CN115925712A

CN115925712A - Targeted antitumor drug compound and preparation method and application thereof

Info

Publication number: CN115925712A
Application number: CN202211576472.2A
Authority: CN
Inventors: 张跃华; 曾琦; 赵梦尧
Original assignee: Nanjing Youyi Medical Technology Co ltd
Current assignee: Nanjing Youyi Medical Technology Co ltd
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-04-07

Abstract

The invention discloses a targeted antitumor drug compound with the systematic name of N- [5- (3-chloro-4-fluorophenylamino) -pyrazole [1,5-a ]]Pyrimidin-3-yl]4- [ bis- (2-chloroethyl) -amino]Phenylalkylamides having the general structural formula I below, in which n = an integer from 0 to 10. The invention also relates to a preparation, a preparation method and application of the pharmaceutical compound.

Description

Targeted antitumor drug compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of antitumor compounds, and relates to a novel targeted antitumor drug compound, a preparation containing the compound, and application of the compound in preparation of an antitumor drug.

Background

The tumor is a disease seriously harming human life health, and is an abnormal pathological change formed by the loss of normal regulation and control of local tissue cells on the gene level under the action of carcinogenic factors of an organism, so that the clonal abnormal hyperplasia of the local tissue cells is caused, and the abnormal pathological change is expressed as the abnormal hyperproliferation and differentiation of the cells. Cancer has now become the first cause of death in humans, and represents the most serious threat to human survival.

The development of tinib drugs successfully brings cancer into the targeted therapy era, and targets of the drugs are human epidermal growth factor tyrosine kinase receptor, vascular endothelial growth receptor and the like, including: erbB1 (EGFR, HER-1), erbB2 (HER-2, neu), erbB3 (HER-3), erbB4 (HER-4), VEGFR, PDGFR, FGFR, c-Kit, met and the like, and the tinib medicaments greatly improve the life quality of a patient while prolonging the life cycle of the patient. Therefore, the tinib small molecule targeted drug develops very hot fire all over the world. Through database query, 45 drugs with 'tinib' in the marketed drug names are available, and more than one hundred drugs are available in the research varieties. From 2001, the time of market continues to the present, the indications are mainly focused on tumors, and skin diseases, blood system diseases, immune system diseases, musculoskeletal and connective tissue diseases are involved, and the targets are almost hot targets. However, the existing clinically used tinib antitumor drugs still have the defects of drug resistance, limited treatment effect and high toxic and side effects, so that the development of novel high-efficiency low-toxicity tinib targeted antitumor drugs has important significance.

Disclosure of Invention

The invention aims to provide a novel targeted anti-tumor medicament compound which has the characteristics of high anti-tumor activity and low toxic and side effects.

The invention also aims to provide a synthesis method of the targeted antitumor drug compound.

The invention also aims to provide a composition of the targeted antitumor drug compound.

Meanwhile, the invention also aims to provide the application of the targeted antitumor drug compound in preparing antitumor drugs.

The targeted antitumor drug compound of the present invention is systematically named as N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- [ bis- (2-chloroethyl) -amino ] -phenylalkylamide, and its molecular formula can be represented by the following general formula I:

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n = an integer of 0-10 in the formula.

Preferably, the targeted antitumor drug compound is N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- [ bis- (2-chloroethyl) -aminophenyl ] -butyramide.

The invention also relates to a preparation method of the antitumor drug compound, which comprises the following steps:

(1) reacting 5-chloro-3-nitropyrazole [1,5-A ] pyrimidine (1) with 3-chloro-4-fluoroaniline (2) to produce 5- (3-chloro-4-fluoroaniline) -3-nitropyrazole [1,5-A ] pyrimidine (3);

(2) Reacting the 5- (3-chloro-4-fluoroaniline) -3-nitropyrazole [1,5-A ] pyrimidine (3) obtained in the step (1) with Fe powder to generate 5- (3-chloro-4-fluoroaniline) -3-aminopyrazole [1,5-A ] pyrimidine (4);

(3) And (3) carrying out N-acylation reaction on the 5- (3-chloro-4-fluoroaniline) -3-aminopyrazole [1,5-A ] pyrimidine (4) obtained in the step (2) and 4- [ bis (2-chloroethyl) amino ] phenylalkyl acid or 4- [ bis (2-chloroethyl) amino ] phenylalkyl acyl chloride (5) to obtain the antitumor drug compound (I).

The preparation method of the antitumor drug compound (I) has a synthetic route shown as formula 1:

formula 1. Synthetic route of antitumor drug compound (I)

Wherein n = an integer of 0-10, and X is OH or Cl.

The invention also relates to a composition of the targeted anti-tumor drug compound, which comprises an effective treatment amount of the targeted anti-tumor drug compound and pharmaceutically acceptable auxiliary materials.

The composition of the antitumor drug compound of the present invention can be prepared into various common preparations including oral preparations, injections or external preparations such as tablets, capsules, liposome agents, emulsions or microemulsions, micelle agents, ointments and the like. The tablet comprises the anti-tumor drug compound and auxiliary materials. The capsule comprises the anti-tumor drug compound and auxiliary materials. Liposomal agents include the antineoplastic compound, phospholipids (most typically lecithin, phosphatidylcholine), cholesterol, and an aqueous phase. The emulsion comprises the antitumor drug compound, one or more surfactants, an oil phase (lipophilic medium) and an aqueous phase. Emulsions may be of the oil-in-water or water-in-oil type. The micelle agent comprises the antitumor drug compound, a cosolvent, one or more surfactants and a water phase. The ointment formula comprises the anti-tumor drug compound and a matrix.

The invention also provides the application of the antitumor drug compound in the preparation of anticancer drugs.

The antitumor drug compounds of the present invention can be used for the treatment of cancers including blood systems, such as leukemia, lymphoma, myeloma; and non-hematologic cancers such as solid tumor cancers (e.g., breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, esophageal cancer, colon cancer, rectal cancer, lung cancer, bladder cancer, gastric cancer, liver cancer, skin cancer), sarcomas, gliomas, and the like.

The therapeutic 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: 50% of the amount of drug that gives rise to a positive reaction in a subject), LD50 (50% lethal dose, half the amount of drug that kills half of the subject), and GI50 (concentration of drug that inhibits the growth of the anti-cancer drug inhibition by 50% of the subject). The ratio of half lethal dose (LD 50)/half effective dose (ED 50) is usually called 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 anti-tumor drug compound aims to improve the treatment index and the safety of the drug and simultaneously improve the treatment effect. The drug dose obtained from in vitro cell experiments and in vivo animal experiments can be used to formulate a range of doses for use in humans. The dosage of such compounds is preferably within the ED50 range where there is little or no toxicity. Dosage will generally vary depending upon the dosage form employed, the sensitivity of the patient, the route of administration, and the like. Reference will generally be made to conventional dosages of the same or similar drugs.

The pharmaceutical compounds of the present invention may be used alone or in combination with one or more other therapeutic agents.

Has the advantages that: the targeted antitumor drug compound has good anticancer activity, and can be used for preparing medicines for treating cancers of a blood system, solid tumor cancers, sarcomas, skin cancers, glioma and the like.

Drawings

FIG. 1 NMR spectrum of the antitumor drug compound N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1).

FIG. 2 mass spectrum of the antitumor drug compound N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1).

Detailed Description

The following examples are intended to illustrate the synthesis, formulation, in vivo pharmacodynamics, etc. of the antitumor drug compounds of the present invention. The examples are set forth to aid in the understanding and practice of the invention and are not intended to limit the invention.

In the following examples, the specific synthesis steps of the targeted antitumor drug compounds of the present invention include (see reaction formula 1):

(1) Reacting 5-chloro-3-nitropyrazole [1,5-A ] pyrimidine 1 with 3-chloro-4-fluoroaniline 2 to generate 5- (3-chloro-4-fluoroaniline) -3-nitropyrazole [1,5-A ] pyrimidine 3;

(2) Reacting the 5- (3-chloro-4-fluoroaniline) -3-nitropyrazole [1,5-A ] pyrimidine 3 obtained in the step (1) with Fe powder to generate 5- (3-chloro-4-fluoroaniline) -3-aminopyrazole [1,5-A ] pyrimidine 4;

(3) And (3) carrying out N-acylation reaction on the 5- (3-chloro-4-fluoroaniline) -3-aminopyrazole [1,5-A ] pyrimidine 4 obtained in the step (2) and 4- [ bis (2-chloroethyl) amino ] phenylalkyl acid or 4- [ bis (2-chloroethyl) amino ] phenylalkyl acyl chloride to generate the antitumor drug compound I.

Preferably, in the N-acylation reaction, the molar ratio of 5- (3-chloro-4-fluoroanilino) -3-aminopyrazolo [1,5-A ] pyrimidine (4) to 4- [ bis (2-chloroethyl) amino ] phenylalkyl acid or 4- [ bis (2-chloroethyl) amino ] phenylalkyl acid chloride (5) is 1:1.5 to 2.5.

Preferably, the condensing agent for the N-acylation reaction is selected from one or more of 2- (7-azabenzotriazole) -N, N, N ', N ' -tetramethylurea Hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), N, N ' -Carbonyldiimidazole (CDI), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) or Diisopropylcarbodiimide (DIC); the acid-binding agent is one or more selected from triethylamine, pyridine, diisopropylethylamine, sodium methoxide, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate or potassium carbonate.

The targeted anti-tumor drug compound is proved to have good anti-cancer activity, and an anti-tumor drug composition can be obtained by combining the targeted anti-tumor drug compound with effective treatment amount and pharmaceutically acceptable auxiliary materials.

The targeted antitumor drug compound can be prepared into various common preparations, including oral preparations, injections or external preparations, such as tablets, capsules, liposome preparations, emulsion or microemulsion preparations, micelle preparations, paste preparations and the like. The selection of various preparations and auxiliary materials containing the targeted antitumor drug compound is described in detail below in combination with common preparation forms.

The tablet of the targeted anticancer drug compound comprises the following components:

(1) The targeted antitumor drug compounds of the present invention;

(2) And (5) auxiliary materials.

Commonly used excipients include: (1) diluents (Diluents), such as starch, powdered sugar, dextrin, lactose, pregelatinized starch (Pregelatinized starch), microcrystalline cellulose (MCC), inorganic calcium salts, such as calcium sulfate, calcium hydrogen phosphate and calcium carbonate for pharmaceutical use, mannitol; (2) binders (Adhesives) such as distilled water, ethanol, starch slurry, sodium carboxymethylcellulose (CMC-Na), hydroxypropylcellulose (HPC), methylcellulose and Ethylcellulose (MC; ethcellulose, EC), hypromellose (hydroxypropyl cellulose, HPMC), other binders (5-20% gelatin solution, 50-70% sucrose solution, 3-5% aqueous or alcoholic solution of polyvinyl pyrrolidone (PVP)); (3) disintegrants (Disintegrants) such as dry starch, sodium Carboxymethyl starch (CMS-Na), low substituted hydroxypropyl cellulose (L-HPC), crospovidone (also known as Cross-linked PVP), croscarmellose sodium (CCNa), which is a Cross-linked Carboxymethyl ether of cellulose (approximately 70% of the carboxyl groups are in the sodium salt form), (4) Lubricants (Lubricants) such as magnesium stearate, hydrogenated vegetable oils, polyethylene glycol, magnesium lauryl sulfate, aerosil, talc, (5) colorants, (6) flavors, etc. whatever excipients are added, they should meet the pharmaceutical requirements, and should not react with, nor interfere with dissolution and absorption of the primary drug.

The tablet can be prepared by wet granulation and tabletting, dry granulation and tabletting and direct tabletting.

Or, the capsule of the anticancer drug compound comprises a hard capsule and a soft capsule. The components of the composition are as follows:

1) The targeted antitumor drug compounds of the present invention;

2) And (5) auxiliary materials.

Common excipients for hard capsules include, but are not limited to: (1) diluent agent: for improving the physical properties of the contents and increasing the volume, often with some compressibility. Common diluents are mannitol, microcrystalline cellulose, lactose, pregelatinized starch 1500, corn starch, and the like. (2) Lubricant: to prevent the adhesion of the powder to the metal material. Magnesium stearate, glyceryl monostearate, stearic acid, pulvis Talci, etc. are commonly used. (3) Glidant preparation: improve the fluidity of the contents. Silica gel micropowder and talcum powder are commonly used. (4) Disintegrating agent: ensuring the disintegration of the content. The common examples include crosslinked cellulose, corn starch, crospovidone, pregelatinized starch 1500, sodium glycyl starch, and alginic acid. (5) Wetting agent: the wettability of the medicine and the dissolution medium is increased, and the efficacy of the preparation is ensured. Tween 80, sodium dodecyl sulfate, etc. are commonly used.

The adjuvant in the soft capsule content includes but is not limited to oily dispersion or PEG dispersion, and the content can be solution, suspension, emulsion, semisolid and the like. Oily dispersed (lipophilic) content excipients include: (1) oily vehicle: soybean oil, castor oil, medium-chain fatty acids, and the like; (2) semi-solids for adjusting viscosity include hydrogenated castor oil, beeswax, and the like; (3) surfactants such as phospholipids can improve the suspension stability of suspensions. Other stabilizers such as antioxidant BHT, etc. may also be added. PEG dispersed (hydrophilic) content adjuvants are usually PEG400 and PEG 600, and low molecular weight PEG200, 300 and high molecular weight PEG4000-10000 can be used together in semisolid.

The hard capsule shell is similar to soft capsule shell, and mainly contains gelatin, acacia, water, plasticizer (such as glycerol, and appropriate amount of propylene glycol and polyethylene glycol 200 can also be added, mannitol or sorbitol can replace glycerol to be used as plasticizer of rubber skin), antiseptic (such as potassium sorbate, nipagin, etc.), opacifier and pigment, etc., wherein water is used as solvent.

Alternatively, an ointment of the antitumor drug compound comprises:

1) The antitumor drug compound of the present invention;

2) A substrate.

Commonly used substrates include: hydrocarbons (such as vaseline, solid paraffin, liquid paraffin, and silicone), lipids (such as lanolin, beeswax and spermaceti wax, and dimethicone), and oils (such as animal and vegetable higher fatty acid glyceride and mixture thereof).

The antitumor drug compound of the present invention is soluble in lipophilic media, and suitable formulations further include liposome agents, emulsions or microemulsion agents, micelle agents, and the like.

Further, the antitumor drug compound liposome preparation comprises the following components:

1) The antitumor drug compound of the present invention;

2) A phospholipid;

3) Cholesterol or vitamin E and derivatives thereof;

4) An aqueous phase.

Alternatively, an emulsion or microemulsion of said anti-neoplastic compound comprises:

1) An oil phase comprising:

a) The antitumor drug compound of the present invention;

b) A biocompatible lipophilic medium;

2) Surfactants and cosolvents;

3) An aqueous phase.

Alternatively, the anti-tumor drug compound micelle agent comprises the following components:

1) The antitumor drug compound of the present invention;

2) A surfactant;

3) A cosolvent;

4) An aqueous phase.

The lipophilic medium (or carrier) may be any biocompatible lipophilic medium, representative biocompatible lipophilic media include:

1) Oils and fats that can be used as lipophilic medium include fatty acids and esters of varying chain length, which are mostly linear but may also be branched, such as capric acid, caprylic acid, caproic acid, lauric acid, myristic acid, stearic acid, oleic acid, linoleic acid, and other saturated or unsaturated fatty acids and esters.

2) Fat-soluble vitamin E and derivatives thereof. Vitamin E refers to the natural or synthetic series of vitamin E commonly referred to as tocopherols and tocotrienols (tocophenols and tocotrienols), and tocopherols include alpha-tocopherol (D, DL, L), beta-tocopherol (D, DL, L), gamma-tocopherol (D, DL, L) and delta-tocopherol (D, DL, L). Tocotrienols are similar in structure to tocopherols, but tocotrienols have three double bonds in the carbon-2 side chain phytyl (phytyl). Tocotrienols include alpha-tocotrienol (D, DL, L), beta-tocotrienol (D, DL, L), gamma-tocotrienol (D, DL, L) and delta-tocotrienol (D, DL, L). The vitamin E derivatives include all derivatives of tocopherol and tocotrienol, such as vitamin E succinate, vitamin E acetate, etc.

3) The mono-, di-or triglycerides formed by the esterification of fatty acids with glycerol, whether synthetic or natural, can be used as lipophilic media, for example, glycerides such as soybean oil, cottonseed oil, rapeseed oil, fish oil, acetylated monoglycerides, glycerol monooleate, glycerol triacetate, and diacetyl tartrate, monoglycerides, castor oil, etc.

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, etc.

2) Phospholipid surfactants (phospholipids), such as lecithin (lecithin), soya lecithin (granulesten or soybean lecithin), polyethylene glycol phospholipids (pegylated phospholipids).

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

4) Polyoxyethylene polyoxypropylene block copolymer: block copolymers of POLOXAMERS or PLURONICS (H (OCH 2CH 2) a (OCH 2CH2CH 2) b (OCH 2CH 2) aOH).

Representative organic cosolvents include: ethanol, polyethylene glycol, propylene glycol, glycerol, N-methylpyrrolidone, etc. Polyethylene glycol (PEG) is hydrophilic and the chemical structure of the repeat unit is-CH ₂ CH ₂ O-with the general formula H- (CH) ₂ CH ₂ ) _n -OH, generally ranging from 200 to 10000 in molecular weight. For example, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, and the like.

As used herein, "emulsion" refers to a heterogeneous liquid dispersion system in which one phase of liquid is dispersed in another phase of liquid in a droplet state by the action of a surfactant, such as droplets of oil and water, and the diameter thereof is generally 0.1 to 3.0 μm.

The emulsion may form a stable microemulsion. The term "microemulsion" refers to two immiscible liquids that form a thermodynamically stable isotropic, transparent or translucent dispersion, such as a microemulsion dispersion of oil and water, that is stabilized by an interfacial film formed by surfactant molecules. The average microemulsion droplet diameter is less than 200nm, typically 10 to 50 nm.

Emulsions or microemulsions comprise an oil phase and an aqueous phase. The emulsion or microemulsion may be oil-in-water emulsion or water-in-oil emulsion.

The homogeneous and transparent solution containing the drug formed by mixing the oil phase, the nonionic surfactant and the co-emulsifier without water is called self-emulsifying drug delivery system (SEDDS), and the self-emulsifying drug delivery system forms an emulsion with the particle size of 100nm to 500nm, which can be used for improving the solubility and oral absorption of lipophilic drugs.

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

In addition to the antineoplastic compounds of the present invention, other components commonly used in pharmaceutical emulsions and microemulsions may be included in the emulsion or microemulsion formulations, including surfactants and cosolvents. Representative surfactants include nonionic surfactants such as polyoxyethylene castor oil EL (Cremophor EL), tween 80 (Tween 80), polyethylene glycol vitamin E derivative surfactants, and other surfactant polymers.

Suitable polyethylene glycol vitamin E derivative surfactants include vitamin E succinic polyethylene glycol derivatives (e.g., vitamin E polyethylene glycol succinate) in which polyethylene glycol is formed by the attachment of succinic acid to the hydroxyl groups of vitamin E, and polyethylene glycols in these vitamin E polyethylene glycol derivatives include polyethylene glycols having various molecular weights (e.g., 200, 300, 400, 600, 1000, etc.). "vitamin E polyethylene glycol succinate" as used herein includes vitamin E polyethylene glycol succinate (e.g., D-alpha tocopheryl polyethylene glycol 1000succinate, TPGS, a nonionic surfactant (HLB = 16-18)) and various ester and ether derivatives of vitamin E polyethylene glycol.

The formulations of the various preparations comprise effective treatment dose of the targeted antitumor drug compound and auxiliary materials.

The tablet of the targeted anti-tumor drug compound comprises the anti-tumor drug compound and auxiliary materials. The content of the anti-tumor drug compound in each tablet can be 1 mg to 1000 mg, and the content of the anti-tumor drug compound in each tablet is 5 mg to 500 mg in a preferred scheme; in a more preferred embodiment, the antitumor drug compound is contained in an amount of 10 mg to 250 mg per tablet.

The capsule of the targeted anti-tumor drug compound comprises the anti-tumor drug compound and auxiliary materials. The content of the anti-tumor drug compound in each capsule can be 1 mg to 1000 mg, and the content of the anti-tumor drug compound in each capsule is 5 mg to 500 mg in a preferred scheme; in a more preferred embodiment, the antitumor compound is contained in an amount of 10 mg to 250 mg per granule.

In the emulsion or microemulsion of the targeted antitumor drug compound, the weight percentage of the antitumor drug compound in the preparation formula is 0.005-5.0%; preferably, the weight percentage of the anti-tumor drug compound in the preparation formula is 0.01 percent to 2.5 percent; in a more preferable scheme, the weight percentage of the anti-tumor drug compound in the preparation formula is 0.1 to 1.5 percent.

In the emulsion or microemulsion, the weight percentage of the lipophilic medium in the preparation formula is 2 to 20 percent; preferably the lipophilic medium is present in the formulation in an amount of from 4% to 12% by weight; more preferably, the lipophilic medium is present in the formulation in an amount of 6 to 10% by weight.

The emulsion or microemulsion contains about 1 to 10%, preferably 2-6%, and more preferably 4-5% by weight of the surfactant in the formulation.

In the emulsion or microemulsion, the cosolvent accounts for 0-20% of the weight of the formula.

The micelle formulation targeting an anticancer drug compound as described above comprises the anticancer drug compound of the present invention, one or more surfactants, one or more co-solvents and an aqueous phase.

In the micelle agent of the antitumor drug compound, the weight percentage of the drug compound in the formulation is about 0.005% to 3.0%, preferably the weight percentage of the drug compound in the formulation is about 0.01% to 2.5%; more preferably, the pharmaceutical compound is present in the formulation in an amount of about 0.1% to about 1.0% by weight.

Suitable surfactants may be present in the micelle formulation of the present invention in an amount of about 1 to 10% by weight, preferably 2-6% by weight, more preferably 4-5% by weight.

Micelle formulations may also include other ingredients, such as the co-solvents mentioned above. In one embodiment, the micelle formulation comprises polyethylene glycol and a lower alkyl alcohol (e.g., ethanol). In the micelle agent, the cosolvent accounts for about 1 to 20 percent of the weight of the formula.

The liposome agent targeting the antitumor drug compound comprises the antitumor drug compound, one or more phospholipids (including PEGylated phospholipid), one or more lipophilic media (such as cholesterol) and an aqueous phase.

In the liposome preparation of the targeted antitumor drug compound, the weight percentage of the drug compound in the formula is about 0.005 to 5.0 percent, and preferably the weight percentage of the drug compound in the formula is about 0.01 to 2.5 percent; more preferably, the pharmaceutical compound is present in the formulation in an amount of about 0.1% to about 1.5% by weight.

Suitable phospholipids may be present in the liposomal formulation of the present invention in an amount of about 1 to about 10% by weight, preferably 2-6% by weight, and more preferably 4-5% by weight.

The liposomal formulation may also include other ingredients such as the lipophilic mediators (e.g., cholesterol) mentioned above. In one embodiment, the liposomal formulation comprises cholesterol or vitamin E. The liposome formulation comprises cholesterol or vitamin E in an amount of about 0.1% to about 20% by weight of the formulation.

The above emulsion, microemulsion, micelle and liposome formulations contain an aqueous phase. In one embodiment, the aqueous phase comprises deionized water. In another embodiment, the aqueous phase comprises physiological saline. In another embodiment, the aqueous phase comprises a buffer of an acid (e.g., succinic acid, citric acid, phosphoric acid).

The paste of the targeted anti-tumor medicine compound comprises the targeted anti-tumor medicine compound and one or more matrixes.

The weight percentage of the anti-tumor drug compound in the paste of the targeted anti-tumor drug compound is about 0.01 to 30 percent, and the weight percentage of the drug compound in the formula is preferably about 0.05 to 20 percent; more preferably, the pharmaceutical compound is present in the formulation in an amount of about 0.1% to about 10% by weight.

The targeted antitumor drug compound or the preparation is used as an anticancer drug for treating cancer, and is used alone or in combination with other drugs. The other drug may be selected from the following drug compounds, including but not limited to: androgen inhibitors such as flutamide (flutamide) and lupperoid (luprolide); antiestrogens, such as tamoxifen (tomoxifen); antimetabolites and cytotoxic drugs such as daunorubicin (daunorubicin), pentafluorouracil (fluorouricin), floxuridine (floxuridine), interferon-alpha (interferon alpha), methotrexate (methotrexate), mithramycin (plicamycin), thiopurine (mecapture), thioguanine (thioguanine), adriamycin (adriamycin), carmustine (carmustine), lomustine (lomustine), cytarabine (cytarabine), cyclophosphamide (cyclophosphamide), adriamycin (doxorubicin), estramustine (estramustine), altretamine (altramine), hydroxyurea (hydroyurea), ifosfamide (ifosfamide), procarbazine (procarbazine), promycin (mitomycin), cyanamide (sultrine), mitomycin (butomycin), mitomycin (bleomycin), mitomycin (imidazole), and platinum (epothilone), carboplatin (cisplatin); hormones, such as megestrol (medroxyprogesterone), ethinylestradiol (ethinyl estradiol), estradiol (estradiol), leuprolide (leuprolide), megestrol (megestrol), octreotide (octreotide), diethylstilbestrol (diethylstilbestrol), chloroestrene (chlorotrianisene), etoposide (etoposide), podophyllotoxin (podophyllotoxin) and goserelin (goserelin); nitrogen mustard derivatives, such as mechlorethamine phenylpropionate (melphalan), chlorambucil (chlorambucil), and thiotepa (thiotepa); steroids, such as betamethasone (betamethasone); and other antitumor agents such as live Mycobacterium bovis (live Mycobacterium bovis), dacarbazine (dicarbazine), asparaginase (aspargine), leucovorin (leucovorin), mitotane (mitotane), vincristine (vincristine), vinblastine (vinblastine), and docetaxel (taxol), etc.

Example 1

N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide and its synthesis comprising the steps of:

(1) Synthesis of 5- (3-chloro-4-fluoroanilino) -3-nitropyrazole [1,5-A ] pyrimidine (3)

The reaction formula is shown as the following formula:

the experimental steps are as follows:

to a 100ml round bottom flask was added 0.8g (4 mmol) of 5-chloro-3-nitropyrazolo [1,5-a ] pyrimidine (1), 60ml of isopropanol as a solvent, stirred, then added 1.2g (8 mmol) of 3-chloro-4-fluoroaniline (2) and 5 drops of concentrated HCl, warmed to 100 ℃ and refluxed until the reaction was complete, cooled to room temperature and TEA was added to adjust PH to weak alkalinity, then the solvent was spin-dried under reduced pressure, added 50ml of dichloromethane, stirred for 0.5h and filtered, and the solid was collected and drained to give 1.06g of 5- (3-chloro-4-fluoroaniline) -3-nitropyrazolo [1,5-a ] pyrimidine (3) in 83.5% yield.

(2) Synthesis of 5- (3-chloro-4-fluoroanilino) -3-aminopyrazolo [1,5-A ] pyrimidine (4)

The reaction formula is shown as the following formula:

adding 1.06g (3.4 mmol) of 5- (3-chloro-4-fluoroanilino) -3-nitropyrazole [1,5-A ] pyrimidine (3), 30ml of ethanol and 15ml of purified water (ethanol: water = 2). The collected organic phases were combined, adjusted to pH with 1N HCl to be slightly acidic (6-7) and stirred for 0.5h, then adjusted to pH 7-8 with saturated aqueous sodium bicarbonate solution and stirred for 0.5h, after ethanol had been spun off, water was added and filtered with stirring, and the solid product was collected to give 1.0g of crude 5- (3-chloro-4-fluoroanilino) -3-aminopyrazolo [1,5-A ] pyrimidine (4) in about 100% yield.

MS(Positive ESI):m/z(M+H ⁺ )＝278.05696，MS(Negative ESI):m/z(M-H ⁺ )＝276.04634。

¹ H NMR(500MHz,DMSO-d ₆ ):δppm:9.5821(s，1H)，8.4550-8.4400(d,J＝7.50Hz，1H),8.1696-8.1519(m,1H)，7.8688-7.8509(m,1H),7.5449(s,1H),7.3484-7.3120(t，1H)，6.2758-6.2602(d,J＝7.80Hz，1H),3.7680(s,2H)。

(3) Synthesis of N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1)

The reaction formula is shown as the following formula:

0.44g (1.44 mmol) of chlorambucil was charged into a 25ml round-bottomed flask, dissolved in 8ml of DMF (dried), followed by addition of 0.65g (1.72 mmol) of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and 0.4ml (2.88 mmol) of TEA in this order, followed by stirring for 40 minutes, addition of 0.2g (0.72 mmol) of 5- (3-chloro-4-fluoroanilino) -3-aminopyrazole [1,5-A ] pyrimidine (4), and completion of the reaction by stirring at 50 ℃ at elevated temperature. Concentrating under reduced pressure, separating column layer (100-200 mesh silica gel), eluting with mixed solution of dichloromethane and ethyl acetate, ethyl acetate and tetrahydrofuran to obtain product 90mg, yield: 22.5 percent.

MS(Positive ESI):m/z(M+Na ⁺ )＝585.10750，MS(Negative ESI):m/z(M-H ⁺ )＝561.11847。

¹ H NMR(500MHz,DMSO-d ₆ ):δppm:9.8319(s，1H)，9.3839(s,1H),8.6180-8.6029(d,J＝7.55Hz，1H),8.3144-8.2960(m,1H)，8.0426(s,1H),7.7476-7.7164(m,1H),7.2806-7.2444(t,1H),7.0656(s,1H),7.0488(s,1H),6.6789(s,1H),6.6620(s,1H),6.4513-6.4362(d,J＝7.55Hz，1H)，3.6944(s，8H),2.5601-2.5299(t,2H),2.3788-2.3489(t,2H),1.9048-1.8751(t,2H)。

The hydrogen nuclear magnetic resonance spectrum and the mass spectrum of the synthesized compound (marked as YY-060-1) are shown in a figure 1 and a figure 2.

Example 2

N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -formamide and its synthesis comprising the steps of:

Same as in step (1) of example 1.

(3) Synthesis of N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -carboxamide

The reaction formula is shown as the following formula:

0.3748g (1.44 mmol) of 4- [ bis- (2-chloroethyl) -amino ] -benzoic acid (6) was added to a 25ml round bottom flask, dissolved in 8ml of DMF (dry) and then 0.65g (1.72 mmol) of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) and 0.4ml (2.88 mmol) of TEA were added in sequence and after stirring for 40 minutes 0.2g (0.72 mmol) of 5- (3-chloro-4-fluoroanilino) -3-aminopyrazole [1,5-A ] pyrimidine (4) was added and the reaction was stirred at 50 ℃ to complete. Concentrating under reduced pressure, separating column layer (100-200 mesh silica gel), eluting with mixed solution of dichloromethane and ethyl acetate, ethyl acetate and tetrahydrofuran to obtain 380mg product (YY-060-0), yield: 50.6 percent.

Example 3

Pharmacodynamic experiment of N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) on inhibition effect of human lung cancer cell A549 nude mouse transplanted tumor

(1) The preparation method of the medicine comprises the following steps:

(1) YY-0-60-1 preparation method

The preparation method comprises the following steps: 14.4mg of YY-060-1 powder medicine is weighed, 2ml of medical grade Tween is added, after the medicine is fully dissolved to be slightly transparent, 18ml of CMCNa aqueous solution (the volume ratio is 0.5 percent) is added, and the medicine is stirred until the medicine is fully dissolved. The administration volume was 0.2ml/20g, i.e., the administration dose was 7.2mg/kg.

(2) Preparation method of positive control medicament Dacomitinib (Dacomitinib)

The preparation method comprises the following steps: weighing 12mg of dacomitinib powder, adding 2ml of pharmaceutical grade Tween, fully dissolving to be slightly transparent, adding 18ml of CMCNa aqueous solution (volume ratio is 0.5%), and stirring until fully dissolving. The administration volume was 0.2ml/20g, i.e., the administration dose was 6mg/kg.

(2) Source, germ line and strain of experimental animal

Source, species, strain: balb/c nude mice, offered by Shanghai Ling Chang Biotech Co., ltd., laboratory animal production licenses: SCXK (Shanghai) 2018-0003, and the qualification number: 20180003022475, laboratory animal use license: SYXK (threo) 2017-0040. Weight: 18-22g, sex: female, feeding conditions: air-conditioning room, temperature 18-24 deg.C, relative humidity 40-70%.

(3) Grouping and dosing regimen for experimental animals

Blank control group (model control group): CMCNa,200ul/20g, was gavaged every other day, data were recorded and observed for 26 days:

positive control drug (dacomitinib): 6 pieces of the Chinese herbal medicine are taken at 6mg/kg, 200ul/20g is taken by intragastric administration, administration is carried out every other day, data are recorded and observed, and the duration is 26 days;

drug treatment group (YY-060-1): 6 patients with 7.2mg/kg, are administrated by intragastric administration of 200ul/20g, are administrated every other day, and data are recorded and observed for 26 days;

(4) Experimental methods

(1) Preparation of the model

Collecting cultured human lung cancer A549 cell suspension at concentration of 1X10 ⁷ One/ml, 0.1ml each, was inoculated subcutaneously in the right axilla of mice.

(2) Administration of drugs

The diameter of the mouse graft tumor was measured with a vernier caliper, and 21 days after the inoculation, the tumor grew to 100mm ³ Animals were randomly grouped into groups of 6 animals each. At the same time, each group of mice is started to be dosed, the dosing scheme is shown in the group and the dosing scheme, and the antitumor effect of the tested sample is dynamically observed by using the method for measuring the tumor size. After the experiment was completed, the mice were sacrificed immediately and the tumor mass was removed by surgery and weighed.

(3) Observation index

The Tumor Volume (TV) is calculated as:

TV＝1/2×a×b ²

wherein a and b represent length and width, respectively.

Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows:

RTV＝V _t /V ₀

wherein V ₀ When administering the medicament in separate cages (i.e. d) ₀ ) Measurement of the resulting tumor volume, V _t For the tumor volume at each measurement.

Evaluation indexes of antitumor activity: the relative tumor proliferation rate T/C (%) was calculated as follows:

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T _RTV : treatment group RTV; c _RTV : model set RTV

Evaluation indexes of antitumor activity: the tumor growth inhibition (%) is calculated as follows:

(4) statistical processing

The mean values are expressed as X + -SD, the inter-group analysis is statistically processed by t-test, and the results are statistically analyzed using SPSS (Standard Package for the Social Science) 17.0.

(5) Results of the experiment

The result of a pharmacodynamic experiment (human non-small cell lung cancer A549) of a nude mouse shows that YY-060-1 and a positive control drug can achieve the obvious inhibition effect (P < 0.01) on the growth of a tumor transplanted in the nude mouse of the human lung cancer A549 compared with a model control group, but the tumor inhibition effect of YY-060-1 is 16.1 percent higher than that of a clinical tinib drug Dacomitinib (Dacomitinib) under the same molar concentration. And the experimental result shows that the new drug has smaller inhibition effect on the weight increase of the mice than the positive control drug Dacomitinib and is close to the model control group, which shows that the new drug has very low toxic and side effects and has lower toxic and side effects than the positive control drug Dacomitinib. Dacomitinib is a second generation multi-target small molecule drug developed by Pfizer, U.S. Peeler, and is approved to be marketed in the U.S. at 9/27.2018, and is a novel tyrosine kinase inhibitor used for first-line treatment of EGFR sensitive mutation local advanced or metastatic non-small cell lung cancer (NSCLC).

TABLE 1 Effect of YY-060-1 on the growth of tumors transplanted from human lung carcinoma cells A549 in nude mice (X + -SD, n = 6)

In comparison with the blank set, the results, ^* P＜0.05， ^** P＜0.01

example 4

Formulations of the anti-tumor drug compound N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) include tablets, capsules, emulsions, micelles, liposomal formulations and ointment formulations.

(1) Capsule (wet granulation) of N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1)

YY-060-1 in the prescribed amount is mixed with sodium starch glycolate, lactose and magnesium stearate in the prescribed amount, and after adding tween 80 in the prescribed amount in water, wet granulation is carried out, the prepared wet material is dried in a fluidized bed, a drying pan or other suitable dryer, the dried granules are milled to a suitable particle size distribution and then mixed with the other components in the prescribed amount, and finally the mixture is filled into two pieces of hard gelatin capsule shells.

Components	Content of each capsule (mg)	Percentage of each component (%)
			YY-060-1	25	40
Tween 80	2.5	2
			Lactose	25	20
Magnesium stearate	2.5	2
			Sodium starch glycolate	45	36
The total weight of each capsule	100

(2) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) tablets (wet granulation)

Granulating a prescribed amount of an aqueous solution of sodium lauryl sulfate with a prescribed amount of YY-060-1, sodium starch glycolate, magnesium stearate, and microcrystalline cellulose, drying the resulting wet material in a fluidized bed, a drying pan, or other suitable dryer, milling the dried granules to the desired particle size distribution, and compressing the mixture into tablets.

Components	Content per tablet (mg)	Percentage of each component (%)
			YY-060-1	30	50
Sodium dodecyl sulfate	1.5	2
			Lactose	4.5	7
Magnesium stearate	3	1
			Sodium starch glycolate	12	20
Microcrystalline cellulose	12	20
			The total weight of each capsule	63

(3) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) tablets (dry granulation)

Firstly, raw material YY-060-1 is crushed and sieved, the particle size is controlled to be less than 80 mu m, then the YY-060-1 with the prescription amount is mixed with superfine silica gel powder, the starch, the cane sugar and the croscarmellose sodium with the prescription amount are added, the mixture is mixed, granulated by a dry method, added with the magnesium stearate with the prescription amount after the granulation, mixed, tableted and coated with film.

(4) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) emulsion

YY-060-1 was dissolved in a mixture of soybean oil, tween 80 and polyethylene glycol PEG (200), and deionized water (DI water) was added, followed by stirring and ultrasonic emulsification or emulsification with a homogenizer, to produce an emulsion having the following composition:

the resulting emulsion drug was filtered through a filter with a pore size of 0.2 μm and filled into a sterile glass bottle.

(5) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) emulsion

YY-060-1 was dissolved in a mixture of D- α -tocopheryl acetate, D- α -tocopheryl polyethylene glycol 1000succinate (TPGS) and polyethylene glycol PEG (200), and deionized water (DI water) was added, followed by stirring and ultrasonic emulsification or emulsification with a homogenizer, and the composition of the produced emulsion was as follows:

the resulting emulsion drug was filtered through a filter with a pore size of 0.2 microns and filled into sterile glass vials.

(6) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) micelle agent

YY-060-1 is dissolved in a mixture of D-alpha-tocopheryl polyethylene glycol 1000succinate (TPGS), ethanol and polyethylene glycol PEG (200) to obtain a transparent liquid, a proper amount of physiological saline is added before use, and then stirring and ultrasonic stirring are carried out to obtain a clear liquid, and the produced micelle agent has the following composition:

the prepared micelle medicine is filtered by a filter with the pore diameter of 0.2 micron for standby.

(7) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) micelle agent

YY-060-1 is dissolved in a mixture of polyoxyethylated castor oil EL (Cremophor EL), ethanol and polyethylene glycol PEG (200) to obtain a transparent liquid, and a proper amount of deionized water (DI water) is added before use, and then stirring and ultrasonic stirring are carried out to obtain a clear liquid, wherein the produced micelle agent has the following composition:

(8) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) micelle agent

YY-060-1 is dissolved in the mixture of polyoxyethylated castor oil EL (Cremophor EL), ethanol and polyethylene glycol PEG (200) to obtain a transparent liquid, and before using, a proper amount of deionized water (DI water) is added, and then stirring and ultrasonic stirring are carried out to obtain a clear liquid, and the composition of the produced micelle agent is as follows:

the prepared micelle agent medicine is filtered by a filter with the pore diameter of 0.2 micron for standby.

(9) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) micelle agent

YY-060-1 is dissolved in a mixture of Tween 80 (Tween 80), ethanol and polyethylene glycol PEG (200) to obtain a transparent liquid, a proper amount of deionized water (DI water) is added before use, then stirring and ultrasonic stirring are carried out to obtain a clear liquid, and the produced micelle agent has the following composition:

(10) Liposomal agent of N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1)

In a round bottom flask, 100 mg YY-060-1, 1600 mg phospholipids (lecithin, phosphatidylcholine) and 110 mg cholesterol were dissolved in 15mL chloroform (CHCl) ₃ ) Slowly heating to 40 deg.C, evaporating solvent under reduced pressure with rotary evaporator to form a thin lipid film, vacuum drying overnight, further removing chloroform from the lipid film, adding 50ml of 5% sucrose solution, stirring and ultrasonic stirring, and filtering the obtained liposome solution with a pore size of 0.2 μmFiltering with a filter, filling into a sterile glass bottle, freezing with dry ice and acetone, and freeze-drying for 24 hr to obtain 4- [ bis (2-chloroethyl) amino]Liposomal agents of 4- (3-chloro-4-fluoroanilino) -7-methoxy-6-aminoquinazoline phenylbutylate (YY-060-1).

(11) N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- {4- [ bis- (2-chloroethyl) -amino ] -phenyl } -butyramide (YY-060-1) ointment

Heating and melting appropriate amount of YY-060-1, stearic acid, glyceryl monostearate, liquid paraffin, polyethylene glycol 200 (PEG 200), and tween-80; heating appropriate amount of glycerol and water to 70-80 deg.C, adding into oil phase under stirring, and stirring to form paste with the following composition:

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Claims

1. a targeted antineoplastic compound, systematically named N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- [ bis- (2-chloroethyl) -amino ] -phenylalkylamide, having the general structural formula of formula I:

wherein n = an integer of 0-10.

2. The targeted antineoplastic compound of claim 1, wherein is N- [5- (3-chloro-4-fluorophenylamino) -pyrazolo [1,5-a ] pyrimidin-3-yl ]4- [ bis- (2-chloroethyl) -aminophenyl ] -butyramide.

3. A method for preparing an antitumor drug compound according to claim 1, comprising the steps of:

4. Use of a targeted antitumor pharmaceutical compound according to claim 1 in the preparation of an anticancer drug.

5. The use according to claim 4, wherein the cancer is a cancer of the hematological system and a solid tumor.

6. An antitumor pharmaceutical composition, comprising a therapeutically effective amount of the targeted antitumor compound of claim 1 and a pharmaceutically acceptable excipient.

7. The antitumor pharmaceutical composition as claimed in claim 6, wherein the pharmaceutical composition is in the form of tablet, capsule, emulsion, micelle, liposome or paste.