeted therapy.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The drug-resistant tumor hypoxia targeting chloroethylnitrosourea combined molecule is characterized in that the structural formula is shown as (I):

R₁is OCH₃，OCH₂CH₃，NH₂，NCH₂CH₂One of (1);

R₄is H, CH₂CH₂NH₂One of (1);

n is an integer of 2 to 6.

2. The chloroethylnitrosourea combination molecule according to claim 1, wherein R is R₁Is OCH₃And NCH₂CH₂，R₂Is H, R₃Is CH₃，CH₂CH₃，CH₂OHOCONH₂，R₄Is H.

3. A chloroethylnitrosourea combination molecule according to claim 1, when R is₁Are respectively OCH₃，R₃Is CH₃I.e. having the following structural formula:

4. a pharmaceutical composition characterized by: the pharmaceutical composition comprises a compound or pharmaceutically acceptable salt of formula (I) as defined in claim 1, and at least one pharmaceutically acceptable carrier;

5. A pharmaceutical composition according to claim 4, wherein: the drug carrier is stabilizer, dispersant, emulsifier, disintegrant, lubricant, water-retaining agent or diluent.

6. A process for preparing a compound according to claim 1, wherein:

(2) amino group of Compound b is (Boc)₂O acid anhydride protectionObtaining a compound c;

(6) reacting the compound g with dihalogenated alkane to obtain a compound h;

(7) reacting the compound h with potassium phthalimide to obtain a compound i;

(12) reacting the compound m with nitrosonium tetrafluoroborate to obtain a compound n;

the method comprises the following steps:

(4) treating the compound d with trifluoroacetic acid or a mixture of trifluoroacetic acid and dichloromethane in a volume ratio of v/v ═ 1:1 at 25 ℃ to obtain a compound e;

7. The method of claim 6, wherein:

the specific operation of the step (1) is that LiAlH is added under the ice bath condition₄Dissolving in anhydrous solvent, stirring for 5-20min, adding dropwise reactant solution a, and adding N₂Reacting for 1-2h at 0-10 ℃ under protection to obtain a compound b; wherein, the compound a and LiAlH₄The molar ratio of (1) to (0.5-1); dissolving LiAlH₄The anhydrous solvent of (1) is Tetrahydrofuran (THF), diethyl ether, ethylene glycol or dimethyl ether; the solvent for dissolving the compound a is anhydrous THF or anhydrous diethyl ether; further comprising a step of purifying the compound b, specifically, after the reaction is finished, under the condition of ice bath stirring, quenching the mixture of water, 10% sodium hydroxide solution by mass percent and water in a v/v/v-1: 2:3 ratio, and carrying out rotary evaporation on the reaction solution at 50 ℃ under reduced pressure; and then separating and purifying the concentrated solution by adopting a column chromatography to obtain a purified compound b.

8. The use of the compound or pharmaceutically acceptable salt of claim 1 and compositions thereof in the preparation of anti-tumor medicaments.

9. The use of claim 8, wherein the tumor is one or more of brain tumor, glioma, myeloma, malignant melanoma, malignant lymphoma, liver cancer, breast cancer, stomach cancer, colon cancer, prostate tumor, leukemia, and lymphoma.