CN1809572B - Substituted quinobenzoxazine analogs - Google Patents

Abstract

The present invention relates to quinobenzoxazines analogs having the general Formula (I), and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein A, U, V, W, X and Z are substituents. The present invention also relates to methods for using such compounds.

Description

Substituted quinobenzoxazine analogs

The mutual reference of related application

This patent claim scope is: U.S. provisional application 60/461,271, put on record on April 7th, 2003; U.S. provisional application 60/463,171, put on record on April 15th, 2003; U.S. provisional application 60/519,535, put on record on November 12nd, 2003; With U.S. provisional application 60/532,727, put on record on December 23rd, 2003.The content of these documents is incorporated into own forces in this with the form of reference.

Invention field

The present invention relates to substituted quinobenzoxazine (quinobenzoxazines) analogue, and the use-pattern of these compounds.

Background

Can form tetramer structure in the nucleic acid chains of some rich purine.In double-strandednucleic acid, some rich purine chain can participate in the equilibrium process at a slow speed between a typical double-spiral structure and open structure and the non-B configuration district.These open structures and non-B configuration can be called as " parallel construction ".Some form is with relevant to the susceptibility of S1 nuclease digestion, and this can be called as " nuclease hypersensitization element " or " NHE." tetramer is a kind of parallel construction, some NHE may adopt tetramer structure.A large amount of circumstantial evidence promptings, tetramer structure may be present in genomic some specific region in the body, comprises chromosomal telomere end and oncogene regulatory region.(Han，et?al.，TrendsPharm.Sci.(2000)21：136-142)。Thereby the tetramer of DNA forms the molecular target that the district can be used as cancer therapy drug.

Summary of the invention

Compound described in the invention forms the district by the tetramer with DNA and interacts, and serves as the less tumor suppression medicine of side effect.These compounds reduce the hyper-proliferative expression of gene, are used to treat cancer.In addition, these compounds have also been showed antibiotic or antiviral activity, can be used to treat bacterium and virus infection.

Different embodiments of the invention are as described below.The present invention also comprises other compound that meets structural formula 1, and these compounds can have from showing independent any substituting group of selecting on the listed compound of 1-3.Thereby the present invention is not limited only to the specified substituent combination of following different embodiment.These compounds have general structure:

And its medicine allows salt, ester and prodrug.

At this V is H, halogen, or NR ¹R ²

A is H, fluorine or NR ¹ ₂

Z is O, S, NR ¹Or CH ₂

U is OR ²Or NR ¹R ²

X is OR ², NR ¹R ², halogen, azido-(azido) or SR ²

N is 1-3;

At this NR ¹R ²In R ¹And R ²Can constitute a bilateral boundary or a ring type structure, every kind of structure all can be optionally substituted;

R ¹Be H or C _1-6Alkyl (C _1-6Alkyl);

R ²Be H; Or one contain one or more non-adjacent heteroatomss (N, O and S) arbitrarily, and the C that is replaced arbitrarily by a carbocyclic ring or heterocycle _1-10Alkyl (C _1-10Alkyl) or C _2-10Thiazolinyl (C _2-10Alkenyl); Or R ²Be a heterocycle that replaces arbitrarily, aryl or heteroaryl;

W is made up of following groups

At this Q, Q ¹, Q ²And Q ³Be independently CH or N;

Y is an independent O, CH ,=O or NR ¹

R ⁵It can be the substituting group on the condensed ring optional position; By halogen, the H that=O or one or more heteroatoms replace arbitrarily, OR ², C _1-6Alkyl (C _1-6Alkyl), C _2-6Thiazolinyl (C _2-6Alkenyl); Perhaps R ⁵It is an inorganic substituting group; Or two adjacent R ⁵Couple together and obtain that 5-6 unit replaces or the carbocyclic ring or the heterocycle structure of non-replacement, these structures can be fused to arbitrarily that another one replaces or the carbocyclic ring or heterocycle of non-replacement on;

Suppose when X be pyrrolidyl (pyrrolidinyl), A is F, Z is O, and W is when being naphthyl (naphthalenyl) or phenylene (phenylene), U is not OR ¹

When X is F or pyrrolidyl (pyrrolidinyl); A is F; Z is O; And W is when being phenylene (phenylene), and U is that right quinoline base (morpholinyl) or 2,4 difluorobenzene amine (2,4-difluoroaniline); With

If further supposition U is OH, then W represents how thick aromatic ring, and X is not a halogen; With X be NH ₂, or do not contain N, or contain the substituting group of 6 above carbon atoms.

In above-mentioned molecular formula 1, A and X can be respectively halogens.For example, A and X can be respectively fluorine.

In above-mentioned molecular formula, V can be H.In addition, V can be NH ₂Or one have molecular formula NR ¹(CR ¹ ₂) _n-NR ³R ⁴The compound of structure;

At this R ¹And R ³Be respectively H or C _1-6Alkyl (C _1-6Alkyl);

N is 1-6; And

R ⁴Be H, by a carbocyclic ring or any C that replaces of heterocycle _1-6Alkyl (C _1-6Or aryl (C alkyl), _1-6Alkyl); At NR ³R ⁴In the group, R ³And R ⁴Can form a ring structure that replaces arbitrarily.

In above-mentioned molecular formula 1, U and X can be respectively NR ¹R ²For example, R ¹Be H and R ²Be a C _1-10Alkyl (C _1-10Alkyl), this alkyl can contain one or more heteroatomss, and by a C _3-6Cycloalkyl (C _3-6Cycloalkyl), aryl or one are contained one or more N, and the 5-14 unit heterocycle of O or S replaces arbitrarily.In another example, R ¹Be H and R ²Be that an aryl or one are contained one or more N, the 5-14 unit heterocycle of O or S, it can be replaced arbitrarily by an amino or another heterocycle.In another example, NR ¹R ²In R ¹And R ²Form one and contain one or more N, the 5-14 unit heterocycle of O or S.In special example, NR ¹R ²Be morpholine (morpholine), thiomorpholine (thiomorpholine), piperazine (piperazine), piperidines (piperidine) or diazepine (diazepine).

In above-mentioned molecular formula 1, U and X can have structural formula respectively

NR ¹-(CR ¹ ₂)n-NR ³R ⁴ (2)

At this R ¹And R ³Be respectively H or C _1-6Alkyl (C _1-6Alkyl);

N is 1-6; And

R ⁴Be that H or one are contained one or more non-adjacent heteroatomss (N, O and S) arbitrarily, and the C that replaces arbitrarily by one or more carbocyclic rings or heterocycle _1-10Alkyl (C _1-10Alkyl) or C _2-10Thiazolinyl (C _2-10Alkenyl); And

At NR ³R ⁴In, R ³And R ⁴Can constitute any substituted ring.

In above-mentioned molecular formula 2, n can be 2-3.For example, NR ³R ⁴Be an aliphatic amide, or a guanidine radicals or its tautomer; Or R ³And R ⁴Can constitute one arbitrarily and contain one or more N, the substituted ring of O or S.In specific example, NR ³R ⁴Be morpholine (morpholine), thiomorpholine (thiomorpholine), imidazoles (imidazole), tetramethyleneimine (pyrrolidine), piperazine (piperazine), pyridine (pyridine) or piperidines (piperidine).

In above-mentioned molecular formula 1, X can be NR ¹R ²U meets molecular formula

NR ¹-(CR ¹ ₂) _n-NR ³R ⁴ (2)

At this R ¹And R ²According to claim 1;

R ³Be H or C _1-6Alkyl (C _1-6Alkyl);

N is 1-6; And

R ⁴Be H or or one contain one or more non-adjacent heteroatomss (N, O and S) arbitrarily, and the C that replaces arbitrarily by one or more carbocyclic rings or heterocycle _1-10Alkyl (C _1-10Alkyl) or C _2-10Thiazolinyl (C _2-10Alkenyl); And

At NR ¹R ²And NR ³R ⁴In, R ¹And R ², and R ³And R ⁴Can distinguish and constitute a substituted ring independently.

In above-mentioned molecular formula, X is NR ¹R ², U meets molecular formula NR ¹-(CR ¹ ₂) _n-NR ³R ⁴(2), NR ¹R ²In R ¹And R ², and NR ³R ⁴In R ³And R ⁴Can constitute one respectively independently and contain one or more N, the substituted ring of O or S.For example, X can be by amino, carbamate, and one contains one or more non-adjacent N, the C of O or S _1-10Alkyl (C _1-10Alkyl) replace arbitrarily, and by a heterocycle, aryl or one 's saturated or unsaturated heterocycle institute replaces arbitrarily.For example, X and NR ³R ⁴Be respectively morpholine (morpholine), thiomorpholine (thiomorpholine), imidazoles (imidazole), tetramethyleneimine (pyrrolidine), piperazine (piperazine), pyridine (pyridine) or piperidines (piperidine).In an example, X and NR ³R ⁴Be respectively tetramethyleneimine (pyrrolidine).In another example, X is the tetramethyleneimine (pyrrolidine) that is replaced by pyrazine (pyrazine).In this embodiment, V is H; A is a fluorine; With W be naphthyl (naphthalenyl).

The example of the assorted unit of 5-6 ring includes but not limited to tetrahydrofuran (THF) (tetrahydrofuran), 1,3-dioxolane (1,3-dioxolane), 2,3-dihydrofuran (2,3-dihydrofuran), tetrahydropyrans (tetrahydropyran), cumarone (benzofuran), isobenzofuran (isobenzofuran), 1,3-dihydro-isobenzofuran (1,3-dihydro-isobenzofuran), isoxzzole (isoxazole), 4,5-dihydro-isoxazole (4,5-dihydroisoxazole), piperidines (piperidine), tetramethyleneimine (pyrrolidine), pyrrolidin-2-one (pyrrolidin-2-one), pyrroles (pyrrole), pyridine (pyridine), pyrimidine (pyrimidine), octahydro-pyrroles [3,4-b] pyridine (octahydro-pyrrolo[3,4-b] pyridine), piperazine (piperazine), pyrazine (pyrazine), morpholine (morpholine), thiomorpholine (thiomorpholine), imidazoles (imidazole), tetrahydroglyoxaline-2,4-diketone (imidazolidine-2,4-dione), benzoglyoxaline (benzimidazole), 1,3-dihydrobenzo imidazoles-2-ketone (1,3-dihydrobenzimidazol-2-one), indoles (indole), thiazole (thiazole), benzothiazole (benzothiazole), thiadiazoles (thiadiazole), thiophene (thiophene), tetramethylene sulfide 1, (tetrahydro-thiophene 1 for the 1-dioxide, 1-dioxide), diazepine (diazepine), triazole (triazole), guanidine (guanidine), diazabicylo [2.2.1] heptane (diazabicyclo[2.2.1] heptane), 2,5-diazabicylo [2.2.1] heptane (2,5-diazabicyclo[2.2.1] heptane), with 2,3,4,4a, 9,9a-six hydrogen-1H-β-Ka Lin (2,3,4,4a, 9,9a-hexahydro-1H-β-carboline).

In above-mentioned molecular formula 1, W can be benzene (benzene), pyridine (pyridine), biphenyl (biphenyl), naphthalene (naphthalene), luxuriant and rich with fragrance (phenanthrene), quinoline (quinoline), isoquinoline 99.9 (isoquinoline), quinazoline (quinazoline), cinnolines (cinnoline), naphthyridine (phthalazine), quinoxaline (quinoxaline), indoles (indole), benzoglyoxaline (benzimidazole), benzoxazoles (benzoxazole), benzothiazole (benzthiazole), cumarone (benzofuran), anthrone (anthrone), xanthone (xanthone), dihydroketoacridine (acridone), Fluorenone (fluorenone), carbazole (carbazolyl), pyrimidine [4,3-b] furans (pyrimido[4,3-b] furan), pyridine [4,3-b] indoles (pyrido[4,3-b] indole), pyridine [2,3-b] indoles (pyrido[2,3-b] indole), diphenylene-oxide (dibenzofuran), acridine (acridine) or azine (acridizine).

In above-mentioned molecular formula 1, U can be OR ², R ²Be the C that replaces arbitrarily by a carbocyclic ring or heterocycle _1-6Alkyl (C _1-6Alkyl).

In above-mentioned molecular formula 1, each substituted radical can be by one or more halogens, OR ², NR ¹R ², carbamate, C _1-10Alkyl (C _1-10Alkyl), C _2-10Thiazolinyl (C _2-10Alkenyl) institute replaces arbitrarily, and these substituting groups again can be by halogen ,=O, aryl (aryl) or one or more heteroatoms; Inorganic substituting group, aryl, carbocyclic ring or heterocycle replace arbitrarily.

The compound that the present invention relates to can have chirality.The chipal compounds of herein mentioning is a kind of compound different with its mirror image, has an enantiomer.The method of the racemic mixture of synthesizing chiral compound and fractionation enantiomer is known by the chemist.These methods can be consulted, for example, and March, " Advanced Organic Chemistry, " John Wiley and Sons, Inc., New York, (1985), this book also is used as reference of the present invention.

The present invention has also comprised the medicine synthetics that contains molecular formula 1 type compound and a kind of medicine permission excipient.

In addition, the invention provides the method for improving cell generation disorders, this method is by giving the compound that meets molecular formula 1 of acceptor effective dose, or its a medicine synthetics, reaches the purpose of improving above-mentioned cell generation disorders.As an example, cell generation disorders can be a cancer.As another example, cell proliferation reduces, or apoptosis is induced.Acceptor can be the mankind or animal.

The present invention also provides the method that reduces cell proliferation or cell death inducing, this method is by giving the compound that meets molecular formula 1 of system's effective dose, or its a medicine synthetics, reach the purpose that makes this system cut down cell proliferation or cell guiding apoptosis.This system can be a cell or tissue.

The present invention further provides and reduced the method that microorganism is tired, this method is by giving the compound that meets molecular formula 1 of system's effective dose, or its a medicine synthetics, reaches to make this system reduce the purpose that microorganism is tired.This system can be a cell or tissue.As an example, it can be that virus, bacterium or fungi are tired that microorganism is tired.

In addition, the invention provides the method that alleviates infected by microbes, this method is by giving the compound that meets molecular formula 1 of acceptor effective dose, or its a medicine synthetics, reaches the purpose that makes this system alleviate infected by microbes.Acceptor can be the mankind or animal.As an example, infected by microbes can be a virus, bacterium or fungi.

Picture specification

Fig. 1 has shown in the Ramos heteroplastic transplantation model, meets the anti-tumor activity of a kind of compound of molecular formula 1.

Fig. 2 has shown in HCT 116 cells, meets the anti-tumor activity of a kind of compound of molecular formula 1.

Invention is described

The present invention relates to meet the quinoline of molecular formula 1, and its medicine allows salt, ester and prodrug. In certain embodiments, the tetramer of these compounds and DNA forms district's interaction. The present invention also relates to use these compounds for treating cancers, the method for bacterium and virus infections.

Because it is adjusting that bioprocess such as oncogene are transcribed that the tetramer of DNA forms the district, so the bioactive Auto-regulator of the tetramer can be used to treatment of cancer. Can form with the tetramer of DNA the interactional molecule in district and for some cell generation disorders and correlation circumstance therapeutic action be arranged. Particularly, the unusual oncogene expression that increases can cause cell generation disorders, and tetramer structure is reduced oncogene expression usually. The example of oncogene can be but be not limited only to MYC, HIF, VEGF, ABL, TGF, PDGFA, MYB, SPARC, HUMTEL, HER, VAV, RET, H-RAS, EGF, SRC, BCL1, BCL2 and the known oncogene of other technical staff.

The molecule that is bonded to DNA tetramer formation district can be brought into play biological agent by different mechanisms. These mechanism comprise as stablizing natural tetramer structure, unwind to suppress a natural tetramer to the conversion of double-stranded DNA by blocking-up, and stable have natural tetramer structure and other sequence-specific that the tetramer takes off stability nucleotides substituted radical and interact. Thereby the compound that is bonded to DNA tetramer formation district herein can act on cell, and tissue, or organ reach the purpose that cell generation disorders was transcribed and therefore treated to the downward modulation oncogene. Term used herein " treatment " and " therapeutic action " refer to reduce or stop a kind of multiplication rate (for example, slow down or stop tumor growth) of cell or reduce the quantity (for example, eliminating part or all of tumour) of propagation cancer cell.

Can judge a cell by monitoring tetramer biologically active, whether the biologically active that can form tetrameric n DNA in tissue or the organ is regulated. The DNA tetramer forms the biologically active in district and can monitor in tissue or the organ at cell, for example, can contact the genetic transcription increase or the minimizing that cause with tetramer formative DNA by detection molecules and reach the monitoring purpose. Transcribe and can detect by direct observation rna transcription basis or the polypeptide of observing by the transcript translation, this all is the method that the professional knows.

It is disorderly to be enough in the molecular energy of tetramer formative DNA and the tetramer formative nucleic acid interaction treatment various kinds of cell propagation. Cell generation disorders comprises, for example, colorectal cancer and hematopoietic system cancer (that is, involve and make haematogenous proliferative/new biological cell, for example from marrow, the cell of lymph or erythron, and the disease of precursor). Disease can arise from low differentiation acute leukemia, for example EBL and acute megakaryoblastic leukemia. Other marrow disorder includes but are not limited to acute promyelocytic leukemia (APML), acute myelocytic leukemia (AML), and chronic granulocytic leukemia (CML) (Vaickus, Crit.Rev.in Oncol./Hemotol 11:267-297 (1991). the lymph malignant tumour comprises, but be not limited to acute lymphoblastic leukemia (ALL), comprise B clone ALL and T clone ALL, chronic lymphatic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell (HLL) and macroglobulinemia Waldenstron (WM). other form of malignant lymphoma also comprises, but be not limited to NHL and modification thereof, lymphoma peripheral T cell, adult T chronic myeloid leukemia/lymthoma (ATL), CTCL (CTCL), large granular lymphocyte leukaemia (LGLL), Hodgkin's disease and Reed-Sternberg disease. cell generation disorders also comprises colorectum, mammary gland, lung, liver, pancreas, lymph node, colon, prostate, brain, neck, skin, the cancer of kidney and heart. forming the interactional compound in district with the DNA tetramer can also take place by the blood vessel that suppresses acceptor, and is used for the treatment of cancer relevant process and situation (increasing such as blood vessel).

The invention provides a kind of method that reduces cell proliferation or treatment or improve cell generation disorders, this method is used the compound that meets molecular formula 1 to having the system that can form tetrameric DNA. This system can be one group of cell or one or more tissue. In one embodiment, this system is the acceptor that needs a kind of cell generation disorders for the treatment of (for example, mammal such as mouse, rat, monkey or the mankind). The present invention also provides a kind of method for the treatment of colorectal cancer, and acceptor is a certain amount of can to form the interactional compound in district with the c-MYC tetramer to this method by giving, and reaches the purpose that reduces rectum cancer cell propagation. In addition, the present invention also provides a kind of method that suppresses blood vessel hyperplasia and selective therapy blood vessel hyperplasia correlation cancer, this method by give acceptor a certain amount of can and VEGF (VEGF) tetramer form the interactional compound in district, reach the purpose that reduces blood vessel hyperplasia and selective therapy blood vessel hyperplasia correlation cancer.

Term used herein " alkyl " refers to a carbon compound, has comprised simultaneously containing one or more heteroatomic compounds. Term " alkyl " has also comprised the compound that is replaced by one or more non-interfering substituent. The example of non-interfering substituent includes, but are not limited to OR¹, amino, amide groups, halogen ,=O, aryl, heterocyclic group or inorganic substituting group, and other does not affect the substituting group of compound activity. Term used herein " carbocyclic ring " is the ring-like compound that only contains carbon atom on the finger ring.

Term used herein " heterocycle " refers to that one contains heteroatomic ring-like compound, comprises monocycle or dicyclo. Term used herein " hetero atom " refers to de-carbon or the outer arbitrary atom of hydrogen atom, such as nitrogen, and oxygen or sulphur. The example of heterocyclic compound includes but not limited to oxirane (oxirane), oxetanes (oxetane), pyrans (pyran), oxinane (tetrahydropyran), dioxane (dioxane), lactone (lactones), aziridine (aziridine), azetidine (azetidine), pyrrolidines (pyrrolidine), piperidines (piperidine), morpholine (morpholine), lactams (lactams) and oxolane (tetrahydrofuran).

Term used herein " dicyclic compound " refers to a compound with dicyclo of sharing a pair of bridge carbon atom. The example of dicyclic compound includes, but are not limited to naphthalane (decalin), norcamphane (norbornane), camphor (camphor) and diazabicyclo [2.2.1] heptane (diazabicyclo[2.2.1] heptane).

Term used herein " heteroaryl " or " hetero-aromatic ring " refer to an aromatic heterocycle. The example of heteroaryl includes, but are not limited to furans (furan), pyrans (pyrrole), pyridine (pyridine), pyrimidine (pyrimidine), imidazoles (imidazole) or triazole (triazole).

Term used herein " treatment " or " therapeutic action " refer to reduce or stop a kind of multiplication rate (for example slow down or stop tumor growth) of cell or reduce the quantity (for example, eliminating part or all of tumour) of proliferative cancer cell. The system that these terms equally also are used to reduce a microbial infection (is cell, the tissue or acceptor) biological potency, the microbial reproduction speed that slows down reduces the impact of symptom quantity or infected by microbes correlation symptom, but and/or removes the microorganism of detection limit from system. The example of microorganism includes, but are not limited to virus, bacterium and fungi.

Can form the interactional compound in district with the DNA tetramer and also can be used to reduce infected by microbes, virus infections for example. retroviruse is considered to the functional element of at least two kinds of secondary structures for G-tetramer targeted therapy provides a large amount of potential target spot .G-tetramer structures, these secondary structures are by the viral RNA of HIV or DNA, dimer syndeton (DLS) and center DNA lobe (CDF) form. in addition, can adopt molecule DNA aptamer (aptamer) interior or intermolecular tetramer structure can suppress virus replication. for example, the DNA aptamer can suppress virus replication by target localization package membrane glycoprotein (putatively). in another example, the DNA aptamer suppresses virus replication by the location of target respectively HIV-integrase, points out the interaction between natural tetramer structure and the integrase.

The dimer syndeton is all very common for all retroviruses, and it combines virus genomic two duplicates by the noncovalent interaction between two the 5 ' ends of two viral RNA sequences. In ripe virion, genome dimer and the albumen (gagprotein) of dehiscing have stable contact. Take HIV as example, the origin of this non-covalent combination can be traced back to 98 base-pair sequences, and this sequence comprises several fragments that have two continuous guanines at least (3 ' end that for example, is used for the external formation of RNA dimer). Can observe the dependence that the external formation of dimer and stability have cation (potassium), and an antisense sequences can not be effectively Dimerized, these two kinds of phenomenons show that most probable integrated structure is an intermolecular G-tetramer.

Before being incorporated into host genome, retroviruse DNA and at least two main virus proteins (integrase and reverse transcriptase) are in conjunction with forming the front compound (PIC) of an integration, and this compound is transported in the nucleus subsequently. Center DNA lobe (CDF) refers to+the strand afterbody of 99 base length of chain, come across the nearly central area of viral double-stranded DNA, and it plays an important role in the nuclear input process of PIC. The oligonucleotide mimetic of CDF can form the intermolecular G-tetramer in cell free system.

Thereby the compound that can identify tetramer formation district can be used to stablize the dimer syndeton, thereby stops the decoupling zero of two RNA chains. And on the tetramer structure that is attached to CDF formation, albumen identification and/or the cohesive process relevant with the transportation of PIC nuclear can be interrupted. In both cases, relative other antiviral therapy, method involved in the present invention has important advantage. Present anti-retroviral treatment (HAART) method relies on unites the medicine that uses for hiv protease and integrase. Polypharmacy is chemical sproof probability minimum to occur in order to make. If use separately these medicines, generally drug resistance can appear very soon. The distortion that this quick chemical sproof reason is reverse transcriptase, this distortion cause an about sudden change in per 10,000 base-pairs. Employing is that chemical sproof generation slowly or not takes place with viral tetramer structure rather than an advantage take protein as target. The tetrameric point mutation of target can damage the integrality of tetramer structure, thereby causes the non-functional of virus to copy. Based on this conception of species, can substitute the polypharmacy of using at present with single medication, thereby when reducing medical expense, eliminate harmful medicine/drug interaction.

The invention provides the method for biological potency in the reduction system, this method has the system in n DNA tetramer formation district with the compound that meets molecular formula 1 of effective dose by giving one, reaches the purpose that makes this system reduce biological potency. This system can be one or more cell or tissues. The example of biological potency includes, but are not limited to virus, bacterium or fungi and tires. As a specific embodiment, this system can be the acceptor (for example, mammal such as mouse, rat, monkey or the mankind) of needs treatment virus infections. The example of virus infections comprises the infection that lower influenza virus causes, such as hepatitis viruse (for example, B-mode or hepatitis C), human immunity lacks virus (HIV), rhinovirus, herpes zoster virus (VZV), herpes simplex virus (for example, HSV-1 or HSV-2), cytomegalovirus (CMV), vaccinia virus, influenza virus, encephalitis viruses, Hantaan virus, arboviruse, West Nile Virus, human papilloma virus (HPV), Epstein-Barr virus and Respiratory Syncytial Virus(RSV). The method that the present invention also provides treatment HIV to infect, this method is by giving the compound that meet molecular formula 1 of an acceptor with effective dose, reach to alleviate the purpose that HIV infects.

Identification can be bonded to the compound that the DNA tetramer forms the district

Compound described herein refers to that the tetramer that can be bonded to DNA forms the compound of distinguishing. Containing or do not containing in the system of these compounds, this regional biologically active (be typically expressed as " signal) difference. Although when surveying a recruit, can measure simultaneously background signal, not require when analyzing a recruit and must measure background signal.

The tetramer forms the example of nucleotide sequence shown in Table A:

Table A

Sequence	Sequence   ID number	The source
			TG 4AG 3TG 4AG 3TG 4AAGG	1	CMYC
GGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGC	2	PDGFA
			G 8ACGCG 3AGCTG 5AG 3CTTG 4CCAG 3CG 4CGCTTAG 5	3	PDGFB/c-  sis
AGGAAGGGGAGGGCCGGGGGGAGGTGGC	4	CABL
			AGGGGCGGGGCGGGGCGGGGGC	5	RET
GGGAGGAAGGGGGCGGGAGCGGGGC
		6	BCL-2
GGGGGGCGGGGGCGGGCGCAGGGGGAGGGGGC				7	Cyclin  D1/BCL-1
	CGGGGCGGGGCGGGGGCGGGGGC	8	H-RAS
AGAGGAGGAGGAGGTCACGGAGGAGGAGGAGAAGGAGGAGG  AGGAA				9	CMYB
	(GGA) 4	10	VAV
AGAGAAGAGGGGAGGAGGAGGAGGAGAGGAGGAGGCGC				11	HMGA2
	GGAGGGGGAGGGG
		12	CPIM
	AGGAGAAGGAGGAGGTGGAGGAGGAGG			13	HER2/neu
AGGAGGAGGAGAATGCGAGGAGGAGGGAGGAGA
		14	EGFR
GGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG
		15	VEGF

Sequence	Sequence   ID number	The source
			CGGGAGGAGGAGGAAGGAGGAAGCGCG	16	CSRC

Whether produce the different signals except measuring tested molecule or tested nucleic acid, the affinity of nucleic acid and compound interaction also can quantitatively be detected. Can compare each interactional IC50, Kd or Ki threshold value and IC50 or Kd measured value, thus identification is as the tested molecule of tetramer interacting molecule or form the tested nucleic acid of nucleic acid as the tetramer. For example, often adopt 10 μ M or lower, 1 μ M or lower, and 100nM or lower as IC50 or Kd threshold value. In another example, the threshold value that identification tetramer interacting molecule and the tetramer form nucleic acid can be 10nM or lower, 1nM or lower, 100pM or lower and 10pM or lower.

Existing many assay methods are used for identification and form the compound that there is compatibility in the district with the DNA tetramer. In some assay methods, biologically active is the tetramer nucleic acid that is bonded to compound, and this combination degree is used as a kind of signal and measures. In other assay method, biologically active is tetrameric polymerase capturing function, and its degree of catching is used as a kind of signal and measures. In some assay method, biologically active is to transcribe, and transcriptional level is used as a kind of signal and measures. In another kind of assay method, biologically active is cell death, what quantitatively detect is just in the quantity of dead cell. The multiplication rate of other assay method monitoring cancer cell. The example of assay method comprises the fluorescence binding assay, gel shift rate fluctuation measurement method (is consulted, Jin ﹠ Pike for example, Mol.Endocrinol. (1996) 10:196-205), polymerase catch assay method is transcribed the report determination method, cancer cell multiplication determination method and Apoptosis determination method (are consulted, for example, Amersham Biosciences (Piscataway, New Jersey)). The embodiment subsequent introduction of these assay methods. Topoisomerase can be used to also judge whether tetramer interacting molecule has topoisomerase path activity (consult, for example, TopoGEN, Inc. (Columbus, Ohio)).

Mobility shift assay fluctuation measurement method (EMSA)

EMSA can be used for judge whether a nucleotides forms the tetramer and a nucleotide sequences and whether be the tetramer and take off stable state. Described the carrying out of EMSA such as document (Jin ﹠ Pike, Mol.Endocrinol.10:196-205 (1996)), but some little changes are arranged. Usually, synthetic single stranded oligonucleotide [γ-³²P] under the existence of ATP (1,000mCi/mmol, Amersham Life Science), with the T4-kinases 5 '-the end mark, then by a cross-linked dextran gel column purifying. Subsequently, containing 10mM Tris pH 7.5,100mM KCl, 5mM dithiothreitol (DTT), 0.1mM EDTA, 5mM MgCl₂, 10% glycerine in 0.05%Nonedit P-40 and 0.1mg/ml poly (dI-dC) buffer solution (Pharmacia), is hatched in the situation that is with or without the tested mixture of variable concentrations³²The oligonucleotides of P-mark (～30,000cpm). After hatching 20 minutes under the room temperature, on 5% polyacrylamide gel in the 0.25xTris borate-EDTA buffer solution (0.25xTBE, 1xTBE are 89mM Tris-borate, and pH 8.0,1mM EDTA), carry out association reaction. Desiccant gel uses phosphorus screen imager (phosphoimager) quantitatively to detect all bands.

The DMS protection determination method that methylates

Chemistry footprint determination method is used for determining tetramer structure. It by which nucleotides of measuring in the nucleic acid is prevented from or is not prevented from by the dressing agent chemical modification, determines tetramer structure. DMS methylation assay method is a kind of chemical footprint determination method. In this determination method, the EMSA band is separated, and that carries out that DMS induces unwinds. From electrophoretic mobility change gel, separate each important band, in 100mM KCl solution (300 μ l), soaking 6 hours under 4 ℃. Filtering solution (little centrifugal), further use 100mM KCl among the 0.1X TE with 30, it is 70 μ l (each reaction) that the dna solution of 000cpm (each reaction) is diluted to cumulative volume. after adding 1 μ l salmon sperm dna (0.1 μ g/ μ l), with reactant mixture at 1 μ l DMS solution (DMS: ethanol; 4: 1; V: hatch a period of time v). Each reacts with 18 μ l stop buffer (b-mercaptoethanol: water: NaOAc (3M); 1: 6: 7; V: v: v) quench. After precipitation with alcohol (twice) and piperidines cracking, reactant mixture separates in preparation gel (16%) and observes with phosphorus screen imager.

Polymerase catch assay method

The catch assay method comprises template nucleic acid, and it can be made up of a tetramer formation sequence and a primer nucleic acid. This primer nucleic acid hybridizes to the template nucleic acid 5 ' end of tetramer formation sequence. Primer prolongs by polymerase (for example Taq polymerase), and it begins to extend along template nucleic acid from primer. In this determination method, tetramer structure can be blocked or the extension of trapped enzyme, thereby produces the short fragment of transcribing. And the catch assay method can be carried out under different temperature (comprise 45 ℃ with 60 ℃) and different ion concentration.

The Taq polymerase stops determination method such as Han, et al., and Nucl.Acids Res. (1999) 27:537-542 is described, and this method is Weitzmann et al., the improvement of J.Biol.Chem. (1996) 271:20958-20964 method therefor. Briefly, with template DNA (50nM), TrisHCl (50mM), MgCl₂(10mM), DTT (0.5mM), EDTA (0.1mM), the tetramer nucleic acid of BSA (60ng) and 5 '-end mark (～18nM) mix after, 90 ℃ of lower heating 5 minutes, in 30 minutes, be cooled to room temperature then. Add Taq polymerase (1 μ l) in reactant mixture, sustained response is 30 minutes under stable temperature. Adding 10 μ l stop buffer (formamides (20ml), 1M NaOH (200 μ l), 0.5M EDTA (400 μ l) and 10mg bromophenol blue) after, reactant mixture separates in preparation gel (12%) and observes with phosphorus screen imager. By the duDNA cycle sequencing system of LifeTechnologies company, carry out adenine order-checking (" A " by the gel top shows). The General Sequences of template strand is

TCCAACTATGTATAC-INSERT-TTAGCGACACGCAATTGCTATAGTGAGTCGTAT TA, " INSERT " refers to a nucleotide sequence (consulting Table A) that contains tetramer formation sequence herein. The slow band of mobility is indicating tetrameric formation in the gel.

High flux polymerase catch assay method

The present invention has formulated a kind of high flux polymerase catch assay method. This method comprises makes a kind of template nucleic acid (often being DNA) contact with a kind of primer (also often being DNA); Primer/template composite is contacted with compound described herein (being also referred to as " test-compound "); Primer/template composite is contacted with a kind of polymerase; Last separated product. This determination method often comprises makes the then step of renaturation of primer/template compound mixture sex change, carries out before tested molecule adding system as these step 1. Many determination methods use multiple test-compound concentration to carry out usually, to obtain IC₅₀The data such as value. Product often comprises the extension primer of different length. When the tetramer structure in a test-compound and the template did not interact significantly, primer often extended to the template end.

When the tetramer structure in a test-compound and the template had significant interaction, primer only extended to the tetramer structure place in the template usually, no longer extended. Therefore, when the tetramer structure in a kind of test-compound and the template interacted, reactant mixture often contained at least two kinds of product, and a kind of have fully the primer that extends, a kind of primer with incomplete extension, these two kinds of each self-separations of reactant. Can use any easily separation means separated product, such as mass spectrum and Capillary Electrophoresis.

But product is identified by a tags detected that is connected on the primer. but tags detected can non-covalently be connected to 5 ' of primer and (for example hold upward, a biotin molecule is covalently bound to primer 5 ' end, but this primer end links to each other in non-covalent mode with the antibiotin molecule that has added a tags detected again) but. tags detected can be connected on the primer in any stage of analyzing, sometimes before with primer adding system, after primer extends, or behind the product separation. but tags detected is general covalently bound to primer by a kind of method, but this method will be selected according to the character of chemical group on the tags detected.

But there are many methods to can be used for the covalently bound of tags detected and nucleic acid, but for example on the succinimide ester derivant of allylamine derivatized nucleotide to a tags detected of chemical coupling, generate then a primer that uses labelled nucleotide. (consulting, for example Nature Biotech (2000) 18:345-348 and network address info.med.yale.edu/genetics/ward/tavi/n_coupling.html). Spacer region (general 5-16 carbon atom grown) but sometimes participated between tags detected and the nucleosides. But operable tags detected include, but are not limited to radio isotope (as¹²⁵I， ¹³¹I， ³⁵S， ³²P， ¹⁴C or³H); The light scattering label is (such as spherical gold or silver label label; Genicon Sciences Corporation, San Diego, CA, U.S. Patent number 6,214,560); Enzyme or protein tag (such as GFP or peroxidase); Or other color development label or dyestuff. Fluorescence labels is (such as amino-methyl cumarin (AMCA); Diethyl amino methylcoumarin (DEAC); Waterfall indigo plant (CB); Fluorescein isothiocynate (FITC); Oregon green (OG); Alexa 488 (A488); Rhodamine green (RGr); Lanthanide chelate (for example, europium); Carboxyl rhodamine 6G (R6G); Tetramethylrhodamin (TAMRA); Dallas Pink (TxR); Cy3; Cy3.5; Cy5, Cy5.5 and carboxylic fluorescent naphthalimide element (CNF), digoxin (DIG); With 2,4-dinitrophenyl (DNP)) also often be used. Other fluorophorre and accompaniment excite and emission wavelength to some extent narration in following document: Anantha, et al., Biochemistry (1998) 37:2709-2714 and Qu﹠ Chaires, Methods Enzymol (2000) 321:353-369.

In one embodiment, the primer tasteless nucleotide of a covalently bound fluorescence labels contacts with template DNA. The compound that obtains contacts with the polymerase that can extend primer with after test-compound contacts. Subsequently, product is by capillary electrophoresis separation and detection. Long primer sequence be used to primer in do not contain link to each other fluorogen or make comparisons without the situation of capillary electrophoresis separation of covalency. Any stage before separation adds deoxynucleotide, generally at primer with in template DNA contacts. Before adding test-compound, template DNA/primer complex is often passed through sex change (for example, by increasing system temperature) and the subsequently operation such as renaturation (for example, by system is cooled off).

To measure the object lesson of implementing below. One 5 '-fluorescently-labeled (FAM) primer (P45,15nM) and template DNA (15nM) are being contained 10mM MgCl₂, mix in Tris-HCL (15mM Tris, the pH 7.5) buffer solution of 0.1mM EDTA and 0.1mM mixing deoxynucleotide triphosphoric acid (dNTP ' s).

The FAM-P45 primer

(5 '-6FAM-AGTCTGACTGACTGTACGTAGCTAATACGACTCACTATAGCAATT-3 ') and template DNA

(5 '-TCCAACTATGTATACTGGGGAGGGTGGGGAGGGTGGGGAAGGTTAGCGACACGCAA TTGCTATAGTGAGTCGTATTAGCTACGTACAGTCAGTCAGACT-3 ') is synthesized and by Applied Biosystems HPLC purifying. Mixture, was hatched under 37 ℃ 15 minutes after being cooled to room temperature 95 ℃ of lower sex change 5 minutes.

Behind the cool to room temperature, add 1mMKCl₂And test-compound (variable concentrations), mixture was at room temperature hatched 15 minutes. (the 2.5U/ reaction Promega), is hatched 30 minutes to finish the prolongation of primer under 70 ℃ to add then 10mM KCl and Taq archaeal dna polymerase. By 1 μ l reactant mixture being joined 10 μ l Hi-Di formamide mixtures and 0.25 μ l LIZ120 size standard, cessation reaction. Hi-Di formamide and LIZ120 size standard are available from Applied Biosystems. It is 61 or 62 bases that the tetramer that part prolongs is caught product length, and all prolonging product length is 99 bases. Product is by capillary electrophoresis separation and analysis. Capillary Electrophoresis adopts ABI PRISM 3100-Avant Genetic Analyzer to carry out. Analyze and adopt above-claimed cpd to carry out, the results are shown in Table 1. The μ M concentration that table 1 is reported is the concentration (short part prolongation DNA (DNA that catches) and the ratio of whole DNA of prolongation are 1: 1) when catching 50% DNA in measuring.

Transcribe the report determination method

In transcribing the report determination method, tested tetramer DNA is coupled on the reporting system, so that tetrameric formation and stabilization can be regulated and control report signal. An example of this system is a report expression system. In this system, polypeptide such as luciferase or green fluorescent protein (GFP) are operatively connected the potential tetramer by one and form gene expression on the nucleic acid, thereby can detect the expression of polypeptide. Term used herein " is operatively connected " and refers to a nucleotide sequence, and it comprises the sequence adjusting that the potential tetramer forms nucleic acid by one. When a sequence as tetramer DNA on identical or different nucleic acid the time, this sequence can be for being operatively connected. What will describe below is a luciferase assay example. At He, et al. has narrated a kind of luciferase promoter determination method among Science (1998) 281:1509-1512, and it often is used to study tetrameric formation. He, the document of et al. (list of references 11) has been illustrated the used media of this law. In this mensuration, use the system (Invitrogen) based on liposome (lipofectamin) 2000 to come transfection HeLa cell. Transfection process uses 0.1 μ g pRL-TK (Renilla luciferase reporting plasmid) and the 0.9 μ g tetramer to form plasmid according to the method for manufacturer. According to the method for manufacturer, in a 96-hole flat board, use Dual-Luciferase report mensuration system (Promega) to analyze the uciferase activity of firefly and sea pansy (Renilla).

The circular dichroism spectra determination method

Circular dichroism spectra (CD) be used to determine other molecule whether with tetramer nucleic acid interaction. CD be particularly useful for judging a PNA or PNA-peptide bond can with the external hybridization of tetramer nucleic acid. Under 37 ℃, contain 10mM potassium phosphate (pH 7.2) and 10 or the buffer solution of 250mM KCl in, the PNA probe is joined tetramer DNA (each 5 μ M). Under identical temperature, left standstill 5 minutes, and recorded subsequently spectrogram. The CD spectrogram divides record on the Photopolarimeter at a Jasco J-715 who is equipped with the unicellular folder of thermoelectric control. CD intensity generally detects between 220nm and 320nm. Produce and only contain tetramer DNA, only contain PNA and the contrast spectrogram that contains PNA and tetramer DNA, to have determined whether interaction (consulting Datta, etal., JACS (2001) 123:9612-9619). Spectrogram record be mean value with eight scannings of speed of 100nm/ minute.

The fluorescence binding assay

An example of fluorescence binding assay is one and comprises tetramer nucleic acid, the system of signaling molecule and tested molecule. In the time of on being attached to tetramer nucleic acid, signaling molecule produces a fluorescence signal (for example, N-methyl mesoporphyrin IX (NMM)). When the competition of test-compound and signaling molecule was attached on the tetramer nucleic acid, the signal that produces will change. When test-compound existed or do not exist, whether the change of signal can be identified test-compound was tetramer interacting compounds.

50 μ l tetramer nucleic acid or a kind ofly can not form tetrameric nucleic acid and be added in the flat board of 96-hole. To the test-compound that wherein adds variable concentrations. Measure usually at 100 μ l 20mM HEPES buffer solutions, pH 7.0, carry out among 140mM NaCl and the 100mM KCl. Subsequently, add 50 μ l signaling molecule NMM, until final concentration is 3 μ M. NMM is from Frontier Scientific Inc, Logan, Utah. Use FluroStar2000 luminoscope (BMG Labtechnologies, Durham, NC), under excitation wavelength 420nm and emission wavelength 660nm, measure fluorescence. The function of fluorescence intensity Chang Zuowei test-compound or tetramer oriented nuclei acid concentration and draw variation diagram, maximum NMM fluorescence signal are measured in the situation of these molecules not having.

The cell proliferating determining method

In the cancer cell multiplication determination method, cell proliferation speed is by adding to the function evaluation of test-compound concentration in the cell culture medium. and this law can be used any cancer cell type. in one embodiment, colon cancer cell is in vitro culture, tested compounds adds culture medium with variable concentrations. and a useful colon carcinoma cell line is colo320, it is that a kind of colon adenocarcinoma cell that NIH preserves is, numbering JCRB0225. uses the parameter of these cells can consult webpage cellbank.nihs.go.jp/cell/data/jcrb0225.htm.

Compound formulas

Term used herein " medicine allows salt, ester and acid amides " includes, but are not limited to carboxylate salt, amino acid addition salt, and the ester of compound and acid amides, and zwitterionic form, above-mentioned form is known by the technician, is suitable for the human and animal and uses.(consult, for example, Gerge, S.M., et al., " Pharmaceutical Salts, " J.Pharm.Sci. (1977) 66:1-19 incorporates in the reference at this.)

Any suitable compound formulas of this paper narration can be produced.When the alkalescence of compound or acid enough strong when forming stable nontoxic acidity or basic salt, perhaps compound is suitable with the form administration of salt.Medicine allows the example of salt to comprise and can form the organic acid salt that physiology allows anionic acid-respons to generate, the physiology that forms allows negatively charged ion to comprise as tosilate mesylate, acetate, Citrate trianion, malonate, tartrate, succinate, benzoate, ascorbate salt, alpha-ketoglutarate, α-glycerophosphate etc.Can also form suitable inorganic acid salt, comprise hydrochloride, vitriol, nitrate, supercarbonate and carbonate.Medicine allows salt to make by well-known standard program, for example makes a q.s basic cpd such as amine and a kind of physiology that provides allow anionic suitable acid-respons.The basic metal (for example, sodium, potassium or lithium) or the carboxylate salt of alkaline-earth metal (as calcium) also can be produced.

A compound can be used as medicinal ingredients and makes preparation, uses for the mammalian hosts that needs treatment.In one embodiment, mammalian hosts is the people.Any suitable route of administration can be used, and includes but not limited to that oral, intestines are outer, vein, muscle, part or subcutaneous administration.

In one embodiment, a compound carries out whole body administration (for example, oral) together with a kind of vehicle such as a kind of inert diluent or a kind of edible carrier that absorbs of medicinal permission.They can be packed into firmly or soft gelatin capsule, are pressed into tablet, or directly put into patient's food.For oral therapeutic administration, activeconstituents need be made with one or more mixed with excipients can absorb tablet, buccal tablet, lozenge, capsule, elixir, suspensoid, syrup, wafer and other similar formulation.Should contain at least 0.1% activeconstituents in these mixtures and the preparation.The ratio of mixture and preparation can change in 2～60% scopes of a unit dosage flexibly.Having in the mixture of medical active, the content of activeconstituents should reach therapeutically effective dosage level.

Tablet, lozenge, pill, capsule and other similar formulation will comprise following material: can add tackiness agent such as tragakanta, gum arabic, W-Gum or gelatin; Vehicle is such as Lin Suanergai; Disintegrating agent is such as W-Gum, yam starch, alginic acid etc.; Lubricant is such as Magnesium Stearate; And sweeting agent is such as sucrose, and fructose, lactose or aspartame or flavouring agent is such as spearmint oil, wintergreen oil or cherry-flavored spices.When the unit form of administration was capsule, except that above material, it also should comprise liquid absorbent, for example vegetables oil or polyoxyethylene glycol.Can also add the physical aspect that many other materials are used as dressing or modify solid preparation.For example, tablet, pill or capsule can be used dressings such as gelatin, beeswax, shellac or sucrose.Should include activeconstituents in syrup or the elixir, sucrose or fructose are used as sweeting agent, and Tegosept M or propyl ester are as sanitas, and tinting material and cherry flavour or flavoring orange essence are as flavouring agent.Used any material all should be suitable medicinal and nontoxic under using dosage in any formulation of preparation.In addition, activeconstituents also can be made in sustained release preparation and the device.

Activeconstituents also can be with the mode administration of intravenous injection or abdominal injection. and the solution of activeconstituents or its salt can be made buffered soln, phosphate buffered saline buffer normally, wherein can be mixed with nontoxic tensio-active agent. dispersion agent can be at glycerine, liquid macrogol, glycerol acetate, or prepare in the mixture of these materials and the oil. under household condition store and use, containing sanitas in these preparations and suppress microbial growth. compound sometimes also can be prepared into the prescription that contains multiple matrix, being used for these route of administration (for example liposome or microsome). liposome is at U.S.Patent No.5,703,055 (Felgner, et al.) and Gregoriadis, be described as an example among the LiposomeTechnology vols.I to III (2nd ed.1993).

The pharmaceutical preparation that is suitable for injecting or import administration can comprise sterile water solution or dispersion agent or the sterilized powder that includes activeconstituents, and these activeconstituentss will be suitable for preparing immediately aseptic injection, transfusion or dispersion agent, also can wrap in the liposome.In all cases, final formulation should be aseptic, liquid and stable under production and storage requirement.Liquid vehicle or solvent can be solution or liquid dispersion media, comprise water, ethanol, polyol (as, glycerine, propylene glycol, liquid polyethylene glycol and similar substance), vegetables oil, nontoxic glyceryl ester, and their suitable mixture.For example pass through to form liposome, or in dispersion agent, keep particle diameter or use tensio-active agent, can keep good flowability.Preventing that microorganism from growing can be by adopting various antiseptic-germicides and anti-mycotic agent, for example, and parabens, trichloro-butyl alcohol, phenol, Sorbic Acid, Thiomersalate and analogue.In many cases, preferably can contain isotonic regulator, for example, sugar, damping fluid or sodium-chlor.Can postpone the material that absorbs by in preparation, using some, for example aluminum monostearate and gelatin, the absorption that prolongs injectable composition.

The preparation of aseptic injection is as required, and the activeconstituents of aequum and above-mentioned various auxiliary material are mixed, and carries out filtration sterilization then.In the preparation injectable sterile powder, prefered method is exactly vacuum-drying and Freeze Drying Technique, and these technology can make activeconstituents and other added ingredients still exist in the sterile solution of preparation.

For topical, these compounds are usually with applied in liquid form.Compound normally with the mode administration of mixture or prescription, will be aided with the carrier that skin can use, normally liquid or solid simultaneously.The dermopathic example of effective treatment that is used for compound is delivered to skin be widely known by the people (consult, for example, Jacquet et al.).(U.S.Pat.No.4,608,392), Geria (U.S.Pat.No.4,992,478), Smith et al. (U.S.Pat.No.4,559,157) and Wortzman (U.S.Pat.No.4,820,508).

Compound also can be made preparation together with solid carrier, and these solid carriers comprise the solid of good distribution such as talcum powder, potter's clay, Microcrystalline Cellulose, tripoli, aluminum oxide and analogue.Practical liquid vehicle comprises water, ethanol, or ethylene glycol or water-ethanol/ethylene glycol mixture, and in these systems, compound can reach the dissolving or the dispersion of level of significance, can select nontoxic tensio-active agent auxiliary simultaneously.Can add auxiliary material such as perfume compound and antimicrobial additive, the performance that earmarks with optimization.Prepared liquid ingredient can be used as absorbable medicine pad, floods bandage or other dressings, or is sprayed onto disease sites with the atomizer of pump or aerosol.Thickening material is such as the synthetic polymkeric substance, lipid acid, soap or ester, Fatty Alcohol(C12-C14 and C12-C18), the Mierocrystalline cellulose of modification or the mineral substance of modification also can make together with liquid vehicle and be used for preparing the ointment that easily is coated with, gelifying agent, ointment, soap and analogue are to be directly used in patient skin.

Compound concentration in the liquid preparation is typically about 0.1wt% to wt25% (mass concentration), is about 0.5wt% to wt10% sometimes.Concentration in semisolid or solid preparation such as gel or the powder then is generally 0.1wt% to wt5%, is about 0.5wt% to 2.5wt% sometimes.The mixture that contains compound can be made unit dosage, and this can prepare according to the known conventional art of pharmacy corporation.Under normal condition, these technology comprise compound are become liquid form with pharmaceutical carrier and/or mixed with excipients, or the solid form of good distribution, or the both has, and product can be made certain shape as required then.The mixture that contains compound can be made any formulation, tablet for example, capsule, soft capsule, syrup, soft gel, suppository, and enema.This mixture also can be made the aqueous solution, the suspensoid of non-aqueous solution or blending agent.Water-soluble suspensoid can further include the material that some can increase viscosity, for example, and cellulose sodium carboxymethyl, sorbyl alcohol, and/or dextran.Can also comprise one or more stablizers in the suspensoid.

Compound or its active salt or derivative required content in treatment not only can change according to the salt of concrete selection, and can change, and finally to judge by make a round of visits doctor or clinician according to the character of route of administration, the disease of controlling and patient's age and self-condition.

A useful compound dosage forms is assessed by its external activity and/or intravital in vivo test of animal in cell or tissue usually.For example, infer that by the effective dose of mouse and other animal human dose is also by (for example, U.S.Pat.No.4,938,949) known to the scientist.This system can be used to judge the LD of a compound ₅₀(medium lethal dose) and ED ₅₀(the effective therapeutic dose of half).The ratio of toxic dose and therapeutic dose is called therapeutic index, and it can be expressed as ED ₅₀/ LD ₅₀The dosage of compound is reaching ED usually ₅₀And low toxicity or the nontoxic interior fluctuation of scope.The variation of dosage is decided by the formulation of administration and the route of administration that is adopted.For the used compound of any method herein, the treatment effective dose can be come according to a preliminary estimate by cell culture test.Dosage will reach IC by reaching the blood circulation concentration range according in vitro tests usually ₅₀Work out (concentration that is test-compound reaches maximum half that suppresses of symptom), and this information often can be judged the effective dose that is used for human body more accurately.Blood level can be measured, and for example, passes through high performance liquid chromatography.

The another one example that detects the acceptor effective dose is exactly " to dissociate " in the direct analysis recipient blood plasma and the measured matter content of " combination ".This analysis means can utilize analog antibody and/or " biosensor " that is obtained by molecular imprinting.With compound as template or " marking molecule ", before polymerisable monomer and polymerization catalyst to its spatial organization.Remove marking molecule subsequently, form and wherein to contain multiple compound " negative mirror image ", and can be under the bioanalysis condition polymer template of selectivity recombine molecule.(for example, Ansell, et al., CurrentOpinion in Biotechnology (1996) 7:89-94and in Shea, Trends in Polymer Science (1994) 2:166-173).This " marking " affine template can be checked with ligand binding assay, and fixed monoclonal antibody composition can be replaced by suitable marking template in the check.(for example, Vlatakis, et al., Nature (1993) 361:645-647).By adopting isotopic labeling, " dissociating " concentration of compound can be monitored easily and is used to calculate IC ₅₀These " marking " affine templates equally also can be designed as and contain fluorophor, and the character of dispersing of its photon can be along with the part of compound or selective binding and produce the variation that can survey.These variations can detect by fibre optical device easily in real time, subsequently according to its IC ₅₀Make the dosage of acceptor reach best rapidly.At Kriz, et al. discusses among Analytical Chemistry (1995) 67:2142-2144 to some extent about the example of " biosensor ".

Typical dosage comprises every kilogram of acceptor or sample weight number milligram or number microgram compound, for example, from every kilogram of about 1 microgram of acceptor to about 500 milligrams, from every kilogram of about 100 micrograms to 5 of acceptor milligram, or from every kilogram of about 1 microgram of acceptor to 50 micrograms.As everyone knows, the suitable dosage of small-molecule substance adjust to express or active aspect depend on the effectiveness of small-molecule substance.When giving animal with one or more these small-molecule substances (for example, human), for adjusting a polypeptide described herein or expression of nucleic acids or activity, doctor, animal doctor or researchist may at first leave a relatively low dosage, increase dosage subsequently again up to obtaining appropriate reaction.In addition, people know that specific dosage level all will depend on series of factors for any particular animals acceptor, the activity that comprises used specific compound, the acceptor age, body weight, healthy state, sex and diet, administration time, route of administration, discharge rate, any medicine unite use, and express or active adjustment degree.

Following Example can be explained, but be not limited to the present invention.

Embodiment

Below for synthesizing the canonical process of substituted quinobenzoxazine analogs.

Example 1

The preparation of substituted quinobenzoxazine analogs

The conventional synthetic schemes of substituted quinobenzoxazine analogs preparation is shown in scheme 1.

Scheme 1

(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl)-the acetic ester ethyl ester (Ethyl-(2 ', 3 ', 4 ', 5 '-tetrafluorobenzoyl)-ethanoate)

Under 10-15 ℃, with 3.66g, the propanedioic acid potassium of 21.5mmol (Potassium ethyl malonate), 2.44g, the MgCl of 25.7mmol ₂And 2.05g, the TEA of 20.3mmol mixed 2.5 hours in the 70ml acetonitrile.In the time of 0 ℃, with 2.00g, 10.3mmol 2,3,4, and the 5-phenyl tetrafluoride formyl chloride (2,3,4,5-tetrafluorobenzoylchloride) be added in 10 milliliters of acetonitriles, add 0.23g once more after 15 minutes, the TEA of 2.3mmol.After being heated to room temperature, mixture was stirred 16 hours.After vacuum is removed volatile component, add the toluene (Toluene) of 30ml, vacuum is removed then.Add toluene (60ml) subsequently, 1.5M HCl (40ml) guarantees that temperature is no more than 25 ℃.Organic phase respectively cleans twice with HCl 25ml and the 25ml water of 1.5M, uses MgSO ₄Dry and under vacuum, be kept to the slightly oil of tangerine look ([M+1] ⁺265,98%).

2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl)-3-(dimethylamino)-propyl group-2-olefin(e) acid ethyl ester (Ethyl-2-(2 ', 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate)

With 0.61g, 5.1mmol dimethylacetal dimethyl formamide (Dimethyl acetal dimethylformamide) dropwise add 0.9g, 3.41mmol ethyl (2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl)-(ethyl-(2 ' for acetic ester, 3 ', 4 ', 5 '-tetrafluorobenzoyl)-ethanoate), and logical argon gas dissolving in 2ml acetic anhydride (acetic anhydride).30 minutes final vacuums remove down and desolvate, and obtain tangerine look oily the product ([M+1 of certain output ⁺] 320).

1,2-two fluoro-4-oxygen-4H-pyrido [3,2,1-kl]-8-phenyl-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate)

With 3.4g, 8.0mmol 2 (2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(dimethylamino)-propyl group-(Ethyl-2-(2 ', 3 ' for 2-olefin(e) acid ethyl ester, 4 ', 5 '-tetrafluorobenzoyl-)-and 3-(dimethylamino)-prop-2-enoate) and 1.5g, the 2-amino of 8.0mmol-4-phenyl-phenol (2-amino-4-phenyl-phenol) places 20mlDMSO, stirs 30 minutes under 60 ℃ of vacuum.The K2CO3 of 5g and the MeCN of 20ml are joined in the suspension, be heated to 80 ℃ to 1 hour.After being cooled to room temperature, also filter in the dilute sulphuric acid that the mixture impouring is excessive a little.Product is tawny solid ([M+1] ⁺420,65%).

Example 2

1,2-two fluoro-4-oxygen-4H-pyrido [3,2,1-kl]-8-phenyl-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylic acid) Preparation

With 2.2g, 5.3mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-phenyl-azophenlyene-5-carboxylic acid, ethyl ester (Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate) mixing refluxed two hours in dense HCl of 20ml and 20ml acetic acid.Behind the cool to room temperature, adding the 40ml frozen water in reaction mixture, with the sedimentation and filtration that obtains, and is 90% tawny solid ([M+1] with the ether washing to obtain productive rate ⁺392).

Example 3

2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(naphthyl-2 ", 3 "-diamino)-propyl group-2-olefin(e) acid ethyl ester (Ethyl-2-(2 ', 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-3-(napthyl-2 ", 3 "-diamino)-prop-2-enoate) Preparation

To be dissolved with 10.53g, the 2-(2 ' of 33mmol, 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(dimethylamino)-propyl group-(Ethyl-2-(2 ' for 2-olefin(e) acid ethyl ester, 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-50ml acetonitrile (acetonitrile) liquid of 3-(dimethylamino)-prop-2-enoate) adds 150ml and is dissolved with 5.22g, 2 of 33mmol, the 3-diaminonaphthalene (2, in acetonitrile 3-diaminonapthalene) (acetonitrile) solution, and under the condition of logical argon gas, maintain 50 ℃.After 3 hours, remove volatile component under the vacuum condition, the residuum silica dehydrator is that the chromatogram of ethyl acetate/normal hexane (EtOAc/Hexane) of 15% is separated with condition again, obtains the yellow product ([M+1] of 55% productive rate ⁺433).

Example 4

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-benzene diazine-5-carboxylic acid, ethyl ester (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phendiazine-5-carboxylate) Preparation

With 600mg 1.4mmol-2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(naphthalene ester-2 "; 3 " diamino)-propyl group-(Ethyl-2-(2 ', 3 ', 4 ' for 2-olefin(e) acid ethyl ester, 5 '-tetrafluorobenzoyl-)-3-(napthyl-2 ", 3 " diamino)-prop-2-enoate) be dissolved in 500ml to be dissolved with K ₂CO ₃The DMF homogenate in, 100 ℃ of these mixtures of following vigorous stirring 1 hour are cooled to room temperature subsequently.Remove by filter K ₂CO ₃And vacuum is removed DMF, obtains the yellowish brown solid of higher yields.([M+1] ⁺393)。

Example 5

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-benzene diazine-5-carboxylic acid (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phendiazine-5-carboxylic acid) system Be equipped with

With KOH solution (1N, 2.54ml, 2.56mmol) join 400ml and be dissolved with 500mg, 1.28mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzo [g]-benzene diazine-5-carboxylic (1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phendiazine-5-carboxylate) ethanol (ethanol) solution in, reflux. after two hours, reaction mixture is cooled to room temperature, and (1N) transfers to neutrality with HCl solution. and filter and obtain yellow solid, productive rate 89%. ([M+1] ⁺365).

Example 6

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-benzene diazine-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate) system Be equipped with

2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(N-aminophenyl disulfide group)-propyl group-2-olefin(e) acid ethyl ester (Ethyl-2-(2 ', 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate)

10ml is dissolved with 17.7g, 55.3mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(dimethylamino)-propyl group-(Ethyl-2-(2 ' for 2-olefin(e) acid ethyl ester, 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-acetonitrile (acetonitrile) solution of 3-(dimethylamino)-prop-2-enoate) joins 100ml and is dissolved with 5.22g, 1 of 33mmol, in acetonitrile (acetonitrile) solution of 2-aminothiophenol dimer (1,2-aminothiophenol dimer).After 3 hours, vacuum is removed volatile component, and the residuum silica dehydrator is used 1% ethanol/methylene (MeOH/DCM) chromatographic separation afterwards, obtains the yellow solid product of productive rate 50%.([M+1] ⁺523)。

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-benzene diazine-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate)

With 2.5g, 3.2mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(N-aminophenyl disulfide group)-propyl group-(Ethyl-2-(2 ', 3 ', 4 ' for 2-olefin(e) acid ethyl ester, 5 '-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate) is dissolved in the dimethyl formamide (DMF) of 120ml reflux six hours.Vacuum is removed DMF, obtains the yellow solid product of productive rate 90%.([M+1] ⁺360)。

Example 7

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-benzene diazine-5-carboxylic acids The preparation of (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylic acid)

With KOH solution (1N, 3.0,3.0mmol) join 400ml and be dissolved with 1000mg, 1 of 2.5mmol, 2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-benzene diazine-5-carboxylic acid, ethyl esters

(ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate) ethanol (ethanol) solution in, reflux.Behind the mixture reaction two hours, be cooled to room temperature, use HCl solution (1N) neutralization subsequently.Filter and collect yellow solid product, productive rate 95%, ([M+1] ⁺332)

Example 8

5,6-two fluoro-9-hydroxyls-3-oxygen-3H-pyridyl [3,2,1-kl] pyrimidyl [g] azophenlyene-2-carboxylic acid (5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl] pyrimido[g] phenoxazine-2-carboxylic Acid) preparation

7-nitro-quinazoline-4, and the 6-glycol (7-nitroquinazoline-4,6-diol)

With 1.4g, 6.3mmol 6-methoxyl group-7-nitro-3, (6-methoxy-7-nitro-3 4-dihydroquinazolin-4-one) joins and contains 20ml 4-dihydroquinazoline-4-ketone, in the solution of 48% hydration HBr and the AcOH of 20ml, again the mixture reflux is spent the night.With the solution evaporation drying that obtains, obtaining residue is that (1.2g 5.8mmol), need not be further purified and can use (M+1,208) the phenol crude product.

7-amido quinazoline-4, the 6-glycol

(1.0g 5.8mmol) with the dilution of 40ml water, adds 3g tin chloride dihydrate (Tin II chloride dihydrate), at room temperature stirring reaction again with the crude product that obtains above.React after 1 hour, use K ₂CO ₃Handle to neutral, use EtOAc (3 * 50mL) extractions again.The organic phase of extraction is merged, use dried over sodium sulfate, solvent removed in vacuo obtains 1.0g, the monoethanolamine of 5.6mmol (amino alcohol) crude product (M+1,178).

56-two fluoro-9-hydroxyls-3-oxygen-3H-pyridyl [32,1-kl] pyrimidyl [g] azophenlyene-2-carboxylic acid, ethyl ester (Ethyl; 5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl] pyrimido[g] phenoxazine-2-carboxylat E)

With 1.0g, 5.6mmol amino phenol (aminophenol) joins 3mL and is dissolved with 2.2g, in the DMSO solution of the tetrafluoro enamine (tetrafluoroenamine) of 6.9mmol, reaction mixture is stirred 20 minutes down in 60 ℃ of vacuum (Rotary Evaporators).Then reaction mixture is cooled to room temperature, adds salt of wormwood (potassium carbonate) again with 200mL acetonitrile (acetonitrile) dilution.With mixture reflux 5 hours, impouring dilute acetic acid/aqueous solution (HOAc/water) then.Solid product was taken out by vacuum and was collected and drying, obtained 1.3g, the pale brown look solid product difluoro ester (difluoroester) (M+1,412) of 3.2mmol.

5,6-two fluoro-9-hydroxyls-3-oxygen-3H-pyridyl [3,2,1-kl] pyrimidyl [g] azophenlyene-2-carboxylic acid (5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl] pyrimido[g] phenoxazine-2-carboxyl Ic acid)

With 1.3g, the difluoro ester of 3.2mmol (difluoroester) is dissolved in 1: 1 mixture of 20mL Glacial acetic acid (glacialacetic acid) and 12M hydrochloric acid (HCl) and refluxed 30 minutes.Then mixture is cooled to room temperature, in the impouring water.Vacuum filtration and the dry solid product of collecting obtain 0.98g, pale brown look solid two fluoric acids (difluoroacid) ([M+1] of 2.5mmol ⁺392).

Example 9

2-(2-(ethoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-9-base oxygen base) acetic acid (2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazin-9-yloxy) aceti C acid) preparation

5,6-two fluoro-9-hydroxyls-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl ester (Ethyl 5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate)

With 2.5g, 15.5mmol 2,4-hydrochloric acid dihydroxy aniline (2,4-dihydroxyaniline hydrochloride) joins 12mL and be dissolved with 5.8g, 18.2mmol dimethyl sulfoxide (DMSO) (DMSO) solution of tetrafluoro enamine (tetrafluoroenamine) in, with mixture under vacuum (Rotary Evaporators), be heated to 60 ℃ 20 minutes. with reaction mixture with the 100mL acetonitrile (acetonitrile) dilution, add 3g salt of wormwood again, then mixture is refluxed and spend the night. mixture is cooled to room temperature, solvent removed in vacuo then. add slight excessive 2MHCl with rapid dissolved carbon hydrochlorate, the solid precipitation that obtains is filtered and drying, obtain 5.0g, 13.9mmol pale brown look solid difluoro ester (difluoroester) (M+1,360).

2-(2-(ethoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-9-base oxygen base) acetic acid (2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazin-9-yloxy) acet Ic acid)

2.0g salt of wormwood (potassium carbonate) is joined 30mL be dissolved with 2.1g, 5.8mmol difluoro ester (difluoroester) and 2.0g, 10.3mmol in the DMF solution to butyl bromide acetic acid (tert-butylbromoacetate), then with mixture heating up to 60 ℃ 1 hour.To react cooling then, and in the impouring 500mL water, with ethyl acetate (ethyl acetate) (3 * 100mL) extractions, normal saline washing, sal epsom (magnesium sulfate) drying, silicagel pad (30 * 50mm) filtrations, ethyl acetate elution.Remove under the vacuum and desolvate, the material that obtains is added normal hexane grind also drying, obtain 2.8g, the pale brown look solid of 5.8mmol is to butyl ester (tert-butyl ester).Material is dissolved in the 40mL tetrafluoro acetate (trifluoroacetic acid), stirred 30 minutes under the room temperature.Solvent removed in vacuo obtains 2.4g, the pale brown look solid acid (M+1,418) of 5.7mmol.

Example 10

9-(methoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acids (9-(Carboxymethoxy)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic Acid) preparation

With 2.4g, 5.7mmol difluoro ester (difluoroester) is dissolved in 1: 1 mixture of 40mL Glacial acetic acid (glacial aceticacid) and 12M hydrochloric acid (HCl), refluxes 1 hour.Then mixture is cooled to room temperature, in the impouring water.Then the solid product vacuum filtration is collected and drying, obtained 2.0g, pale brown look solid two fluoric acids (difluoroacid) (M+1,390) of 5.1mmol.

Example 11

1,2,3-three fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acid, ethyl ester (Ethyl 1,2,3-trifluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylate) preparation

With 3.5g, 21.9mmol 3-amino-beta naphthal (3-amino-2-naphthol) is dissolved in 12mL and is dissolved with 8g, 23.7mmol in the DMSO solution of five fluorine enamines (pentafluoroenamine) (preparation process and tetrafluoro enamine (tetrafluoroenamine) are similar), under the vacuum (Rotary Evaporators) with mixture heating up to 60 ℃ to two hours.Reaction mixture with 200mL acetonitrile (acetonitrile) dilution, is added 8.0g salt of wormwood (potassium carbonate) again, mixture is refluxed spend the night then.Mixture is cooled to room temperature, solvent removed in vacuo.Add slight excessive 2M HCl with rapid dissolved carbon hydrochlorate, solid precipitation is filtered and drying, obtain 1.3g, the pale brown look solid difluoro ester (difluoroester) (M+1,412) of 3.2mmol.

Example 12

1,2,3-three fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acids (1,2,3-Trifluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylic acid) Preparation

With 1.3g, 3.2mmol trifluoto ester (trifluoroester) is dissolved in the 5mL acetic acid (acetic acid), and adds the 12M HCl of 5mL, then with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 1.0g, the white solid trifluoto ester (trifluoroacid) (M+1,384) of 2.6mmol.

Example 13

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylate) Preparation

Under the room temperature with 10g, 63mmol 3-amino-beta naphthal (3-amino-2-naphthol) joins 100mL and is dissolved with 30g, in the ethyl acetate of 94mmol enamine (enamine) (ethyl acetate) solution, immediately mixture is placed Rotary Evaporators then, at 0 ℃ after following more than two hours, (having ice to form on the flask) removed to desolvate and obtained yellow solid.In this solid, add 200mL ether (ether), homogenate is filtered obtain yellow solid.Subsequently solid is dissolved among the 200mL DMF, and adds 16.5g, 120mmol salt of wormwood (potassium carbonate), then with mixture heating up to 90 ℃ to 1 hour.Mixture is cooled to room temperature, adds 500mL water then, with the solid filtering that obtains, washing is also dry, obtains 12.2g, the pale brown look solid difluoro ester (difluoroester) (M+1,394) of 30.8mmol.

Example 14

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylic acid) system Be equipped with

With 5g, 12.7mmol difluoro ester (difluoroester) is dissolved in the 50mL methyl alcohol (methanol), adds the dense HCl of 20mL again, then mixture is refluxed 12 hours.Mixture is cooled to room temperature, and vacuum filtration is collected solid, and uses methanol wash, obtains 3.6g, light brown yellow powder two fluoric acids (difluoroacid) (M+1,366) of 9.9mmol.

Example 15

1-fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acid, ethyl ester (Ethyl 1-fluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylate) Preparation

Under the room temperature with 5.0g, 31.2mmol 3-amino-beta naphthal (3-Amino-2-naphthol) joins 100mL and is dissolved with 14g, 46.3mmol in ethyl acetate (ethylacetate) solution of enamine (enamine) (preparation is similar to the tetrafluoro enamine), mixture is placed Rotary Evaporators immediately, at 0 ℃ after following more than two hours, (having ice to form on the flask) removed to desolvate and obtained yellow solid. in this solid, add 200mL methyl alcohol (methanol), the homogenate filtration is obtained yellow solid. then solid is dissolved in the 200mL acetonitrile (acetonitrile), add 10.0g again, 72.5mmol salt of wormwood (potassium carbonate), then with mixture heating up to 80 ℃ to 1 hour.Mixture is cooled to room temperature, adds 500mL water, with the solid filtering that obtains, washing is also dry, obtains 6.0g, the pale brown look solid fluorine ester (fluoroester) (M+1,376) of 16.0mmol.

Example 16

1-fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [g]-azophenlyene-5-carboxylic acid (1-Fluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[g]-phenoxazine-5-carboxylic acid) preparation

With 6.0g, 16.0mmol fluorine ester (fluoroester) was dissolved in the 10mL acetic acid (acetic acid), and adds 10mL 12MHCl, with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and desciccate, obtains 4.8g, the white solid fluoric acid (fluoroacid) (M+1,348) of 13.8mmol.

Example 17

9-chloro-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl esters (Ethyl 9-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate) Preparation

With 5.0g, 34.8mmol 5-chloro-2-amino-phenol (5-chloro-2-aminophenol) joins 200mL and is dissolved with 14.4g, 45.3mmol in the ethyl acetate of enamine (enamine) (ethyl acetate) solution, use the Rotary Evaporators vacuum to revolve and steam two hours, do not heat except that desolvating.Add methyl alcohol (Methanol), with phenol enamine (phenolic enamine) vacuum filtration that obtains.The 7.0g solid that obtains is dissolved in adding salt of wormwood (potassium carbonate) in the acetonitrile (acetonitrile), with the mixture reflux that obtains two hours. then mixture is cooled to room temperature, be poured among rare HCl. the solid vacuum filtration that obtains is collected and drying, obtain 5.0g, 13.3mmol faint yellow solid difluoro ester (difluoroester) (M+1,378).

Example 18

9-chloro-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acids The system of (9-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid) Be equipped with

With 5.0g, 13.3mmol difluoro ester (difluoroester) is dissolved in the 45mL acetic acid (acetic acid), adds 30mL 12M HCl again, then with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and is dry, obtains 4.0g, white solid two fluoric acids (difluoroacid) (M+1,350) of 10.6mmol.

Example 19

10-chloro-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl esters (Ethyl 10-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate) preparation

With 5.0g, 34.8mmol 4-chloro-2-amino phenol (4-chloro-2-aminophenol) joins 200mL and is dissolved with 14.4g, 45.3mmol in the ethyl acetate of enamine (enamine) (ethyl acetate) solution, revolve two hours solvent removed in vacuo of steaming, do not heat with Rotary Evaporators.Add methyl alcohol (Methanol), with phenol enamine (phenolic enamine) vacuum filtration that obtains.The 7.5g solid that obtains is dissolved in the acetonitrile (acetonitrile), adds salt of wormwood (potassium carbonate) then, and with the mixture reflux that obtains two hours.Then mixture is cooled to room temperature, among the rare HCl of impouring.The solid vacuum filtration that obtains is collected and drying, obtained 5.0g, the faint yellow solid difluoro ester (difluoroester) (M+1,378) of 13.3mmol.

Example 20

10-chloro-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acids (10-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid) Preparation

2.5g difluoro ester (difluoroester) is dissolved in the 25mL acetic acid (acetic acid), adds 20mL 12M HCl, then with mixture reflux two hours.Then mixture is cooled to room temperature, in the impouring water, the solid product vacuum filtration that obtains is collected and drying, obtain 2.0g, 5.3mmol white solid two fluoric acids (difluoroacid) (M+1,350).

Example 21

5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl esters The preparation of (Ethyl 5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate)

Under the room temperature with 1.9g, 17.43mmol 2-amino phenol (2-aminophenol) joins 50mL and is dissolved with 5.7g, 17.9mmol in the ethyl acetate of enamine (enamine) (ethyl acetate), immediately mixture is placed Rotary Evaporators afterwards, at 0 ℃ after following more than two hours, (having ice to form on the flask) removed to desolvate and obtained yellow solid.In this solid, add 25mL ether (ether), homogenate is filtered obtain yellow solid again.Then this solid is dissolved among the 20mL DMF, adds 2.9g again, 21mmol salt of wormwood (potassiumcarbonate), with mixture heating up to 90 ℃ to 1 hour.Mixture is cooled to room temperature, adds 200mL water, with the solid filtering that obtains, washing, drying obtains 2.9g, the pale brown look solid azophenlyene (phenoxazine) (M+1,344) of 8.45mmol.

Example 22

5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acids The preparation of (5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid)

With 5.0g, 14mmol difluoro ester (difluoroester) is dissolved in the 50mL methyl alcohol (methanol), adds the dense HCl of 20mL, and mixture was refluxed two hours.Mixture is cooled to room temperature, and vacuum filtration is collected solid, with methyl alcohol (methanol) flushing, obtains 4.2g, light brown yellow powder two fluoric acids (difluoroacid) of 13.3mmol, productive rate 91% (M+1,316).

Example 23

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [h]-azophenlyene-5-carboxylic acid, ethyl ester (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[h]-phenoxazine-5-carboxylate) preparation

With 5.0g, 31.3mmol 1-amino-beta naphthal (1-amino-2-naphthol) joins 200mL and is dissolved with 14.0g, 45.3mmol (enamine) in the ethyl acetate of enamine (ethyl acetate) solution, revolve two hours solvent removed in vacuo of steaming, do not heat with Rotary Evaporators.Add methyl alcohol (Methanol), with phenol enamine (phenolic enamine) vacuum filtration that obtains.Solid is dissolved in the acetonitrile (acetonitrile), adds 10g salt of wormwood (potassium carbonate), with mixture reflux two hours.Then mixture is cooled to room temperature, in the impouring dilute hydrochloric acid.The solid vacuum filtration that obtains is collected and drying, obtained 5.0g, the faint yellow solid difluoro ester (difluoroester) (M+1,376) of 13.3mmol.

Example 24

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [h]-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[h]-phenoxazine-5-carboxylic acid) system Be equipped with

5.5g difluoro ester (difluoroester) is dissolved in the 25mL acetic acid (acetic acid), adds 20mL 12M HCl, with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 5.0g, white solid two fluoric acids (difluoroacid) (M+1,348) of 14.4mmol.

Example 25

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [f]-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[f]-phenoxazine-5-carboxylate) Preparation

With 15.0g, 93.8mmol 2-amino-1-naphthols (2-amino-1-naphthol) joins 500mL and is dissolved with 45g, in the ethyl acetate of 141mmol enamine (enamine) (ethyl acetate), revolve two hours solvent removed in vacuo of steaming with Rotary Evaporators, do not heat. add methyl alcohol (Methanol), the phenol enamine that vacuum filtration obtains (phenolic enamine).The solid that obtains is dissolved in the 400mL acetonitrile (acetonitrile), adds 25g salt of wormwood (potassium carbonate), with mixture reflux 2.5 hours.Then mixture is cooled to room temperature, the rare HCl of impouring.The solid vacuum filtration that obtains is collected and drying, obtained 19.69g, the faint yellow solid difluoro ester (difluoroester) (M+1,394) of 50.1mmol.

Example 26

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [f]-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[f]-phenoxazine-5-carboxylic acid) system Be equipped with

With 15.0g, 38.1mmol difluoro ester (difluoroester) is dissolved in the 60mL acetic acid (acetic acid), adds 60mL 12M HCl, then with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, the solid product vacuum filtration is collected and drying, obtain 11.7g, white solid two fluoric acids (difluoroacid) (M+1,366) of 32mmol.

Example 27

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [b] furans [2,3-g]-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[b] furan[2,3-g]-phenoxazine-5-carboxylat E) preparation

3-ADP and furans-2-alcohol (3-Aminodibenzofuran-2-ol)

In the time of 0 ℃, will be dissolved in CH with dropping funnel ₂Cl ₂200mL, 1MBBr ₃Be added to 500mL and be dissolved with 15g, in the monochloro methane of 70.4mmol diphenylene-oxide (dibenzofuran) (methylene chloride).Wait to dropwise, mixture was placed room temperature condition following 1 hour, use water cooling then, add 40g salt of wormwood (potassium carbonate) subsequently.Vacuum filtration obtains solid again, and drying obtains 13.2g, the white solid hydroxyl diphenylene-oxide (hydroxyl dibenzofuran) (M+1,200) of 199mmol.

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [b] furans [2,3-g]-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[b] furan[2,3-g]-phenoxazine-5-carboxylat E)

With 12.0g, 60mmol hydroxyl diphenylene-oxide (hydroxyldibenzofuran) joins 30mL and is dissolved with 15.0g, in the DMSO solution of 47mmol tetrafluoro enamine (tetrafluoroenamine), mixture is heated to 60 ℃ to 20 minutes under vacuum (Rotary Evaporators).Then reaction mixture is diluted with 200mL acetonitrile (acetonitrile), add 17g salt of wormwood (potassium carbonate), again mixture was refluxed 2.5 hours.Mixture is cooled to room temperature, solvent removed in vacuo.Add the rapid dissolved carbon hydrochlorate of excessive in a subtle way 2M HCl, solid precipitation is filtered, drying obtains 15.0g, the pale brown look solid difluoro ester (difluoroester) (M+1,434) of 34.6mmol.

Example 28

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl] benzos [b] furans [2,3-g]-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl] benzo[b] furan[2,3-g]-phenoxazine-5-carboxylic Acid) preparation

With 15.0g, 34.6mmol difluoro ester (difluoroester) was dissolved in the 60mL acetic acid (acetic acid), adds 60mL 12M HCl, with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 13.7g, 34mmol white solid two fluoric acids (difluoroacid) (M+1,406).

Example 29

2-(ethoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-10-carboxylic acid, ethyl esters (Ethyl 2-(ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-10-carboxylate) Preparation

With 3.0g, 19.6mmol the amino M-nitro benzoic acid of 4-hydroxyl-3-(4-hydroxy-3-amino benzoic acid) joins 25mL and is dissolved with 7.0g, 21.9mmol in the DMSO solution of tetrafluoro enamine (tetrafluoroenamine), with mixture under vacuum (Rotary Evaporators), be heated to 60 ℃ two hours.Then reaction mixture is diluted with 200mL acetonitrile (acetonitrile), add 8.0g salt of wormwood (potassium carbonate), mixture is refluxed spend the night.Mixture is cooled to room temperature, solvent removed in vacuo.Add excessive a little 2MHCl with rapid dissolved carbon hydrochlorate,, obtain 6.2g, the pale brown look solid difluoro ester (difluoroester) (M+1,388) of 16.0mmol the excessive drying of solid precipitation.

Example 30

2-(ethoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-10-carboxylic acids (2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-10-carboxylic Acid) preparation

With 6.2g, the 16.0mmol difluoro ester was dissolved in 25mL (difluoroester) acetic acid (acetic acid), adds 20mL 12M HCl, with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 5.3g, the white solid difluoro diacid (difluorodi-acid) (M+1,360) of 14.8mmol.

Example 31

5,6-two fluoro-10-nitros-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl ester (Ethyl 5,6-difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate) Preparation

To be dissolved with 6.0g, 18.7mmol enamine (enamine) and 3.5g, acetonitrile (acetonitrile) solution of 23.3mmol 2-amino-4-naphthols be heated to 80 ℃ 15 minutes.Add 8.0g salt of wormwood (Potassiumcarbonate) then, the mixture reflux is spent the night.With the reaction mixture filtered while hot, solvent removed in vacuo obtains 5.0g then, the crude product nitric ether (nitroester) (M+1,389) of 12.8mmol.

Example 32

5,6-two fluoro-10-nitric acid-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid The system of (5,6-Difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid) Be equipped with

With 5.0g, 12.8mmol crude product difluoro ester (difluoroester) was dissolved in the 25mL acetic acid (acetic acid), adds 20mL 12M HCl, with reaction mixture refluxed heating two hours.Then reaction mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 2.0g, white solid two fluoric acids (difluoroacid) (M+1,361) of 5.5mmol.

Example 33

5,6-two fluoro-3-oxygen-10-phenyl-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid, ethyl ester (Ethyl 5,6-difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylate) preparation

To be dissolved with 5.4g, 16.9mmol enamine (enamine) and 3.5g, acetonitrile (acetonitrile) solution of 18.9mmol 3-amino-4-hydroxy biphenyl (3-amino-4-hydroxybiphenyl) be heated to 80 ℃ 90 minutes.Add 8.0g salt of wormwood (Potassium carbonate) then, the mixture reflux is spent the night.With the reaction mixture filtered while hot, solvent removed in vacuo obtains 3.9g then, the crude product difluoro ester (difluoroester) (M+1,420) of 9.3mmol.

Example 34

5,6-two fluoro-3-oxygen-10-phenyl-3H-pyridyl [3,2,1-kl] azophenlyene-2-carboxylic acid (5,6-Difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl] phenoxazine-2-carboxylic acid) Preparation

With 3.6g, 8.6mmol crude product difluoro ester (difluoroester) was dissolved in the 10mL acetic acid (acetic acid), adds 10mL 12M HCl, with reaction mixture refluxed heating two hours.Then mixture is cooled to room temperature, in the impouring water, vacuum filtration is collected and dried solid product, obtains 2.6g, white solid two fluoric acids (difluoroacid) (M+1,392) of 6.6mmol.

Example 35

1,2-two fluoro-4-oxygen-4H-11-sulfonic acid-pyridyl [3,2,1-kl] benzos [h]-azophenlyene-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-11-sulfonic-acid-pyrido[3,2,1-kl] benzo[h]-phenoxazine-5-carboxy Late) preparation

With 4.8g, 20mmol 1-amino-2-hydroxyl-4-naphthene sulfonic acid (1-amino-2-hydroxy-4-naphthalenesulfonic acid) joins 30mL and is dissolved with 5.4g, 16.9mmol among the DMSO of tetrafluoro enamine (tetrafluoroenamine), mixture is heated to 60 ℃ to two hours under vacuum (Rotary Evaporators).In reaction mixture, add 10.0g salt of wormwood (potassiumcarbonate), with mixture heating up to 60 ℃ 1 hour.Mixture is cooled to room temperature, adds excessive a little 2M HCl with rapid dissolved carbon hydrochlorate (carbonate).Water layer is transferred to other container, and remaining organic layer is dissolved in 100mL methyl alcohol (methanol), uses 200mL ethyl acetate (ethyl acetate) to make its precipitation again, with the solid precipitation filtration drying, obtain 3.1g, the brown solid sulfonic acid of 6.5mmol (sulfonic acid) (M+1,474).

Example 36

1,2-two fluoro-4-oxygen-4H-11-sulfonic group-pyridyl [3,2,1-kl] benzos [h]-azophenlyene-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-11-sulfonic-pyrido[3,2,1-kl] benzo[h]-phenoxazine-5-carboxylic Acids) preparation

With 1.5g, 3.2mmol crude product difluoro ester (difluoroester) was dissolved in the 10mL acetic acid (acetic acid), adds 10mL 12M HCl, with reaction mixture refluxed heating 30 minutes.Solvent removed in vacuo obtains 1.1g, the brown solid sulfonic acid of 2.5mmol (sulfonic acid) (M+1,446)

Example 37

2-(ethoxycarbonyl)-5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-9-carboxylic acids (2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-9-carboxylic Acid) preparation

To be dissolved with 5.2g, 16.3mmol difluoro enamine (difluoroenamine) and 4.0g, the DMSO solution of 26.1mmol 4-amino-3-hydroxy formic acid (4-amino-3-hydroxybenzoic acid) stirred under room temperature 1.5 hours.Add 8g salt of wormwood (Potassium carbonate) then, (Rotary Evaporators) under the reaction mixture vacuum stirred 1 hour.Then with mixture heating up to 100 ℃ 1 hour, cool to room temperature again.Then with reaction mixture impouring 500mL 1M H ₂SO ₄In, vacuum filtration obtains solid again.With the gained solid drying, obtain 5.0g, pale brown look solid crude product two fluoric acids (difluoroacid) (M+1,388) of 12.9mmol.

Example 38

5,6-two fluoro-3-oxygen-3H-pyridyl [3,2,1-kl] azophenlyene-2,9-dicarboxylic acid The preparation of (5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl] phenoxazine-2,9-dicarboxylic acid)

With 5.0g, 12.9mmol crude product difluoro ester (difluoroester) was dissolved in the 20mL acetic acid (acetic acid), adds 20mL 12M HCl, with reaction mixture refluxed heating 1 hour.Room temperature is reduced in reaction, add entry.Vacuum filtration obtains solid, and dried overnight obtains 1.9g, the pale brown look solid diacid (di-acid) (M+1,360) of 5.3mmol.

Example 39

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-Fluorenone-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate) preparation

3-nitro-2-hydroxyl Fluorenone (3-Nitro-2-hydroxyfluorenone)

Under the normal temperature, 100ml is dissolved with 3.52g, 25.5mmol NO ₂BF ₄Acetonitrile (acetonitrile) drips of solution be added to 400ml and be dissolved with 5g, 25.5mmol in acetonitrile (acetonitrile) solution of 2-hydroxyl Fluorenone (2-hydroxyfluorenone). reaction mixture is cooled to 0 ℃ then, add 100ml water again and make contamination precipitation. after the filtration, add 200ml water, sedimentation and filtration is obtained red solid, productive rate 68% (M+1,242).

3-amino-2-hydroxyl Fluorenone (3-amino-2-hydroxyfluorenone)

To contain 1.6g, 6.6mmol 3-nitro-2-hydroxyl Fluorenone (3-nitro-2-hydroxyfluorenone) and 3g, 6.6mmol SnCl ₂100ml acetic acid (acetic acid)-dense HCl equal to reflux 1 hour in 1: 1 the mixture.Mixture is cooled to room temperature, adds ammoniacal liquor (ammonium hydroxide) neutralization.After 100ml ethyl acetate (EtOAc) extraction 3 times, the organic phase of merging sal epsom (magnesium sulfate) drying, evaporating water obtains the brown solid product, productive rate 65% (M+1,212).

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-Fluorenone-5-carboxylic acid second Ester (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate)

50ml is contained 0.9g, 4.26mmol 3-amino-2-hydroxyl Fluorenone (3-amino-2-hydroxyfluorenone) and 1.36g, 4.26mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(dimethylamino)-propyl group-(ethyl-2-(2 ', 3 ' for 2-olefin(e) acid ethyl ester, 4 ', 5 '-tetrafluorobenzoyl-)-the DMSO mixture of 3-(dimethylamino)-prop-2-enoate) heated 18 hours under vacuum.Product with the extraction of ethyl acetate/salt solution (EtOAc/Brine) liquid, is merged organic phase, and drying obtains the red solid product.Solid is dissolved in 40ml contains excessive K ₂CO ₃DMSO solution in, be heated to 100 ℃ to 30 minutes.Behind the cool to room temperature, add 30ml salt solution, collecting precipitation obtains yellow solid, and two step productive rates are 60% (M+1,446).

Example 40

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-Fluorenone-5-carboxylic acids The preparation of (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylic acid)

To be dissolved with 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-Fluorenone-5-carboxylic acid, ethyl ester (ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate) acetic acid (aceticacid) and each 50ml of dense HCl, by 1: 1 blended mixture reflux two hours.After being cooled to room temperature, add 50ml water, collect product, obtain yellow solid, productive rate 94% (M+1,418).

Example 41

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-anthraquinone-5-carboxylic acid, ethyl ester (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylate) preparation

Will about 10ml DMSO, wherein contain 5.54g, 23.2mmol 3-amino-2-hydroxyanthraquinone (3-amino-2-hydroxyanthraquinone) and 8.7g, 34.8mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-3-(dimethylamino)-propyl group-(ethyl-2-(2 ' for 2-olefin(e) acid ethyl ester, 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-mixture of 3-(dimethylamino)-prop-2-enoate) heated 24 hours under vacuum.Add 50ml water and make the product precipitation.Place the vacuum drying oven inner drying to spend the night solid, be dissolved in 40ml and contain excessive K ₂CO ₃DMSO solution in, be heated to 100 ℃ 30 minutes.After being cooled to room temperature, add 30ml salt solution, the yellow solid product precipitation that obtains is collected, two step productive rates 60% (M+1,474).

Example 42

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-anthraquinone-5-carboxylic acids The preparation of (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylic acid)

To contain 3.5g, 6.8mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-(ethyl 1 for 8-anthraquinone-5-carboxylic acid, ethyl ester, 2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinonenone-5-carboxylate) acetic acid (acetic acid) and each 50ml of dense HCl be by 1: 1 blended mixture reflux two hours.After being cooled to room temperature, add 50ml water, collect product and get yellow solid, productive rate 94% (M+1,446).

Example 43

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-phenyl-isoxazolyl benzenesulfonamide-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate) preparation

Amino Resorcinol (the 2-amino (t-butoxy carbonyl)-5-amino of 2-amino (to tertbutyloxycarbonyl)-5- Hydroquinone)

At room temperature, 20ml is contained 7.17g, the tertbutyloxycarbonyl acid anhydride of 33mmol (Boc anhydride) and 17ml, the DMSO drips of solution of 99mmol DIEA is added to the 7g under stirring, and 33mmol 1,4-dihydroxyl-2, (1,4-dihydroxy-2 is 5-diaminobenzene) in the solution for the 5-diaminobenzene.Stir after 18 hours, separated product in ethyl acetate and salt solution merges organic phase, and uses MgSO ₄Dry.Except that after desolvating, residuum is crossed silica gel chromatographic column, and with the elution of 25-50%EtOAc normal hexane (hexane) solution, obtain clean product, productive rate 45% (M+1,239).

4-hydroxyl-3-amino (to tertbutyloxycarbonyl)-isoxazolyl benzenesulfonamide (4-hydroxy-3-amino (t-butoxy Carbonyl)-phenoxazole)

Excessive sodium hydrosulfite 90min (Na hydrosulfite) is joined 20ml contain 4.69g, 23.3mmol2-the amino Resorcinol of amino (to tertbutyloxycarbonyl)-5-(in 1: 1 solution of the acetonitrile/water (acetonitrile/water) of 2-amino (t-butoxy carbonyl)-5-aminohydroquinone), will stir 15 minutes under the mixture room temperature.Remove acetonitrile under the vacuum, the aqueous solution extracts 3 times with ethyl acetate (EtOAc) 20ml.Merge organic phase, use MgSO ₄Drying is removed under the vacuum and is desolvated.Residuum absorbs with the pure triethyl orthoformate of 100ml (triethyl orthoformate), stirs 16 hours, and reflux is 10 minutes then.Be cooled to room temperature subsequently, add entry and make product precipitation, productive rate 83% (M+1,251).

4-hydroxyl-2-amino-benzene oxazole (4-hydroxy-2-amino phenoxazole)

With 3g, (4-Hydroxy-3-amino (t-butoxy carbonyl)-phenoxazole) is dissolved in the pure TFA solution of 100ml 12mmol 4-hydroxyl-3-amino (to tertbutyloxycarbonyl)-isoxazolyl benzenesulfonamide, stirs 1 hour under the room temperature.Remove trifluoroacetic acid under the vacuum, obtaining final product is trifluoroacetate (quantitatively) (M-1,149) 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-(N-(4-hydroxyl-2-amino-benzene oxazole))-propyl group-(Ethyl-2-(2 ', 3 ' for 2-olefin(e) acid ethyl ester for 3-, 4 ', 5 '-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-aminophenoxazole))-prop-2-enoate)

Under vacuum, 20ml is contained 10ml triethylamine (triethylamine) and is dissolved with 7.34g, 23mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-(ethyl-2-(2 ' for 3-(dimethylamino)-propyl group-2-ethyl ester, 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-and 3-(dimethylamino)-prop-2-enoate) and 3.45g, ethyl acetate (EtOAc) solution of 23mmol 2-amino-4-phenyl-phenol (2-amino-4-phenyl-phenol) stirred 3 hours with Rotary Evaporators.Vacuum is removed ethyl acetate (EtOAc), and residuum is crossed silica gel chromatographic column, and with the elution of the normal hexane that contains 50% ethyl acetate (EtOAc) (hexane) solution, obtains clean product, productive rate 72% (M+1,425).

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-phenyl-isoxazolyl benzenesulfonamide-5-carboxylic acid, ethyl esters (Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate)

50ml is contained 3.5g, 8.25mmol 2-(2 ', 3 ', 4 ', 5 '-the tetra fluoro benzene formyl-)-(N-(4-hydroxyl-2-amino-benzene oxazole))-propyl group-(ethyl-2-(2 ' for 2-olefin(e) acid ethyl ester for 3-, 3 ', 4 ', 5 '-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-aminophenoxazole))-prop-2-enoate) and excessive K ₂CO ₃DMSO solution be heated to 80 ℃ 10 minutes.After being cooled to room temperature, adding entry and form precipitation, be yellow solid product, productive rate 82% (M+1,385).

Example 44

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(3 '-hydroxyl-4 '-aminophenyl)-5-carboxylic acid (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3 '-hydroxy-4 '-amino phenyl)-5-carboxylic Acid) preparation

To be dissolved with 2.3g, 6mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-phenyl-isoxazolyl benzenesulfonamide-5-carboxylicesters (ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate) acetic acid (acetic acid): dense HCl was by 1: 1 blended 100ml mixing solutions reflux 30 minutes.After being cooled to room temperature, vacuum is removed volatile component, obtains the brown solid product, productive rate 82% (M+1,347).

Example 45

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(nitro-azophenlyene)-5-carboxylic acids The system of (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylic acid) Be equipped with

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(3 '-hydroxyl-4 '-amino-(N-2 "-fluoro-4 "-oil of mirbane Base)-phenyl)-the 5-carboxylic acid (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3 '-hydroxy-4 '-amino-(N-2 "-fluoro-4 "-nitr O phenyl)-phenyl)-5-carboxylic acid)

To contain 0.5g, 1.44mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(3 '-hydroxyl-4 '-aminophenyl)-5-carboxylic acid (1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3 '-hydroxy-4 '-aminophenyl)-5-carboxylic acid), 0.5ml 4.3mmol 3,4-two fluoro-oil of mirbane (3,4-difluoro-nitrobenzene) and the solution of 1ml DIEA place 50mlNMP, be heated to 90 ℃ 30 minutes.Behind the cool to room temperature, add entry and make the product precipitation, filter productive rate 63% (M+1,486).

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(nitro-azophenlyene)-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylic acid)

50ml is contained 0.3g, 0.6mmol 2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(3 '-hydroxyl-4 '-aminophenyl)-5-carboxylic acid (2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3 '-hydroxy-4 '-amino phenyl)-5-carboxylicacid) and excessive K ₂CO ₃The DMSO solution stirring and be heated to 110 ℃ 1 hour.After being cooled to room temperature, adding 3M HCl and make the product precipitation, filter productive rate 71% (M+1,465).

Example 46

1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(amino-azophenlyene)-5-carboxylic acids (1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(amino-phenoxazine)-5-carboxylic acid) Preparation

To contain 0.1g, 0.2mmol 1,2-two fluoro-4-oxygen-4H-pyridyl [3,2,1-kl]-8-(nitro-azophenlyene)-5-carboxylic acid (1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylicacid) and 0.15g, the acetic acid (acetic acid) of 0.6mmol tin protochloride (Tin (II) chloride): dense HCl was by 1: 1 blended solution 50ml reflux four hours.After being cooled to room temperature, adding entry and make the product precipitation, filter productive rate 72% (M+1,435).

Example 47

The preparation of amino (amide) the derivative analogue of substituted quinobenzoxazine (quinobenzoxazine)

Under the room temperature with 0.5-2.0mmol amine (amines) NHR ₁R ₂Joining 3.6mL contains in the NMP series solution of 0.5mmol fluoric acid (fluoroacid). container sealing, place on 90 ℃ of well heaters and continue and stirred 1 to two hour, can detect by enough HPLC/MS methods analysts up to reaction. reaction mixture is cooled to room temperature, add 20mL water. the precipitation that vacuum filtration is collected and vacuum-drying obtains. under the situation of using 1.0 equivalent amine (amine), the reaction mixture that obtains can use in next step, " will not change ". solid that obtains or solution are handled with the NMP that 2.5 equivalent HBTU and 3.6mL are dissolved with DIEA, and in inert environments stirring at room 30 minutes. solution is joined 2.5 equivalent amine (amines) NHR ₃R ₄In, (Whatman Uniplate, 2mL) middle reaction is two hours at 96 hole flat boards.Add 50-100 μ L methyl alcohol (Methanol) then, plate is filtered (Whatman Unifilter polypropylene (Polypropylene)).The liquid that obtains directly enters and carries out stratographic analysis (Waters Xterra 19 * 50mm), and a large amount of directly collect (Micromass ZQ, Waters FCII) in the reversed-phase HPLC.Small portion is used for purity check, and (MS TIC is UV) and with the dry mean yield 5-10mg of vacuum-evaporation instrument (Savant).Table 1 and 2 has been listed quinobenzoxazine (quinobenzoxazines) analogue that typical ester replaces (ester-substituted) and amine replacement (amide-substituted) respectively.

Example 48

Anticancer data

Make the two class heteroplastic transplantation models that are used to inoculate, and to be diluted to concentration be 50 * 10 ⁶Individual cell/mL or 100 * 10 ⁶Individual cell/mL.Get the nude mice in four to six ages in week, injection 0.1mL contains 5 * 10 ⁶With 10 * 10 ⁶The cell suspension of individual cell.When tumour was grown to suitable size, beginning quantitatively gave compound.In whole therapeutic processes, measure the size of tumour and detect body weight with calipers.

The antitumour activity of Compound C X-3092 (1204) and CX-1535 (148) is seen Fig. 1 and Fig. 2, and its anticancer effect (delaying tumor propagation) and less toxic side effect (several no changes of body weight) have been described.Fig. 1 shows the antitumour activity (used model be congenital leukemia of children) of compound 494 in the Ramos cell.Fig. 2 shows the antitumour activity (used model be colorectal carcinoma) of compound 516 in the HCT-116 cell.

Example 49

Cell proliferation and/or cytotoxicity analysis

Cell cultures

People's cervical epithelial cells (HeLa cell, HeLa cells) from U.S. typical case culture collection center (Manassas, VA).Containing 5%CO ₂37 ℃ of moist environments under, cell is adding 2mM glutamine, 0.1mM non-essential amino acid, 1mM Sodium.alpha.-ketopropionate, 1.5g/L NaHCO ₃, 50mg/L gentamicin and 10% calf serum (Hyclone, the U.S.) the MEM substratum on grow.

MTS measures

The antiproliferative effect of cancer therapy drug can be by CellTiter 96AQ _UeousAssay (Promega, WI) tester detects, and this instrument detects the number of survivaling cell by colorimetry.(consulting Wang, L., et al., Methods Cell Sci (1996) 18:249-255).

The 0th day, with cell (4,500cells/well) be inoculated in 96 hole flat boards (Corning, NY) in (every hole 100 μ l do not contain the substratum of cancer therapy drug).The 1st day, change the substratum that contains the different concns cancer therapy drug.After hatching 3 days under the normal growth condition (the 4th day), once with PBS damping fluid washing monolayer.The former substratum of the dull and stereotyped replacement in 96 holes that contains 100 μ l PBS damping fluids subsequently with every hole.In each hole of 96 orifice plates, add 20 μ l MTS/PMS (20: 1) solution, containing 5%CO ₂37 ℃ of moist environments in hatched 4 hours.(BMG Labtechnologies Germany) measures light absorption ratio under 490nm by FLUOstar Galaxy 96 hole plate reader.μ M concentration (MTS data) provides in table 1-2.Cancer therapy drug concentration when this concentration is 50% cell inhibition growth rate.IC ₅₀Compound greater than 5 μ M will not be reported.

Example 50

MRNA value in the raji cell assay Raji detects

Real-time quantitative PCR (QPCR) method is usually used in accurately surveying the variation of target gene c-myc and endogenous contrast phosphoglyceraldehy-de dehydrogenase (GAPDH) gene copy number in the same test tube.Cell (15,000cells/well) be inoculated in 96 hole flat boards (Corning, NY), overnight incubation under normal growth conditions.Next day, change the substratum that contains the different concns cancer therapy drug, containing 5%CO ₂37 ℃ of moist environments in hatched 4 hours.(QIAGEN CA) extracts total RNA (tRNA) with RNeasy 96 test kits.Total rna concentration can record by RiboGreen RNA quantitative reagent (Molecular Probes, OR).

In 25 μ l reaction systems, carry out reverse transcription (RT) reaction with 50ng tRNA.Wherein every pipe contains 1x TaqMan RT damping fluid, and 2.5uM is sexamer at random, 5.5mM MgCl ₂, every kind of deoxynucleoside triphosphate (dNTP) 0.5mM, 30U MultiScribe ThermoScript II and 10U RNA enzyme inhibitors.The reverse transcription reaction system was hatched under 25 ℃ 10 minutes, carried out reverse transcription 30 minutes subsequently under 48 ℃, 95 ℃ of following inactivations 5 minutes, preserved down and be placed on 4 ℃.All reverse transcription reagent is all purchased the Biosystems in Applied, CA.

In real time QPCR is reflected at and contains 5 μ l cDNA, the universal PCRMasterMix reagent of 1x, and 1xc-myc cultivates primer and probe unit in advance, and 0.8x GAPDH cultivates in advance in the 50 μ l reaction systems of primer and probe unit and carries out.Because the GAPDH gene content is abundant relatively in the Hela cell, therefore GAPDH primer and concentration and probe concentration are transferred to a desired value to obtain the accurate threshold circulation (C of two genes in the same reaction tubes _T) value.Threshold circulation (C _T) value shows that the part cycle number of the destination number that increases reaches a fixed threshold value.In view of the above, the amplification of GAPDH is terminated before it can limit the suitable common response thing of c-myc amplification, causes the Δ Rn value of GAPDH to reduce, but does not influence its C _TValue, and the expanding effect of two genes is identical.The signal that on behalf of standard instrument, Δ Rn value detect deducts background signal.Δ Rn value increases with the increase of amplicon copy number in PCR, reaches plateau up to reaction.

C-myc probe 6FAM ^TMPainted-the MGB mark, GAPDH probe VIC ^TMPainted-the MGB mark.Cultivate in advance and under 50 ℃, carry out two minutes, keep 10 minutes down to activate the AmpliTaqDNA polysaccharase at 95 ℃ subsequently to activate AmpErase UNG enzyme.40 cycles of DNA cloning need 15 seconds under 95 ℃, need 1 minute under 60 ℃.Human c-myc and GAPDH cDNA are amplified, detect and by ABI Prism 7000 sequence detection systems (Applied Biosystems, CA) real-time quantitative detects, this system can detect 6-FAM simultaneously and body is dyed in the VIC report.

Data are analyzed with ABI PRISM sequence detection system and Microsoft Excel.Carry out relevant detection by quantitative by typical curve simultaneously with the method for CT value relatively, the result that two kinds of methods obtain is equal.C _TThe cycle number of amplification figure can the relative mRNA value of accurate response in the value.(consult Heid, et al., GenomeRes. (1996) 6:986-994; Gibson, et al., Genome Res. (1996) 6:995-1001).Every part of cDNA sample is carried out three QPCR reactions, get three C _TThe mean value of value.All reagent comprises pre-cultivation primer and probe unit, and from Applied Biosystems, CA buys.The μ M concentration of listing among the table 1-2 (STOP data) is the concentration of 50% inhibition c-mycmRNA level.IC ₅₀Compound greater than 5 μ M will not be reported.

As everyone knows, foregoing detailed description and subsidiary example only are explanations, can not be as the restriction of category of the present invention.Disclosed embodiment is carried out various changes and modifies apparent to the professional person.These variations and modification include but not limited to chemical structure, replace, derive, and intermediate product, synthetic, preparation and/or using method etc., they can change arbitrarily and without prejudice to primary condition of the present invention and category.Incorporate the United States Patent (USP) of reference and publication into own forces for your guidance at this.

Claims (44)

1. compound that meets structural formula 1

Or its medicine allows salt;

At this V is H, halogen, NR ¹R ², NH ₂Or NR ¹-(CR ¹ ₂) _n-NR ³R ⁴

A is H, fluorine or NR ¹ ₂

Z is O;

U is NR ¹R ²Or NR ¹-(CR ¹ ₂) _n-NR ³R ⁴

X is OR ², NR ¹R ², halogen, azido-or SR ²

N is 1-6;

At this NR ¹R ²In R ¹And R ²With N can constitute together one be optionally substituted contain one or more N, the 5-14 unit ring type structure of O or S;

R ¹Be H or C _1-6Alkyl;

R ²Be one and contain one or more N of being selected from arbitrarily, the non-adjacent heteroatoms of O or S, and by a C _3-6Cycloalkyl or contain one or more N, the C that the 5-14 unit heterocycle of O or S replaces arbitrarily _1-10Alkyl; Or R ²It is a heterocycle, described heterocycle is selected from trimethylene oxide, pyrans, tetrahydropyrans, dioxane, lactone, ethylenimine, azetidine, tetramethyleneimine, piperidines, morpholine, lactan or tetrahydrofuran (THF), or is selected from the heteroaryl of furans, pyrroles, pyridine, pyrimidine, imidazoles or triazole;

R ₃Be H or C _1-6Alkyl;

R ⁴Be H or C _1-6Alkyl, and NR wherein ³R ⁴In R ³And R ⁴Can constitute morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or a piperidine ring;

W is made up of following groups

R ⁵It can be the substituting group on the condensed ring optional position; By halogen, the H that=O or one or more heteroatoms replace arbitrarily, OR ², C _1-6Alkyl, C _2-6Thiazolinyl;

Wherein each part that is optionally substituted is by one or more halogens, OR ₂, NR ¹R ², carbamate, C _1-10Alkyl, C _2-10Alkenyl substituted, these groups each by halogen ,=O or one or more heteroatoms replace arbitrarily;

Suppose that working as X is F or pyrrolidyl; A is F; Z is O; And W is when being phenylene, U is that right quinoline base or 2,4 difluorobenzene amine.

2. compound according to claim 1, wherein X is a halogen.

3. compound according to claim 1, wherein A is a fluorine.

4. compound according to claim 1, its V is H.

5. compound according to claim 1, its U and X are respectively NR ¹R ²

6. compound according to claim 5, its R ¹Be H and R ²Be a C _1-10Alkyl, this alkyl can contain one or more heteroatomss, and by a C _3-6Cycloalkyl or contain one or more N, the 5-14 unit heterocycle of O or S replaces arbitrarily.

7. compound according to claim 6, the first heterocycle of described 5-14 can be selected from following material group: tetrahydrofuran (THF), 1,3-dioxolane, 2, the 3-dihydrofuran, tetrahydropyrans, cumarone, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxzzole, 4,5-dihydro-isoxazole, piperidines, tetramethyleneimine, pyrrolidin-2-one, pyrroles, pyridine, pyrimidine, octahydro-pyrroles [3,4-b] pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazoles, tetrahydroglyoxaline-2,4-diketone, benzoglyoxaline, 1,3-dihydrobenzo imidazoles-2-ketone, indoles, thiazole, benzothiazole, thiadiazoles, thiophene, tetramethylene sulfide 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo [2.2.1] heptane, 2,5-diazabicyclo [2.2.1] heptane and 2,3,4,4a, 9,9a-six hydrogen-1H-β-Ka Lin.

8. compound according to claim 5, its R ¹Be H and R ²Be one and contain one or more N, the 5-14 unit heterocycle of O or S, it can be replaced arbitrarily by an amino or another heterocycle.

9. compound according to claim 8, the first heterocycle of described 5-14 can be selected from following material group: tetrahydrofuran (THF), 1,3-dioxolane, 2, the 3-dihydrofuran, tetrahydropyrans, cumarone, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxzzole, 4,5-dihydro-isoxazole, piperidines, tetramethyleneimine, pyrrolidin-2-one, pyrroles, pyridine, pyrimidine, octahydro-pyrroles [3,4-b] pyridine, piperazine, pyrazine, morpholine, thiomorpholine, imidazoles, tetrahydroglyoxaline-2,4-diketone, benzoglyoxaline, 1,3-dihydrobenzo imidazoles-2-ketone, indoles, thiazole, benzothiazole, thiadiazoles, thiophene, tetramethylene sulfide 1,1-dioxide, diazepine, triazole, guanidine, diazabicyclo [2.2.1] heptane, 2,5-diazabicyclo [2.2.1] heptane and 2,3,4,4a, 9,9a-six hydrogen-1H-β-Ka Lin.

10. compound according to claim 5, its NR ¹R ²In R ¹And R ²Form one and contain one or more N, the 5-14 unit heterocycle of O or S.

11. compound according to claim 10, its NR ¹R ²Be morpholine, thiomorpholine, piperazine, piperidines or diazepine.

12. compound according to claim 1, its U and X can have structural formula respectively

NR ¹-(CR ¹ ₂)n-NR ³R ⁴ (2)

At this R ¹And R ³Be respectively H or C _1-6Alkyl;

N is 1-6; And

R ⁴Be H or C _1-10Alkyl; And

At NR ³R ⁴In, R ³And R ⁴Can constitute morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or a piperidine ring.

13. compound according to claim 12, its n is 2-3.

14. compound according to claim 12, its NR ³R ⁴Be an aliphatic amide, or guanidine radicals or its tautomer.

15. compound according to claim 12, its NR ³R ⁴Be morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or piperidines.

16. compound according to claim 1, its X is NR ¹R ²U meets molecular formula

NR ¹-(CR ¹ ₂) _n-NR ³R ⁴ (2)。

17. compound according to claim 16, its NR ¹R ²In R ¹And R ², and NR ³R ⁴In R ³And R ⁴Can distinguish and constitute morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or a piperidine ring independently.

18. compound according to claim 17, its X can be by amino, carbamate, and one contains one or more non-adjacent N, the C of O or S _1-10Alkyl replaces arbitrarily.

19. compound according to claim 15, wherein U is NR ¹R ², R wherein ¹Be H, R2 is by C _3-6Cycloalkyl or contain the C of the 5-14 unit heterocyclic substituted of one or more N, O or S _1-10Alkyl, X are NR ¹R ², R wherein ¹, R ²The 5-14 unit that forms one or more N of containing of any replacement, O or S with N together encircles.

20. compound according to claim 19, its X is a morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or piperidines.

21. compound according to claim 20, its X is a tetramethyleneimine.

22. compound according to claim 21, wherein X is the tetramethyleneimine that is replaced by pyrazine.

23. compound according to claim 22, its W is a naphthyl.

24. compound according to claim 1, its compound has chirality.

25. compound according to claim 1, its V is NH ₂Or NR ¹-(CR ¹ ₂) _n-NR ³R ⁴

At this R ¹And R ³Be respectively H or C _1-6Alkyl;

N is 1-6; And

R ⁴Be H, or C _1-6Alkyl; At this NR ³R ⁴In R ³And R ⁴Can form morpholine, thiomorpholine, imidazoles, tetramethyleneimine, piperazine, pyridine or a piperidine ring.

26. compound according to claim 19, its V is H.

27. compound according to claim 19, its A is a fluorine.

28. compound according to claim 19, its W is a naphthyl.

29. compound according to claim 19, its V are H and A is fluorine.

30. compound as claimed in claim 1, wherein said compound is selected from

Or its pharmacy acceptable salt.

31. compound as claimed in claim 30, wherein said compound is

Or its pharmacy acceptable salt.

32. compound as claimed in claim 30, wherein said compound is

Or its pharmacy acceptable salt.

33. pharmaceutical composition that contains the described compound of claim 1 and a kind of medicine permission excipient.

34. the described compound of claim 1, or its pharmaceutical composition is used for improving the purposes of the medicine of cell generation disorders in preparation.

35. the described application of claim 34, the cell generation disorders of mentioning is a cancer.

36. the described application of claim 34, the cell generation disorders of mentioning is alleviated, or necrocytosis is induced.

37. the described compound of claim 1, or its pharmaceutical composition is used for making this system to reduce the application of the medicine of cell proliferation or cell guiding apoptosis in preparation.

38. the described application of claim 37, wherein said system is a cell or tissue.

39. the described compound of claim 1, or its pharmaceutical composition is used for making system to reduce the application of the medicine that microorganism tires in preparation.

40. the described application of claim 39, wherein system is a cell or tissue.

41. the described application of claim 39, wherein microorganism to tire be that virus, bacterium or fungi are tired.

42. the described compound of claim 1, or its pharmaceutical composition is used for making system to alleviate the application of the medicine of infected by microbes in preparation.

43. the described application of claim 42, wherein said infected by microbes can be virus, bacterium or fungi infestation.

44. a compound, described compound are selected from following compounds or its pharmacy acceptable salt:

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