CN101941953B - Nitrogenous heterocyclic substituted hydrazide compounds as well as preparation methods and applications thereof - Google Patents

Abstract

The invention relates to nitrogenous heterocyclic substituted hydrazide compounds as well as preparation methods and applications thereof, in particular to novel nitrogenous heterocyclic substituted hydrazide compounds shown in formulas (I, II and III) or pharmaceutically acceptable salts, solvate, stereomers or prodrugs thereof, wherein various symbols are shown in the specification. The invention also relates to preparation methods of the compounds shown in the formulas (I, II and III), the applications of the compounds in the formulas (I, II and III) to preparing medicines for treating and/or preventing tumors and/or cancers, methods for treating and/or preventing tumors and/or cancers by using the compounds in the formulas (I, II and III) and effective amounts of pharmaceutical compositions of the compounds in the formulas (I, II and III) for treatment and/or prevention. The compounds in the formulas (I, II and III) in the invention can selectively kill a certain cancer cells, have strong anti-cancer activity and can inhibit the growth of tumors.

Description

Nitrogen heterocyclic ring substituted hydrazide compound and preparation method and application thereof

Technical Field

The invention relates to a novel nitrogen heterocyclic ring substituted hydrazide compound, a method for preparing the nitrogen heterocyclic ring substituted hydrazide compound, application of the nitrogen heterocyclic ring substituted hydrazide compound in preparing a medicament for treating and/or preventing tumors and/or cancers, and a pharmaceutical composition containing the nitrogen heterocyclic ring substituted hydrazide compound.

Background

Tumor, a worldwide disease, has become an important disease species that threatens human health and survival and causes human death. At present, chemotherapy is mainly adopted clinically for treating advanced cancers. However, the chemotherapy drugs used at present basically have no selectivity to cancer cells, and kill a large number of normal cells while killing the cancer cells, thereby bringing great harm to the mind and body of cancer patients. If the chemotherapeutic drug only selectively acts on cancer cells, the personalized treatment can be provided for cancer patients, the toxic and side effects are reduced, and the life of the patients is prolonged.

The molecular targeted therapy is a new way for selectively treating tumors, and the molecular targeted drug provides feasibility for personalized treatment of cancer patients. In recent years, an important protein procaspase-3 is discovered in cells, and the activation of procaspase-3 into caspase-3 can cause the metabolic death of the cells. The literature reports (e.g., Clin Cancer Research 2004, 6807; PNAS 2001, 6132) that the concentration of procaspase-3 in many Cancer cells is hundreds of times higher than in corresponding normal cells, indicating that procaspase-3 cannot be activated to caspase-3 in tumor cells, and Cancer cells cannot be metabolically killed and transformed to tumors. Modern molecular pharmacology clarifies the action mechanism of procaspase-3 in the tumor development process, and provides that a compound capable of activating procaspase-3 can be used as a targeting molecule to induce cancer cell death. Therefore, the procaspase-3 activator has good application prospect.

Professor Paul j. hergenother et al at the university of illinois obtained the small molecule compound PAC-1(4- (phenylmethyl) -1-piperazineacetic acid [ [ 2-hydroxy-3- (2-propenyl) phenyl ] methylene ] hydrazide, CAS No: 315183-21-2), which directly activated Procaspase-3 to caspase-3 to induce apoptosis by screening 22000 for a variety of compounds. The expression of Procaspase-3 in tumor cells is many times higher than that of normal cells, so PAC-1 can selectively kill tumor cells without damaging the normal cells.

A series of novel nitrogen heterocyclic ring substituted hydrazide compounds are designed and synthesized, and the activity results show that the compounds have the advantages of strong activity for inhibiting tumor cells and small side effect.

Disclosure of Invention

The object of the first aspect of the invention is to provide a novel class of nitrogen heterocyclic ring substituted hydrazide compounds. The second aspect of the invention aims to provide a preparation method of the novel nitrogen heterocyclic ring substituted hydrazide compound. The third aspect of the invention aims to provide the application of the novel nitrogen heterocyclic ring substituted hydrazide compound in pharmacy. In addition, the invention provides a pharmaceutical composition containing the nitrogen heterocyclic ring substituted hydrazide compound. The present inventors have surprisingly found that the novel nitrogen-containing heterocyclic substituted hydrazide compounds provided by the present invention have potent anticancer activity, and the present invention has been completed based on the above findings.

In general, in a first aspect the invention provides compounds of formula I, formula II and formula III:

wherein,

ar is or is

Radical part of]Selected from substituted or unsubstituted aryl radicals [ e.g. substituted or unsubstituted phenyl, especially substituted phenyl]And substituted or unsubstituted heteroaryl group [ e.g., substituted or unsubstituted nitrogen-containing aryl group]Wherein said substituents are selected from hydroxy, halogen (e.g. fluorine, chlorine, bromine, iodine), C₂-C₆Alkenyl (e.g. C)₂-C₄Alkenyl, e.g. vinyl, propenyl, allyl), C₁-C₆Alkyl (e.g. C)₁-C₄Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₆Alkyloxy (e.g. C)₁-C₄Alkyloxy groups such as methoxy, ethoxy, propoxy), nitro, cyano, mercapto, amino, carboxyl, sulfonamido;

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂- [ namely at

Radical part of]；

Z is a nitrogen atom or a carbon atom;

n is selected from 1, 2, or 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl (e.g. C)₂-C₄Alkenyl, e.g. vinyl, propenyl, allyl), C₁-C₆Alkyl (e.g. C)₁-C₄Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₆Alkyloxy (e.g. C)₁-C₄Alkyloxy groups such as methoxy, ethoxy, propoxy), nitro, cyano, sulfonamide,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl, preferably substituted or unsubstituted phenyl;

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is selected from 1, 2, or 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom;

n is selected from 1, 2, or 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is 1; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is selected from 2; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is selected from 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is selected from 1, 2, or 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from the group consisting of hydrogen, hydroxy, halogen, vinyl, allyl, 2-butenyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, methoxy, ethoxy, tert-butyloxy,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is 1;

R₁is a hydroxyl group;

R₂is an allyl group; and

R₃、R₄and R₅Is a hydrogen atom, and is a hydrogen atom,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl [ e.g., substituted or unsubstituted phenyl, particularly substituted phenyl ], substituted or unsubstituted heteroaryl [ e.g., substituted or unsubstituted nitrogen-containing aryl ];

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is 1;

R₅is a hydroxyl group;

R₄is an allyl group; and

R₁、R₂and R₃Is a hydrogen atom, and is a hydrogen atom,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention, which has the characteristics of any one or more of:

1) ar is selected from aryl [ e.g. phenyl ]]Or heteroaryl [ e.g. nitrogen-containing aryl)]Wherein said aryl or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of: hydroxy, halogen (e.g. fluorine, chlorine, bromine, iodine), C₂-C₆Alkenyl (e.g. C)₂-C₄Alkenyl, e.g. vinyl, propenyl, allyl), C₁-C₆Alkyl (e.g. C)₁-C₄Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₆Alkyloxy (e.g. C)₁-C₄Alkyloxy groups such as methoxy, ethoxy, propoxy), nitro, cyano, sulfonamide groups; in one embodiment, Ar is phenyl, optionally substituted with 1 to 3 (e.g., 1 to 2, e.g., 1) substituents selected from: hydroxy, halogen (e.g. fluorine, chlorine, bromine, iodine), C₁-C₆Alkyl (e.g. C)₁-C₄Alkyl radicals, e.g. C₁-C₂Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₆Alkyloxy (e.g. C)₁-C₄Alkyloxy groups such as methoxy, ethoxy, propoxy), nitro, cyano, sulfonamide groups; in one embodiment, Ar is phenyl, optionally substituted with C₁-C₄Alkyl (e.g. C)₁-C₂Alkyl, e.g., methyl, ethyl);

2) x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-; in one embodiment, X is selected from-CH₂-, and-SO₂-；

3) Z is a nitrogen atom or a carbon atom;

4) n is selected from 1, 2, or 3; in one embodiment, said n is 1 or 2; and

5)R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl (e.g. C)₂-C₄Alkenyl, e.g. vinyl, propenyl, allyl), C₁-C₆Alkyl (e.g. C)₁-C₄Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₆Alkyloxy (e.g. C)₁-C₄Alkyloxy groups such as methoxy, ethoxy, propoxy), nitro, cyano, sulfonamide groups; in one embodiment, said R is₁、R₂、R₃、R₄And R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₄Alkenyl (e.g. ethenyl, propenyl, allyl), C₁-C₄Alkyl (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl), C₁-C₄Alkyloxy (e.g., methoxy, ethoxy, propoxy), nitro, cyano, sulfonamido; in one embodiment, said R is₁、R₂、R₃、R₄And R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₄Alkenyl (e.g. ethenyl, propenyl, allyl), C₁-C₄Alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl).

A compound according to any one of the first aspect of the invention wherein:

ar is selected from substituted or unsubstituted aryl, preferably substituted or unsubstituted phenyl;

x is selected from-CH₂-、-CH₂CH₂-, and-SO₂-；

Z is a nitrogen atom or a carbon atom;

n is selected from 1, 2, or 3; and

R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl, halogen, C₂-C₆Alkenyl radical, C₁-C₆Alkyl radical, C₁-C₆An alkyl-oxy group, a carboxyl group,

or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention which is:

1. 3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenebenzene) hydrazine

2.4- (4-benzyl-piperazin-1-yl) -butyryl (3-allyl-2-hydroxy-methylenebenzene) hydrazine

3. 3- (4-benzyl-piperidin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine

4. 3- (4-p-toluenesulfonyl-piperazin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine

5. 3- (4-benzyl-piperazin-1-yl) -propyleneimido (3-allyl-2-hydroxy-methylenebenzene) hydrazine

6. 2- (4-benzyl-homopiperazin-1-yl) -ethylimido (3-allyl-2-hydroxy-methylenebenzene) hydrazine

Or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

A compound according to any one of the first aspect of the invention which is 3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenebenzene) hydrazine.

In a second aspect, the present invention provides a process for the preparation of a compound of formula I according to the first aspect of the invention, comprising the steps of:

(1) reacting ethyl 3-bromopropionate, ethyl 4-bromobutyrate or ethyl 5-bromovalerate with a compound of formula Ia below in the presence of a base,

to form a compound of formula Ib:

(2) reacting hydrazine hydrate with the compound of formula Ib above in an organic solvent to produce a compound of formula Ic:

(3) reacting the compound of formula Ic obtained in step (2) with a compound of formula Id,

to produce the compound represented by the following formula I:

wherein each symbol is as defined in any one of the first aspect of the invention.

The process according to the second aspect of the present invention, wherein the step (1) is carried out in a solvent, which may be an organic solvent including, but not limited to, dichloromethane, toluene, benzene, tetrahydrofuran, or a mixture of any two or more thereof.

The method according to any one of the second aspect of the present invention, wherein the base used in step (1) includes, but is not limited to, triethylamine, potassium carbonate.

The method according to any one of the second aspect of the present invention, wherein the steps (2) and (3) are carried out in a solvent, the solvent used in the two steps may each independently be an organic solvent, which may each independently include but is not limited to methanol, ethanol, and the preferred organic solvent is ethanol.

The process according to any one of the second aspect of the invention, wherein the compound of formula 1d of step (3)

In (1), the R₁、R₂、R₃、R₄And R₅Each as defined in any one of the first aspect of the invention. In a preferred embodiment of the compound of formula 1d, R₁Is hydroxy, R₂Is allyl, R₃、R₄And R₅Is a hydrogen atom. In a preferred embodiment of the compound of formula 1d, R₅Is hydroxy, R₄Is allyl, R₁、R₂And R₃Is a hydrogen atom.

In a second aspect, the present invention provides a process for the preparation of a compound of formula II according to the first aspect of the invention, comprising the steps of:

(1) reacting hydrogen chloride and ethanol in an organic solvent under anhydrous conditions with 3-bromoacetonitrile, 4-bromobutyronitrile or 5-bromovaleronitrile to produce a compound of formula IIa;

(2) reacting hydrazine hydrate with the following IIb compound in a suitable organic solvent,

to form the following compound of formula IIc:

(3) reacting a compound of the formula IIa with a compound of the formula IIc in the presence of a base in a suitable organic solvent to form a compound of the formula IId

(4) Reacting the compound IId obtained in step (3) with a compound of the following formula IIe in a suitable organic solvent in the presence of a base,

to produce the following compounds shown in II: :

wherein each symbol is as defined in any one of the first aspect of the invention.

The method according to the second aspect of the present invention, wherein the steps (1) and (2) are performed in a solvent, and the solvent may be an organic solvent including, but not limited to, diethyl ether, dichloromethane, toluene, benzene, ethanol, methanol, or a mixture of any two or more thereof.

The method according to any one of the second aspect of the present invention, wherein the base used in the steps (3) and (4) includes, but is not limited to, triethylamine, potassium carbonate.

The process according to any one of the second aspect of the invention, wherein the compound of formula lib of step (2)

In (1), the R₁、R₂、R₃、R₄And R₅Each as defined in any one of the first aspect of the invention. In a preferred embodiment of the compounds of formula IIb, R₁Is hydroxy, R₂Is allyl, R₃、R₄And R₅Is a hydrogen atom. In a preferred embodiment of the compound of formula 1d, R₅Is hydroxy, R₄Is allyl, R₁、R₂And R₃Is a hydrogen atom.

In a second aspect, the present invention provides a process for the preparation of a compound of formula III according to the first aspect of the invention, comprising the steps of:

(1) reacting hydrogen chloride and ethanol in an organic solvent under anhydrous conditions with 2-chloroacetonitrile, 3-chloropropionitrile or 4-chlorobutyronitrile to generate a compound shown in the following formula IIIa;

(2) reacting hydrazine hydrate with the following IIIb compound in a suitable organic solvent,

to produce the following compound of formula IIIc:

(3) reacting a compound of formula IIIa with a compound of formula IIIc in the presence of a base in a suitable organic solvent to form a compound of formula IIId

(4) Reacting the compound IIId obtained in step (3) with a compound of formula IIIe below in a suitable organic solvent in the presence of a base,

to produce the following compounds of formula III: :

wherein each symbol is as defined in any one of the first aspect of the invention.

The method according to the second aspect of the present invention, wherein the steps (1) and (2) are performed in a solvent, and the solvent may be an organic solvent including, but not limited to, diethyl ether, dichloromethane, toluene, benzene, ethanol, methanol, or a mixture of any two or more thereof.

The method according to any one of the second aspect of the present invention, wherein the base used in the steps (3) and (4) includes, but is not limited to, triethylamine, potassium carbonate.

The process according to any one of the second aspect of the invention, wherein the compound of formula IIIb of step (2)

In (1), the R₁、R₂、R₃、R₄And R₅Each as defined in any one of the first aspect of the invention. In a preferred embodiment of the compounds of formula IIb, R₁Is hydroxy, R₂Is allyl, R₃、R₄And R₅Is a hydrogen atom. In a preferred embodiment of the compound of formula 1d, R₅Is hydroxy, R₄Is allyl, R₁、R₂And R₃Is a hydrogen atom.

The method according to any one of the second aspect of the invention, wherein the compound of formula I is: 3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenebenzene) hydrazine, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof.

In one embodiment of the process according to the second aspect of the present invention, the compound of formula I is synthesized as follows:

in one embodiment of the process according to the second aspect of the present invention, the compound of formula II is synthesized as follows:

in one embodiment of the process according to the second aspect of the present invention, the compound of formula III is synthesized as follows:

the process according to the second aspect of the present invention, wherein each of the starting materials or intermediates (e.g. the compound of formula Ia) is commercially available or can be synthesized by a person skilled in the art according to the prior knowledge.

In a third aspect, the present invention provides the use of a compound according to any one of the first aspect of the present invention in the manufacture of a medicament for the treatment and/or prophylaxis of tumours and/or cancers.

The use according to the third aspect of the present invention, wherein said tumor and/or cancer may be any tumor and/or cancer known medically. Preferably, the tumors and/or cancers include, but are not limited to:

malignancies including, but not limited to, bladder cancer, breast cancer, colon cancer, renal cancer, liver cancer, lung cancer (including small cell lung, non-small cell carcinoma), head and neck cancer, esophageal cancer, gallbladder cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma);

hematopoietic tumors of the lymphatic system including, but not limited to, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, and Brukett's lymphoma;

hematopoietic tumors of the myeloid system including, but not limited to, acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia;

tumors of mesenchymal origin, including but not limited to fibrosarcoma and rhabdomyosarcoma;

tumors of central origin, including but not limited to fibrosarcoma and rhabdosarcoma;

tumors of the central and peripheral nervous system, including astrocytomas, fibroblastic neuromas, gliomas and schwannomas; and

other tumors, including but not limited to melanoma, seminoma, teratocarcinoma, osteosarcoma, erythropolima (xenoderoma pimentosum), thyroid follicular cancer, and kaposi's sarcoma.

In a fourth aspect, the present invention provides a method of treating and/or preventing a tumour and/or cancer in a subject in need thereof, which method comprises administering to said subject a therapeutically and/or prophylactically effective amount of a compound according to any one of the first aspects of the present invention.

The method according to the fourth aspect of the present invention, wherein said tumor and/or cancer may be any tumor and/or cancer known medically. Preferably, the tumors and/or cancers include, but are not limited to:

malignancies including, but not limited to, bladder cancer, breast cancer, colon cancer, renal cancer, liver cancer, lung cancer (including small cell lung, non-small cell carcinoma), head and neck cancer, esophageal cancer, gallbladder cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma);

hematopoietic tumors of the lymphatic system including, but not limited to, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, and Brukett's lymphoma;

hematopoietic tumors of the myeloid system including, but not limited to, acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia;

tumors of mesenchymal origin, including but not limited to fibrosarcoma and rhabdomyosarcoma;

tumors of central origin, including but not limited to fibrosarcoma and rhabdosarcoma;

tumors of the central and peripheral nervous system, including astrocytomas, fibroblastic neuromas, gliomas and schwannomas; and

other tumors, including but not limited to melanoma, seminoma, teratocarcinoma, osteosarcoma, erythropolima (xenoderoma pimentosum), thyroid follicular cancer, and kaposi's sarcoma.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound according to any one of the first aspect of the present invention and optionally a pharmaceutically acceptable diluent, carrier, excipient, adjuvant or vehicle.

The pharmaceutical composition according to the fifth aspect of the present invention may be used for the treatment and/or prevention of tumors and/or cancers. The pharmaceutical composition according to the fifth aspect of the present invention, wherein the tumor and/or cancer may be any tumor and/or cancer known medically. Preferably, the tumors and/or cancers include, but are not limited to:

malignancies including, but not limited to, bladder cancer, breast cancer, colon cancer, renal cancer, liver cancer, lung cancer (including small cell lung, non-small cell carcinoma), head and neck cancer, esophageal cancer, gallbladder cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer (including squamous cell carcinoma);

hematopoietic tumors of the lymphatic system including, but not limited to, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, and Brukett's lymphoma;

hematopoietic tumors of the myeloid system including, but not limited to, acute and chronic myelogenous leukemias, myelodysplastic syndrome, and promyelocytic leukemia;

tumors of mesenchymal origin, including but not limited to fibrosarcoma and rhabdomyosarcoma;

tumors of central origin, including but not limited to fibrosarcoma and rhabdosarcoma;

tumors of the central and peripheral nervous system, including astrocytomas, fibroblastic neuromas, gliomas and schwannomas; and

other tumors, including but not limited to melanoma, seminoma, teratocarcinoma, osteosarcoma, erythropolima (xenoderoma pimentosum), thyroid follicular cancer, and kaposi's sarcoma.

Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

As used herein, for example in the definition of Ar (or

) As used in the context of the indicated moiety, the term "substituted aryl" such as "substituted phenyl" and the term "substituted heteroaryl" such as "substituted heteroaryl-containing" are intended to include substituents selected from the group consisting of: hydroxy, nitro, cyano, mercapto, amino, carboxyl, sulfonamido, halogens such as fluorine, chlorine, bromine, straight or branched C₁-C₆Alkyl groups such as, but not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, straight or branched C₂-C₆Alkenyl groups such as but not limited to vinyl, propenyl, allyl, 2-butynyl.

The term "alkyl" as used herein refers to straight and branched chain saturated hydrocarbon radicals containing the specified number of carbon atoms, typically exemplified by methyl, ethyl, and straight chain propyl, butyl, pentyl, hexyl and the like. The term "alkyl" also includes cycloalkyl, i.e., cyclic C₃-C₆Hydrocarbon radicals, e.g. cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl group. Preferably, the term "alkyl" as used herein refers to straight and branched chain alkyl groups including the specified number of carbon atoms.

The term "alkenyl" as used herein refers to straight and branched chain alkenyl groups containing the specified number of carbon atoms, typically such as vinyl, allyl, propenyl, and straight and branched butenyl, pentenyl, hexenyl and the like containing one or more double bonds and the double bonds being in any feasible position.

The term "alkoxy" as used herein, alone or in combination, refers to an alkyl ether group, wherein the term "alkyl" is as defined above. Examples of suitable alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

The term "halogen" is defined herein to include fluorine, chlorine, bromine or iodine, and may also include isotopes thereof.

The various starting materials for the reaction are either prepared by the skilled worker on the basis of their knowledge, or can be prepared by methods known from the literature, or are commercially available. The intermediates, starting materials, reagents, reaction conditions, etc. used in the above reaction schemes may be appropriately modified according to the knowledge of those skilled in the art. Alternatively, one skilled in the art can prepare other compounds of formula I, formula II and formula III not covered by the process of the present invention based on the teachings of the present invention.

The compounds of the formulae I, II and III according to the invention may exist in stereoisomeric forms. The present invention includes all possible stereoisomers, i.e. cis or trans single stereoisomers, or mixtures of the two in any desired ratio. The present invention contemplates all such isomers (e.g., enantiomers and diastereomers) in purified and mixed forms, including racemic mixtures. The enol form is also included within the scope of the invention.

The compounds of the formulae I, II and III according to the invention can be used both as such and in the form of their pharmaceutically acceptable salts or solvates. Pharmaceutically acceptable salts of the compounds of formula I, formula II and formula III include conventional salts formed with pharmaceutically acceptable inorganic or organic acids or bases. Examples of suitable acid addition salts include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid, and the like. Pharmaceutically acceptable salts include inorganic or organic acid salts thereof, including but not limited to: hydroiodide, bisulfate, biphosphate, butyrate, oxalate, pivalate, adipate, alginate, picrate, aspartate, gluconate, ethanesulfonate, p-toluenesulfonate, pamoate, pyruvate, glycolate, trifluoroacetate, p-aminosalicylate, pamoate, ascorbate and the like. Examples of suitable base addition salts include salts with sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, and the like. Reference herein to the compounds of the invention includes compounds of formula I, formula II and formula III and pharmaceutically acceptable salts or solvates thereof. The free base forms of the compounds of the invention differ slightly from their respective salt forms in certain physical properties (e.g., solubility in polar solvents), but for purposes of the present invention, each acid salt is equivalent to their respective free base forms. See, e.g., s.m. berge, et al, "pharmaceutical salts," j.pharm. sci, 66: 1-19(1977), which is incorporated herein by reference.

The term "composition" as used herein is intended to encompass a product comprising the specified amounts of each of the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified amounts of each of the specified ingredients, the meaning of "pharmaceutical composition" being similarly understood by those skilled in the art in view of this explanation, and "composition" and "pharmaceutical composition" in certain instances may be used interchangeably. Depending on the mode of administration, the compositions of the invention may contain 0.1% by weight, or more suitably 10-60% by weight, of the active ingredient. However, where the components comprise unit doses, each unit preferably contains from 1 to 500 mg of active ingredient.

The compounds of the invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. The phrase "pharmaceutically acceptable salt" refers to salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, s.m.berge, et al, "Pharmaceutical Salts," j.pharm.sci., 66: 1-19(1977), wherein pharmaceutically acceptable salts are described in detail. The salts may be prepared in situ or separately during the final isolation and purification of the compounds of the invention by reacting the free base functionality of the compounds of the invention with a suitable organic acid. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, isothionate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Likewise, basic nitrogen-containing groups can be quaternized with; lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long chain halides such as decyl, dodecyl, tetradecyl and octadecyl chlorides, bromides and iodides; arylalkyl halides such as benzyl bromide and phenethyl bromide and others. Thus obtaining a product that is soluble or dispersible in water or oil. Examples of acids which may be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acids, and organic acids such as oxalic, maleic, succinic and citric acids.

Base addition salts can be prepared in situ during the final isolation and purification of the compounds of the invention by reacting the carboxylic acid-containing moiety of the compounds of the invention with a suitable base, such as the hydroxide, carbonate and bicarbonate salts of a pharmaceutically acceptable metal cation, or ammonia or an organic primary, secondary or tertiary amine.

Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, as well as non-toxic quaternary amines and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, ethylammonium, and the like. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

The compounds of formula I, formula II and formula III of the present invention, or pharmaceutically acceptable salts thereof, may also form solvates, such as hydrates, alcoholates and the like. The compounds may also be prodrugs or forms which release the active ingredient upon metabolic change in vivo. The selection and preparation of appropriate pro-derivatives is well known to those skilled in the art. In general, for the purposes of the present invention, the solvate forms with pharmaceutically acceptable solvents such as water, ethanol, etc. are equivalent to the non-solvate forms.

Dosage forms for topical administration of the compounds of the present invention include powders, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants. Ophthalmic formulations, ophthalmic ointments, powders, and solutions are also contemplated as being within the scope of the present invention.

When used in the above treatments or other treatments, a therapeutically effective amount of one of the compounds of the present invention may be used in pure form, or in the form of a pharmaceutically acceptable salt, ester or prodrug, where such forms are present. Alternatively, the compounds may be administered in a pharmaceutical composition comprising the compound of interest together with one or more pharmaceutically acceptable excipients. The phrase "therapeutically effective amount" of a compound of the present invention refers to a sufficient amount of the compound to treat a disorder at a reasonable benefit/risk ratio applicable to any medical treatment. It will be appreciated, however, that the total daily amount of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dose level will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the particular compound employed; the duration of treatment; drugs used in combination or concomitantly with the specific compound employed; and similar factors known in the medical arts. For example, it is common in the art to start doses of the compound at levels below those required to achieve the desired therapeutic effect and to gradually increase the dose until the desired effect is achieved. In general, the dosage of the compounds of the invention for use in mammals, particularly humans, may be in the range of 0.0001 to 1000mg/kg body weight/day, such as 0.001 to 100mg/kg body weight/day, such as 0.01 to 10mg/kg body weight/day.

The inventors have found that the novel compounds of formula I, formula II and formula III provided by the present invention have potent anti-tumor and/or anti-cancer activity. On this basis, the present invention provides a method of treating and/or preventing tumors and/or cancers in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I, formula II and formula III according to any one of the first aspects of the present invention, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof. The term "subject" refers to an animal, preferably e.g. a vertebrate, more preferably e.g. a mammal, still more preferably in particular e.g. a human, which has or will have or may have had a tumor and/or cancer as described herein. The term "therapeutically effective amount" is a dose which, upon administration to the subject, produces a desired physiological response, particularly a physiological response associated with the tumor and/or cancer of the present invention.

The present invention also provides pharmaceutical compositions comprising a compound of the invention, optionally formulated together with one or more non-toxic pharmaceutically acceptable diluents, carriers, excipients, adjuvants or vehicles. The pharmaceutical compositions may be specifically formulated for oral administration, for parenteral injection or for rectal administration in solid or liquid form.

The pharmaceutical compositions of the present invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, or drops), bucally to humans and other mammals, or as an oral or nasal spray. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In another aspect, the present invention provides a pharmaceutical composition comprising an ingredient of the invention and a physiologically tolerable diluent. Included in the invention are one or more of the above compounds formulated in compositions for parenteral injection, intranasal delivery, oral administration in solid or liquid form, rectal or topical administration, and the like, together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles, which are collectively referred to herein as diluents.

Compositions suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous or nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, and suitable mixtures thereof.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the action of microorganisms can be determined by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of substances delaying absorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, or mixtures of these substances, and the like.

In some cases, to prolong the duration of action of a drug, it is desirable to slow the absorption of the drug, either subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. Thus, the rate of absorption of the drug is dependent on its rate of dissolution, which in turn may be dependent on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot formulations may be prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer employed. Examples of other biodegradable polymers also include polyorthoesters (poly (orthoesters)) and polyanhydrides (polyanhydrides)). Injectable depot formulations may also be prepared by embedding the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable agents can be sterilized, for example, by filtration through a bacterial filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silica gel; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; c) insulating agents such as glycerin; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type, using excipients such as lactose and high molecular weight polyethylene glycols and the like, can also be used as fillers in soft and hard capsules.

Solid dosage forms of tablets, dragees (dragees), capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known for pharmaceutical preparations. These solid dosage forms may optionally contain opacifying agents and may be of a composition such that they release the active ingredient(s) only, or preferentially, at a site in the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. If appropriate, the active compounds can also be formulated in microencapsulated form with one or more of the abovementioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms may contain, in addition to the active compound, inert diluents commonly used in the art such as, for example, water or other solutions, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl ester, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of sorbitol with esters of fatty acids and esters of fatty acids of glue and mixtures thereof. Oral compositions may contain, in addition to inert diluents, adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Compositions for rectal or vaginal administration are preferably suppositories. Suppositories can be prepared by mixing the compounds of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at room temperature but liquid at body temperature and therefore melt rectally or vaginally to release the active compound.

The compounds of the invention may also be administered in the form of liposomes. As is well known in the art, liposomes are typically made from phospholipids or other lipid materials. Liposomes are formed from single or multiple layers of aqueous liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The compositions of the present invention in liposome form may contain, in addition to the compound of the present invention, stabilizers, preservatives, excipients and the like. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholines (lecithins), which may be used alone or together. Methods of forming liposomes are well known in the art. See, e.g., Prescott, ed., Methods in Cell Biology, Volume XIV, academic press, New York, n.y. (1976), p.33.

The term "pharmaceutically acceptable pro-salt" as used herein, represents a prodrug of a compound of the present invention which is, within the scope of sound medical judgment, suitable for use in tissue contact with humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for its intended use, and also represents, where possible, the zwitterionic form of the compound of the present invention. Prodrugs of the invention may be rapidly converted in vivo to the parent compound described above, for example, by hydrolysis in blood. A full discussion is provided in T.Higuchi and V.Stella, Pro-drugs as Novel delivery systems, V.14 of the A.C.S.Symphosium Series and Edward B.Roche, ed., Bioreversible Carriers in Drug Design, American pharmaceutical Association and Pergamon Press (1987), which are incorporated herein by reference.

The compounds of formula I, formula II and formula III of the present invention, and of course the pharmaceutical compositions formed therefrom, are useful in the treatment of tumors and/or cancers.

The inventor conducts research on the anti-tumor/anti-cancer activity test on the novel compounds I, II and III, and the result shows that the compounds I, II and III can selectively kill some cancer cells, have strong anti-cancer activity and can inhibit the growth of tumors.

Drawings

FIG. 1 is a graph showing the effect of SM-3 (code for the compound of example 1 of the present invention) on Hela cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, SM-3 (1. mu.M), SM-3 (10. mu.M), SM-3 (20. mu.M), SM-3 (40. mu.M), SM-3 (60. mu.M), SM-3 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). The experimental result shows that SM-3 has obvious inhibition effect on the growth of Hela cells.

FIG. 2 is a graph of the effect of SM-3 on HPF cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, SM-3 (1. mu.M), SM-3 (10. mu.M), SM-3 (20. mu.M), SM-3 (40. mu.M), SM-3 (60. mu.M), SM-3 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). Experimental results show that SM-3 has obvious inhibition effect on HPF cell growth.

FIG. 3 is the effect of SM-3 on the survival of A549 cells. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, SM-3 (1. mu.M), SM-3 (10. mu.M), SM-3 (20. mu.M), SM-3 (40. mu.M), SM-3 (60. mu.M), SM-3 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). The experimental result shows that SM-3 has obvious inhibition effect on the growth of A549 cells.

FIG. 4 is a graph of the effect of SM-3 on HepG-2 cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, SM-3 (1. mu.M), SM-3 (10. mu.M), SM-3 (20. mu.M), SM-3 (40. mu.M), SM-3 (60. mu.M), SM-3 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). Experimental results show that SM-3 has obvious inhibition effect on the growth of HepG-2 cells.

FIG. 5 is a graph showing the effect of SM-3 on survival of human peripheral blood mononuclear cells. The abscissa in the figure is the processing time, and the processing time is 24h and 48h, respectively, each time point has seven columns, which are respectively represented from left to right: blank control, DMSO control, SM-3 (10. mu.g/ml), SM-3 (20. mu.g/ml), SM-3 (40. mu.g/ml), SM-3 (80. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). The experimental result shows that SM-3 has small inhibition effect on the growth of the mononuclear cells.

FIG. 6 is a graph showing the effect of PAC-1 on Hela cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, PAC-1 (1. mu.M), PAC-1 (10. mu.M), PAC-1 (20. mu.M), PAC-1 (40. mu.M), PAC-1 (60. mu.M), PAC-1 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). The experimental result shows that PAC-1 has obvious inhibition effect on the growth of Hela cells.

FIG. 7 is a graph showing the effect of PAC-1 on HPF cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, PAC-1 (1. mu.M), PAC-1 (10. mu.M), PAC-1 (20. mu.M), PAC-1 (40. mu.M), PAC-1 (60. mu.M), PAC-1 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). The experimental result shows that PAC-1 has no obvious inhibition effect on the growth of HPF cells after 24 hours of action, but the effect is obviously enhanced after 48 hours. .

FIG. 8 is the effect of PAC-1 on the survival of A549 cells. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, PAC-1 (1. mu.M), PAC-1 (10. mu.M), PAC-1 (20. mu.M), PAC-1 (40. mu.M), PAC-1 (60. mu.M), PAC-1 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). Experimental results show that PAC-1 has obvious inhibition effect on the growth of A549 cells.

FIG. 9 is a graph of the effect of PAC-1 on HepG-2 cell survival. The abscissa in the figure is the processing time, and the processing time is three time points, namely 24h, 48h and 72h, each time point has ten columns, which are respectively represented from left to right: blank control, DMSO control, PAC-1 (1. mu.M), PAC-1 (10. mu.M), PAC-1 (20. mu.M), PAC-1 (40. mu.M), PAC-1 (60. mu.M), PAC-1 (80. mu.M), cisplatin (5. mu.g/ml), cisplatin (10. mu.g/ml). The ordinate represents the cell survival rate (%). Experimental results show that PAC-1 has obvious inhibition effect on the growth of HepG-2 cells.

In each of the above figures, at each test time point, a plurality of bars corresponding from left to right correspond to corresponding icons on the right side of the coordinate graph from top to bottom respectively. For example, in FIG. 1, 10 bars at 24 hours, from left to right, correspond to the top to bottom right of the graph (i.e., 10 icons from blank (0M) to cisplatin (10 ug/ml)).

Detailed Description

The present invention is further illustrated by the following specific examples and/or experimental examples, but it should be understood that these examples and/or experimental examples are only for the purpose of more detailed explanation and are not to be construed as limiting the present invention in any way.

The present invention generally and/or specifically describes the materials used in the tests and the test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well known in the art, unless otherwise specified.

Example 1, 3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenebenzene) hydrazine Preparation of

The reaction process is as follows:

and (1).To a reaction flask containing 1.76g (10mmol) of 1-benzylpiperazine, 1.66g (12mmol) of potassium carbonate and 10ml of acetone was added dropwise 2.17g (12mmol) of ethyl 3-bromopropionate at room temperature with electromagnetic stirring. After the dropwise addition, stirring is carried out for 5min at room temperature, then the temperature is raised to 60 ℃, stirring is carried out for 5 hours at constant temperature, after the reaction is finished, filtration is carried out, a filter cake is washed by a small amount of acetone, filtrates are combined, and concentration is carried out to obtain 2g of orange liquid, wherein the yield is 72.5%. Mass spectrum: 277(M + 1).

And 2. step 2.5ml of ethanol and 2g (7.24mmol) of ethyl 3- (4-benzyl-piperazin-1-yl) -propionate were added to a reaction flask under electromagnetic stirring at room temperature, then the temperature was raised to 70 ℃, 1.28g (21.74mmol) of 85% hydrazine hydrate was slowly added dropwise, the reaction was refluxed for 6 hours, and the reaction was stopped after completion of the detection reaction. The reaction mixture was concentrated, 5ml of water was added thereto, and extraction was performed twice with methylene chloride. The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated to give 1.36g of a pale yellow oil in 66.3% yield. Mass spectrum: 285(M + 23).

And 3. step 3.5ml of ethanol and 1.31g (5mmol) of 3- (4-benzyl-piperazin-1-yl) -propionylhydrazide are added to a reaction flask with electromagnetic stirring at room temperature, and after stirring, 0.81g (5mmol) of 2-hydroxy-3-allylbenzaldehyde is slowly added dropwise. After the reaction is carried out for 20 minutes, a solid is continuously precipitated, and the reaction is stopped after the detection reaction is finished. The solid was collected by filtration and the resulting product was recrystallized biphasically from ethyl acetate and n-hexane to give 1.45g of the product 3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenephenyl) hydrazine (SM-3) in 71.4% yield, m.p. 132-. Hydrogen spectrum (400MHz, DMSO): 11.88(s, 1H); 11.76(s, 1H); 8.28(s, 1H); 7.1-7.33(m, 7H); 6.84-6.9(m, 1H); 6.00(m, 1H); 5.03(m, 2H); 3.44(s, 2H); 3.35(d, 2H); 2.4-2.65(m, 12H). Mass spectrum: 407(M + 1).

Example 2, (4-benzyl-piperazin-1-yl) -butyryl (3-allyl-2-hydroxy-methylenebenzene) hydrazine Preparation of

The reaction process is as follows:

and (1).To a reaction flask containing 1.76g (10mmol) of 1-benzylpiperazine, 1.66g (12mmol) of potassium carbonate and 10ml of acetone was added dropwise 2.34g at room temperature with electromagnetic stirring(12mmol) of ethyl 4-bromobutyrate. After the dropwise addition, stirring is carried out for 5min at room temperature, then the temperature is raised to 60 ℃, stirring is carried out for 5 hours at constant temperature, after the reaction is finished, filtration is carried out, a filter cake is washed by a small amount of acetone, filtrates are combined, and the orange liquid is obtained by concentration, wherein the yield is 110%. Mass spectrum: 291(M + 1).

And 2. step 2.At room temperature, under the condition of electromagnetic stirring, 5ml of ethanol and 3.2g (10mmol theoretically) of the product obtained in the first step, namely 4- (4-benzyl-piperazine-1-yl) -ethyl butyrate, are added into a reaction bottle, then the temperature is raised to 70 ℃, 1.76g (30mmol) of 85% hydrazine hydrate is slowly and dropwise added, the reflux reaction is carried out for 6 hours, and the reaction is stopped after the detection reaction is finished. The reaction mixture was concentrated, 5ml of water was added thereto, and extraction was performed twice with methylene chloride. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to give 2.4g of a pale yellow oil in 87% yield. Mass spectrum: 277(M + 1).

And 3. step 3.5ml of ethanol and 2g (7.24mmol) of 4- (4-benzyl-piperazin-1-yl) -butyrylhydrazine were added to a reaction flask at room temperature with electromagnetic stirring, the temperature was raised to 70 ℃ and 1.17g (7.24mmol) of 2-hydroxy-3-allylbenzaldehyde was slowly added dropwise. The reaction was carried out at constant temperature for 1 hour, and the reaction was stopped after the completion of the detection reaction. The reaction solution was concentrated, and the obtained product was subjected to two-phase recrystallization using ethyl acetate and n-hexane to obtain 1.96g of 4- (4-benzyl-piperazin-1-yl) -butyryl (3-allyl-2-hydroxy-methylenebenzene) hydrazine, yield 66.58%. Hydrogen spectrum (400MHz, DMSO): 11.93(s, 1H); 11.65(s, 1H); 8.35(s, 1H); 7.1-7.33(m, 7H); 6.84-6.9(m, 1H); 5.95(m, 1H); 5.03(m, 2H); 3.4(s, 2H); 3.3(d, 2H); 2.4-2.65(m, 12H); 1.86(m, 2H). Mass spectrum: 421(M + 1).

Example 3 preparation of 3- (4-benzyl-piperidin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine

The reaction process is as follows:

and (1).To a reaction flask containing 1.76g (10mmol) of 1-benzylpiperazine, 1.66g (12mmol) of potassium carbonate and 10ml of acetone was added dropwise 2.17g (12mmol) of ethyl 3-bromopropionate at room temperature with electromagnetic stirring. After the dropwise addition, stirring is carried out for 5min at room temperature, then the temperature is raised to 60 ℃, stirring is carried out for 5 hours at constant temperature, after the reaction is finished, filtration is carried out, a filter cake is washed by a small amount of acetone, filtrates are combined, and concentration is carried out to obtain orange liquid 2.5g, and the yield is 91%. Mass spectrum: 276(M + 1).

And 2. step 2.5ml of ethanol and 2.5g (9.1mmol) of ethyl 3- (4-benzyl-piperidin-1-yl) -propionate were added to a reaction flask under electromagnetic stirring at room temperature, then the temperature was raised to 70 ℃ and 1.6g (27.3mmol) of 85% hydrazine hydrate was slowly added dropwise, the reaction was refluxed for 6 hours, and the reaction was stopped after completion of the detection reaction. The reaction mixture was concentrated, 5ml of water was added thereto, and extraction was performed twice with methylene chloride. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to give 1.85g of a pale yellow oil in 78% yield. Mass spectrum: 262(M + 1).

And 3. step 3.5ml of ethanol and 1.31g (5mmol) of 3- (4-benzyl-piperidin-1-yl) -propionylhydrazide are added to a reaction flask under electromagnetic stirring at room temperature, and stirred uniformly, and then 0.61g (5mmol) of 2-hydroxybenzaldehyde is slowly added dropwise. After the reaction is carried out for 20 minutes, a solid is continuously precipitated, and the reaction is stopped after the detection reaction is finished. The solid was collected by filtration and the resulting product was recrystallized diphasic from ethyl acetate and n-hexane to give 1.4g of the product 3- (4-benzyl-piperidin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine in 76.9% yield. Hydrogen spectrum (400MHz, DMSO): 11.82(s, 1H); 11.34(s, 1H); 8.31(s, 1H); 7.1-7.33(m, 8H); 3.4(s, 2H); 3.3(d, 2H); 2.4-2.65(m, 12H). Mass spectrum: 366(M +1)

Example 4, 3- (4-p-toluenesulfonyl-piperazin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine Preparation of

The reaction process is as follows:

and (1).Under ice bath and electromagnetic stirring, 1.9g (10mmol) of p-toluenesulfonyl chloride was added dropwise to a reaction flask containing 6g (60mmol) of anhydrous piperazine and 10ml of tetrahydrofuran. And after the dropwise addition is finished within half an hour, the reaction is allowed to naturally warm to room temperature, stirred and reacted overnight, after the reaction is finished, the filtration is carried out, a filter cake is washed by a small amount of tetrahydrofuran, the filtrates are combined, and a small amount of liquid is obtained by concentration. To the residue was added 20ml of water, extracted with ethyl acetate, and dried over anhydrous magnesium sulfate. Concentration and separation of the residual liquid by silica gel column gave 2g of 1-p-toluenesulfonylpiperazine as the product in 83.3% yield. Mass spectrum: 241(M + 1).

And 2. step 2.To a reaction flask containing 2g (8.3mmol) of 1-p-toluenesulfonylpiperazine, 1.38g (10mmol) of potassium carbonate and 10ml of acetone was added dropwise 1.81g (10mmol) of ethyl 3-bromopropionate at room temperature with electromagnetic stirring. After the dropwise addition, stirring is carried out for 5min at room temperature, then the temperature is raised to 60 ℃, stirring is carried out for 5 hours at constant temperature, after the reaction is finished, filtration is carried out, a filter cake is washed by a small amount of acetone, filtrates are combined, and concentration is carried out to obtain orange liquid 2.5g, and the yield is 88%. Mass spectrum: 341(M + 1).

And 3. step 3.5ml of ethanol and 2.5g (7.35mmol) of ethyl 3- (4-p-toluenesulfonyl-piperazin-1-yl) -propionate were added to a reaction flask under electromagnetic stirring at room temperature, the temperature was then raised to 70 ℃, 1.30g (22.1mmol) of 85% hydrazine hydrate was slowly added dropwise, the reaction was refluxed for 6 hours, and the reaction was stopped after completion of the detection reaction. The reaction mixture was concentrated, 5ml of water was added thereto, and extraction was performed twice with methylene chloride. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to give 2g of a pale yellow oil in 83% yield. Mass spectrum: 327(M + 1).

And 4. step 4.Adding 5ml of ethanol and 2g (6.1mmol) of 3- (4-p-toluenesulfonyl-piperazin-1-yl) -propionylhydrazide into a reaction flask at room temperature under the condition of electromagnetic stirring, stirring uniformly, heating to 70 ℃, and then slowly adding0.74g (6mmol) of 2-hydroxybenzaldehyde was slowly added dropwise. After the completion of the dropping, the reaction was carried out at a constant temperature for 1 hour, and after the completion of the detection reaction, the reaction was stopped. The reaction solution was concentrated, and the obtained product was recrystallized with ethyl acetate and n-hexane in two phases to obtain 2.05g of the product 3- (4-p-toluenesulfonyl-piperazin-1-yl) -propionyl (2-hydroxy-methylenebenzene) hydrazine, in a yield of 77%. Hydrogen spectrum (400MHz, DMSO): 11.82(s, 1H); 11.34(s, 1H); 8.31(s, 1H); 7.1-7.33(m, 8H); 3.3(d, 2H); 2.4-2.65(m, 12H). Mass spectrum: 431(M + 1).

Example 5, 3- (4-benzyl-piperazin-1-yl) -propieimide (3-allyl-2-hydroxy-methylene) Benzene) hydrazine preparation

The reaction process is as follows:

(1) and 3-bromopropionyl ethylidene hydrochloride:

13.4g (100mmol) of 3-bromopropionitrile and 4.6g (100mmol) of absolute ethanol were dissolved in 100ml of absolute ethyl ether under magnetic stirring in an ice bath, and dried hydrogen chloride gas was introduced into the reaction solution, and after about 40 minutes, a large amount of white solid was precipitated, and the solid was filtered off, washed three times with absolute ethyl ether, and then dried by suction, to obtain 17.6g of 3-bromopropionylacetylidene hydrochloride in a yield of 81.5%. Hydrogen spectrum (400MHz, DMSO): 4.45(t, 2H); 4.28(q, 2H); 3.5(t, 2H); 1.24(t, 3H).

(2) And 2-allyl-6- (N-amino imino methyl) phenol preparation:

15ml of methanol, 14.7g (250mmol) of an 85% hydrazine hydrate solution are added to the reaction flask at room temperature under magnetic stirring, and 8.1g (50mmol) of 2-hydroxy-3-allylbenzaldehyde are added dropwise, the solution becoming yellow. After the dripping is finished, the reaction is stirred for 10 min. After completion of the reaction, methanol was removed under reduced pressure. To the residue were added DCM (20ml) and water (40 ml). Stirring and standing. The organic layer was then separated, washed twice with water, and finally dried over anhydrous magnesium sulfate (4g) for 0.5 hour, filtered, and the filtrate was concentrated to give 7.9g of 2-allyl-6- (N-aminoiminomethyl) phenol as a yellow oil in 90% yield. Mass spectrum: 177.1(M + 1).

(3) And 3-bromopropylimido (N-amino imino methyl-3-hydroxybenzene) hydrazine preparation:

10.8g (50mmol) of 3-bromopropionylidene ethyl ester hydrochloride are added to the reaction flask at room temperature with magnetic stirring, the rubber stopper is sealed and 70ml of absolute ethanol are injected. 6.06g (60mmol) of dried triethylamine was then injected and the reaction stirred for 10min to dissolve the solid gradually, then 8.8g (50mmol) of 2-allyl-6- (N-aminoiminomethyl) phenol was added and the reaction stirred overnight. After the reaction was completed, ethanol was removed under reduced pressure. Then, the mixture was dissolved in DCM, and an appropriate amount of water was added thereto to separate a DCM layer, which was dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column separation to obtain 11g of 3-bromopropylimidoyl (N-iminomethyl-3-hydroxyphenyl) hydrazine in a yield of 71%. Mass spectrum: 311.2(M + 1).

(4) Preparation of 3- (4-benzyl-piperazin-1-yl) -propieimide (3-allyl-2-hydroxy-methylenebenzene) hydrazine:

7.93g (25mmol) of 3-bromopropylimidoyl (N-aminoiminomethyl-3-hydroxyphenyl) hydrazine, 20ml of acetone, 2.76g (20mmol) of K were added to a reaction flask at room temperature under magnetic stirring₂CO3 and 3.52g (20mmol) 1-benzylpiperazine. The reaction is stirred for 10min, and then the temperature is raised to 60 ℃ for reflux reaction. About 4-5 hours, after which filtration, the filter cake is washed with acetone and the acetone is evaporated off under reduced pressure. Adding DCM, washing with water, separating DCM layer, and anhydrous MgSO₄And (5) drying. Concentration and silica gel column separation of the residue gave 7.5g of a pale yellow oil in 74.2% yield. Hydrogen spectrum (400MHz, D)₂O): 11.24 (1H); 10.01 (1H); 8.44 (1H); 7.20-7.32 (5H); 7.10 (2H); 6.82 (1H); 5.04-5.07 (2H); 3.46 (4H); 3.19 (2H); 2.63 (8H). Mass spectrum: 406.6(M +1)

Example 6, 2- (4-benzyl-homopiperazin-1-yl) -ethylimido (3-allyl-2-hydroxy-methylene) Benzene) hydrazine preparation

The reaction process is as follows:

(1) preparation of 2-chloroacetoethylene ester hydrochloride:

under ice bath and magnetic stirring, dissolving 10g (130mmol) of chloroacetonitrile and 6.1g (130mmol) of absolute ethyl alcohol in 100ml of anhydrous ethyl ether, introducing dry hydrogen chloride gas into the reaction solution, precipitating a large amount of white solid after about 40 minutes, filtering the solid, washing the solid with the anhydrous ethyl ether for three times, and pumping to dryness to obtain 16g of 2-chloroacetimide ethyl ester hydrochloride with the yield of 98%; melting point 84-86 ℃. Hydrogen spectrum (400MHz, DMSO): 4.40(s, 2H); 4.20(q, 2H); 1.24(t, 3H).

(2) And 2-allyl-6- (N-amino imino methyl) phenol preparation:

15ml of methanol, 14.7g (250mmol) of an 85% hydrazine hydrate solution are added to the reaction flask at room temperature under magnetic stirring, and 8.1g (50mmol) of 2-hydroxy-3-allylbenzaldehyde are added dropwise, the solution becoming yellow. After the dripping is finished, the reaction is stirred for 10 min. After completion of the reaction, methanol was removed under reduced pressure. To the residue were added DCM (20ml) and water (40 ml). Stirring and standing. The organic layer was then separated, washed twice with water, and finally dried over anhydrous magnesium sulfate (4g) for 0.5 hour, filtered, and the filtrate was concentrated to give 7.9g of 2-allyl-6- (N-aminoiminomethyl) phenol as a yellow oil in 90% yield. Mass spectrum: 177.1(M +1)

(3) And 2-chloroethyl imido methyl-3-hydroxybenzene hydrazine preparation:

7.9g (50mmol) of ethyl 2-chloroacetimide hydrochloride are added to the reaction flask at room temperature with magnetic stirring, the rubber stopper is sealed and 70ml absolute ethanol is injected. 6.06g (60mmol) of dried triethylamine was then injected and the reaction stirred for 10min to dissolve the solid gradually, then 8.8g (50mmol) of 2-allyl-6- (N-aminoiminomethyl) phenol was added and the reaction stirred overnight. After the reaction was completed, ethanol was removed under reduced pressure. Then, the mixture was dissolved in DCM, and an appropriate amount of water was added thereto to separate a DCM layer, which was dried over anhydrous magnesium sulfate, concentrated, and subjected to silica gel column separation to obtain 11g of 2-chloroacetimidoyl (N-iminomethyl-3-hydroxyphenyl) hydrazine in a yield of 87.3%. Mass spectrum: 252.2(M +1)

(4) Preparation of 2- (4-benzyl-homopiperazin-1-yl) -ethylimido (3-allyl-2-hydroxy-methylenebenzene) hydrazine:

5.04g (25mmol) 2-chloroethylimido (N-aminoiminomethyl-3-hydroxyphenyl) hydrazine, 20ml acetone, 2.76g (20mmol) K were added to the flask at room temperature with magnetic stirring₂CO3 and 3.8g (20mmol) of 1-benzylhomopiperazine. The reaction is stirred for 10min, and then the temperature is raised to 60 ℃ for reflux reaction. About 4-5 hours, after which filtration, the filter cake is washed with acetone and the acetone is evaporated off under reduced pressure. Adding DCM, washing with water, separating DCM layer, and anhydrous MgSO₄And (5) drying. Concentration and silica gel column separation of the residue gave 7g of a pale yellow oil in 69.3% yield. Hydrogen spectrum (400MHz, D)₂O): 11.24 (1H); 10.01 (1H); 8.44 (1H); 7.20-7.32 (5H); 7.10 (2H); 6.82 (1H); 5.04-5.07 (2H); 3.46 (2H); 3.19 (2H); 2.63 (8H); 1.41 (2H). Mass spectrum: 406.5(M +1)

Experimental example 1, biological test

In vitro activity screening, an MTT method is adopted to detect the growth inhibition effect of SM-3 and PAC-1 on lung cancer cells A549, breast cancer cells Hela, lung fibroblast HPF, liver cancer cells HepG-2 and human peripheral blood mononuclear cells, and the growth inhibition effect is compared with a PAC-1 medicament for experiments. The specific method comprises the following steps: cells in logarithmic growth phase were taken, counted after conventional digestion, seeded in 96-well plates at a density of 3000/well and grown overnight adherent thereto. Cells were treated with 1, 10, 20, 40, 60, 80 μ M SM-3 (mother liquor in DMSO) and PAC-1, respectively, in duplicate wells set at each concentration, with the highest dose of DMSO as a negative control and 5, 10 μ g/ml cisplatin as a positive control. After 24, 48 and 72 hours of treatment, 10. mu.l of MTT (5mg/ml) was added to each well, and the culture was continued for 2 to 4 hours. And absorbing the culture solution, adding 100 mu l of DMSO into each hole, shaking and dissolving for 5 minutes, detecting the light absorption value at 570nm by using a microplate reader, and calculating the inhibition rate of SM-3 and PAC-1 on cell growth.

In addition, mononuclear cells were isolated from normal human peripheral blood using a lymphocyte separation medium, seeded on a 96-well plate at a density of 5X 104, and grown overnight. Cells were treated with 1, 10, 20, 40, 60, 80 μ M SM-3 (stock in DMSO) at 3 replicate wells per concentration, with the highest dose of DMSO as a negative control and 10 μ g/ml of cisplatin as a positive control. After 24 and 48 hours of treatment, adding 10 mul MTT (5mg/ml) into each hole, continuously culturing for 2-4 hours, centrifuging the whole plate at 1000rpm for 5 minutes, removing the culture solution, adding 100 mul DMSO into each hole, shaking and dissolving for 5 minutes, detecting the light absorption value at 570nm by using a microplate reader, and calculating the inhibition rate of SM-3 on cell growth. PAC-1 was tested in a similar manner.

The experimental results show that the inhibition effect of SM-3 on several cells is positively correlated with the concentration and the action time. After 24 hours of action, the effect of the medicament on HPF cells and A549 cells is not obvious, but the inhibition rate is greatly increased by 48 hours, and is more obvious by 72 hours, but the difference among a plurality of concentrations of 10, 20 and 40 mu M is not large. Compared with the prior art, the SM-3 has stronger inhibiting effect on Hela cells and HepG-2 cells, and has more obvious dose-effect and time-effect relationship, particularly, the inhibiting rate of the Hela cells reaches 77 percent when the concentration is 60 mu M and the inhibiting rate of other cells is only about 40 percent when the concentration is 24 hours. The effect of SM-3 on monocytes was mild, with no significant difference over time, and the dosing concentration was much higher than that of several other cells, with no significant difference between 40. mu.g/ml and 80. mu.g/ml. However, SM-3 inhibited several cells more strongly than PAC-1 and the concentration gradient was more pronounced. PAC-1 has no obvious influence on HPF cells and A549 cells after 24 hours of action, and the effect is gradually obvious along with the prolonging of the action time. The test results are described in detail with reference to fig. 1 to 9 and the accompanying description, respectively.

In addition, the IC50 values for the inhibition rates of SM-3 and PAC-1 on several cells at different time points were calculated, and the results are shown in tables 1 and 2.

Table 1: IC50 values for SM-3 on various cells

Table 2: IC50 values for PAC-1 on various cells

From the results in Table 1, the IC50 values of the inhibition rate of SM-3 on several cells at different time points can be seen, wherein the inhibition rate of HPF cells and A549 cells is lower when the medicine acts for 24 h.

From the results in Table 2, it can be seen that PAC-1 has IC50 values for inhibition rates of several cells at different time points, wherein the inhibition rates of HPF cells, A549 cells and HepG-2 are all low at 24h of drug action.

The inventors have also found that other compounds of the present invention, particularly the compounds of the other several examples, also have the same or similar results as the compound of example 1 above.

Claims (15)

1. A compound of formula I:

wherein,

1) ar is phenyl;

2) x is-CH₂-；

3) Z is a nitrogen atom;

4) n is selected from 1 or 2; and

5)R₁、R₂、R₃、R₄and R₅Each independently selected from hydrogen atom, hydroxyl group, C₂-C₆Alkenyl, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein said C₂-C₆Alkenyl is selected from C₂-C₄An alkenyl group.

3. The compound of claim 2, wherein said C₂-C₄Alkenyl is selected from vinyl, propenyl, allyl.

4. The compound of claim 1 which is the following:

3- (4-benzyl-piperazin-1-yl) -propionyl (3-allyl-2-hydroxy-methylenebenzene) hydrazine

4- (4-benzyl-piperazin-1-yl) -butyryl (3-allyl-2-hydroxy-methylenebenzene) hydrazine

Or a pharmaceutically acceptable salt thereof.

5. A process for the preparation of a compound of formula I as claimed in claim 1, which comprises the steps of:

(1) reacting ethyl 3-bromopropionate or ethyl 4-bromobutyrate with a compound of formula Ia below in the presence of a base,

to form a compound of formula Ib:

(2) reacting hydrazine hydrate with the compound of formula Ib above in an organic solvent to produce a compound of formula Ic:

(3) reacting the compound of formula Ic obtained in step (2) with a compound of formula Id,

to produce the compound represented by the following formula I:

wherein each symbol is as defined in claim 1.

6. Use of a compound according to any one of claims 1 to 4 for the preparation of a medicament for the treatment and/or prophylaxis of tumours.

7. The use of claim 6, wherein the neoplasm is cancer.

8. The use of claim 6, wherein the tumor is selected from the group consisting of:

bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, head and neck cancer, esophageal cancer, gallbladder cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, burkitt's lymphoma, myelodysplastic syndrome, fibrosarcoma, rhabdomyosarcoma, astrocytoma, fibroblastic tumor, glioma, schwannoma, melanoma, seminoma, teratocarcinoma, osteosarcoma, ectoid purple neck tumor, thyroid follicular cancer, and kaposi's sarcoma.

9. The use of claim 8, wherein the lung cancer is small cell lung cancer or non-small cell cancer.

10. The use of claim 8, wherein the skin cancer is squamous cell carcinoma.

11. The use of claim 8, wherein the leukemia is acute lymphocytic leukemia, acute lymphoblastic leukemia, acute and chronic myelogenous leukemia or promyelocytic leukemia.

12. A pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound according to any one of claims 1-4 and optionally a pharmaceutically acceptable adjuvant or vehicle.

13. The pharmaceutical composition of claim 12, wherein said excipient is a carrier.

14. The pharmaceutical composition of claim 13, wherein the carrier is an excipient.

15. The pharmaceutical composition of claim 14, wherein the excipient is a diluent.

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