WO2015110040A1 - Chlorambucil derivative, preparation method and application - Google Patents

Abstract

Disclosed are a chlorambucil derivative provided with a structure of formula I, II, or III and a pharmaceutically acceptable salt thereof. The present invention also relates to a preparation method of a preceding compound, a preparation and an application.

Description

Chlorambucil derivative, preparation method and application thereof Technical field

The present invention relates to a benzoic acid nitrogen mustard derivative, a pharmaceutically acceptable salt thereof, a preparation method, a preparation, and use as an antitumor drug.

Background technique

At present, targeted therapy has become an important direction for cancer treatment, using a single drug-targeted treatment mode, such as irinotecan (CPT-11) for the treatment of advanced colorectal cancer acting on DNA topoisomerase I. And paclitaxel (Taxol) or the like for treating ovarian cancer and breast cancer acting on intracellular microtubules. However, tumors are different from general diseases. Its growth and survival depend not only on the conduction of a receptor or a signaling pathway, but the strategy of acting on a single target does not completely kill tumor cells and is easy to produce. Drug resistance. Therefore, the combination of multiple drugs has become the main means of clinical treatment of cancer. Although it can achieve the expected therapeutic effect to a certain extent, it is easy to interact with each other, such as the absorption and metabolism of drugs, even if Without interaction, these drugs are usually not combined with the doses when they are used alone. To this end, drug researchers draw on the principle of multiple drug combinations, using a combination of pharmacophores, two or more The pharmacophores are combined into one molecule to cause the molecule itself or its metabolite to act on two or more targets, thereby producing a synergistic effect to improve the therapeutic effect. Therefore, the design and synthesis of anticancer drugs acting on two or more targets can not only improve the therapeutic effect, but also avoid side effects such as drug interactions caused by a combination of drugs, thereby reducing toxic side effects.

Camptothecin (20(s)-camptothecin, formula 1-1) and its derivatives have excellent antitumor activity and are an important class of DNA topoisomerase I (Top I) inhibitors. The I-DNA cleavable complex binds to form a CPT-Top I-DNA ternary complex, thereby stabilizing the cleavable complex, resulting in cell death. However, due to its poor solubility and toxicity in water and other biocompatible solvents, it eventually failed to enter the clinic. Many camptothecin derivatives have been synthesized in order to increase the water solubility of the drug and preserve the anti-tumor properties of the parent compound. However, only the derivatives Topotecan (formula 1-4) and irinotecan (Irinotrcan, formula 1-5) have been approved by the US Food and Drug Administration (FDA) for clinical use, respectively, for the treatment of ovarian cancer. , lung cancer and rectal cancer. However, topotecan and irinotecan have obvious shortcomings, including short half-life in the body, large side effects and limited therapeutic effects. Currently, several camptothecin derivatives are in clinical research.

1.20(S)-camptothecin, R=R ₁ =R ₂ =H

2.10-hydroxycamptothecin, R=OH, R ₁ =H

3.7-ethyl-10-hydroxycamptothecin, R=OH, R ₁ =H, R ₂ =CH ₃ H ₂ -

4.topotecan, R=OH, R ₁ =(CH ₃ ) ₂ NCH ₂ -, R ₂ =H

5.irinotecan, R ₁ =H, R ₂ =Et,

Formula 1. The chemical structure of camptothecin and its derivatives, topotecan and irinotecan.

Nitrogen mustard compounds (including nitrogen mustard, cyclophosphamide, and chlorambucil) are anticancer drugs for clinical use and achieving outstanding therapeutic effects. It is a highly active compound which is a dichloroethylamine alkylating agent. After entering the body, nitrogen mustard compounds form a highly active ethyleneimine ion by intramolecular ring formation, and rapidly react with nucleophilic groups of various organic substances under neutral or weak base conditions (such as carboxyl and amino groups of proteins). The alkyl group is bonded to the thiol group, the amino group of the nucleic acid, and the hydroxyl group and the phosphate group. The most important reaction of nitrogen mustard compounds is to covalently bind to the 7th nitrogen of guanine, resulting in cross-linking within the double strand of DNA or cross-linking of different bases within the same strand of DNA.

Summary of the invention

The object of the present invention is to provide a chlorambucil derivative having the chemical structure of formulas I, II and III, which is 4-[bis(2-chloroethyl)amino]phenylbutyric acid with camptothecin or hi A new compound in which a tree base derivative molecule is bonded via an ester bond:

Wherein: R is H or a C1-C6 linear or branched saturated or unsaturated hydrocarbon group, preferably H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ or CH(CH ₃ ) ₂ , more preferably H or CH ₂ CH ₃ .

Another object of the present invention is to provide a method for synthesizing the chlorambucil derivative compounds I, II and III.

The above compound I, II or III can be produced by esterification of 4-[bis(2-chloroethyl)amino]phenylbutyric acid with camptothecin or camptothecin derivatives. Two preparation methods are preferred,

Method 1: The following steps are included:

1) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid with an acid chloride reagent (such as thionyl chloride, (SOCl ₂ ), oxalyl chloride ((COCl) ₂ ), phosphorus oxychloride (POCl _{3 )} The reaction produces 4-[bis(2-chloroethyl)amino]phenylbutanoyl chloride;

2) 4-[Bis(2-chloroethyl)amino]phenylbutanoyl chloride is further combined with 7-R-10-hydroxycamptothecin or 10-hydroxycamptothecin (10-) Hydroxycamptothecin) or camptothecin reaction, under the action of a base (such as triethylamine, pyridine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, cesium carbonate, etc.) to form a structure of the formula I or II a compound wherein R in 7-R-10-hydroxycamptothecin is a C1-C6 straight or branched saturated or unsaturated hydrocarbon group, preferably R is CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ or CH(CH ₃ ) ₂ .

The schematic diagram of the reaction route is shown below:

Or, method two:

Preferred is 4-dimethylaminopyridine (DMAP) and 2-chloro-1-methylpyridinium iodide (CMPI) or N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine. (DMAP) or other esterification reagents are coupling agents, 4-[bis(2-chloroethyl)amino]phenylbutyric acid directly with 7-R-10-hydroxycamptothecin or 10-hydroxycamptothecin or hi The base is reacted to form a compound having the structure of the formula I or II or III, wherein R of the 7-R-10-hydroxycamptothecin is a C1-C6 linear or branched saturated or unsaturated hydrocarbon group, preferably R is CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ or CH(CH ₃ ) ₂ .

The schematic diagram of the reaction route is shown below:

Another object of the present invention is to provide the use of the chlorambucil derivatives: compounds I, II and III for the preparation of anticancer drugs. The cancer includes, but is not limited to, a cancer of the blood system, such as leukemia, lymphoma, myeloma; 2 non-blood cancer, such as solid tumor cancer (such as breast cancer, ovarian cancer, pancreatic cancer, colon cancer, rectal cancer, non-small Cell lung cancer, bladder cancer, stomach cancer, liver cancer, etc.), sarcoma, skin cancer and glioma.

In the above use, the medicament comprises a therapeutically effective amount of a compound of formula I or II or III and a pharmaceutically acceptable pharmaceutical carrier. It may be an injection, an oral preparation or an external preparation. Preferred are emulsions, microemulsions, micelles, liposomes, tablets or capsules, and ointments.

Camptothecin and its derivatives have excellent antitumor activity and are an important class of DNA topoisomerase I (Top I) inhibitors, which can bind to Top I-DNA cleavable complexes to form CPT-Top. I-DNA ternary complexes, thereby stabilizing the cleavable complex, leading to cancer cell death.

4-[Bis(2-chloroethyl)amino]phenylbutyric acid (chlorambucil) is a nitrogen mustard derivative which is a dichloroethylamine alkylating agent which is compatible with the 7th nitrogen of guanine of DNA. The combination of valences results in cross-linking within the double strand or cross-linking of different bases within the same strand, causing cancer cells to die. Similar to cyclophosphamide, it has inhibitory effects on various tumors and is clinically used for chronic lymphocyte white Blood diseases, lymphosarcoma, He Jinjie's disease, ovarian cancer, breast cancer, villus epithelioma, multiple myeloma, etc.

The molecules of the compounds I, II and III of the present invention simultaneously contain a dichloroethylamine alkylation group which can cross-link DNA bases and a camptothecin structure which inhibits DNA topoisomerase I (Top I), an expected molecule The drug itself or its metabolites are produced in two types of cells for use in cancer cells, thereby producing a synergistic effect of inhibiting and killing cancer cells to improve efficacy and reduce toxic side effects.

The invention further relates to formulations of the novel chlorambucil derivatives, including emulsion or microemulsions, micelles, liposomes, tablets, capsules and ointments. The emulsion comprises the chlorambucil derivative of the invention, one or more surfactants, an oil phase (lipophilic medium) and an aqueous phase. The emulsion can be of the oil-in-water or water-in-oil type. The micelle comprises the chlorambucil derivative of the present invention, a co-solvent and one or more surfactants and an aqueous phase. Liposomal agents include the chlorambucil derivatives of the present invention, phospholipids (most notably lecithin, phosphatidylcholine), cholesterol and aqueous phases. Tablets include the chlorambucil derivatives and adjuvants of the present invention. Capsules include the chlorambucil derivatives and adjuvants of the present invention. The ointment formulation includes the chlorambucil derivative and matrix of the present invention.

The technical solution adopted is an emulsion or microemulsion of the chlorambucil derivative of the present invention, and the composition thereof comprises:

1) Oil phase, including:

a) a compound having the structure of formula I or II or III;

b) a biocompatible lipophilic medium;

2) surfactants and co-solvents;

3) Water phase.

Alternatively, a benzoic acid mustard derivative micelle of the present invention, the composition of which comprises:

1) a compound having the structure of formula I or II or III;

2) a surfactant;

3) a co-solvent;

4) Water phase.

Alternatively, a phenylbutyrate mustard derivative liposome preparation of the present invention, the composition of which comprises:

1) a compound having the structure of formula I or II or III;

2) phospholipids;

3) cholesterol;

4) Water phase.

Alternatively, a tablet of the chlorambucil derivative of the present invention, the composition of which comprises:

1) a compound having the structure of formula I or II or III;

2) Excipients.

Commonly used excipients include: 1 diluents, such as starch, powdered sugar, dextrin, lactose, pregelatinized starch, microcrystalline cellulose (MCC), inorganic calcium salts, such as calcium sulfate. , calcium hydrogen phosphate and medicinal calcium carbonate, mannitol; 2 adhesive (Adhesives), such as distilled water, ethanol, starch slurry, sodium carboxymethyl cellulose sodium (CMC-Na), hydroxypropyl cellulose ( Hydroxypropylcellulose, HPC), methylcellulose and ethylcellulose (Methylcellulose, MC; Ethylcellulose, EC), Hydroxypropylmethylcellulose (HPMC), other binders (5% to 20% gelatin solution, 50% to 70% sucrose solution, 3% to 5% aqueous solution of polyvinylpyrrolidone (PVP) or alcohol solution); 3 Disintegrants such as dry starch, sodium carboxymethyl starch (Carboxymethyl) Starch sodium, CMS-Na), low-substituted hydroxypropyl cellulose (L-HPC), cross-linked polyvinyl pyrrolidone (also known as cross-linked PVP), cross-linked carboxymethyl cellulose Sodium (Croscarmellose sodium CCNa is a crosslinked cellulose carboxymethyl ether (approximately 70% of the carboxyl groups are sodium salt type); 4 Lubricants such as magnesium stearate, hydrogenated vegetable oil, polyethylene glycol, and hydroxy alcohol Magnesium sulfate, silica gel (Aerosil), talc powder; 5 coloring agent; 6 flavoring agents, etc. No matter what kind of excipients are added, they should meet the requirements of medicinal requirements, and they cannot react with the main drug, nor should they interfere with the main drug. Dissolution and absorption.

Tablets can be prepared by wet granulation tableting, dry granulation tableting and direct compression.

Alternatively, a capsule of the chlorambucil derivative of the present invention comprises a hard capsule and a soft capsule. Its ingredients include:

1) a compound having the structure of formula I or II or III;

2) Excipients.

Common excipients for hard capsules include, but are not limited to: 1 diluent: used to improve the physical properties of the contents and increase the volume, often with a certain compressibility. Commonly used diluents are mannitol, microcrystalline cellulose, lactose, pregelatinized starch 1500, corn starch and the like. 2 Lubricant: To prevent adhesion of powder to metal materials. Commonly used are magnesium stearate, glyceryl monostearate, stearic acid, talc, and the like. 3 Glidant: Improve the fluidity of the contents. Commonly used are micro-silica gel and talcum powder. 4 disintegrant: to ensure the disintegration of the contents. Commonly used are crosslinked cellulose, corn starch, crospovidone, pregelatinized starch 1500, glycyl starch sodium, alginic acid and the like. 5 Wetting agent: increase the wettability of the drug and the dissolution medium to ensure the efficacy of the preparation. Commonly, there are Tween 80, sodium lauryl sulfate and the like.

Excipients in the soft capsule contents include, but are not limited to, oily dispersion or PEG dispersion, and the contents may be solutions, suspensions, emulsions, semi-solids, and the like. Oily dispersion (lipophilic) content excipients include: 1 oily carrier: soybean oil, Castor oil, medium chain fatty acid, etc.; 2 semi-solids for viscosity adjustment include hydrogenated castor oil, beeswax, etc.; 3 surfactants such as phospholipids can improve the suspension stability of the suspension. Other stabilizers such as antioxidant BHT and the like may also be added. The PEG-dispersed (hydrophilic) content excipients are typically PEG 400 and 600, and the semi-solid can be used with both low molecular weight PEG 200, 300 and polymeric PEG 4000-10000.

The hard capsule shell is similar to the soft capsule shell. It mainly contains gelatin, gum arabic, water, plasticizer (such as glycerin, and can also be added with proper amount of propylene glycol and polyethylene glycol 200. Mannitol or sorbitol can replace glycerin as plastic for plasticizing. Agents, preservatives (such as potassium sorbate, paraben, etc.), sunscreens and pigments, etc., wherein water acts as a solvent.

Alternatively, a paste of the chlorambucil derivative of the present invention, the composition of which comprises:

1) a compound having the structure of formula I or II or III;

2) Matrix.

Commonly used substrates include: hydrocarbons (such as petrolatum, paraffin wax, liquid paraffin, silicone), lipids (such as lanolin, beeswax and cetyl, dimethicone), oils and fats (such as animal and plant higher fatty acid glycerides) Its mixture).

The chlorambucil derivatives of the present invention are soluble in a lipophilic medium. The lipophilic medium (or carrier) can be any one of a biocompatible lipophilic medium, and representative biocompatible lipophilic media include:

1) Fats and oils that can be used as lipophilic media, including fatty acids and esters of different chain lengths, which are mostly linear, but may also be branched, such as capric acid, caprylic acid, caproic acid, lauric acid, nutmeg, hard Fatty acid, oleic acid, linoleic acid, and other saturated or unsaturated fatty acids and esters.

2) Fat-soluble vitamin E and derivatives. Vitamin E refers to the natural or synthetic vitamin E series, commonly referred to as tocopherols and tocotriols (tocopherols and tocotrienols), tocopherols including alpha-tocopherol (D, DL, L), beta - Tocopherol (D type, DL type, L type), γ-tocopherol (D type, DL type, L type) and δ-tocopherol (D type, DL type, L type). Tocotrienol is structurally similar to tocopherol, but tocotrienol has three double bonds on the side chain phytyl of carbon-2. Tocotrienols include α-tocotrienol (D type, DL type, L type), β-tocotrienol (D type, DL type, L type), γ-tocotrienol (D type, DL) Type, L type) and δ-tocotrienol (D type, DL type, L type). Vitamin E derivatives include all tocopherol and tocotrienol derivatives such as vitamin E succinate, vitamin E acetate and the like.

3) Monoglycerides, diglycerides or triglycerides formed by esterification of fatty acids with glycerol, whether they are synthetic or natural, can be used as lipophilic media, for example, glycerides such as soybean oil, cottonseed oil, Rapeseed oil, fish oil, acetylated monoglyceride, glycerol monooleate, triacetin, and diacetyl tartaric acid ester, monoglyceride, castor oil and the like.

4) Fatty alcohols such as benzyl alcohol, stearyl alcohol, lauryl alcohol, etc., or their esters or ethers, such as benzyl benzoate.

Representative surfactants include:

1) Polyethylene glycol surfactants, such as polyoxyethylene castor oil EL (Cremophor EL), Tween series surfactants, and the like.

2) Phospholipids such as lecithin and pegylated phospholipids.

3) Polyethylene glycol vitamin E derivatives, such as vitamin E succinate polyethylene glycol (d-α-tocopherol polyethylene glycol 1000succinate, TPGS).

4) Polyoxyethylene polyoxypropylene block copolymer: block copolymer of POLOXAMERS or PLURONICS (H(OCH2CH2)a(OCH2CH2CH2)b(OCH2CH2)aOH).

Representative organic co-solvents include:

Ethanol, polyethylene glycol, propylene glycol, glycerin, N-methylpyrrolidone, and the like. Polyethylene glycol (PEG) is hydrophilic, and the chemical structure of the repeating unit is -CH ₂ CH ₂ O-, the formula is H-(CH ₂ CH ₂ ) _n -OH, and the molecular weight range is generally from 200 to 10,000. . For example, polyethylene glycol 200, PEG-300, polyethylene glycol 400, and the like.

Emulsions, microemulsions, micelles and liposomes, tablets, capsules and ointments of the invention. The chlorambucil derivatives of the present invention are included in the formulation.

As used herein, "emulsion" refers to a droplet formed by a heterogeneous liquid dispersion system, such as oil and water, formed by the dispersion of one phase liquid in a droplet state in another phase liquid under the action of a surfactant. Its diameter is generally from 0.1 to 3.0 microns.

The emulsion can form a stable microemulsion. The term "microemulsion" means that two immiscible liquids form a thermodynamically stable isotropic, transparent or translucent dispersion. For example, a microemulsion dispersion of oil and water is an interface formed by surfactant molecules. The membrane is stable. The microemulsion has an average droplet diameter of less than 200 nm, typically 10 to 50 nm.

The oil phase and the water phase are included in the emulsion or microemulsion. The emulsion or microemulsion may be an oil-in-water emulsified or water-in-oil type.

In the absence of water, a homogeneous, transparent drug-containing solution formed by the mixing of an oil phase, a nonionic surfactant, and a co-emulsifier is called a self-emulsifying drug delivery system (SEDDS). Spontaneous emulsification to form an emulsion having a particle diameter of 100 nm to 500 nm, which can be used to improve the solubility of lipophilic drugs and oral absorption.

In the emulsion or microemulsion, the phenylbutyric acid mustard derivative of the present invention accounts for 0.005% to 5.0% by weight of the formulation; preferably, the weight percentage in the formulation is 0.01% to 2.5%; in a more preferred embodiment, the chlorambucil derivative of the present invention accounts for 0.1% to 1.5% by weight of the formulation.

In the emulsion or microemulsion, the lipophilic medium accounts for 2% to 20% by weight of the formulation; preferably the lipophilic medium accounts for 4% to 12% by weight of the formulation; more preferably In the solution, the lipophilic medium accounts for 6% to 10% by weight of the formulation.

In one embodiment of the emulsion or microemulsion, the lipophilic medium comprises soybean oil and the aqueous medium is water. In another embodiment of the emulsion and microemulsion, the lipophilic medium comprises an oil soluble vitamin E. In another embodiment of the emulsion or microemulsion, the lipophilic medium comprises an oil soluble vitamin E derivative.

In addition to the chlorambucil derivatives of the present invention, the emulsion or microemulsion formulation may also include other ingredients commonly used in pharmaceutical emulsions and microemulsions, including surfactants and co-solvents. Representative surfactants include nonionic surfactants such as Cremophor EL, Tween 80, polyethylene glycol vitamin E derivative surfactants and other surfactants. polymer.

Suitable polyethylene glycol vitamin E derivative surface activities include vitamin E succinic polyethylene glycol derivatives (such as vitamin E polyethylene glycol succinate), in the vitamin E derivative molecule, polyethylene glycol is Succinic acid is formed by linking the hydroxyl group of vitamin E. The polyethylene glycol in the polyethylene glycol derivative of these vitamin E includes polyethylene glycol having various molecular weights (for example, 200, 300, 400, 600, 1000, etc.). alcohol. "Vitamin E polyethylene glycol succinate" herein includes vitamin E polyethylene glycol succinate (such as D-alpha tocopherol polyethylene glycol 1000 succinate, TPGS, a nonionic surfactant) (HLB = 16-18)) and various ester and ether derivatives of vitamin E polyethylene glycol.

In the emulsion or microemulsion, the surfactant is present in the formulation in an amount of from about 1% to about 10%, preferably from 2% to 6%, more preferably from 4% to 5% by weight.

In the emulsion or microemulsion, the co-solvent comprises from about 0% to about 20% by weight of the formulation.

In another aspect, the present invention provides a micelle preparation of the chlorambucil derivative of the present invention comprising the chlorambucil derivative of the present invention, one or more surfactants, One or more co-solvents and an aqueous phase.

In the micelle of the chlorambucil derivative of the present invention, the pharmaceutical compound is present in the formulation in an amount of from about 0.005% to about 3.0% by weight, preferably the pharmaceutical compound is present in the formulation in an amount of from about 0.01% by weight to 2.5%; more preferably, the pharmaceutical compound is present in the formulation in an amount of from about 0.1% to about 1.0% by weight.

Suitable surfactants are present in the micellar formulations of the invention in an amount of from about 1% to about 10%, preferably from 2% to 6%, more preferably from 4% to 5% by weight.

The micelle formulation also includes other ingredients such as the cosolvents mentioned above. In one embodiment, the micelle formulation comprises polyethylene glycol and a lower alkyl alcohol (such as ethanol). In the micelle, the co-solvent comprises from about 1% to about 20% by weight of the formulation.

In another aspect, the present invention provides a liposome agent of the chlorambucil derivative of the present invention comprising the chlorambucil derivative of the present invention, one or more phospholipids (including PEG) Phospholipids), one or more lipophilic mediators (such as cholesterol) and an aqueous phase.

In the liposome of the chlorambucil derivative of the present invention, the weight percentage of the pharmaceutical compound in the formulation From about 0.005% to about 5.0%, preferably the pharmaceutical compound is present in the formulation at a weight percent of from about 0.01% to about 2.5%; more preferably, the pharmaceutical compound is present in the formulation at a weight percent of from about 0.1% to about 1.5%.

Suitable phospholipids are present in the liposome formulation of the invention in an amount of from about 1% to about 10%, preferably from 2% to 6%, more preferably from 4% to 5% by weight.

The liposome formulation also includes other ingredients such as the lipophilic medium (e.g., cholesterol) as mentioned above. In one embodiment, the liposomal formulation comprises cholesterol or vitamin E. In the liposome, cholesterol or vitamin E is about 0.1% to 20% by weight of the formulation.

The aqueous phase is included in the emulsion, microemulsion, micelle, and liposome formulations. In one embodiment, the aqueous phase comprises deionized water. In another embodiment, the aqueous phase comprises physiological saline. In another embodiment, the aqueous phase contains an acid such as succinic acid, citric acid, phosphoric acid) buffer.

In another aspect, the present invention provides a tablet of the chlorambucil derivative of the present invention comprising the chlorambucil derivative and the adjuvant of the present invention. The chlorambucil derivative of the present invention may be contained in an amount of from 1 mg to 1000 mg per tablet. In a preferred embodiment, the chlorambucil derivative of the present invention has a content of 10 mg per tablet. Up to 500 mg; in a more preferred embodiment, the chlorambucil derivative of the present invention is contained in an amount of from 20 mg to 250 mg per tablet.

In another aspect, the present invention provides a capsule of the chlorambucil derivative of the present invention comprising the chlorambucil derivative and the adjuvant of the present invention. The content of the chlorambucil derivative of the present invention may be from 1 mg to 1000 mg per capsule. In a preferred embodiment, the content of the chlorambucil derivative of the present invention in each capsule is 10 mg to 500 mg; in a more preferred embodiment, the chlorambucil derivative of the present invention is contained in an amount of 20 mg to 250 mg per one.

In another aspect, the present invention provides a cream of the chlorambucil derivative of the present invention comprising the chlorambucil derivative of the present invention, one or more matrices.

The phenylbutyric acid mustard derivative of the present invention is present in an amount of from 0.01% to 30% by weight in the paste, preferably from 0.05% to 20% by weight of the pharmaceutical compound in the formulation; more preferably, The pharmaceutical compound is present in the formulation in an amount of from about 0.1% to about 10% by weight.

The invention also provides the use of the newly invented pharmaceutical compound, that is, the use of the chlorambucil derivative of the invention in the preparation of an anticancer drug.

For example, the pharmaceutical compounds of the invention are useful in the manufacture of a medicament for the treatment of cancer. The pharmaceutical compound of the present invention can be used for the treatment of cancer including the blood system such as leukemia, lymphoma, myeloma; and non-blood cancer such as solid tumor cancer (such as breast cancer, ovarian cancer, pancreatic cancer, colon cancer, rectal cancer, non- Small cell lung cancer, bladder cancer, stomach cancer, liver cancer, etc.), meat Tumor, skin cancer and glioma.

The efficacy and toxicity of the pharmaceutical compounds of the present invention are determined by in vitro cell or in vivo animal experiments, for example, ED50 (50% effective dose, half effective amount: 50% of the dose when the subject is positive), LD50 (50% lethal) Dose, half-lethal dose, killing half of the test subject) and GI50 (concentration of the anti-cancer drug that inhibits the growth of cancer cells by 50%, inhibiting the concentration of the drug in 50% of the subjects). The ratio of the median lethal dose (LD50) / half effective dose (ED50) is usually referred to as the therapeutic index to indicate the safety of the drug. Drugs with a large therapeutic index are safer than drugs with a small therapeutic index.

The newly invented anticancer drug compound aims to improve the therapeutic index and the safety of the drug while also improving the therapeutic effect. The dose of the drug obtained from in vitro cell experiments and in vivo animal experiments can be used to formulate a dose range for the human body. The dose of such a compound is preferably in the range of ED50 with little or no toxicity. The dosage change will generally depend on the dosage form employed, the sensitivity of the patient, and the route of administration. Reference can be made to conventional doses of the same or similar drugs, such as irinotecan and topotecan. For example, a conventional dose of topotecan is 0.2-1.5 mg/m ² , and a conventional dose of irinotecan is 100 mg-350 mg/m ² .

The pharmaceutical compounds of the invention may be used alone or in combination with one or more other therapeutic agents. For example, in the treatment of cancer, these pharmaceutical compounds can be used with the following therapeutic agents including, but not limited to, androgen inhibitors such as flutamide and luprolide; antiestrogens, such as Tamoxifen; antimetabolites and cytotoxic drugs such as daunorubicin, fluorouracil, floxuridine, interferon alpha, Methotrexate, plicamycin, mecaptopurine, thioguanine, adriamycin, carmustine, lomustine Lomustine), cytarabine, cyclophosphamide, doxorubicin, estramustine, altretamine, hydroxyurea, ifosfamide (ifosfamide), procarbazine, mutamycin, busulfan, mitoxantrone, carboplatin, cisplatin, streptozotocin (streptozocin), bleomycin, release line Dactinomycin, and idamycin; hormones such as medroxyprogesterone, ethinyl estradiol, estradiol, leuprolide, and adidia Megestrol, octreotide, diethylstilbestrol, chlorotrianisene, etoposide, podophyllotoxin, and goserelin; nitrogen mustard Such as melphalan, chlorambucil and thiotepa; steroids such as betamethasone; and other antitumor drugs such as M. bovis (live Mycobacterium bovis), dacarbazine, asparagine Asparaginase, leucovorin, mitotane, vincristine, vinblastine, and taxotere.

DRAWINGS

Figure 1. Nuclear magnetic resonance spectrum of the chlorambucil derivative XBB-001 of the present invention.

Figure 2. Mass spectrum of the chlorambucil derivative XBB-001 of the present invention.

Figure 3. Liquid chromatography of the chlorambucil derivative XBB-001 of the present invention.

Figure 4. Nuclear magnetic resonance spectrum of the chlorambucil derivative XBB-002 of the present invention.

Figure 5. Mass spectrum of the chlorambucil derivative XBB-002 of the present invention.

Figure 6. Liquid chromatography of the chlorambucil derivative XBB-002 of the present invention.

Figure 7. Nuclear magnetic resonance spectrum of the chlorambucil derivative XBB-003 of the present invention.

Figure 8. Mass spectrum of the chlorambucil derivative XBB-003 of the present invention.

Figure 9. Liquid chromatography of the chlorambucil derivative XBB-003 of the present invention.

Figure 10. Nuclear magnetic resonance spectrum of the chlorambucil derivative XBB-004 of the present invention.

Figure 11. Mass spectrum of the chlorambucil derivative XBB-004 of the present invention.

Figure 12. Liquid chromatography of the chlorambucil derivative XBB-004 of the present invention.

detailed description

The synthesis, preparation, in vitro cell experiments and the like of the chlorambucil derivatives of the present invention are described below by way of examples.

The examples are intended to be illustrative of the invention and are not to be construed as limiting.

Example 1.4 Synthesis of [bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001, compound of formula II, R=H)

The reaction formula is as follows:

Experimental steps:

To a 100 mL round bottom flask, 0.348 g (1 mmol) of camptothecin and 40 mL of anhydrous DMF were added and dissolved by heating.

To the solution was added 0.334 g (1.1 mmol) of 4-[p-bis(2-chloroethyl)amino]phenylbutyric acid, followed by 0.501 g (2 mmol). 2-Chloro-1-methylpyridinium iodide, 0.489 g (4 mmol) of 4-dimethylaminopyridine, stirred at room temperature, and allowed to react overnight until the reaction was completed. 200 mL of ethyl acetate was added to the reaction mixture, and the mixture was stirred for 15 minutes, and transferred to a separating funnel. The mixture was washed three times with 100 mL of brine, and the organic phase was dried over 20 g of anhydrous magnesium sulfate for 50 min. Solvent ethyl acetate. The column was separated, and 100-200 mesh silica gel was used as a stationary phase, and a mixture of dichloromethane and ethyl acetate was used as a eluent to give a pale yellow solid, 0.436 g, yield 68.8%. The XBB-001 nuclear magnetic resonance spectrum is shown in Figure 1, and the mass spectrum is shown in Figure 2.

MS (Positive Ion mode ESI): m/z = 634.2 (M + H) ⁺ , MS (Negtive Ion Mode ESI): m/z = 632.2 (MH) ^- .

_{^{1 H NMR (300MHz, CDCl 3}} ): δppm: 8.4132 (s, 1H), 8.2582-8.2296 (d, J = 8.58Hz, 1H), 7.9683-7.9411 (d ,, J = 8.16Hz, 1H), 7.8813- 7.8295 (t, J = 7.77 Hz, 1H), 7.7100-7.6603 (t, J = 7.455 Hz, 1H), 7.2789 (s, 1H), 7.0978-7.0707 (d, J = 8.13 Hz, 2H), 6.6334-6.6073 (d, J = 7.83, 2H), 5.7199-5.1624 (d, J = 17.25 Hz, 1H), 5.4473-5.3893 (d, J = 17.4 Hz, 1H), 5.2961 (s, 2H), 3.6773-3.6576 (m) , 4H), 3.6217-3.6016 (m, 4H), 2.6152-2.5912 (t, J = 7.5 Hz, 2H), 2.5329-2.4670 (q, J = 6.2 Hz, 2H), 2.3347-2.2651 (m, 1H), 2.1936-2.1218 (m, 1H), 1.9870-1.8902 (m, 2H), 1.0085-0.9592 (t, J = 7.4 Hz, 3H).

The liquid chromatogram of XBB-001 is shown in Figure 3, purity: 99.07%; chromatographic conditions: column: C18 column (5 μm, 150 mm × 5 mm); mobile phase: CH ₃ CN: IPA: HAc (95: 5: 0.1); detection wavelength: 254 nm; flow rate: 1.0 ml/min; injection amount: 5 μL; column temperature: 40 °C.

Example 2.4 Synthesis of [Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin (XBB-002, compound of formula I, R is ethyl)

The reaction formula is as follows:

Experimental steps:

To a 100 mL round bottom flask was added 0.456 g (1.5 mmol) of 4-[p-bis(2-chloroethyl)amino] phenylbutyric acid, 30 mL of anhydrous toluene, 600 μL of thionyl chloride and 2 drops of anhydrous DMF. Stir at room temperature for 6 hours, and evaporate off excess under reduced pressure The thionyl chloride and anhydrous toluene were then added to 10 mL of chloroform to dissolve the residue to obtain a solution A.

0.392 g (1 mmol) of 7-ethyl-10-hydroxycamptothecin was weighed and added to another 100 mL round bottom flask.

20 mL of anhydrous DMF was added, dissolved by heating and stirring, and 250 μL of anhydrous triethylamine was added. To the above solution, the solution A was slowly added dropwise through a dropping funnel, and the dropwise addition was completed for 60 minutes, and the mixture was stirred at room temperature, and the reaction was allowed to stand overnight until the reaction was completed. A part of the solvent was evaporated off, and 200 mL of ethyl acetate was added to the reaction mixture, and the mixture was stirred for 15 minutes, and then transferred to a separating funnel, and the mixture was washed three times with 100 mL of brine, and the organic phase was dried over 20 g of anhydrous magnesium sulfate for 50 min. The solvent ethyl acetate was removed by rotary evaporation. The column was separated, and 100-200 mesh silica gel was used as a stationary phase. A mixture of dichloromethane and ethyl acetate was used as a solvent. The product was dried in vacuo to give a pale yellow solid (0.460 g, yield: 67.8%).

The nuclear magnetic resonance spectrum of XBB-002 is shown in Fig. 4, and the mass spectrum is shown in Fig. 5.

MS (Positive Ion mode ESI): m/z = 678.2 (M+H) ⁺ , 700.2 (M+Na) ⁺ , MS (Negtive Ion Mode ESI): m/z = 676.3 (MH) ^- .

¹ H NMR (300 MHz, CDCl ₃ ): δ ppm: 8.2788-8.2484 (d, J = 9.12 Hz, 1H), 7.8243-7.8165 (d, J = 2.34 Hz, 1H), 7.6913 (s, 1H), 7.5533-7.5149 (dd, J = 2.31 and 9.15 Hz, 1H), 7.1614-7.1330 (d, J = 8.52 Hz, 2H), 6.7171-6.6885 (d, J = 8.58 Hz, 2H), 5.7798-5.7252 (d, J = 16.38) , 1H), 5.3353-5.2804 (d, J = 16.47 Hz, 1H), 5.2654 (s, 2H), 3.7442-3.7020 (m, 4H), 3.6598-3.6096 (m, 4H), 3.1981-3.1229 (q, J =7.52 Hz, 2H), 2.738-2.6549 (q, J = 7.79 Hz, 4H), 2.1576-2.0578 (m, 2H), 1.9476-1.8278 (m, 2H), 1.4273-1.3766 (t, J = 7.60 Hz, 3H), 1.0656-1.0166 (t, J = 7.35 Hz, 3H).

The liquid chromatogram of XBB-002 is shown in Figure 6, purity: 98.8%; chromatographic conditions: column: C18 column (5μm, 150mm × 5mm); mobile phase: acetonitrile: water (70:30); detection wavelength: 254 nm; flow rate: 1.0 ml/min; injection amount: 5 μL; column temperature: 40 °C.

Example 3.4 - Synthesis of [bis(2-chloroethyl)amino] phenylbutyric acid 10-hydroxycamptothecin (XBB-003, compound of formula I, R = H)

The reaction formula is as follows:

Experimental steps:

To a 100 mL round bottom flask was added 0.456 g (1.5 mmol) of 4-[p-bis(2-chloroethyl)amino]phenylbutyric acid, 30 mL of anhydrous toluene, 600 μL of thionyl chloride and 2 drops of anhydrous DMF. After stirring at room temperature for 6 hours, excess thionyl chloride and anhydrous toluene were evaporated off under reduced pressure, and then 10 mL of chloroform was added to dissolve the residue to obtain a solution A.

0.364 g (1 mmol) of 10-hydroxycamptothecin was weighed, added to another 100 mL round bottom flask, and 20 mL of anhydrous DMF was added thereto, dissolved by heating and stirring, and 250 μL of anhydrous triethylamine was added. To the above solution, the solution A was slowly added dropwise through a dropping funnel, and the dropwise addition was completed for 60 minutes, and the mixture was stirred at room temperature, and the reaction was allowed to stand overnight until the reaction was completed. A part of the solvent was evaporated off, and 200 mL of ethyl acetate was added to the remaining reaction mixture, and the mixture was stirred for 15 minutes, and the mixture was transferred to a separating funnel, and washed three times with 100 mL of brine, and the organic phase was dried over 20 g of anhydrous magnesium sulfate for 50 minutes. Magnesium sulfate was removed by filtration, and the solvent ethyl acetate was removed by rotary evaporation. The column layer was separated, and 100-200 mesh silica gel was used as a stationary phase, and a mixture of dichloromethane and ethyl acetate was used as a eluent to give a pale yellow solid (0.462 g).

The XBB-003 nuclear magnetic resonance spectrum is shown in Fig. 7, and the mass spectrum is shown in Fig. 8.

The liquid chromatogram of XBB-003 is shown in Figure 9, purity: 94.55%; chromatographic conditions: column: C18 column (5μm, 150mm × 5mm); mobile phase: acetonitrile: water (80:20); detection wavelength: 254 nm; flow rate: 1.0 ml/min; injection amount: 5 μL; column temperature: 40 °C.

MS (Positive Ion mode ESI): m/z = 650.2 (M + H) ⁺ , MS (Negtive Ion Mode ESI): m/z = 648.2 (MH) ^- .

_{^{1 H NMR (300MHz, CDCl 3}} ): δppm: 8.3330 (s, 1H), 8.2554-8.2246 (d, J = 9.24Hz, 1H), 7.6727 (s, 1H), 7.6601 (s, 1H), 7.5611-7.5225 (dd, J = 2.49 and 9.15 Hz, 1H), 7.1623-7.1338 (d, J = 8.55 Hz, 2H), 6.7314-6.7029 (d, J = 8.55 Hz, 2H), 5.7770-5.7224 (d, J = 16.38) , 1H), 5.3342-5.2799 (d, J = 16.29 Hz, 1H), 5.2949 (s, 2H), 3.7428-3.7009 (m, 4H), 3.6599-3.6167 (m, 4H), 2.7304-2.6438 (m, 4H) ), 2.1502-2.0502 (m, 2H), 1.9731-1.8315 (m, 2H), 1.0707-1.0216 (t, J = 7.36 Hz, 3H).

Example 4.7 Synthesis of ethyl-10-hydroxycamptothecin two {4-[bis(2-chloroethyl)amino]phenylbutyric acid ester (XBB-004)

The reaction formula is as follows:

Experimental steps:

Into a 100 mL round bottom flask, 0.392 g (1 mmol) of 7-ethyl-10-hydroxycamptothecin and 40 mL of anhydrous DMF were added and dissolved by heating. To the solution was added 0.639 g (2.1 mmol) of 4-[p-bis(2-chloroethyl)amino] phenylbutyric acid, followed by 1.002 g (4 mmol) of 2-chloro-1-methylpyridinium iodide, 0.978. g (8 mmol) 4-dimethylaminopyridine, stirred at room temperature, and allowed to react overnight until the reaction was completed. 200 mL of ethyl acetate was added to the reaction mixture, and the mixture was stirred for 15 minutes, and transferred to a separating funnel. The mixture was washed three times with 100 mL of brine, and the organic phase was dried over 20 g of anhydrous magnesium sulfate for 50 min. Solvent ethyl acetate. The column was separated, and 100-200 mesh silica gel was used as a stationary phase, and a mixture of dichloromethane and ethyl acetate was used as a eluent to give a pale yellow solid (0.690 g, yield 71.5%).

The XBB-004 nuclear magnetic resonance spectrum is shown in Figure 10, and the mass spectrum is shown in Figure 11.

The liquid chromatogram of XBB-004 is shown in Figure 12, purity: 96.82%; chromatographic conditions: column: C18 column (5 μm, 150 mm × 5 mm); mobile phase: acetonitrile: water (85: 15); detection wavelength: 254 nm; flow rate: 1.0 ml/min; injection amount: 5 μL; column temperature: 40 °C.

MS (Positive Ion mode ESI): m/z = 963.30 (M+H) ⁺ .

_{^{1 H NMR (500MHz, CDCl 3}} ): δppm: 8.2322-8.2139 (d, J = 15.2Hz, 1H), 7.8266-7.8224 (d, J = 2.1Hz, 1H), 7.5618-7.5395 (dd, J = 2.1 and 9.15 Hz, 1H), 7.2198 (s, 1H), 7.1442-7.1273 (d, J = 8.45 Hz, 2H), 7.0801-7.0632 (d, J = 8.45 Hz, 2H), 6.6851-6.6681 (d, J = 8.50) Hz, 2H), 6.6112-6.5943 (d, J = 8.45 Hz, 2H), 5.7015-5.6671 (d, J = 17.2, 1H), 5.4330-5.3987 (d, J = 17.15 Hz, 1H), 5.2508-5.2454 ( d, J = 2.7 Hz, 2H), 3.7335-3.5823 (m, 16H), 3.1594-3.1440 (m, 2H), 2.7176-2.6611 (m, 4H), 2.5951-2.5656 (m, 2H), 2.4968-2.4743 ( m, 2H), 2.3400-2.110 (m, 1H), 2.1573-2.0910 (m, 3H), 1.9465-1.9169 (m, 2H), 1.4058-1.3752 (t, J = 7.65 Hz, 3H), 0.9916-0.9619 ( t, J = 7.43 Hz, 3H).

Example 5. Formulation of the chlorambucil derivative of the present invention, including emulsion, micelle, liposome, tablet, capsule and ointment formulation

In this embodiment, an emulsion, a micelle, a liposome, a tablet, a capsule, and a paste formulation of the chlorambucil derivative of the present invention are included. The emulsion, micelle, liposome, tablet, capsule and ointment formulation contains the chlorambucil derivative of the present invention, optionally one or more of the compounds of formula I, II or III .

1) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin emulsion 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7-B The base 10-hydroxycamptothecin (XBB-002) is dissolved in a mixture of soybean oil, Tween 80 and polyethylene glycol PEG (200), followed by deionized water (DI water), then stirred and emulsified or Emulsified with a homogenizer, the composition of the emulsion produced is as follows:

The prepared emulsion drug was filtered through a 0.2 micron filter and placed in a sterile glass bottle.

2) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin emulsion

4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin (XBB-002) dissolved in D-α-tocopheryl acetate, D-α-fertility a mixture of phenol polyethylene glycol 1000 succinate (TPGS) and polyethylene glycol PEG (200), followed by deionized water (DI water), then stirred and emulsified or emulsified with a homogenizer, produced The composition of the emulsion is as follows:

The resulting emulsion drug was filtered through a 0.2 micron filter and placed in a sterile glass vial.

3) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid camptothecin emulsion

4-[Bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001) is dissolved in a mixture of soybean oil, Tween 80 and polyethylene glycol PEG (200), and then deionized water is added. (DI water), then stirred and emulsified or emulsified with a homogenizer. The composition of the emulsion produced is as follows:

The prepared emulsion drug was filtered through a 0.2 micron filter and placed in a sterile glass bottle.

4) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 10-hydroxycamptothecin emulsion

4-[Bis(2-chloroethyl)amino]benzophenone 10-hydroxy acid camptothecin (XBB-003) is dissolved in a mixture of soybean oil, Tween 80 and polyethylene glycol PEG (200), and then added Deionized water (DI water), then stirred and emulsified or emulsified with a homogenizer. The composition of the emulsion produced is as follows:

The prepared emulsion drug was filtered through a 0.2 micron filter and placed in a sterile glass bottle.

5) 7-ethyl-10-hydroxycamptothecin two {4-[bis(2-chloroethyl)amino] phenylbutyrate ester emulsion

7-Ethyl-10-hydroxycamptothecin 2{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB-004) is dissolved in soybean oil, Tween 80 and polyethylene glycol PEG ( In the mixture of 200), add DI water, then stir and emulsified or emulsified with a homogenizer. The composition of the emulsion produced is as follows:

The prepared emulsion drug was filtered through a 0.2 micron filter and placed in a sterile glass bottle.

6) micelles of 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin

4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin (XBB-002) was dissolved in Tween 80, ethanol and polyethylene glycol PEG ( A transparent liquid is obtained from the mixture of 200), and an appropriate amount of DI water is added before use, followed by stirring and ultrasonic stirring to obtain a clear liquid. The composition of the micelle produced is as follows:

The prepared micelle drug was filtered through a 0.2 micron filter and used.

7) micelles of 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin

4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin (XBB-002) dissolved in D-α-tocopherol polyethylene glycol 1000 succinate ( TPGS), a mixture of ethanol and polyethylene glycol PEG (200) to obtain a transparent liquid, add appropriate amount of physiological saline before use, then stir and ultrasonically stir to obtain a clear liquid, the composition of the produced micelle as follows:

The prepared micelle drug was filtered through a 0.2 micron filter and used.

8) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid camptothecin ester micelle

4-[Bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001) dissolved in a mixture of polyoxyethylene castor oil EL (Cremophor EL), ethanol and polyethylene glycol PEG (200) A clear liquid is obtained, and an appropriate amount of DI water is added before use, and then stirred and ultrasonically stirred to obtain a clear liquid. The composition of the produced micelle is as follows:

The prepared micelle drug was filtered through a 0.2 micron filter and used.

9) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 10-hydroxycamptothecin ester micelle

4-[Bis(2-chloroethyl)amino]phenylbutyric acid 10-hydroxycamptothecin (XBB-003) dissolved in polyoxyethylene castor oil EL (Cremophor EL), ethanol and polyethylene glycol PEG (200 a clear liquid obtained in the mixture, plus before use A suitable amount of DI water is added, followed by stirring and ultrasonic agitation to obtain a clear liquid. The composition of the micelle produced is as follows:

The prepared micelle drug was filtered through a 0.2 micron filter and used.

10) 7-ethyl-10-hydroxycamptothecin two {4-[bis(2-chloroethyl)amino]phenylbutyric acid} ester micelle

7-Ethyl-10-hydroxycamptothecin two {4-[bis(2-chloroethyl)amino]phenylbutyric acid ester (XBB-004) dissolved in polyoxyethylene castor oil EL (Cremophor EL), ethanol A transparent liquid is obtained in a mixture with polyethylene glycol PEG (200), and an appropriate amount of deionized water (DI water) is added before use, followed by stirring and ultrasonic stirring to obtain a clear liquid, and the composition of the produced micelle is obtained. as follows:

The prepared micelle drug was filtered through a 0.2 micron filter and used.

11) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin liposome In a round bottom flask, 0.01 mmol of 7-ethyl-10 - Hydroxycamptothecin 4-[bis(2-chloroethyl)amino]phenylbutyrate (XBB-002) and 0.5 mmol of phospholipid (lecithin, phosphatidylcholine) dissolved in 15 mL of chloroform (CHCl3), slow Slowly heat to 40 ° C, evaporate the solvent under reduced pressure on a rotary evaporator to form a thin lipid film, vacuum dry overnight, further remove the chloroform in the lipid film, add 10 mL of distilled water, then stir and ultrasonically stir, the resulting lipid The body fluid was filtered through a filter with a pore size of 0.2 μm, placed in a sterile glass bottle, frozen with dry ice and acetone, and then freeze-dried for 24 hours to obtain 7-ethyl-10-hydroxycamptothecin 4-[double (2 Liposomal agent of -chloroethyl)amino] phenylbutyrate.

In the same way, 4-[bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001), 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7- Ethyl-10-hydroxycamptothecin (XBB-003), 7-ethyl-10-hydroxycamptothecin-{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB- a liposome agent of 004).

12) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin capsule ((wet granulation) The prescribed amount of XBB-002 is mixed with the prescribed amount of sodium starch glycolate, and then the wetted granulation is carried out by adding a prescribed amount of Tween 80 aqueous solution, and the obtained wet material is dried in a fluidized bed, a drying tray or other suitable drier. The dried granules are ground to a suitable particle size distribution, mixed with the other ingredients of the formulation, and finally the mixture is filled into two hard gelatin capsule shells.

Component	Content of each capsule (mg)	Percentage of each component (%)
			XBB-002	100	40
Tween 80	5	2
			lactose	50	20
Magnesium stearate	5	2
			Sodium starch glycolate	90	36
Total weight of each capsule	250

In the same way, 4-[bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001), 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7- Ethyl-10-hydroxycamptothecin (XBB-003), 7-ethyl-10-hydroxycamptothecin-{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB- 004) capsules.

13) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin tablets (wet granulation) prescribed amount of sodium lauryl sulfate aqueous solution and prescription The XBB-002, sodium starch glycolate and microcrystalline cellulose are granulated, and the obtained wet material is dried in a fluidized bed, a drying tray or other suitable drier, and the dried granules are ground to a desired particle size. Distribute and then compress the mixture into tablets.

Component	Content per tablet (mg)	Percentage of each component (%)
			XBB-002	300	50
Sodium dodecyl sulfate	12	2
			lactose	42	7
Magnesium stearate	6	1
			Sodium starch glycolate	120	20
Microcrystalline cellulose	120	20
			Total weight of each capsule	600

In the same way, 4-[bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001), 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7- Ethyl-10-hydroxycamptothecin (XBB-003), 7-ethyl-10-hydroxycamptothecin-{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB- Tablets of 004) (wet granulation).

14) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin tablets (dry granulation) First, the XBB-002 raw material is sifted and sieved to control the particle size. Less than 80μm, then mix the prescribed amount of XBB-002 with micro-silica gel, add the prescribed amount of starch, sucrose, croscarmellose sodium, mix, dry granulation, after granulation, add a prescription amount of hard Magnesium citrate, mixed, compressed, coated with film.

Component	Content per tablet (mg)	Percentage of each component (%)
			XBB-002	100	50
starch	52	26
			sucrose	15	7.5
Croscone sodium	15	7.5
			Microsilica gel	15	7.5
Magnesium stearate	3	1.5
			Total weight of each capsule	200

In the same way, 4-[bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001), 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7- Ethyl-10-hydroxycamptothecin (XBB-003), 7-ethyl-10-hydroxycamptothecin-{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB- Tablets of 004) (dry granulation).

15) 4-[Bis(2-chloroethyl)amino]phenylbutyric acid 7-ethyl-10-hydroxycamptothecin ester, take an appropriate amount of 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7 -ethyl-10-hydroxycamptothecin (XBB-002), stearic acid, glyceryl monostearate, liquid paraffin, polyethylene glycol 200 (PEG 200), Tween-80 heating and melting; Appropriate amount of glycerin and water are heated to 70-80 ° C, added to the oil phase under stirring, and stirring is continued until molding. The composition of the produced paste is as follows:

In the same way, 4-[bis(2-chloroethyl)amino]phenylbutyric acid camptothecin (XBB-001), 4-[bis(2-chloroethyl)amino]phenylbutyric acid 7- Ethyl-10-hydroxycamptothecin (XBB-003), 7-ethyl-10-hydroxycamptothecin-{4-[bis(2-chloroethyl)amino]phenylbutyrate} (XBB- 004) paste.

Example 6. In vitro cytotoxicity assay of the chlorambucil derivatives of the present invention

1) Experiment 1

This experiment compares the chlorambucil derivative of the present invention with the anticancer drug irinotecan to inhibit A549 (human non-small cell lung cancer), BGC-823 (human gastric adenocarcinoma), HepG2 cells (human liver cancer cells). ), GI50 values of K562 cells (human chronic myeloid leukemia cells), HT-29 cells (human colon cancer cells) (drug concentrations inhibiting 50% growth of cancer cells), and evaluation of in vitro cytotoxicity of the drug compounds. Adherent cells adopt SRB method, suspension cells adopt CCK-8 law.

The IC50 values are shown in Table 1. The experimental results show that the chlorambucil derivatives of the present invention have obvious proliferation inhibition effects on the above five kinds of cancer cells, and the inhibitory activities are higher than that of irinotecan, especially XBB-002. It has a strong inhibitory activity on cancer cells. The IC50 value of irinotecan at 48 hours is about 10 μM, while the IC50 value of XBB-002 is about 0.1 μM, which is much lower than the IC50 value of irinotecan, indicating that the inhibitory activity of XBB-002 on cancer cells is much higher than that of irinotecan. And XBB-002 has a high inhibition rate of 100% at high concentrations, which can kill cancer cells.

Experimental steps:

a. Take a bottle of cells in a good condition in the exponential growth phase, add 0.25% trypsin digest, digest and let the adherent cells fall off, and count to make a cell suspension;

B.5% FBS, cultured in a constant temperature CO ₂ incubator for 24 hours;

c. Add the drug compound, 10 μL/well; the adherent cell T0 group is fixed with trichloroacetic acid (TCA);

d. After 48 hours, the suspension cells were added with CCK-8, 10 μL/well, and after incubation at 37 ° C for 2 hours, the absorbance at 450 nm was read to calculate the inhibition rate;

e. Adherent cells were fixed with trichloroacetic acid (TCA), and each well was fixed with 25 μL of pre-cooled 50% TCA solution (the final concentration of TCA was 10%). When adding TCA, it must be gently added to the surface of the culture solution. After standing for 5 min, the plate was moved to 4 ° C for 1 h, so that the cells were fixed at the bottom of the culture well;

f. Pour off the fixing solution, wash the small holes with deionized water for 5 times, dry and air dry;

g. SRB was formulated into a 0.4% solution with 1% acetic acid, 100 μL of RBRB solution was added to each well, and allowed to stand at room temperature for 10 min. SRB not bound to protein was washed 5 times with 1% acetic acid and air dried;

h. The combined SRB was dissolved in 150 μL of 10 mmol/L unbuffered Tris lye (pH 10.5);

i. Read the absorbance at 515 nm and calculate the inhibition rate.

Table 1. Comparison of the chlorambucil derivatives of the present invention with the anticancer drug irinotecan IC50 (μM) (48 hours)

drug	A549	BGC-823	HepG2	HT-29	K562
						Irinotecan	9.43	28.25	5.63	17.46	10.81
XBB-002	0.044	0.037	0.024	0.195	0.043

2) Experiment 2

In this experiment, the inhibitory effects of XBB-001, XBB-002 and XBB-004 on human colon cancer cell line Caco-2 were examined by MTT assay, and compared with the anticancer drug irinotecan (irinotican). In vitro cytotoxicity of the compounds.

The IC50 values are shown in Table 2. The experimental results show that the chlorambucil derivatives of the present invention have a significant proliferation inhibitory effect on human colon cancer cell Caco-2, and the cells are increased with the increase of drug concentration. The proliferation inhibition is enhanced and cancer cells can be killed.

Table 2. Comparison of the chlorambucil derivative of the present invention with the anticancer drug irinotecan IC50 (μM) (48 hours)

Experimental steps:

The Caco-2 cell line was cultured to a good state and viable, and the log phase cells were collected, seeded on a 96-well culture plate, and plated so that the density of the cells to be tested was 8 × 10 ³ per well, and the marginal wells were filled with sterile PBS. Incubate in a 5% CO ₂ , 37 ° C cell incubator, and start testing after the cells are attached to 60%-70% of the cells. Each group was given a blank control group, a negative control group and a drug administration group. The blank control group contained no cells, the negative control group did not add the drug, and the concentration (final concentration) gradient of the administration group was set as shown in 3.1.

After dissolving each compound, it was sterilized by filtration and diluted to the corresponding concentration with a medium. The injection volume per well was 150 μL (the control DMSO content was 0.5% in the final volume), and 6 replicate wells were set for each administration dose. After 5% CO _2, 37 ℃ incubator incubated 48h, the drug-containing hole media was aspirated, washed twice with PBS, sterile filtered after adding 200μL of MTT solution (0.5mg / mL), placed in cell culture Continue to culture for 4 hours in the box. Stop the culture and discard the supernatant. 150 μL of DMSO was added to each well, and the mixture was shaken at 37 ° C for 10 min at a low speed to sufficiently dissolve the methyl violet crystal, and the OD value was measured at a wavelength of 490 nm.

200 μl was added to each well and a blank control group (n=6) containing 0.5% DMSO was provided. After 48 hours of culture, the supernatant medium was discarded, and the cells were washed twice with PBS, and a 0.5 mg/ml MTT solution was added. After 4 h, the supernatant was discarded and the reaction was stopped by the addition of 150 ul/well DMSO. After mixing for 10 min on a shaker, the mixture was shaken and mixed for 10 min. After the crystals were completely dissolved, the absorbance (D) of each well at a wavelength of 490 nm was detected on a microplate reader. The growth inhibition rate of the cells was calculated according to the following formula: % inhibition rate = (negative control group - administration group) / negative control group × 100%.

Claims (12)

1. A chlorambucil derivative having the structure of the following formula I or II or III and a pharmaceutically acceptable salt thereof:

   

   Wherein: R is H or a C1-C6 linear or branched saturated or unsaturated hydrocarbon group.
2. The chlorambucil derivative according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R is H, CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ or CH(CH ₃ ) ₂ .
3. The method for preparing a chlorambucil derivative according to claim 1 or 2, comprising the steps of:

   (1) reacting 4-[bis(2-chloroethyl)amino]phenylbutyric acid with an acid chloride reagent to form 4-[bis(2-chloroethyl)amino]phenylbutanoyl chloride;

   (2) 4-[bis(2-chloroethyl)amino]phenylbutanoyl chloride obtained in the step (1), respectively, and 7-R-10-hydroxycamptothecin or 10-hydroxyl The reaction of gibberellin or camptothecin produces a compound having the structure of the formula I or II or III under the action of a base, and the R of the 7-R-10-hydroxycamptothecin is a C1-C6 straight chain or Branched saturated or unsaturated hydrocarbon group.
4. The method for preparing a chlorambucil derivative according to claim 1 or 2, characterized in that 4-[bis(2-chloroethyl)amino]phenylbutyric acid and 7 are respectively subjected to a coupling agent. -R-10-hydroxycamptothecin or 10-hydroxycamptothecin or camptothecin to form a compound having the structure of formula I or II or III, wherein R of 7-R-10-hydroxycamptothecin is C1-C6 straight or branched saturated or unsaturated hydrocarbon group.
5. The method for producing a chlorambucil derivative according to claim 3 or 4, wherein R of the 7-R-10-hydroxycamptothecin is CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ or CH(CH ₃ ) ₂ .
6. The method for producing a chlorambucil derivative according to claim 4, wherein the coupling agent is 4-dimethylaminopyridine and 2-chloro-1-methylpyridinium iodide or N,N. '-Dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
7. Use of the chlorambucil derivative according to claim 1 or 2 for the preparation of an anticancer drug.
8. The use according to claim 7, characterized in that the cancer comprises cancer of the blood system, solid tumor cancer, sarcoma or glioma.
9. The use according to claim 8, wherein the cancer comprises leukemia, lymphoma, myeloma, breast cancer, ovarian cancer, pancreatic cancer, colon cancer, rectal cancer, non-small cell lung cancer, bladder cancer, stomach cancer, liver cancer , sarcoma, skin cancer or glioma.
10. The use according to claim 7, characterized in that the medicament comprises a therapeutically effective amount of a compound of formula I or II or III and a pharmaceutically acceptable pharmaceutical carrier.
11. The use according to claim 10, characterized in that the drug is an injection, an oral preparation or an external preparation.
12. The use according to claim 11, characterized in that the medicament is an emulsion, a microemulsion, a micelle, a liposome, a tablet, a capsule, or a paste.

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