CN1934101B - Dehydrophenylahistine and its analogues and synthesis of dehydrophenylahistine and its analogues - Google Patents

Abstract

The invention discloses a compound shown in a structural formula and a preparation method thereof: wherein the method comprises: reacting a diacylpiperazinedione with a first aldehyde to prepare an intermediate compound; reacting the intermediate compound with a second aldehyde to produce a compound having the general structure, wherein the first aldehyde and the second aldehyde are selected from the group consisting of oxazole formaldehyde, imidazole formaldehyde, benzaldehyde, imidazole formaldehyde derivatives, and benzaldehyde derivatives, thereby forming the compound, wherein R is₁、R₁'、R₁''、R₂、R₃、R₄、R₅And R₆，X₁And X₂，Y、Z、Z₁、Z₂、Z₃And Z₄The definitions of (A) and (B) are respectively consistent with the specification. The invention also discloses a composition and a method for treating vascular proliferation.

Description

Synthesizing of dehydrophenylahistin and analogue thereof and dehydrophenylahistin and analogue thereof

Related application

The application is the U.S. Patent application of submitting on August 1st, 2003 the 10/632nd, No. 531 part continuation application, and require the U.S. Provisional Application the 60/542nd of submission on February 4th, 2004, the U.S. Provisional Application the 60/624th that No. 073 and on November 1st, 2004 submit to, No. 262 right of priority, the denomination of invention of all these applications are " synthesizing of dehydrophenylahistin (dehydrophenylahistins) and analogue thereof and dehydrophenylahistin and analogue thereof "; Be incorporated herein by reference at this full content each application.

Technical background

Technical field

The present invention relates to the compound and the synthesis preparation method of chemistry and field of medicaments.More specifically, the method that the present invention relates to be used for the treatment of the compound of cancer and fungi infestation and prepare this compounds.

The general introduction of association area

The investigator thinks the celelular mechanism of the adjusting that has single, general purpose control eukaryotic cell cycle progression.Referring to, Hartwell for example, L.H. etc., Science (1989), 246:629-34.Simultaneously, known when appearance in the control mechanism of cell cycle is unusual, just may produce cancer or immunologic derangement.Therefore, as known, anti-tumor agent comprising salmosin and immunosuppressor can be to regulate the material of cell cycle.Therefore, need the novel method of preparation eukaryotic cell cycle inhibitor (as antitumor and compound enhancing immunity), and these inhibitor can be used as chemotherapy, anti-tumor agent comprising salmosin is used for the treatment of human cancer.Referring to, Roberge for example, M. etc., Cancer Res. (1994), 54,6115-21.

Fungi, particularly pathogenic agent fungi and infections relating increase day by day to clinical medical challenge.Existing anti-mycotic agent effect is limited and toxic, constantly develops and/or is found and the medicine in existing medicine or the exploitation is had chemical sproof pathogenic agent fungal bacterial strain.For example, can comprise by morbific fungi in human body among others: Candida (Candida spp.) comprises Candida albicans (C.albicans), Oidium tropicale (C.tropicalis), Candida kefyr (C.kefyr), candida krusei (C.krusei) and Candida glabrata (C.galbrata); Aspergillus (Aspergillus spp.) comprises Aspergillus fumigatus (A.fumigatus) and flavus (A.flavus); Cryptococcus neoformans (Cryptococcusneoformans); Blastomycete (Blastomyces spp.) comprises Blastomyces dermatitidis (Blastomycesdermatitidis); Pneumocystis carinii (Pneumocystis carinii); Posadasis spheriforme (Coccidioidesimmitis); The living frog excrement of the frog mould (Basidiobolus ranarum); Conidiobolus (Conidiobolus spp.); Histoplasma capsulatum (Histoplasma capsulatum); Rhizopus (Rhizopus spp.), for example Rhizopus oryzae (R.oryzae) and Rhizopus microsporus (R.microsporus); Cunninghammella (Cunninghamellaspp.); Root Acarasiales belongs to (Rhizomucor spp.); Blastomyces brasiliensis (Paracoccidioidesbrasiliensis); The swollen bacterium (Pseudallescheriaboydii) of Podbielniak foot; Rhinosporidium seeberi (Rhinosporidium seeberi) and sporotrichosis schenckii (Sporothrixschenckii) (Kwon-Chung, K.J.﹠amp; Bennett, J.E.1992 Medical Mycology, Leaand Febiger, Malvern, PA).

Recently, it is reported that other the piperazinedione of structurally associated of tryprostatin A and B (its piperazinedione for being made up of the tryptophan residue of proline(Pro) and isoprenylation (isoprenylated)) and five kinds has suppressed the carrying out of the cell cycle in the m period (M phase), referring to Cui, 1996 J Antibiotics 49:527-33 such as C.; Cui, 1996 J Antibiotics 49:534-40 such as C., and these compounds have also influenced the microtubule assembling, referring to Usui, and 1998 Biochem J333:543-48 such as T.; Kondon, 1998 J Antibiotics 51:801-04 such as M..In addition, reported that natural and synthetic compound can be by combine inhibition mitotic division with colchicine binding site (CLC-site) on the tubulin (tubulin), thereby suppress the eukaryotic cell cycle, wherein said tubulin is a kind of macromole of being made up of two 50kDa subunits (α and β tubulin) and is the main composition composition of microtubule.Referring to, Iwasaki for example, S., 1993 Med Res Rev 13:183-198; Hamel, E.1996 Med ResRev 16:207-31; Weisenberg, R.C. etc., 1969 Biochemistry 7:4466-79.Microtubule has been considered to participate in multiple basic cell function, and for example axonal transport, cell movement and cellular form determines.Therefore, the microtubule depressant of functions can have wide biological activity, and can be used for medicine and agrochemicals purpose.Also possible, colchicine (CLC) site part can be proved to be the value that has as the eukaryotic cell cycle inhibitor, therefore and can be used as chemotherapy agents, wherein said part such as CLC, steganacin are (referring to Kupchan, S.M. etc., 1973 J Am Chem Soc95:1335-36), podophyllotoxin is (referring to Sackett, D.L, 1993 Pharmacol Ther59:163-228) and combretastatin (Kang Burui Taka spit of fland) (referring to Pettit, 1995 J MedChem 38:166-67 such as G.R.).

Though from various fungies, isolated piperazinedione type meta-bolites as mycotoxin (referring to Horak R.M. etc., 1981 JCS Chem Comm 1265-67; Ali M. etc., 1898Toxicology Letters 48:235-41) or as secondary metabolite (referring to Smedsgaard J. etc., 1996JMicrobiol Meth 25:5-17), but to the concrete structure of this piperazinedione type meta-bolites or derivatives thereof and anti-tumor activity thereof particularly activity in vivo know little.These compounds not only are separated as mycotoxin, and Hayashi etc. are at J. Org.Chem. (2000) 65, have also described the chemical synthesis process of a kind of piperazinedione type meta-bolites-phenyl ahistins (phenylahistin) on the 8402nd page.In this area, by parent phenyl ahistins being carried out derivative-dehydrophenylahistin that the enzyme catalysis dehydrogenation has prepared a kind of this class piperazinedione type meta-bolites.Along with the rising of cancer incidence, press for by chemical process prepare a class substantially pure, have the special proliferation inhibition activity of zooblast and high anti-tumor activity and a piperazinedione type meta-bolites derivative optionally.Therefore, press for a kind of effectively, by synthetic preparation piperazinedione type meta-bolites derivative substantially pure, that have structure and biological characteristics.

Simultaneously, to be that the PCT application (July 26 calendar year 2001) of WO/0153290 has been described a kind of by phenyl ahistins or specific phenyl ahistins analogue being exposed to the non-synthetic method that the desaturase that obtains from streptomyces albus (Streptomyces albulus) prepares dehydrophenylahistin for international publication number.

Summary of the invention

The invention discloses a class has the compound of general formula (I) structure and passes through synthetic method of producing this compounds:

Disclosed compound has the structure of general formula (I), wherein:

Each R ₁, R ₄And R ₆Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-COO-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

R ₁' and R ₁" be independently selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-COO-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

Each R ₂, R ₃And R ₅Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₁₂Alkyl, undersaturated C ₁-C ₁₂The alkylsulfonyl group of the nitryl group of the amino of the heteroaryl of the aryl of thiazolinyl, acyl group, cycloalkyl, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro and replacement, alkylsulfonyl and replacement;

X ₁And X ₂Be selected from Sauerstoffatom, nitrogen-atoms and sulphur atom respectively, and all can be unsubstituted or by R as defined above ₅Group replaces;

Y is selected from nitrogen-atoms, by above-mentioned R ₅The methylene group of sulphur atom, methylene group and the replacement of the nitrogen-atoms that group replaces, Sauerstoffatom, sulphur atom, oxidation;

N equals 0,1 or 2 integer;

Each is each Z and each Z of non-zero n correspondence independently ₁, Z ₂, Z ₃And Z ₄Be independently selected from carbon atom, sulphur atom, nitrogen-atoms or Sauerstoffatom respectively; And

The key that dotted line is represented can be singly-bound or two key;

In specific compound, need to satisfy, if R ₁, R ₁', R ₂, R ₃, R ₄And R ₅Be hydrogen atom, then X ₁And X ₂Can not be Sauerstoffatom, and R ₆Can not be 3,3-dimethyl-1-butylene and hydrogen atom.

Described method comprises the steps:

The diacyl piperazinedione and first aldehyde reaction are prepared midbody compound; And

The described midbody compound and second aldehyde reaction are prepared the compounds with described formula, wherein:

Described first aldehyde and the second aldehyde Xuan Zi oxazole formaldehyde (oxazolecarboxaldeyhyde), imidazole formaldehyde, phenyl aldehyde, imidazole formaldehyde derivative and benzaldehyde derivative, thus compound formed, wherein

Disclosed compound has the structure of general formula (I), wherein:

Each R ₁, R ₄And R ₆Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-COO-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

R ₁' and R ₁" be independently selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-COO-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

Each R ₂, R ₃And R ₅Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₁₂Alkyl, undersaturated C ₁-C ₁₂The alkylsulfonyl group of the nitryl group of the amino of the heteroaryl of the aryl of thiazolinyl, acyl group, cycloalkyl, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro and replacement, alkylsulfonyl and replacement;

X ₁And X ₂Be selected from Sauerstoffatom, nitrogen-atoms and sulphur atom respectively, and all can be unsubstituted or by R as defined above ₅Group replaces;

Y is selected from nitrogen-atoms, by above-mentioned R ₅The methylene group of sulphur atom, methylene group and the replacement of the nitrogen-atoms that group replaces, Sauerstoffatom, sulphur atom, oxidation;

N equals 0,1 or 2 integer;

Each is each Z and each Z of non-zero n correspondence independently ₁, Z ₂, Z ₃And Z ₄Be selected from carbon atom, sulphur atom, nitrogen-atoms or Sauerstoffatom respectively; And

The key that dotted line is represented can be singly-bound or two key.

In the preferred embodiment of The compounds of this invention and method, described imidazole formaldehyde is 5-(1, a 1-dimethyl-2-ethyl) imidazoles-4-formaldehyde, and described phenyl aldehyde comprises independent methoxy group.Other preferred embodiment of The compounds of this invention comprise have tertiary butyl groups, the compound of dimethoxy group, cl radical and thiotolene group and prepare the method for these compounds, and the compound shown in the table 2,3 and 4 and prepare the method for these compounds.

The invention also discloses method and material that treatment tumor tissues or preventing cancer or pathogenic fungi infect.These methods and material are specially adapted to treat mammalian subject, more are specially adapted to the mankind, and comprise described individual dehydrophenylahistin or its analogue administration.Described method comprises and gives the dehydrogenation ahistins that individuality comprises effective antitumor or antimycotic dosage or the composition of its analogue.

Another embodiment relates to the method for the morbid state for the treatment of animal, and this method can comprise with the amount of effective minimizing blood vessel hyperplasia or the amount that effectively reduces vessel density and gives described animal with compound of the present invention.Exemplary morbid state comprises the vegetation such as cancer, and other morbid state relevant with vascularization or that depend on vascularization, for example comprise kapillary hyperplasia, osteoporosis or the like in immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, Terry's sign disease, RI, the atherosclerotic plaque.In some embodiments, described disease is not a cancer.

Other embodiment relates to the method for blood vessel atrophy in the induced animal body.Described method for example can comprise the described animal of compounds for treating with the general formula of the present invention (I) of treatment significant quantity.The described compound of treatment significant quantity can cause the tubulin depolymerization in the vascular system.

Described animal can be preferably the mankind.Preferred disease can be tumour, diabetic retinopathy, macular degeneration relevant with the age or the like.In some respects, described disease is not a cancer, perhaps especially cancer is got rid of outside described method and purposes.Preferably, described compound is KPU-02.

Other embodiment also relates to the pharmaceutical composition that is used for the treatment of or prevents blood vessel hyperplasia, and said composition comprises the compound disclosed by the invention and the medicine acceptable carrier of medicine effective quantity.Described blood vessel hyperplasia can be the symptom of disease, for example, and cancer, macular degeneration and the diabetic retinopathy relevant with the age.

Some embodiments relate to the vascular system with respect to nonneoplastic tissue, the method for preferential target tumor vascular system.Described method can comprise the step that the compound with general formula (I) structure of the present invention is given animal (being preferably the mankind).Described nonneoplastic tissue can be, for example, and skin, muscle, brain, kidney, heart, spleen, intestines or the like.The vascular system of described tumour relatively nonneoplastic tissue vascular system preferentially, for example with about degree of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% by target.

Other embodiment relates to the vascular system with respect to nonneoplastic tissue, and the method for the vascular system of preferential target tumor, this method can comprise that with the vascular system with respect to nonneoplastic tissue the reagent of the vascular system of preferential target tumor gives animal.

Other embodiments relate to the application of compound in medication preparation of (I) structure that has general formula, the vascular system morbid state relevant or that depend on vascular system that this medicine is used for the treatment of and increases.In some aspects, described morbid state can be a cancer, and in others, does not especially comprise the cancer of particular type or all cancers.Described morbid state can be any other with blood vessel excessively form relevant, with vascular system morbid state that be correlated with or that depend on vascular system.Example comprises blood capillary proliferation, osteoporosis or the like in immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, Terry's sign disease, RI, the atherosclerotic plaque.

Brief description of drawings

This specification sheets is introduced and is constituted its a part of accompanying drawing just for some preferred embodiment of the present invention is described.The other parts of itself and specification sheets are used for illustrating to those skilled in the art the optimal way of some compound of preparation the present invention jointly.In described accompanying drawing:

Figure 1 shows that the reaction scheme figure of preparation dehydrophenylahistin, comprise, again midbody compound 3 and phenyl aldehyde 4 reactions are obtained dehydrophenylahistin diacyl piperazinedione 1 and imidazole formaldehyde 2 prepared in reaction midbody compounds 3.

Figure 2 shows that the HPLC spectrogram of synthetic dehydrophenylahistin crude product.

Figure 3 shows that the reaction scheme figure of preparation dehydrophenylahistin, comprise, again midbody compound 17 and imidazole formaldehyde 15 reactions are obtained dehydrophenylahistin diacyl piperazinedione 1 and phenyl aldehyde 4 prepared in reaction midbody compounds 17.

Figure 4 shows that HPLC spectrogram by the synthesizing tertiary butyl-dehydrophenylahistin crude product of route A and B preparation.

Fig. 5 has shown dehydrogenation PLH modified makes it have the active two kinds of strategies of effective cell toxin.

Fig. 6 has shown the infer activity conformation of dehydrogenation PLH at phenyl moiety.

The Cytochrome P450 metabolism of Fig. 7 phenyl ahistins.

The Z-E that Figure 8 shows that the tertiary butyl-dehydrogenation PLH changes.

Fig. 9 has shown the image of the synthetic and prodrug of the acyl group-E-tertiary butyl-dehydrogenation PLH.

Figure 10 has shown 3-Z-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-Z-base-methylene radical]-piperazine-2, the thermograde of 5-diketone.

Figure 11 has shown 3-Z-benzylidene-6-[5 "-tertiary butyl-1H-imidazoles-4 "-Z-base-methylene radical]-piperazine-2, the thermograde of 5-diketone.

Figure 12 has shown with colchicine and has compared with taxol (taxol) that KPU-2, KPU-35 and the tertiary butyl-phenyl ahistins is to the effect of the HuVEC individual layer perviousness of FITC-Dextran.

Figure 13 has shown when KPU-2 makes up separately or with CPT-11 the effect to the tumor growth of estimating in HT-29 human colon tumor heterograft (the HT-29 Human Colon Tumor Xenograft) model.

Figure 14 has shown the effect of the tumor weight that when KPU-2 makes up separately or with CPT-11 individual mouse corpse from HT-29 human colon tumor xenograft models is excised.

Figure 15 has shown when KPU-2 makes up separately or with CPT-11 the effect to the tumor growth of estimating in the HT-29 human colon tumor xenograft models.

Figure 16 has shown the effect of the tumor weight that when KPU-2 makes up separately or with CPT-11 individual mouse corpse from HT-29 human colon tumor xenograft models is excised.

Figure 17 has shown when A.KPU-2, B.KPU-35 and the C. tertiary butyl-phenyl ahistins make up separately or with CPT-11 the effect to the tumor growth of estimating in the HT-29 human colon tumor xenograft models.

Figure 18 has shown the effect of the tumor weight that when A.KPU-2, B.KPU-35 and the C. tertiary butyl-phenyl ahistins make up separately or with CPT-11 the individual mouse corpse from HT-29 human colon tumor xenograft models is excised.

Figure 19 has shown when KPU-2 makes up separately or with CPT-11 the effect to tumor propagation in the HT-29 human colon tumor xenograft models: the comparison of three researchs.

Figure 20 has shown when KPU-2 make up separately or with CPT-11 the finally effect of tumor weight from the HT-29 human colon tumor xenograft models: the comparison of three researchs.

Figure 21 has shown when KPU-2 makes up separately or with taxotere (Taxotere) effect to the tumor propagation of estimating in the DU-145 human prostate tumor xenogeneic graft model.

Figure 22 has shown when A.KPU-2, B.KPU-35 and the C. tertiary butyl-phenyl ahistins make ups separately or with taxotere that the tumor growth of wherein said estimation is to obtain to the effect of the tumor growth of estimating on the observation basis that the survival of DU-145 human prostate tumor xenogeneic graft (DU-145 Human Prostate TumorXenograft) model is partly carried out.

Figure 23 has shown when KPU-2 makes up separately or with taxotere the effect to the individual tumors weight of excising from corpse in the DU-145 human prostate tumor xenogeneic graft model.

Figure 24 has shown when KPU-35 makes up separately or with taxotere the effect to the individual tumors weight of excising from corpse in the DU-145 human prostate tumor xenogeneic graft model.

Figure 25 has shown the effect in MCF-7 human breast tumor xenogeneic graft (MCF-7 Human BreastTumor Xenograft) model when A.KPU-2, B.KPU-35 and the C. tertiary butyl-phenyl ahistins make up separately or with taxotere.

Figure 26 has shown when KPU-2 makes up separately or with taxotere the effect to the tumor propagation of estimating in A549 human lung cancer heterograft (the A549 Human Lung Tumor Xenograft) model.

Figure 27 has shown when KPU-2 makes up separately or with taxotere the effect to the tumor weight that excises from corpse in the A549 human lung cancer xenograft models.

Figure 28 has shown when KPU-2 makes up separately or with Paclitaxel (Paclitaxel) effect to the tumor weight of estimating in the mouse breast buccal pad of having transplanted MDA-231 human breast tumor model.

Figure 29 has shown the effect in mouse melanoma B16 F10 metastatic tumo(u)r model (MurineMelanoma B16 F10 Metastatic Tumor Model) when A.KPU-2, B.KPU-35 and the C. tertiary butyl-phenyl ahistins make up separately or with Paclitaxel.

Figure 30 has shown KPU-35 and the KPU-02 effect to the tumor vascular system in the skin of back gauffer chamber of Figure 30.

Figure 31 has shown that KPU-02 and CPT-11 make up the effect to the tumor weight of assessing in the HT-29 human colon tumor xenograft models.

Figure 32 has shown that KPU-02 and CPT-11 make up the effect to the tumor weight that exsomatizes in the HT-29 human colon tumor xenograft models.

Figure 33 has shown in the HuVEC cell by KPU-02 and the tubulin unzipping fast of KPU-35 inductive.

Figure 34 has shown the effect of KPU-02 to mono-layer osmotic in the HuVEC cell.

Figure 35 has shown the effect of KPU-02 to tumor blood flow amount in the P22 rat sarcoma model of using the 125I-IAP technology.

Figure 36 has shown after the administration 1 hour and 24 hours, and the 15mg/kgKPU-02 of abdominal injection (IP) (being expressed as the % vehicle Control) is to the effect of volume of blood flow in the different tissues.

Figure 37 has shown in P22 rat sarcoma model, by the 7.5mg/kg of abdominal injection and the KPU-02 inductive neoplasm necrosis of 15.0mg/kg.

Figure 38 has listed the activity of multiple derivative in the HT-29 cell of the tertiary butyl-dehydrogenation-PLH.

Figure 39 has shown that the 3D QSAR (CoMFA) of the tertiary butyl-dehydrogenation-PLH derivative analyzes.

Figure 40 has shown the X-radiocrystallgraphy analysis of the tertiary butyl-dehydrogenation-PLH derivative.

Figure 41 has shown with colchicine and has compared the biological activity of multiple phenyl ahistins derivative.

Figure 42 has shown the tertiary butyl-dehydrogenation-PLH (KPU-2) and the KPU-35 effect to the cell cycle progression of HeLa cell.

Figure 43 shown with Paclitaxel and compared, and dehydrogenation-PLH and the tertiary butyl-dehydrogenation-PLH (KPU-2) is to medicaments insensitive and effect drug-fast tumor cell line.

Figure 44 A has shown in the presence of DMSO pharmaceutical carrier (◇), 1.25 μ M (), 2.5 μ M (-) and 5 μ M (zero) KPU-02, the turbidity spectrum of tubulin (microtubule protein) polymerization.

Figure 44 B has shown in the presence of DMSO pharmaceutical carrier (◇), 1.25 μ M (), 2.5 μ M (-) and 5 μ M (zero) CA4, the turbidity spectrum of tubulin polymerization effect.

Figure 44 C has shown in the presence of DMSO pharmaceutical carrier (◇), 1.25 μ M (), 2.5 μ M (-) and 5 μ M (zero) CLC, the turbidity spectrum of tubulin polymerization effect.

Figure 45 has shown the restraining effect of MT when not having or existing a series of KPU-02 (zero), CA4 () and colchicine (◇) concentration.

Figure 46 A has shown in the presence of KPU-02, when stable state, at the frequency histogram of external microtubule mean length.

Figure 46 B has shown in the presence of CA4, when stable state, at the frequency histogram of external microtubule mean length.

Figure 46 C has shown in the presence of CLC, when stable state, at the frequency histogram of external microtubule mean length.

Figure 47 A shown in the presence of KPU-02, when stable state, and the electron microscope picture of the microtubule that is rich in MAP of external formation.

Figure 47 B shown in the presence of CA4, when stable state, and the electron microscope picture of the microtubule that is rich in MAP of external formation.

Figure 47 C shown in the presence of CLC, when stable state, and the electron microscope picture of the microtubule that is rich in MAP of external formation.

Figure 48 has shown in the presence of KPU-02, CA4 and colchicine, the graphic summary that MT length reduces when stable state.

Figure 49 A has shown in the presence of the KPU-02 that increases, the fluorescence emission spectrum of tubulin.

Figure 49 B has shown in the peaked matched curve of the fluorescent emission of 487nm, to obtain the Kd value for KPU-02 of tubulin.Illustration has shown residue.

Figure 49 C has shown the conversion two reciprocal of binding data.

Figure 50 shown and do not had (◇) or having 10 μ M KPU-02 (zero) or during 10 μ M CA4 (), contain multiple concentration [ ³H] the diagram result that analyzes in conjunction with the competitive inhibition of tubulin of the colchicine of CLC.

Figure 51 has shown that KPU-02, CA4 and CLC are to inhibiting logarithm [compound] response curve of mitotic division process in the MCF7 cell of cultivating in the presence of KPU-02 (zero), CA4 (a) and colchicine (0).

Figure 52 has shown the immunofluorescence micro-image of MCF7 cell.A-d: in the contrast-(a) tubulin, (b) KPU-02, (c) CA4 and (d) tubulin in the cell handled of CLC in the contrast; E-h: in the contrast-(e) DNA, (f) KPU-02, (g) CA4 in the contrast and (h) DNA in the cell handled of CLC.

Figure 53 A has shown the immunofluorescence micro-image of the MCF7 cell of handling with KPU-02.

Figure 53 B has shown the immunofluorescence micro-image of the MCF7 cell of handling with CA4.

Figure 53 C has shown the immunofluorescence micro-image of the MCF7 cell of handling with CLC.

Figure 54 A has shown the immunofluorescence micro-image of the MCF7 cell of handling with KPU-02.

Figure 54 B has shown the immunofluorescence micro-image of the MCF7 cell of handling with A4.

Figure 54 C has shown the immunofluorescence micro-image of the MCF7 cell of handling with CLC.

In some accompanying drawing, shown compound is represented with interchangeable title.The complete list of the conversion of these replaceable titles is as follows:

Interchangeable title	Title used herein
		NPI-2350	(-)-phenyl ahistins
NPI-2352	KPU-01

NPI-2353	KPU-03
		NPI-2354	KPU-04
NPI-2355	KPU-05
		NPI-2356	KPU-06
NPI-2357	KPU-07
		NPI-2358	KPU-02
NPI-2359	KPU-08
		NPI-2360	KPU-09
NPI-2361	KPU-10
		NPI-2362	KPU-11
NPI-2363	KPU-12

NPI-2364	KPU-13
		NPI-2365	KPU-14
NPI-2366	KPU-15
		NPI-2367	KPU-16
NPI-2368	KPU-17
		NPI-2369	KPU-18
NPI-2370	KPU-19
		NPI-2371	KPU-21
NPI-2372	KPU-22
		NPI-2373	KPU-23
NPI-2374	KPU-24
		NPI-2375	KPU-25

NPI-2376	KPU-28
		NPI-2377	KPU-26
NPI-2378	KPU-27
		NPI-2379	KPU-29
NPI-2380	KPU-20
		NPI-2381	KPU-30
NPI-2382	KPU-31
		NPI-2383	KPU-32
NPI-2384	KPU-33
		NPI-2385	KPU-34
NPI-2386	KPU-35
		NPI-2387	KPU-36
NPI-2388	KPU-37
		NPI-2389	KPU-38
NPI-2390	KPU-39
		NPI-2391	KPU-40
NPI-2392	KPU-41
		NPI-2393	KPU-42

NPI-2394	KPU-43
		NPI-2395	KPU-44
NPI-2396	KPU-45
		NPI-2397	KPU-46
NPI-2398	KPU-47

NPI-2399	KPU-48
		NPI-2400	KPU-49
NPI-2401	KPU-50
		NPI-2402	KPU-51
NPI-2403	KPU-52
		NPI-2404	KPU-53
NPI-2405	KPU-54
		NPI-2406	KPU-55
NPI-2407	KPU-56
		NPI-2408	KPU-57
NPI-2409	KPU-58
		NPI-2410	KPU-59
NPI-2411	KPU-60
		NPI-2412	KPU-61
NPI-2413	KPU-62
		NPI-2414	KPU-63
NPI-2415	KPU-64
		NPI-2416	KPU-65
NPI-2417	KPU-66
		NPI-2418	KPU-67
NPI-2419	KPU-68
		NPI-2420	KPU-69
NPI-2421	KPU-70
		NPI-2422	KPU-71

NPI-2423

KPU-72

NPI-2424	KPU-73
		NPI-2425	KPU-74
NPI-2426	KPU-75
		NPI-2427	KPU-76
NPI-2428	KPU-77
		NPI-2429	KPU-79
NPI-2430	KPU-80
		NPI-2431	KPU-81
NPI-2432	KPU-82
		NPI-2433	KPU-83
NPI-2434	KPU-84
		NPI-2435	KPU-86
NPI-2436	KPU-87
		NPI-2437	KPU-88
NPI-2438	KPU-89
		NPI-2439	KPU-90
NPI-2440	KPU-91
		NPI-2441	KPU-92
NPI-2442	KPU-80
		NPI-2455	KPU-94
NPI-2456	KPU-95
		NPI-2457	KPU-96

NPI-2458	KPU-97
		NPI-2459	KPU-98
NPI-2460	The tert-butyl-phenyl ahistins

Detailed description of preferred embodiments

In allowed by law scope, each reference that this paper quotes comprises that United States Patent (USP) all is considered as introducing in full the reference as this specification sheets.At this with the 10/632nd of U.S. Patent application, No. 531 and PCT apply for that PCTUS03/24232 number full content is incorporated herein by reference, two applications are all submitted on August 1st, 2003, and title is " synthesizing of dehydrophenylahistin and analogue thereof and dehydrophenylahistin and analogue thereof ".

The invention provides the method that comprises the novel cpd of dehydrophenylahistin and dehydrophenylahistin derivative by synthetic preparation, method with the high relatively acceptable cell cycle inhibitor of produced in yields medicine, anti-tumor agent comprising salmosin and antifungal agents also is provided, and wherein said compound and/or its derivative belong to the activeconstituents of these cell cycle inhibitors, anti-tumor agent comprising salmosin and anti-mycotic agent.Other purpose of the present invention comprises provides the novel cpd that can not obtain by existing non-synthetic method.Purpose of the present invention also comprises provides the particularly method of human cancer of treatment cancer, and this method comprises the step with some compound administration in the class new antitumoral compounds of inhibition tumor growth effective dose.The present invention also provides the method for the pathogenic fungi in prevention or the treatment individuality, comprise some compound in the class new class of antifungal of effective antimycotic dosage described individual administration, for example with the dosage that can produce required antifungic action and mode with dehydrophenylahistin or its analogue administration.These targets are achieved in the preferred embodiment of the method for described compound disclosed by the invention and preparation and this compounds of use, and needn't all realize in all embodiments of the present invention.

The method that the invention also discloses compound and produce this compounds, wherein said compound is shown in general formula (I):

(I)

Wherein:

Each R ₁, R ₄And R ₆Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-CO-O-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R7, wherein R ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

R ₁' and R ₁" be independently selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-CO-O-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

R, R ₁' and R ₁" or covalent attachment or non-covalent each other combination each other;

Each R ₂, R ₃And R ₅Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₁₂Alkyl, undersaturated C ₁-C ₁₂The alkylsulfonyl group of the nitryl group of the amino of the heteroaryl of the aryl of thiazolinyl, acyl group, cycloalkyl, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro and replacement, alkylsulfonyl and replacement;

X ₁And X ₂Be selected from Sauerstoffatom, nitrogen-atoms and sulphur atom respectively, and all can be unsubstituted or by R as defined above ₅Group replaces;

Y is selected from nitrogen-atoms, by above-mentioned R ₅The methylene group of sulphur atom, methylene group and the replacement of the nitrogen-atoms that group replaces, Sauerstoffatom, sulphur atom, oxidation;

N equals 0,1 or 2 integer;

Each is each Z and each Z of non-zero n correspondence independently ₁, Z ₂, Z ₃And Z ₄Be selected from carbon atom, sulphur atom, nitrogen-atoms or Sauerstoffatom respectively; And

The key that dotted line is represented can be singly-bound or two key.

Described method comprises the method for preparing general formula (I) compound as follows:

The diacyl piperazinedione and first aldehyde reaction are prepared midbody compound; With

The described midbody compound and second aldehyde reaction are prepared the compounds with described formula, wherein

Described first aldehyde and the second aldehyde Xuan Zi oxazole formaldehyde, imidazole formaldehyde, phenyl aldehyde, imidazole formaldehyde derivative and benzaldehyde derivative, thereby the compound of formation general formula (I), wherein

Each R ₁, R ₄And R ₆Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-CO-O-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

R ₁' and R ₁" be independently selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement, hydroxyl, carboxyl ,-CO-O-R ₇, cyano group, alkylthio, the haloalkyl that comprises multi-haloalkyl, halo carbonyl and carbonyl-CCO-R ₇, R wherein ₇Be selected from hydrogen atom, halogen atom and saturated C ₁-C ₂₄Alkyl, undersaturated C ₁-C ₂₄The phenyl group of the nitro of the amino of the heteroaryl of the aryl of thiazolinyl, cycloalkyl, cycloalkenyl group, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro, azido-, replacement, phenyl and replacement;

Each R ₂, R ₃And R ₅Be selected from hydrogen atom, halogen atom and saturated C respectively ₁-C ₁₂Alkyl, undersaturated C ₁-C ₁₂The alkylsulfonyl group of the nitryl group of the amino of the heteroaryl of the aryl of thiazolinyl, acyl group, cycloalkyl, alkoxyl group, cycloalkyloxy, aryl, replacement, heteroaryl, replacement, amino, replacement, nitro and replacement, alkylsulfonyl and replacement;

X ₁And X ₂Be selected from Sauerstoffatom, nitrogen-atoms and sulphur atom respectively, and

Y is selected from nitrogen-atoms, by above-mentioned R ₅The methylene group of sulphur atom, methylene group and the replacement of the nitrogen-atoms that group replaces, Sauerstoffatom, sulphur atom, oxidation;

Each is each Z and each Z of non-zero n correspondence independently ₁, Z ₂, Z ₃And Z ₄Be selected from carbon atom, sulphur atom, nitrogen-atoms or Sauerstoffatom respectively; And

The key that dotted line is represented can be singly-bound or two key.

The present invention also provides the acceptable salt of medicine and the ester class prodrug of general formula (I) and formula (II) compound, and the method for synthesizing this compounds by method disclosed by the invention.

Term " ester class prodrug " particularly when referring to pass through the ester class prodrug of the inventive method synthetic general formula (I) compound, refers to the chemical derivative of described compound, and it can transform (for example hydrolysis in blood or tissue) in vivo rapidly and obtain described compound.Term " ester class prodrug " refers to the derivative of compound disclosed by the invention, and it can be by adding multiple can formation by any group in the one-tenth ester group of hydrolysis under the physiological condition.The example of the ester group of prodrug comprises pivoyloxymethyl, acetoxyl methyl, 2-phthalidylidene, 2,3-indanyl and methoxymethyl and other this class group well known in the art (comprising (5-R-2-oxygen-1,3-Dioxol-4-yl) methyl group).Other example of the ester group of prodrug can be at for example T.Higuchi and V. Stella " Pro-drugs asNovel Delivery Systems " volume 14, A.C.S.Symposium Series, AmericanChemical Society (1975); " Bioreversible Carriers in Drug Design:Theoryand Application ", E.B.Roche edits, Pergamon Press:New York finds among the 14-21 (1987) (example of the ester that can be used as prodrug is provided for the compound that contains carboxyl).

Term used herein " ester class prodrug " also refers to the chemical derivative of described compound, and it can transform (for example hydrolysis in blood) in vivo rapidly and obtain described compound.Term " ester class prodrug " refers to the derivative of compound disclosed by the invention, and it can be by adding multiple can formation by any group in the one-tenth ester group of hydrolysis under the physiological condition.The example of the ester group of prodrug comprises pivoyloxymethyl, acetoxy-methyl, 2-phthalidylidene, 2,3-indanyl and methoxymethyl and other this class group well known in the art (comprising (5-R-2-oxygen-1,3-Dioxol-4-yl) methyl group).Other example of the ester group of prodrug can be at for example T.Higuchi and V. Stella " Pro-drugs as Novel Delivery Systems " volume 14, A.C.S.SymposiumSeries, American Chemical Society (1975); " Bioreversible Carriers in DrugDesign:Theory and Application ", E.B.Roche edits, Pergamon Press:NewYork finds among the 14-21 (1987) (example of the ester that can be used as prodrug is provided for the compound that contains carboxyl).

Term " the acceptable salt of medicine " especially when the medicine acceptable salt of finger by method synthetic general formula disclosed by the invention (I) compound, refers to the acceptable salt of any medicine of compound, preferably refers to the acid-adducting salt of compound.The example of the preferred acceptable salt of medicine is an alkali metal salt (sodium or potassium), alkaline earth salt (calcium or magnesium) or from ammonia or such as C ₁-C ₇The acceptable organic amine deutero-of the medicine ammonium salt of alkylamine, cyclo-hexylamine, trolamine, quadrol or three (methylol) aminomethane.For with described method synthetic basic amine compound, the example of the preferred acceptable salt of medicine is the acceptable inorganic or organic acid acid-adducting salt of medicine, for example haloid acid, sulfuric acid, phosphoric acid or aliphatics or aromatic carboxylic acid or sulfonic acid, for example acetate, succsinic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, xitix, nicotinic acid, methylsulfonic acid, tosic acid or naphthene sulfonic acid.

Term used herein " the acceptable salt of medicine " also refers to the acceptable salt of any medicine of compound, preferably refers to the acid-adducting salt of compound.The example of the preferred acceptable salt of medicine is an alkali metal salt (sodium or potassium), alkaline earth salt (calcium or magnesium) or from ammonia or such as C ₁-C ₇Alkylamine, cyclo-hexylamine, trolamine, quadrol or three-(the acceptable organic amine deutero-of the medicine of hydroxymethyl)-aminomethane ammonium salt.For with described method synthetic basic amine compound, the example of the preferred acceptable salt of medicine is acceptable mineral acid of medicine or organic acid acid-adducting salt, for example haloid acid, sulfuric acid, phosphoric acid or aliphatics or aromatic carboxylic acid or sulfonic acid, for example acetate, succsinic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, xitix, nicotinic acid, methylsulfonic acid, tosic acid or naphthene sulfonic acid.

Preferred pharmaceutical compositions disclosed by the invention comprises by the acceptable salt of medicine of the inventive method synthetic general formula (I) compound and ester class prodrug.Therefore, if comprise described activeconstituents thorough mixing with drug excipient and salt form in the production process of pharmaceutical preparation, then preferred what use non-alkalescence is acidity or neutral vehicle.

In the preferred embodiment of the method for compound of the present invention, can form relative rigidity, the pseudo-tricyclic structure in plane.For stablize this class relative rigidity, the pseudo-tricyclic structure in plane, R ₃Be preferably hydrogen.

In other preferred embodiment of the method for compound of the present invention, n equals 0 or 1, and more preferably 1, and each Z ₂, Z ₃And Z ₄Be selected from Sauerstoffatom, nitrogen-atoms and carbon atom respectively, more preferably Z ₂, Z ₃And Z ₄In at least one is carbon atom, most preferably Z ₂, Z ₃And Z ₄In at least two be carbon atom.All Z can be carbon atom simultaneously.

Other preferred embodiment of Compounds and methods for of the present invention comprises having the following general formula (Ia) and (Ib) compound of structure:

Wherein each group definition is as described herein.

Term used herein " halogen atom " refers to the stable atom of radiation of the arbitrary periodic table of elements the 7th row, i.e. fluorine, chlorine, bromine or iodine, preferably fluorine and chlorine.

Term used herein " alkyl " refers to any straight or branched, replacement or unsubstituted saturated hydrocarbyl, preferred C ₁-C ₆Straight chain, saturated, unsubstituted alkyl, most preferable, ethyl, isobutyl-and the tertiary butyl.In the saturated hydrocarbyl of described replacement, preferred C ₁-C ₆Single halogen replaces and two halogens replace and the plain saturated hydrocarbyl that replaces of perhalogeno and the amino alkyl that replaces, most preferably perfluoro-methyl, perchloro-methyl, the perfluor tertiary butyl and the perchloro-tertiary butyl.Term " replacement " has its implication originally, described in the of the same period various patents in the association area.Referring to, for example United States Patent (USP) the 6th, 583, and 143,6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443 and 6,350, No. 759.Especially, the definition of described " replacement " and United States Patent (USP) the 6th, 583, defined equally wide in range in No. 143, wherein being defined as the term replacement arbitrarily, at least one hydrogen atom is substituted basic institute alternate group, for example alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclic radical and heterocyclic radical alkyl.Term " replacement " also with United States Patent (USP) the 6th; 509; defined equally wide in range in No. 331; wherein term " alkyl of replacement " is defined as finger and preferably contains 1-10 carbon atom; have 1-5 substituting group and preferably have 1-3 substituent alkyl group, wherein said substituting group is selected from alkoxyl group; the alkoxyl group that replaces; cycloalkyl; the cycloalkyl that replaces; cycloalkenyl group; the cycloalkenyl group that replaces; acyl group; acyl amino; acyloxy; amino; the amino that replaces; aminoacyl; amino acyloxy; the oxygen acyl amino; cyano group; halogen; hydroxyl; carboxyl; carboxyalkyl; ketone group; the sulfo-ketone group; thiol group; thio alkoxy; the thio alkoxy that replaces; aryl; aryloxy; heteroaryl; heteroaryl oxygen base; heterocyclic radical; the heterocyclyloxy base; hydroxyl amino; alkoxy amino; nitro;-SO-alkyl; the alkyl that-SO-replaces;-SO-aryl;-SO-heteroaryl;-SO ₂-alkyl ,-SO ₂The alkyl of-replacement ,-SO ₂-aryl and-SO ₂-heteroaryl.Other above-mentioned patent of enumerating also provides the standard definition of the known term of those skilled in the art " replacement ".Term " cycloalkyl " refers to that the hydrocarbon ring of any non-aromatic, preferred described ring are by 5-12 atomic building.Term " acyl group " refers to by oxygen acid deutero-alkyl or aryl group, preferred ethanoyl.

Term used herein " thiazolinyl " refers to comprise any straight or branched, replacement or the unsubstituted unsaturated alkyl of many unsaturated alkyls, preferred C ₁-C ₆Straight chain, single unsaturated and diunsaturated, unsubstituted alkyl.The alkyl most preferably monounsaturated, that two halogens replace.R at general formula (I) compound ₁And R ₄The position is preferably z-isoprene part especially.Term " cycloalkenyl group " refers to the hydrocarbon ring of any non-aromatic, and preferred described ring is by 5-12 atomic building.

Term used herein " aryl ", " aryl of replacement ", " heteroaryl " and " heteroaryl of replacement " refer to the aromatic hydrocarbons ring, preferably have 5,6 or 7 atoms, most preferably have 6 described rings of atomic building.The aromatic hydrocarbons ring that has at least one heteroatoms (for example oxygen, sulphur or nitrogen-atoms) and at least one carbon atom in " heteroaryl " and " heteroaryl of replacement " finger ring.

Term " alkoxyl group " refer to any straight or branched, replacement or do not replace, saturated or undersaturated ethers, preferred C ₁-C ₆Straight chain, saturated, unsubstituted ether, more preferably methoxyl group, equally preferably dimethyl, diethyl, methyl-isobutyl-and methyl-tertbutyl ether.Term " cycloalkyloxy " refers to any non-aromatic hydrocarbon ring, preferably by 5-12 atomic building ring.

Term used herein " purifying ", " basic purifying " or " isolating " refer to not contain the compound of other dissimilar compound of being correlated with usually under its native state, therefore The compounds of this invention constitute the sample quality of giving (referring to weight) at least 0.5%, 1%, 5%, 10% or 20%, more preferably at least 50% or 75%.

General formula (I) compound can carry out chemosynthesis or preparation by reagent known in this field and that can get.For example, as Loughlin etc., 2000 Bioorg MedChem Lett 10:91 or Brocchini etc. are diacyl piperazinedione (diacetyl piperazinedione) modifying method described in the international application of WO95/21832 at publication number.Described diacyl piperazinedione (diacetyl piperazinedione) can pass through for example with 2 of cheapness, and (TCl Cat.No.G0100 25g) prepares with sodium-acetate and acid anhydrides sodium (sodium anhydride) diacetylation the 5-piperazinedione.The diacetyl structure of the active piperazinedione of described tool can be substituted by other carboxyl groups, thereby comprises the carbamate such as Boc (tert-butoxycarbonyl), Z (benzoyloxy carbonyl).

Described imidazole formaldehyde can be according to for example Hayashi etc., disclosed method (as follows) preparation among the 2000 J OrganicChem 65:8402:

Synthetic method disclosed herein preferably in DMF, as in the presence of the cesium carbonate of alkali and deoxidation atmosphere in carry out.Watanabe etc. for example, 18 ^ThInternational Congress of HeterocyclicChemistry inYokohama, Japan (July 30 calendar year 2001), Abstract, reported in 225 pages that inert atmosphere can be avoided the oxidation (as follows) with the active alpha-carbon atom of contingent piperazinedione ring in the cesium carbonate treating processes.

There is the atmospheric oxidation of active carbonyl compound down in cesium salt

Other embodiment of described synthetic method comprises the modification of the compound that the synthetic middle institute of compound shown in the mutual-through type (I) uses or relates to.This analog derivative can comprise the change of the modification to phenyl ring, the position of introducing other aromatic ring, aromatic nucleus, imidazo ring systems and/or further to the modification of described imidazole ring 5-position.The example that this class is modified is for example being discussed among the embodiment 4.The result that this class is modified comprises the rising of nitrogen content in phenyl ring and/or the described compound, and it can strengthen the solvability of compound.Other modification can be in conjunction with the derivative of known tubulin inhibitor, thus the activity of simulation tubulin inhibitor.Other modification can be simplified the synthetic of 'beta '-ketoester, and it is applied to prepare the imidazole formaldehyde that uses in the method disclosed herein.

Pharmaceutical composition

The present invention also is applied to pharmaceutical composition with compound disclosed herein (at random or preferably being prepared by method disclosed herein), this pharmaceutical composition is included as the medicine acceptable carrier of storage and follow-up administration preparation, and described pharmaceutical composition comprises the above-mentioned product of the medicine effective quantity that is contained in medicine acceptable carrier or the thinner.The acceptable carrier or the thinner that are used for medical use are that pharmaceutical field is known, and at for example Remington ' s Pharmaceutical Science, are described among the MackPublishing Co. (A.R.Gennaro edit.1985).Sanitas, stablizer, dyestuff even seasonings also can be used in the described pharmaceutical composition.For example Sodium Benzoate, xitix and p-Hydroxybenzoate can be used as sanitas.In addition, can use antioxidant and suspension agent.

Described dehydrophenylahistin or dehydrophenylahistin analogue composition can be made into or use with following form: the tablet, capsule or the elixir that are used for oral administration; The suppository that is used for rectal administration; The sterile solution, the suspension that are used for drug administration by injection; The paster that is used for transdermal administration; And subcutaneous settling or the like.Injection can be made into conventional form, as liquor or suspension, be applicable at injection or the preceding solid form that is dissolved or suspended in the liquid of infusion (infusion), or emulsion.The vehicle that is fit to is for example water, salt solution, glucose, mannitol, lactose, Yelkin TTS, albumin, Sodium Glutamate, cysteine hydrochloride, human serum albumin or the like.In addition, if desired, described medicinal composition for injections can contain the nontoxic auxiliary substance of trace, as wetting agent, pH buffer reagent or the like.If desired, can use absorption to strengthen preparation (for example liposome).

The pharmaceutical preparation that is used for parenterai administration comprises the aqueous solution of the described active compound of water-soluble form.In addition, the suspension of described active compound can be prepared into suitable oily injection suspensions.Lipophilic solvent that is fit to or carrier comprise the fatty oil such as sesame oil, or other is such as the organic oil of soybean oil, shaddock or Prunus amygdalus oil, or such as the Acrawax of ethyl oleate or triglyceride level, or liposome.Water injection suspension liquid can contain the material that increases described suspension viscosity, for example Xylo-Mucine, Sorbitol Powder or dextran.At random, described suspension also can contain suitable stablizer or strengthen the reagent of described compound dissolution, the feasible solution that can prepare high density.

Being used for oral pharmaceutical preparation can be by combining described active compound, at random the gained mixture being ground, reaches described mixture is made particle with solid excipient, after the auxiliary material that add to be fit to (if desired), thereby obtain the core of tablet or drageeing.Particularly suitable vehicle is a weighting agent, for example such as the sugar of lactose, sucrose, mannitol or Sorbitol Powder; Such as the cellulose preparation of W-Gum, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose gum, hydroxypropyl methyl-Mierocrystalline cellulose, Xylo-Mucine or the like, and/or polyvinylpyrrolidone (PVP).Can add disintegrating agent if desired, for example cross-linked polyvinylpyrrolidone, agar or alginic acid or such as the alginate of sodiun alginate.The drageeing core is suitably wrapped quilt.For reaching this purpose, can use spissated sugar soln, organic solvent or mixed solvent that it optionally contains gum arabic, talcum, polyvinylpyrrolidone, carbopol gel, polyoxyethylene glycol and/or titanium dioxide, lacquer (lacquer) solution and is fit to.In order to differentiate or represent the various combination of active compound doses, can in described tablet or drageeing dressing, add coating or pigment.For reaching this purpose, can use spissated sugar soln, it optionally contains gum arabic, talcum, polyvinylpyrrolidone, carbopol gel, polyoxyethylene glycol and/or titanium dioxide, lacquer solution and suitable organic solvent or mixed solvent.In order to differentiate or represent the various combination of active compound doses, can in described tablet or drageeing dressing, add coating or pigment.Can use method well known in the art prepare this class preparation (referring to, for example United States Patent (USP) the 5th, 733, No. 888 (composition for injection); 5,726, No. 181 (being insoluble in the compound of water); 5,707, No. 641 (albumen or peptide that therapeutic activity is arranged); 5,667, No. 809 (lipophilicity reagent); 5,576, No. 012 (solubilising polymerization agent); 5,707, No. 615 (anti-virus formulation); 5,683, No. 676 (drug particles); 5,654, No. 286 (topical formulations); 5,688, No. 529 (oral suspension agent); 5,445, No. 829 (sustained release preparation); 5,653, No. 987 (liquid preparation); 5,641, No. 515 (controlled release preparation) and 5,601, No. 845 (spherical preparation)).

This paper discloses also that various pharmacy fields are known to be used to comprise in intraocular, the nose and the pharmaceutical composition that (intraauricular) carries in the ear.Pharmaceutical preparation comprises the water-based ophthalmic solution of described active compound, it can be water-soluble form (as eye drop), or gelling gum (gellan gum) form (Shedden et al., 2001 Clin Ther 23 (3): 440-50) or form of hydrogels (Mayer et al., 1996 Ophthalmologica 210:101-3); Ophthalmic ointment; The eye suspension agent, particulate (being suspended in the little aggregated particles that contains medicine in the liquid carrier medium) (Joshi for example, A., 1994 J OculPharmacol 10:29-45), fat-soluble preparation (Alm etc., 1989 Prog ClinBiol Res312:447-58) and microballoon (Mordenti, 1999 Toxicol Sci 52:101-6); And eye intercalating agent (ocular insert).For stable and comfortable, that the pharmaceutical preparation that this class is fit to is made usually and preferably is aseptic, etc. open and buffered.Pharmaceutical composition also can comprise drops and sprays, and it is prepared into usually simulates nasal discharge in many aspects, thereby guarantees to keep normal eyelashes motion.As Remington ' sPharmaceutical Science (Mack Publishing, 18 ^ThEdition) described in, and those skilled in the art are known, the preparation that is fit to usually and be preferably wait open, slight buffering to be keeping pH5.5-6.5, and is and common and preferably include antibiotic antiseptic and suitable medicine stablizer.The pharmaceutical preparation that is used for carrying in the ear is included in the suspension agent and the ointment of ear topical application.The general solvent that is used for this class aural preparations comprises G ﹠ W.

When being used as cell cycle inhibitor, when tumor growth inhibitor or fungal growth inhibitor compound, described general formula (I) compound can pass through oral or non-oral administration.When oral administration, can capsule, tablet, granule, sprays, syrup or suchlike form administration.When non-oral administration, can the aqueous suspension agent, Oily preparation or similar type administration, or with form administrations such as instillation, suppository, ointment, ointment, when by injection or infusion administration, can carry out at subcutaneous, intraperitoneal, intravenously, intramuscular etc.Similarly, can carry out part, rectum or vagina administration, make described compound and tumour obtain optimal the contact, thereby suppress this growth of tumor according to the mode that those skilled in the art think fit.It is also conceivable that tumor resection is front or rear at the knub position topical, for example the preparation of controllable release, depot formulation and infusion pump are carried.

Medication

The present invention also comprises the method for preparation and described disclosed compound of administration and pharmaceutical composition.Among others, this class disclosed method comprises: (a) by oral route administration comprises with capsule, tablet, granule, sprays, syrup or suchlike form administration; (b) by non-oral administration, comprise with aqueous suspension agent, Oily preparation or the like administration, or with form administrations such as instillation, suppository, ointment, ointment; Can inject or the infusion administration at subcutaneous, intraperitoneal, intravenously, intramuscular, intradermal or the like; And carry out (c) topical according to the mode that those skilled in the art think fit, and (d) rectal administration or (e) vagina administration, thus described compound is contacted with biological tissue; And (f) by controllable release preparation, depot formulation and infusion pump conveying administration.As other example and other disclosed administering mode of this class administering mode, herein disclosed is the method for described disclosed compound of various administrations and pharmaceutical composition, comprise mode by administration in intraocular, the nose and in the ear.

The dosage of the required described dehydrophenylahistin or the medicine effective quantity of dehydrophenylahistin analogue composition depends on route of administration, the constitutive character of animal (comprising the people) type of being treated and the concrete animal of being considered.Described dosage can be adjusted to and can reach required effect, but can depend on such as the factor of body weight, diet, the pharmacological agent carried out simultaneously and the factor that the described technician of other field of medicaments generally acknowledges.

In putting into practice the process of described method, described compound or composition can be used alone or in conjunction with each other, or are used in combination with other treatment or diagnostic reagent.For example, as disclosed herein, when with other active substance (especially other chemotherapeutics) of biological example preparation and specific chemotherapeutic agent CPT-11, taxotere (docataxel) and Paclitaxel when being used in combination, compound disclosed herein can effectively be treated cancer.When being used in combination with other active substance, compound disclosed herein also can effectively be treated cancer, these active substances comprise anti-angiogenic dose (anti-vascular agents), the angiogenesis inhibitor reagent such as Erbuitus (Imclone/bristol-Myers) and Iressa (AstraZeneca), other VEGF inhibitor and biotechnological formulation, more particularly comprise at least a anti-VEGF antibodies, particularly to the monoclonal antibody of vegf receptor, it comprises DC101, the rat monoclonal antibody of a kind of blocking-up mouse vegf receptor 2 (flk-1).This class combination can be applicable in the body, in mammalian body, preferably in human body, perhaps can be applicable to external usually.It is being carried out the body planted agent time spent, described disclosed compound (separately or with other chemotherapy agents or the combination of other biotechnological formulation) can use various dosage forms, by number of ways to the Mammals administration, described approach comprises non-enteron aisle, intravenously, by infusion or injection, subcutaneous, intramuscular, colon, rectum, vagina, nose or intraperitoneal.These class methods also can be applicable to chemically reactive in the test body.

As to those skilled in the art apparently, useful vivo medicine-feeding dosage and concrete administering mode can be according to the mammal species of being treated, body weight and ages, employed particular compound and use the specific purposes of these compounds and change.Those skilled in the art can determine effective dosage level (promptly reaching the necessary dosage level of required effect) according to the pharmacological method of routine.Usually, the human clinical of product uses from lower dosage level, improves constantly dosage level subsequently up to reaching required effect.Selectively, can adopt acceptable in vitro study to set up the useful dosage and the route of administration of the composition of present method evaluation by existing pharmacological method.

In inhuman zooscopy, the application of potential product reduces dosage till can't realizing that required effect or harmful side effect disappear subsequently at first from higher dosage.Rely on required effect and described treatment performance (indication), described dosage can change in very wide scope.Usually, used dosage can be between about 10 micrograms/kg body weight and 100mg/kg body weight, preferably between about 100 micrograms/kg body weight and 10mg/kg body weight.Selectively, it should be understood by those skilled in the art that used dosage can calculate according to patient's surface-area.Oral administration can per three days once, every other day once, once a day, twice of every day or every day three times mode carry out.

Each doctor can select definite prescription, route of administration and dosage according to patient's situation.Referring to, the The Pharmacological Basis of Therapeutics of Fingl etc. for example, 1975.It is pointed out that described doctor should be able to judge how to reach when stop, interrupting or adjust administration according to toxicity or the not normal situation of organ dysfunction.On the contrary, when the clinical response deficiency when (not comprising toxicity) described doctor also will be appreciated that treatment transferred to higher level.The dosage value fibrous root that is adopted when the disease that control is paid close attention to changes according to the seriousness and the route of administration of morbid state to be treated.For example can partly estimate the seriousness of described morbid state by the prognostic evaluation method of standard.In addition, described dosage and dose frequency roughly also can be adjusted according to age, body weight and the reaction of individual patient.Can use the scheme suitable to veterinary medicine with above-mentioned discussion.

According to the disease specific state of being treated, this class reagent can carry out preparation or administration at whole body or part.Can be in Remington ' s Pharmaceutical Sciences, 18 ^ThEd., MackPublishing Co., Easton finds the technology of various prescriptions and administration among the PA (1990).That the route of administration that is fit to can comprise is oral, administration in rectum, transdermal, vagina, saturating film or the intestines; Non-enteron aisle is carried, comprise in intramuscular, subcutaneous, intramedullary injection and the sheath, directly in the ventricle, intravenously, by in infusion, intraperitoneal, the nose or intraocular injection.

To injection or infusion, but described reagent prescription is in the aqueous solution, for example in the physiological compatibility damping fluid such as Hank ' s solution, Ringer ' s solution or normal saline buffer solution.To those saturating film administrations, in described preparation, use the permeate agent that is suitable for permeating barrier.This class permeate agent is well known in the art.The scope of the invention also comprises uses the medicine acceptable carrier that compound disclosed herein is carried out prescription, thereby the present invention is made the formulation that is suitable for the whole body administration.The preparation method who selects appropriate carriers and be fit to, composition disclosed herein (particularly making the composition of solution) can pass through parenterai administration, for example by intravenous injection or infusion.Described compound can use medicine acceptable carrier well known in the art to make the formulation that is suitable for oral administration at an easy rate.This class carrier makes described compound can be made into tablet, pill, capsule, liquid, gel, syrup, slurries, suspension or the like, and it is oral to be convenient to patient to be treated.

Need the reagent of administration in the cell can adopt those skilled in the art's technique known to carry out administration.For example, this class reagent can be encapsulated in the liposome, then administration as stated above.All molecules in the liposome forming process in the aqueous solution all are incorporated in its internal water solution.The interior composition of liposome all is protected and not influenced by outside microenvironment, and because liposome and cytolemma merge, therefore described composition is imported in the tenuigenin effectively.In addition, because its hydrophobicity, the small molecules organic molecule can directly carry out administration in the cell.

To effective dose determine it is that those skilled in the art are known, especially according to concrete disclosed guidance provided herein.Except that described activeconstituents, these pharmaceutical compositions also can contain suitable medicine acceptable carrier (comprising vehicle and auxiliary material), and it can make the preparation that is used for medicinal use with described active compound easily.The preparation that is used for oral administration can be the form of tablet, drageeing, capsule or solution.Described pharmaceutical composition can adopt known mode to be prepared, for example by conventional mixing, dissolving, granulating, drageeing making, suspension, emulsification, encapsulated, collecting (entrap), lyophilization.

Can use known method that compound disclosed herein is carried out drug effect and toxicity assessment.For example, can set up the toxicology of the compound subclass of particular compound or shared some chemical part by the in vitro toxicity of measuring pair cell system (as mammiferous, preferred people's clone).The result of this class research can be used for the prediction toxicity of (for example Mammals or more specifically interior for human body) in animal body.Selectively, can use known method to determine that particular compound is such as the toxicity in the animal model of mouse, rat, rabbit or monkey.Can use multiple art-recognized method to determine the drug effect of particular compound, these methods are in vitro method, animal model or human clinical trial for example.Basically generally acknowledged to all capable territory of each class morbid state in vitro method, described morbid state comprises the morbid state of available compounds for reducing disclosed herein, comprises cancer, cardiovascular disorder and various fungi infestation.Similarly, can use acceptable animal model to determine the drug effect of the chemical substance of this class morbid state of treatment.When selecting to determine the model of drug effect, those skilled in the art can select suitable model, dosage and route of administration and scheme according to the guidance of this area regulation.Certainly, also can utilize the human clinical trial to determine the drug effect of compound to the people.

When as antitumor and anticancer agent or tumor growth when suppressing compound, compound disclosed herein can pass through oral or non-oral administration.During oral administration, it can capsule, tablet, granule, sprays, syrup or suchlike form administration.During non-oral administration, it can waterborne suspension, Oily preparation and similar type thereof or instillation, suppository, ointment, ointment or the like form administration, when by injection or during the infusion administration, can adopt subcutaneous, intraperitoneal, intravenously, intramuscular, transdermal or the like mode to carry out.Similarly, the mode of can those skilled in the art thinking fit is carried out part, rectum or vagina administration, and described compound is well contacted with tumour, thereby suppresses tumor growth.Also can consider before or after tumor resection, at the position of tumour or other morbid state topical, or with its part as the methods of treatment of art-recognized described morbid state.Similarly, also can consider controllable release formulation, storage formulation and infusion pump conveying.

When as antitumor and anticancer agent or anti-tumor agent comprising salmosin, can arrive about 7 in 0.0007mg/ days, 000mg/ days described activeconstituents dosage carries out oral or non-oral administration to human patients, 0.07mg/ days to about 70mg/ days described activeconstituentss more preferably from about, preferably once a day, or less preferred every day 2 to 10 times.Can select and equally preferably, described compound preferably the mode by for example intravenous drip with the consistent dose successive administration.Therefore, patient for 70 kilograms of body weight, the preferred per daily dose of described active antineoplastic component is about 0.0007mg/kg/ days to about 35mg/kg/ days (comprising 1.0mg/kg/ days and 0.5mg/kg/ days), more preferably from 0.007mg/kg/ days to about 0.050mg/kg/ days (comprising 0.035mg/kg/ days).Yet will be understood by those skilled in the art that may need administration to surpass even the described antineoplastic compound of super above-mentioned preferred dose scope far away in some cases, thereby treats specific late period or lethality tumour effectively and energetically.

When the anti-mycotic agent, described to treatment or prevent the preferred dose of the effective dehydrophenylahistin of concrete fungal pathogens or its analogue to need part according to the characteristic of described fungi and the degree of infection, and can determine by the clinical technology of standard.Can select to adopt external or the definite best dosage range of body inner analysis help.Can be from determining effective dose by analyzed in vitro or the preferred dose-response curve extrapolation that obtains by animal model.Definite dosage level should be determined that this class factor comprises age, body weight, sex and comprehensive physique of route of administration, individuality by the doctor who uses or other health worker according to known factor; The character, seriousness and the clinical stage that infect; Use (or not using) to follow treatment.

The effective dosage ranges of described dehydrophenylahistin or its analogue usually every day about 0.01mg/kg to about 50mg/kg weight of mammal, be preferably about 0.1mg/kg and arrive about 10mg/kg, but single administration or multiple dosing.Usually, described compound can each patient's every day about 1mg to the dosage of about 2000mg the patient of this treatment of needs is carried out administration.

To comprise the preparation as the compound that suppresses tumor growth disclosed herein in order preparing, can to use known tensio-active agent, vehicle, smooth agent (smoothing agent), suspension reagent and the acceptable filmogen of medicine and bag by auxiliary material and analogue thereof.Preferably use alcohol, ester, sulphated fatty alcohol or the like as tensio-active agent; Sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, slight anhydrous silicate, magnesium aluminate, aluminic acid methyl siliconic acid magnesium (Magnesiummethasilicate aluminate), synthetic aluminium silicate, lime carbonate, acid sodium carbonate, secondary calcium phosphate, calcium carboxymethylcellulose or the like are as vehicle; Magnesium Stearate, talcum, winterized stearin or the like are as smooth agent; Oleum Cocois, sweet oil, sesame oil, peanut oil, soybean oil are as suspension reagent or lubricant; Cellulose acetate-phthalate (derivative of carbohydrate (as Mierocrystalline cellulose and sugar)) or ritalin-methylacrylic acid ester copolymer (polythene derivative) are as suspension reagent; And such as the softening agent of phthalic ester or the like as suspension reagent.Except that aforementioned preferred component, sweetener, spices, tinting material, sanitas or the like also can add in the drug-delivery preparation of described compound, especially when described compound adopts oral administration.

Also can comprise the medicine acceptable carrier in the pharmaceutical composition disclosed herein.Can be storage and follow-up administration purpose and prepare this based composition.The acceptable carrier or the thinner that are used for medical treatment are that pharmaceutical field is known, and at for example Remington ' s Pharmaceutical Sciences, are described among the MackPublishing Co. (A.R.Gennaro edit.1985).For example, this based composition can be made into or uses with following form: the tablet, capsule or the solution that are used for oral administration; The suppository that is used for rectum or vagina administration; The sterile solution or the suspension that are used for drug administration by injection.Injection can be made into conventional form, as liquor or suspension, be applicable to the solid form that before injection or infusion, is dissolved or suspended in the liquid, or emulsion.The vehicle that is fit to includes but not limited to salt solution, glucose, mannitol, lactose, Yelkin TTS, albumin, Sodium Glutamate, cysteine hydrochloride or the like.In addition, if desired, described medicinal composition for injections can contain the nontoxic auxiliary substance of trace, as wetting agent, pH buffer reagent or the like.If desired, can use absorption to strengthen preparation (for example liposome).

The dosage of the medicine effective quantity of required described composition depends on route of administration, the type of animal of being treated and the constitutive character of the concrete animal considered.Described dosage can be adjusted to and reach required effect, but can depend on such as the factor of body weight, diet, the pharmacological agent carried out simultaneously and the factor that the described technician of other field of medicaments generally acknowledges.

Aforesaid compound or composition can be used alone or in conjunction with each other, or are used in combination with other treatment or diagnostic reagent.Particularly, described disclosed compound product can use separately or be used in combination with other chemotherapy agents or biotechnological formulation (comprising antibody) when being used for the treatment of cancer, or is used in combination with other anti-infection agent when being used for the treatment of fungi infestation.These products or composition can be in vivo or external use.The useful dosage of administration and more useful mode can be according to body weight, age and the animals of being treated, employed particular compound and use the concrete purposes of this composition or these compositions and change.The dose value that is adopted when control or treatment disease specific needs to change according to the seriousness and the route of administration of morbid state to be treated, and according to described morbid state and seriousness thereof, described composition can carry out preparation or administration at whole body or part.Can be in Remington ' sPharmaceutical Sciences, 18 ^ThEd., Mack Publishing Co., Easton finds the technology of various preparations and administration among the PA (1990).

For general formula (I) compound (preferably according to the synthetic preparation of method disclosed herein) as cell cycle inhibitor, tumor growth inhibition or antifungal compound is carried out preparation, can use known tensio-active agent, vehicle, smooth agent, suspension reagent and the acceptable filmogen of medicine and bag by auxiliary material and analogue thereof.Preferably use alcohol, ester, sulphated fatty alcohol or the like as tensio-active agent; Sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, slight anhydrous silicate, magnesium aluminate, aluminic acid methyl siliconic acid magnesium, synthetic aluminium silicate, lime carbonate, acid sodium carbonate, secondary calcium phosphate, calcium carboxymethylcellulose or the like are as vehicle; Magnesium Stearate, talcum, winterized stearin or the like are as smooth agent; Oleum Cocois, sweet oil, sesame oil, peanut oil, soybean oil are as suspension reagent or lubricant; Cellulose acetate-phthalate (derivative of carbohydrate (as Mierocrystalline cellulose and sugar)) or ritalin-methylacrylic acid ester copolymer (polythene derivative) are as suspension reagent; And such as the softening agent of phthalic ester or the like as suspension reagent.Except that aforementioned preferred component, sweetener, spices, tinting material, sanitas or the like also can add in the drug-delivery preparation of the compound for preparing by described method, especially when described compound adopts oral administration.

Cell cycle inhibitor, anti-tumor agent comprising salmosin and the anti-mycotic agent of available described method preparation can be by oral or non-oral form to the human patients administrations, used dosage arrived about 10 for about 0.001mg/kg/ days, 000mg/kg/ days described activeconstituentss, 0.1mg/kg/ days to about 100mg/kg/ days described activeconstituentss more preferably from about, preferably with three days once be one-period, two days once, once a day, every day twice, or surpass less preferred every day 2 to about 10 times.Selectively and equally preferably, by the compound of described method preparation preferably the mode by for example intravenous drip with the consistent dose successive administration.Therefore, for the patient of 70 kilograms of body weight for example, the preferred per daily dose of described active antineoplastic component is about 0.07mg/ days to about 700g/ days, more preferably from 7mg/ days to about 7g/ days.Yet, those skilled in the art are appreciated that, may need administration to surpass in some cases even the described antineoplastic compound of the described method preparation of usefulness of super above-mentioned preferred dose scope far away, to treat specific late period or lethality tumour effectively and energetically.

When using the cell cycle inhibitor for preparing by described method as biochemical test reagent, the carrying out in the compound cycle capable of inhibiting cell when being dissolved in organic solvent or water-containing organic solvent by the inventive method preparation, and it can be directly applied to various cultured cells systems.The available organic solvent comprises, for example methyl alcohol, methyl sulfoxide or the like.Described preparation can be for example pulvis, granule or other solid inhibitor, or with an organic solvent or the liquid inhibitor of anhydrous organic solvent preparation.Although the compound by the inventive method preparation as the preferred concentration of cell cycle inhibitor usually at about 1 μ g/ml to about 100 μ g/ml scopes, but will be understood by those skilled in the art that optimal dosage depends on the kind of application target and cultured cells system.Simultaneously, in some applications, the dosage that those skilled in the art may need or preferably use surpasses above-mentioned scope.

From medical angle, some embodiment provides fungi infestation in prevention or the treatment individuality and/or the method for pathogenic agent fungi, comprise the composition that described individual administration is comprised dehydrophenylahistin or its analogue, for example dosage and the described dehydrophenylahistin of mode administration or its analogue so that required anti-mycotic efficiency can be provided.

Other embodiment comprise the treatment or the prevention patient by the fungus-caused infection of all that enumerated as mentioned or hereinafter related pathogenic agent.

Another embodiment relate to treat or prevent the patient by the fungus-caused infection of pathogenic agent, wherein said pathogenic agent fungi can anti-one or more other anti-mycotic agents (reagent that particularly is different from dehydrophenylahistin or its analogue), comprise for example amphotericin B or its analogue or derivative (hydrazides and other derivative that comprise 14 (s)-hydroxyl AmBMEs, amphotericin B and 1-amino-4-methylpiperazine) or other polyene macrolide microbiotic, comprise for example nystatin (nystatin), candicidin, pimaricin and natamycin; Flucytosine; Grisovin (griseofulvin); Echinocandin (echinocandin) or aureobasidin comprise natural or semisynthetic analogue; Dihydrobenzo [a] four benzoquinones (dihydrobenzo[a] naphacenequinone); Nucleosides peptide anti-mycotic agent comprises polyoxin (polyoxin) and Buddhist nun's Mycosporin (nikkomycin); Propylamine and other squalene epoxidase (epoxidase) inhibitor such as naftifungin (naftifine); And pyrroles, imidazoles and triazole, for example clotrimazole, miconazole, KETOKONAZOL, econazole, butoconazole (butoconazole), oxiconazole (oxiconazole), Triaconazole (terconazole), itraconazole (itraconazole) or fluconazole (fluconazole) or the like.To other conventional anti-mycotic agent and the novel agent developed referring to for example Turner and Rodriguez, 1996 Current PharmaceuticalDesign, 2:209-224.Another embodiment comprise treatment or prevention patient by the fungus-caused infection of pathogenic agent, wherein said patient is to one or more other anti-mycotic agent allergy, or do not tolerate or reactionless, perhaps described patient forbidden other anti-mycotic agent.Among others, these other antiseptic-germicides also comprise above or the disclosed antiseptic-germicide of this paper other parts.

In aforementioned therapies or prevention method, with effective antibiotic dosage with dehydrophenylahistin or its analogue to individual administration.

Other embodiment relate to treat or prevent the patient by the fungus-caused infection of pathogenic agent, it passes through dehydrophenylahistin or its analogue administration, simultaneously in conjunction with one or more other anti-mycotic agents of administration (for example comprise aforementioned agents or types of agents arbitrarily, as with use amphotericin B (preferably with lipoid or Liposomal formulation form); For example pyrroles or triazole (as fluconazole); Aureobasidin; Dihydrobenzo [a] four benzoquinones; Or the therapeutic combination of echinocandin) and different dehydrophenylahistins or its analogue.

Described dehydrophenylahistin or its analogue can be before described other antiseptic-germicide administrations, afterwards or administration simultaneously.In certain embodiments, described combined therapy makes and compares and can reduce relatively when a kind of or whole consumption of two kinds of antibacterial components uses separately with it.

Other embodiment is related to treatment or prevents by the fungus-caused infection of pathogenic agent individual administration dehydrophenylahistin or its analogue, wherein said individuality is immunosuppression or immunocompromised host, for example the result who is caused by following factor: genetic flaw, such as the disease of diabetes or HIV or other infection, to the chemotherapy of cancer or other disease or radiotherapy or drug-induced immunosuppression or with the relevant inductive immunosuppression of treatment of tissue or organ transplantation or autoimmunization obstacle.When described patient is accepting maybe will accept the treatment (for example relevant with tissue or organ transplantation) of immunosuppressor, can be with dehydrophenylahistin or its analogue and described immunosuppressor co-administered, with treatment or the fungi infestation of prevention pathogenic agent.

Another aspect of the present invention relate to treatment or prevention HIV infect (or suspect and infect) patient by the fungus-caused infection of pathogenic agent, comprise antimycotic dehydrophenylahistin or its analogue administration, simultaneously in conjunction with one or more anti-HIV therapeutical agents of administration (comprising for example hiv protease inhibitor, reverse transcriptase inhibitors or antiviral agent).Described dehydrophenylahistin or its analogue can be before the administrations of described anti-HIV reagent, afterwards or administration simultaneously.

Another aspect of the present invention relate to treat or prevent the patient by the fungus-caused infection of pathogenic agent, comprise antimycotic dehydrophenylahistin or its analogue administration, simultaneously in conjunction with one or more other Antibiotique compositions of administration, one or more antiseptic-germicides particularly are preferably with the effective dose or the scheme administration of treatment or prevention infectation of bacteria.Equally, described dehydrophenylahistin or its analogue can be before described other reagent administration, afterwards or administration simultaneously.

Among others, the pathogenic agent fungi infestation of available method disclosed herein treatment or prevention also comprises: aspergillosis comprises the invasive pulmonary aspergillosis; Blastomycosis is included in the degree of depth in the central nervous system or the type that develops rapidly infects and blastomycosis; Moniliosis, comprise urinary tract degeneration moniliosis (as block at urinary stone disease, urinary tract, among renal transplantation or the not good diabetic subject of control); Coccidioidomycosis comprises the not good chronic disease of other chemotherapy reaction; Torulosis; Histopolasmosis; Mucormycosis comprises for example cranium face mucormycosis and pulmonary mucormycosis; Brazilian blastomycosis; And sporotrichosis.It is to be noted, the composition administration that will comprise one or more dehydrophenylahistins of antibiotic dosage or its analogue is specially adapted to treat or prevent the pathogenic agent fungi infestation of mammalian subject, anti-one or more other antibacterial therapy agent of wherein said fungi or wherein one or more antifungal therapy agent are forbidding (for example mentioned above).

The invention also discloses and put into practice the antifungal medicine composition that comprises at least a antimycotic dehydrophenylahistin or its analogue that described open method is adopted.These pharmaceutical compositions can be packed together with suitable packing plug-in part (among others, comprising the tips and information relevant with its antifungal application).The invention also discloses the pharmaceutical composition that contains one or more dehydrophenylahistins or its analogue and second anti-mycotic agent.

The method of treatment fungi infestation

Some embodiment disclosed herein relates to the method for the treatment of or preventing pathogenic agent fungi infestation, and described infection comprises that for example aspergillosis comprises the invasive pulmonary aspergillosis; Blastomycosis is included in the degree of depth in the central nervous system or the type that develops rapidly infects and blastomycosis; Moniliosis, comprise urinary tract degeneration moniliosis (as block at urinary stone disease, urinary tract, among renal transplantation or the not good diabetic subject of control); Coccidioidomycosis comprises the chronic disease not good to other therapeutic response; Torulosis; Histopolasmosis; Mucormycosis comprises for example cranium face mucormycosis and pulmonary mucormycosis; Brazilian blastomycosis; And sporotrichosis.Described method can comprise at least a aforesaid antimycotic dehydrophenylahistin or its analogue human individual's administration, thus treatment or prevention fungi infestation.In certain embodiments, can be with described dehydrophenylahistin or its analogue administration the time in conjunction with the antifungal agents of one or more non-dehydrophenylahistins of administration or its analogue, for example amphotericin B or all imidazoles as the aforementioned or triazole reagent.

Described pathogenic agent fungi infestation can be partial (for example except that other biology, cause by Candida, a moss Pseudomonas, microsporum or Epiderinophyton (Epidermophyton) kind), or (for example the causing by Candida albicans (for example mycodermatitis and vaginal candidiasis)) of mucous membrane.Described infection can be a general, and is for example microbial by Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Coccidiode, Paracocciciode, Histoplasma (Histoplasma) or blastogenesis.Described infection also can comprise fungi mycetoma, chromoblastomycosis (chromoblastomycosis), cryptococcal meningitis (cryptococcal meningitits) or phycomycosis (phycomycosis).

Other embodiments relate to the method for the treatment of or preventing pathogenic agent fungi infestation, and wherein said fungi is selected from: Candida comprises Candida albicans, Oidium tropicale, Candida kefyr, candida krusei and Candida glabrata; Aspergillus comprises Aspergillus fumigatus and flavus; Cryptococcus neoformans; Blastomycete comprises Blastomyces dermatitidis; Pneumocystis carinii; Posadasis spheriforme; The living frog excrement of the frog is mould; Conidiobolus; Histoplasma capsulatum; Rhizopus, for example Rhizopus oryzae and Rhizopus microsporus; Cunninghammella; Root Acarasiales belongs to; Blastomyces brasiliensis; The swollen bacterium of Podbielniak foot; Rhinosporidium seeberi and sporotrichosis schenckii.Equally, described method can comprise the non-immunosuppressant antimycotic dehydrophenylahistin of the individual administration that needs are arranged or its analogue, thus treatment or prevention fungi infestation and can not induce negative immunosuppressive action.

Other embodiments relate to the method for the treatment of or preventing the pathogenic agent fungi infestation of anti-other antifungal therapy agent, described infection comprises the pathogenic agent fungi infestation of the described anti-mycotic agent of anti-one or more this paper other parts, these anti-mycotic agents are amphotericin B, flucytosine, a kind of imidazoles or triazole (comprising, for example fluconazole, KETOKONAZOL, itraconazole gram and other previous example) for example.Described method can comprise one or more antimycotic dehydrophenylahistins or its analogue with the dosage of the fungi infestation that can treat or prevent individual anti-other antifungal therapy agent and scheme described patient's administration.

Other embodiments relate to the method for the treatment of or preventing patient's pathogenic agent fungi infestation, wherein said patient is to other antifungal therapy allergy, or do not tolerate or reactionless, perhaps described patient is forbidden other anti-mycotic agent, described other anti-mycotic agent comprises other anti-mycotic agent that one or more this paper other parts are mentioned, for example amphotericin B, flucytosine, a kind of imidazoles or triazole (comprising, for example fluconazole, KETOKONAZOL, itraconazole gram and other previous example).Described method can comprise one or more antimycotic dehydrophenylahistins or its analogue with the dosage that can treat or prevent fungi infestation this class patient administration.

Dehydrophenylahistin or its analogue of packing

Some embodiment relates to dehydrophenylahistin or its analogue of packing, the antimycotic dehydrophenylahistin of non-inhibitive ability of immunity or its analogue of preferred packaging, the implication of this term is meant and comprises at least a above-mentioned dehydrophenylahistin or its analogue of packing with specification sheets that described instructions direct does not cause negative immunosuppressive effect as anti-mycotic agent to human individual's administration with described dehydrophenylahistin or its analogue.In certain embodiments, the antimycotic dehydrophenylahistin of described non-inhibitive ability of immunity or its analogue are the members of a preferred subset of above-claimed cpd.Described dehydrophenylahistin or its analogue can be packed with described specification sheets separately, or can be with other dehydrophenylahistin or its analogue, rapamycin or other composition or additive (for example composition of one or more described pharmaceutical compositions) packing.Described packing can comprise one or more containers that are contained with one or more described pharmaceutical composition compositions.At random identify the notice of government bodies' prescribed form of production, use or sale that management medicine or biology product are arranged on this class container, this notice shows that above-mentioned machine-operated approval is human administration purpose manufacturing, uses or sale.

The embodiment purpose of following indefiniteness is to describe the preferred method that uses some preferred embodiment.To used concrete grammar and in practice the change carried out of the details of the specific chemical combination of gained undoubtedly be that those skilled in the art are conspicuous.

Embodiment 1

A. dehydrophenylahistin is synthetic

As shown in fig. 1, synthesize dehydrophenylahistin according to following primitive reaction route by polycondensation:

N, N '-diacetyl-2,5-piperazinedione

To be dissolved in 100mL acetic anhydride (Ac ₂O) 2 of 25.0g, 5-piperazinedione 1[2,5-piperazinedione (Aldrich G640-6), 25.0g, 0.218mol] (17.96g 0.218mol) mixes with sodium-acetate (NaOAc).Described mixture was heated 8 hours at 110 ℃ under ar gas environment with double plate condenser (double coiled condenser).Remove Ac by evaporation ₂Behind the O, resistates is dissolved with AcOEt, with 10% citric acid, 10%NaHCO ₃With saturated NaCl washing (each is three times), Na ₂SO ₄Drying, and under vacuum, concentrate.Resistates grinds the formation solid with ether.With ether-hexane this solid recrystallization from EtOAc is come out, obtain the N of 26.4g (61%), N '-diacetyl-2,5-piperazinedione 1.

1-ethanoyl-3-{ (Z)-1-[5-(1,1-dimethyl-2-propenyl)-1H-4-imidazolyl] methylene radical }-2, the 5-piperazine Piperazine diketone 2

To 5-(1,1-dimethyl-2-propenyl) imidazoles-4-formaldehyde (100mg, add in DMF 0.609mmol) (2mL) solution compound 1 (241mg, 1.22mmol), at short notice gained solution is carried out the repetition emptying to remove oxygen and to charge into Ar, add Cs subsequently ₂CO ₃(198mg 0.609mmol), and repeats the process of emptying-inflation once more.The gained mixture is at room temperature stirred 5h.Except that after desolvating, this resistates is dissolved in EtOAc and 10%Na by evaporation ₂CO ₃Mixture in, the gained organic phase is used 10%Na once more ₂CO ₃Wash and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.Residual oily matter with CHCl ₃-MeOH (100: 0 to 50: 1) is the eluent purification by silica gel column chromatography, obtains 60mg (33%) faint yellow solid 2.

Dehydrophenylahistin

(30mg, (51mL, 0.496mol 5eq), carry out the repetition emptying to remove oxygen and to charge into Ar to gained solution at short notice, add Cs subsequently to add phenyl aldehyde in DMF 0.099mmol) (0.8mL) solution to compound 2 ₂CO ₃(53mg, 0.149mmol 1.5eq), and repeat the process of emptying-inflation once more.(temperature must slowly promote at 80 ℃ of heating 2.5h with the gained mixture.Intensification can cause the increase at the E-at benzylidene position isomer growing amount rapidly)., except that after desolvating described resistates is dissolved among the EtOAc by evaporation, washes twice and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.On TLC, use CHCl ₃-MeOH (10: 1) can be observed bright orange green emitting point under 365nmUV.HPLC analyzes the purity of learning this crude product and surpasses 75%.The gained resistates is dissolved in 90% the MeOH aqueous solution and adds reversed-phase HPLC post (YMC-Pack, ODS-AM, in 20 * 250mm), and the MeOH aqueous solution of using from 70% to 74% linear gradient about 16 minutes with 12mL/ minute flow rate wash-out, collect required part and, obtain the yellow dehydrophenylahistin of 19.7mg (60%) by evaporation concentration.The HPLC spectrogram of the thick dehydrophenylahistin of institute's synthetic as shown in Figure 2.

In the dehydrophenylahistin of purifying (as shown in Figure 4), main peak is the Z-formalization compound of required dehydrophenylahistin.The E-isomer of observing formation is accessory constituent (about 10%), and its wash-out becomes the polarity peak bigger than Z-isomer.Also observe another accessory peak, it represents the Z-that the dimethyl-allyl of dehydrophenylahistin partly is reduced and the reduzate of E-compound.The formation of described reducing compound is because described aldehyde 2 contains reductive impurity, and it is carrying out generation and not separated in subsequent process in the reductive process with DIBAL-H.

These less important compounds can be removed by preparation HPLC purifying, obtain the dehydrophenylahistin that has the Z-conformation at described benzylidene position, yield 60% (yield in two steps is 20%), and purity surpasses 95%.In this HPLC spectrogram, do not observe the compound that has the E-conformation in the imidazoles side of described piperazinedione ring, show first reaction shown in Figure 1 from compound 1 to 3 be Z-optionally.

B. Chemical property

Above-mentioned dehydrophenylahistin compound is a faint yellow solid.By standard NMR analysis confirmation its structure.

Embodiment 2

Synthetic and the physical properties of the tertiary butyl-dehydrophenylahistin derivative

The structural derivative that has synthesized dehydrophenylahistin according to the reaction scheme that is prepared as follows the tertiary butyl-dehydrophenylahistin.The synthetic (see figure 1) of pass course A in some aspects with embodiment 1 in dehydrophenylahistin synthetic similar.

Route A:

Prepare N according to method described in the embodiment 1, N '-diacetyl-2,5-piperazinedione 1.

1) 1-ethanoyl-3-{ (the Z)-1-[5-tertiary butyl-1H-4-imidazolyl] methylene radical }-2,5-dioxopiperidine (16)

(3.02g, (5.89g 29.72mmol), carries out the repetition emptying to remove oxygen and to charge into Ar to gained solution at short notice, adds Cs subsequently to add compound 1 in DMF 19.8mmol) (30mL) solution to 5-t-butyl imidazole-4-formaldehyde 15 ₂CO ₃(9.7g 29.72mmol), and repeats the process of emptying-inflation once more.The gained mixture is at room temperature stirred 5h.Except that after desolvating, described resistates is dissolved in EtOAc and 10%NaHCO by evaporation ₃Mixture in, the gained organic phase is used 10%Na once more ₂CO ₃Wash and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.Residual oily matter with CHCl ₃-MeOH (100: 0 to 50: 1) is the eluent purification by silica gel column chromatography, obtains 1.90g (33%) faint yellow solid 16. ¹H?NMR(270MHz，CDCl ₃)，12.14(d，br-s，1H)，9.22(br-s，1H)，7.57(s，1H)，7.18(s，1H)，4.47(s，2H)，2.65(s，3H)，1.47(s，9H)。

2) the stupid basic ahistins of the tertiary butyl-dehydrogenation

To 1-ethanoyl-3-{ (the Z)-1-[5-tertiary butyl-1H-4-imidazolyl] methylene radical }-2, (11mg adds phenyl aldehyde (19 in DMF 0.038mmol) (1.0mL) solution to 5-dioxopiperidine (16)

0.19mmol, 5eq), at short notice gained solution is carried out the repetition emptying to remove oxygen and to charge into Ar, add Cs subsequently ₂C0 ₃(43mg, 0.132mmol 3.5eq), and repeat the process of emptying-inflation once more.The gained mixture is heated 2.5h at 80 ℃., except that after desolvating described resistates is dissolved among the EtOAc by evaporation, washes twice and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.The gained resistates is dissolved in 90% the MeOH aqueous solution and adds reversed-phase HPLC post (YMC-Pack, ODS-AM, in 20 * 250mm), and the MeOH aqueous solution of using from 70% to 74% linear gradient about 16 minutes with 12mL/ minute flow velocity wash-out, collect required part and, obtain the yellow tertiary butyl dehydrophenylahistin of 6.4mg (50%) by evaporation concentration. ¹H?NMR(270MHz，CDCl ₃)，12.34(br-s，1H)，9.18(br-s，1H)，8.09(s，1H)，7.59(s，1H)，7.31-7.49(m，5H)，7.01(s，2H)，1.46(s，9H)。

Identical among the dehydrophenylahistin reaction of the preparation tertiary butyl-dehydrophenylahistin and the embodiment 1.

The total recovery of the tertiary butyl-dehydrophenylahistin that reclaims is 16.5%.

Route B:

Prepare N according to method described in the embodiment 1, N '-diacetyl-2,5-piperazinedione 1. 1) 1-ethanoyl-3-[(Z)-benzylidene-1]-2,5-dioxopiperidine (17)

(0.54g, (2.0g 10.1mmol), carries out the repetition emptying to remove oxygen and to charge into Ar to gained solution at short notice, adds Cs subsequently to add compound 1 in DMF 5.05mmol) (5mL) solution to phenyl aldehyde 4 ₂CO ₃(1.65g 5.05mmol), and repeats the process of emptying-inflation once more.The gained mixture is at room temperature stirred 3.5h.Except that after desolvating, described resistates is dissolved in EtOAc and 10%Na by evaporation ₂CO ₃Mixture in, the gained organic phase is used 10%Na once more ₂CO ₃Wash and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.The gained resistates obtains 17 white-yellowish solid with MeOH-ether recrystallization; Product 1.95g (79%).

2) tertiary butyl-dehydrophenylahistin

To 1-ethanoyl-3-[(Z)-benzylidene-1]-2; 5-dioxopiperidine (17) (48mg; 0.197mmol) DMF (1.0mL) solution in add 5-t-butyl imidazole-4-formaldehyde 15 (30mL; 0.197mmol); at short notice gained solution is carried out the repetition emptying to remove oxygen and to charge into Ar, add Cs subsequently ₂CO ₃(96mg 0.296mmol), and repeats the process of emptying-inflation once more.The gained mixture is heated 14h at 80 ℃., except that after desolvating described resistates is dissolved among the EtOAc by evaporation, washes twice and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.The gained resistates is dissolved in 90% the MeOH aqueous solution and adds reversed-phase HPLC post (YMC-Pack, ODS-AM, in 20 * 250mm), and the MeOH aqueous solution of using from 70% to 74% linear gradient about 16 minutes with 12mL/ minute flow velocity wash-out, collect required part and, obtain the yellow tertiary butyl dehydrophenylahistin of 0.8mg (1.2%) by evaporation concentration.

The total recovery of the tertiary butyl-dehydrophenylahistin that reclaims is 0.9%.

The HPLC spectrogram of pass course A and the thick tertiary butyl-dehydrophenylahistin of route B synthetic as shown in Figure 4.

The two kinds of tertiary butyl-dehydrophenylahistin derivatives have in addition been synthesized according to the method for route A.In above-mentioned other tertiary butyl-dehydrophenylahistin derivative synthetic, described benzaldehyde compound 4 is changed.

Fig. 4 has shown the similarity (chromatographic column: YMC-Pack ODS-AM (20 * 250mm) of the HPLC spectrogram of the representational tertiary butyl-dehydrophenylahistin that embodiment 1 synthetic dehydrophenylahistin (Fig. 2) and above-mentioned pass course A prepare; The methanol-water system wash-out of gradient: 65%-75% 20 minutes was used 100% methanol system wash-out 10 minutes subsequently; Flow rate: 12mL/ minute; O.D.230nm).

The addition sequence of described aldehyde is influential to yield, thereby also is aspect of synthetic.In contrast or model, synthesized the analogue of dehydrophenylahistin, the 5-position that wherein said dimethyl-allyl group is transformed at described imidazole ring has similar spaces sterically hindered tertiary butyl groups.

Use " route A " to synthesize this " tertiary butyl (tBu)-dehydrophenylahistin " as mentioned above.Especially, follow dehydrophenylahistin synthetic aldehyde addition sequence fully and obtained 16.5% tBu-dehydrophenylahistin total recovery.The yield of this yield and dehydrophenylahistin (20%) is similar.Use " route B ", the reversed in order of synthetic dehydrophenylahistin among the addition sequence of wherein said aldehyde and " the route A ", have to the required tBu-dehydrophenylahistin of the trace of total recovery 0.9%, although obtain the midbody compound 17 of 76% yield when adding first phenyl aldehyde 4.This result shows and may be difficult to be introduced in imidazoles-4-formaldehyde 15 that 5-position adjacent on the imidazole ring is connected with the substituent comparatively large vol of quaternary carbon on midbody compound 17, illustrates that the addition sequence of aldehyde is an important aspect to dehydrophenylahistin or the dehydrophenylahistin analogue that uses the synthetic high yield of the open method of this paper.

As shown in Figure 4, from analyzing, the HPLC to the final crude product of gained as seen, in the crude samples of described route A (left side), observes high-load tBu-dehydrophenylahistin and a spot of by product.Yet, in the sample that pass course B (right side) obtains, observe required tBu-dehydrophenylahistin and multiple other by product of relatively small amount.

Embodiment 3

Other of dehydrophenylahistin and analogue is synthetic on a large scale

3-Z-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene]-piperazine-2,5-diketone [dehydrophenylahistin] (1) synthetic

Reagent: a) LDA, CH ₃CHO; B) Tos-Cl, pyridine; C) DBU; D) NaOH; E) C ₂Cl ₂O ₂F) KOOCCH ₂COOEt, BuLi; G) SO ₂Cl ₂H) H ₂NCHO, H ₂O; I) LiAlH ₄J) MnO ₂K) 1,4-diacetyl-piperazine-2,5-diketone, Cs ₂CO ₃L) phenyl aldehyde, Cs ₂CO ₃3-hydroxyl-2,2-dimethyl-methyl-butyrate

Under ar gas environment, (0.39mol) (45ml in THF 0.39mol) (270ml) solution, stirs the gained mixture 30 minutes solution adding methyl isobutyrate for 2M, 196ml in-60 ℃ of hexanes with LDA/THF/ ethylbenzene.Slowly adding is chilled to-60 ℃ acetaldehyde in advance (27ml, THF 0.48mol) (45ml) solution makes gained solution continue to stir 30 minutes.Add saturated ammonium chloride (50ml) and make gained solution be warming up to room temperature.Reaction mixture ethyl acetate extraction, extract are successively used HCl (2M), sodium bicarbonate and salt water washing.Organic layer is with dried over mgso, filtration, evaporation obtains clarifying oily matter (52.6g) then.Obtain purified 3-hydroxyl-2,2-dimethyl-methyl-butyrate (42.3g, 74%) in 76-82 °/30mmHg distillation.(Burk?et?al.，J.Am.Chem.Soc.，117：4423-4424(1995))。

¹H NMR (400MHz, CDCl ₃) δ 1.15 (d, J=6.2Hz, 3H); 1.17 (s, 6H); 2.66 (d, J=6.2Hz, 1H ,-OH); 3.71 (s, 3H ,-OMe); 3.87 (the app quintet, J=6.4Hz, 1H, H3).

2,2-dimethyl-3-(toluene-4-sulfonyloxy)-methyl-butyrate

To the cooling (0 ℃) 3-hydroxyl-2,2-acid dimethyl methyl esters (52.0g, add gradually in pyridine 0.36mol) (100ml) solution p-methyl benzene sulfonic chloride (69.0g, 0.36mol).Make the gained mixture be warming up to room temperature and stir 60h.In ice, reactant cooled off once more and carry out acidifying by adding HCl (2M).Gained solution ethyl acetate extraction, the gained extract is successively washed with HCl and salt solution, and dry, evaporation obtains oily matter, leaves standstill the back and produces white precipitate., add sherwood oil subsequently and obtain white precipitate this mixture dissolving with minimum acetic ester, collecting precipitation is also used more petroleum ether.Filtrate is carried out the part evaporation, collects once more in the crystallization and the crystallization with its adding collection for the first time that obtains, and obtains 2,2-dimethyl-3-(toluene-4-sulfonyloxy) methyl-butyrate (81.276%).

¹H NMR (400NMz, CDCl ₃) δ 1.12 (s, 3H); 1.13 (s, 3H); 1.24 (d, J=Hz, 3H); 2.45 (s, 3H ,-PhMe); 3.58 (s, 3H ,-OMe); 4.94 (quartet, J=6.41H, H3), 7.33 (d, J=8.0Hz, 2H), 7.78 (d, J=8.0Hz, 2H).

Evaporate remaining filtrate obtain extra thick 2,2-dimethyl-3-(toluene-4-sulfonyloxy) fourth methyl esters (19.0g, 18%).

2,2-dimethyl-3-M Cr

With 2, (18.06g, DBU 0.06mol) (15ml) solution is at 140-160 ° of heating 3.5h for 2-dimethyl-3-(toluene-4-sulfonyloxy) methyl-butyrate.Make the gained mixture be cooled to room temperature, dilute with ether subsequently.The gained mixture is successively used HCl (1M), sodium bicarbonate and salt water washing.With gained ether layer dry and part evaporation obtain spissated 2,2-dimethyl-3-M Cr solution (10g).(Savu?and?katzenellenbogen，J.Org.Chem，46：239-250(1981))。Because the volatility of products therefrom (102 ° of bp) should be avoided further evaporation.(Tsaconas?et?al.，Aust.J.Chem.，53：435-437(2000))。

¹H?NMR(400MHz，CDCl ₃)δ1.31(s，6H)；3.68(s，3H)；5.06(d，J＝17.1Hz，1H，-CH＝CH ₂)；5.11(d，J＝10.7Hz，1H，-CH＝CH ₂)；6.03(dd，J＝17.1，10.7Hz，1H，-CH＝CH ₂)。

2,2-dimethyl-3-butenoic acid

With above-mentioned 2, the ethereal solution of 2-dimethyl-3-M Cr (10g) is with alcohol dilution (25ml), and (4M stirs 22ml) and with the gained mixture and to spend the night to add sodium hydroxide.Gained solution partly evaporated removes ethanol, and with the gained mixture add HCl (1M, 100ml) in.The products therefrom ethyl acetate extraction obtains 2,2-dimethyl-3-butenoic acid (6.01g, 88% liang of step) with the dry also evaporation of extraction liquid.(Hayashi?et?al.，J.?Org.Chem.，65：8402-8405(2000))。

¹H?NMR(400MHz，CDCl ₃)δ1.33(s，6H)；5.11(d，J＝10.8Hz，1H，-CH＝CH ₂)；5.15(d，J＝17.2Hz，1H，-CH＝CH ₂)；6.05(dd，J＝17.2，10.8Hz，1H，-CH＝CH ₂)。

Monoethyl malonate (Wierenga and Skulnick, " Aliphatic and Aromatic β-ketoEstersfrom Monoethyl Malonate:Ethyl 2-Butyrylacetate; " OrganicSynthesesCollectiveVolume 7,213).

(25.0g 0.15mol) is suspended in the water (15.6ml) and cools off in ice bath with the sylvite of monoethyl malonate.Drip concentrated hydrochloric acid (12.5ml) in 30 minutes, then the gained mixture is continued to stir 10 minutes.Filter the gained precipitation and use the ether washed twice.Isolate filtrate, the water extracted with diethyl ether.With the ethereal solution drying (MgSO that merges ₄) and evaporation, obtain buttery monoethyl malonate (19.2g, 99%), before using with it in dried overnight under the vacuum (or under the 50 °/1mm 1h).

4,4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester

With oxalyl chloride (3.83ml 43.9mmol) is added dropwise to 2 of cooling (0 ℃), 2-dimethyl-3-butenoic acid (5.0g, 43.9mmol) with anhydrous methylene chloride (25ml) solution of DMF (1) in.The gained mixture is stirred 1h at 0 ℃, at room temperature stir 16h then.Fractionation (121 °/760mmHg) obtain 2,2-dimethyl-3-crotonyl chloride (4.1g, 71%).

With monoethyl malonate (7.2g, 0.05mol) and dipyridyl (several milligrams) be dissolved among the THF (90ml), and in the gained system, charge into nitrogen.Gained solution is cooled to-70 ℃, add subsequently BuLi (2.5M in hexane, 37ml, 0.09mol).After adding about 10ml BuLi, described solution becomes pink, need add THF (15ml) again for keeping magnetic agitation simultaneously.Remove cooling bath and add remaining BuLi, temperature is reached to-10 °, this moment, reaction soln became colorless.Mixture is cooled to once more-60 ℃, and drips 2,2-dimethyl-3-crotonyl chloride (4.1g, THF 0.03mol) (12ml) solution.After being added dropwise to complete, make the gained mixture be warming up to 0 ° and stir 3h, it is added in ether/1M HCl (260ml) mixture of 1: 1 and continue to stir 1.5h at 0 ° subsequently.Separate organic layer, successively use HCl (1M), sodium hydrogen carbonate solution, salt water washing, dry then and evaporation obtains 4,4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester (5.6g, 98%).(Hayashi?et?al.，J.Org.Chem.，65：8402-8405(2000))。With (160 °/1mmHg) obtain pure material of Kugelrohr stove distillations.

¹H?NMR(400MHz，CDCl ₃)δ1.26(s，6H)；1.27(t，J＝6.9Hz，3H，-CH ₂CH ₃)；3.51(s，2H)；4.18(q，J＝6.9Hz，2H，-CH ₂CH ₃)；5.20(d，J＝17.7Hz，1H，-CH＝CH ₂)；5.21(d，J＝9.6Hz，1H，-CH＝CH ₂)；5.89(dd，J＝17.7，9.6HZ，1H，-CH＝CH ₂)。

2-chloro-4,4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester

(0.84ml 10.4mmol) adds 4 of cooling (0 °), and (18.3g is in chloroform 9.93mmol) (7ml) solution for 4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester with SULPHURYL CHLORIDE.Make the gained mixture be warming up to room temperature and stirred 30 minutes, be heated backflow 2h subsequently.After being cooled to room temperature, the gained reaction mixture is diluted with chloroform, use sodium bicarbonate, water and salt water washing subsequently successively.The dry also evaporation of organic phase is obtained brown buttery 2-chloro-4,4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester (2.01g, 93%).(Hayashi?et?al.，J.?Org.Chem.，65：8402-8405(2000))。

¹H?NMR(400MHz，CDCl ₃)δ1.28(t，J＝7.0Hz，3H，-CH ₂CH ₃)；1.33(s，3H)；1.34(s，3H)；4.24(q，J＝7.0Hz，2H，-CH ₂CH ₃)；5.19(s，1H)；5.28(d，J＝16.9Hz，1H，-CH＝CH ₂)；5.29(d，J＝10.9Hz，1H，-CH-CH ₂)；5.96(dd，J＝16.9，10.9Hz，1H，-CH＝CH ₂)。

LC/MS?t _R＝8.45(219.3[M(Cl ³⁷)+H] ⁺)min。

Using this material directly to react need not to be further purified.

5-(1,1-dimethyl-allyl group)-3H-imidazoles-4-carboxylic acid, ethyl ester

To be suspended in the 2-chloro-4 in the methane amide (36.8ml), 4-dimethyl-3-oxygen-5-hexenoic acid ethyl ester (19.4g, 0.09mol) and water (1.94ml, suspension 0.11mol) slightly shakes, in the bottle of the 15 * 18ml that packs into then.With the bottle sealing and at 150 ° of heating 5h.To be cooled to room temperature, thoroughly extract with the mixing of the composition in described each bottle and with chloroform.The dry also evaporation of extract is obtained spissated formamide soln (14.7g).Its adding is soaked in 1%MeOH/1%Et ₃In the silicon-dioxide chromatography post in the chloroformic solution of N (7cm diameter, 11cm height).With this mixture of 2L described chromatography column is carried out wash-out, use the 2%MeOH/1%Et of 2L then ₃The chloroformic solution wash-out of N in the part than wash-out morning, obtains suspecting being the compound of 5-(1, the 1-dimethyl-allyl)-oxazoles-4-carboxylic acid, ethyl ester (1.23g, 7%).

HPLC(214nm)t _R＝8.68(50.4％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.40(t，J＝7.2Hz，3H，-CH ₂CH ₃)；1.54(s，6H)；4.38(t，J＝7.2Hz，2H，-CH ₂CH ₃)；5.03(d，J＝17.4Hz，1H，-CH＝CH ₂)；5.02(d，J＝10.4Hz，1H，-CH＝CH ₂)；6.26(dd，J＝17.4，10.4Hz，1H，-CH＝CH ₂)；7.83(s，1H)。

LCMS?t _R＝8.00(210.1[M+H] ⁺，361.1[2M+H] ⁺)min。

Reclaim the part after and obtain required 5-(1,1-dimethyl-allyl group)-3H-imidazoles-4-carboxylic acid, ethyl ester (3.13 g, 17%).(Hayashi?et?al.，J.Org.Chem.，65：8402-8405(2000))。

HPLC(214nm)t _R＝5.52(96.0％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.38(t，J＝7.0Hz，3H)；1.57(s，6H)；4.35(q，J＝7.0Hz，2H)；5.04-5.14(m，2H，-CH＝CH ₂)；6.28(dd，J＝18.0，10.4Hz，1H，-CH＝CH ₂)；7.52(s，1H)。

LC/MS?t _R＝5.30(209.1[M+H] ⁺，417.2[2M+H] ⁺)min。

Also can from described chromatography column, reclaim more 5-(1,1-dimethyl-allyl group)-3H-imidazoles-4-carboxylic acid, ethyl ester (3.59g, 19%), satisfy the needs of subsequent reactions though its purity is lower.

In similar reaction (on a small scale), by using the 5%MeOH/1%Et that is dissolved in chloroform ₃N carries out further wash-out separation to described chromatography column and obtains other by product, suspects that it is 5-(1,1-dimethyl-allyl group)-3H-imidazoles-4-carboxylic acid (0.27g, 9%).

HPLC(245nm)t _R＝5.14(68.9％)min。

¹H?NMR(400MHz，CD ₃OD)δ1.45(s，6H)；4.97(d，J＝10.6Hz，1H，-CH＝CH ₂)；5.01(d，J＝17.7Hz，1H，-CH＝CH ₂)；6.28(dd，J＝17.7，10.6Hz，1H，-CH＝CH ₂)；7.68(s，1H)。

LCMS?t _R＝4.72(181.0[M+H] ⁺，361.1[2M+H] ⁺)min。

[5-(1,1-dimethyl-allyl group)-3H-imidazol-4 yl]-methyl alcohol

With 5-(1,1-dimethyl-allyl group)-and 3H-imidazoles-4-carboxylic acid, ethyl ester (3.13g, THF 15.0mmol) (60ml) drips of solution adds lithium aluminum hydride, and (95% suspends 1.00g, 25.0mmol) be suspended in the suspension of gained among the THF (40ml), and the gained mixture is at room temperature stirred 4h.Add water till no longer producing gas, reaction mixture was stirred 10 minutes, use sintering (sintered) funnel to filter subsequently.Precipitation is successively with THF and methanol wash, with filtrate and washings merge, evaporation, freeze-drying then, obtain [5-(1,1-dimethyl-allyl group)-3H-imidazol-4 yl] methyl alcohol (2.56g, 102%).Before subsequent reactions by removing remaining water with the chloroform azeotropic.(referring to, Hayashi etc., J. Org.Chem., 65:8402-8405 (2000)).

HPLC(240nm)t _R＝3.94(56.8％)min。

¹H?NMR(400MHz，CD ₃OD)δ1.43(s，6H)；4.57(s，2H)；5.01(d，J＝10.5Hz，1H，-CH＝CH ₂)；5.03(d，J＝17.7Hz，1H，-CH＝CH ₂)；6.10(dd，J＝17.7，10.5Hz，1H，-CH＝CH ₂)；7.46(s，1H)。

LC/MS?t _R＝3.77(167.3[M+H] ⁺)min。

5-(1,1-dimethyl-allyl group)-3H-imidazoles-4-formaldehyde

(20g, (2.56g in acetone 0.02mol) (300ml) solution, at room temperature stirs 5h with the gained mixture 0.23mol) to add [5-(1, the 1-dimethyl-allyl)-3H-imidazol-4 yl] methyl alcohol with Manganse Dioxide.With the reaction mixture filter paper filtering, the resistates washing with acetone.With filtrate and washings merging and evaporation, obtain 5-(1, the 1-dimethyl-allyl)-3H-imidazoles-4-formaldehyde (1.82g, 51%).(Hayashi?et?al.，J.Org.Chem.，65：8402-8405(2000))。

HPLC(240nm)t _R＝4.08(91.5％)min。

¹H?NMR(400?MHz，CDCl ₃)δ1.56(s，6H)；5.16(d，J＝10.6Hz，1H，-CH＝CH ₂)；5.19(d，J＝17.3Hz，1H，-CH＝CH ₂)；6.22(dd，J＝17.3，10.6Hz，1H，-CH＝CH ₂)；7.75(s，1H)，10.02(s，1H，HCO)。

LC/MS?t _R＝3.75(165.2[M+H] ⁺)min。

1-ethanoyl-3-[5 '-(1, the 1-dimethyl-allyl)-1 H-imidazoles-4 '-Z-methylene] piperazine-2, the 5-diketone

To 5-(1, the 1-dimethyl-allyl)-3H-imidazoles-4-formaldehyde (1.78g adds 1 in DMF 0.01mol) (35ml) solution, 4-diacetyl piperazine-2, (8.59g 0.04mol), carries out emptying to described mixture to the 5-diketone, charges into argon gas subsequently.Emptying-gas replenishment process is repeated twice again, add subsequently cesium carbonate (3.53g, 0.01mol).With emptying-gas replenishment process triplicate again, subsequently with reaction mixture at 45 ° of heating 5h.Reaction mixture is partly evaporated (under high vacuum, heating) up to the residue a little volume, the gained drips of solution is added in the frozen water (50ml).Collect the yellow mercury oxide that produces, wash freeze-drying then with water, obtain 1-ethanoyl-3-[5 '-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 '-Z-methylene] piperazine-2,5-diketone (1.18g, 36%).(Hayashi，Personal?Communication(2001))。

HPLC(214nm)t _R＝6.01(72.6％)min。

¹H NMR (400MHz, CDCl ₃) δ 1.53 (s, 6H); 2.64 (s, 3H); 4.47 (s, 2H); 5.19 (d, J=17.3Hz, 1H ,-CH=CH ₂); 5.23 (d, J=10.7Hz, 1H ,-CH=CH ₂); 6.06 (dd, J=17.3,10.7Hz, 1H ,-CH=CH ₂); 7.16 (s, 1H), 7.59 (s, 1H), 9.47 (bs, 1H); 12.11 (bs, 1H) [observe～2%1,4-diacetyl piperazine-2, and 5-diketone impurity δ 2.59 (s, 6H); 4.60 (s, 4H).]

LC/MS?t _R＝6.65(303.3[M+H] ⁺，605.5[2M+H] ⁺)min。(n.b. uses different systems).

3-Z-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene] piperazine-2, the 5-diketone

To 1-ethanoyl-3-[5 '-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 '-methylene] piperazine-2,5-diketone (2.91g; 9.62mmol) DMF (70ml) solution in add phenyl aldehyde (4.89ml; 48.1mmol), described solution is carried out emptying, charge into argon gas subsequently.Emptying-gas replenishment process is repeated twice again, add subsequently cesium carbonate (4.70g, 14.4mmol).With emptying-gas replenishment process triplicate again, subsequently reaction mixture is heated under following thermograde.

After reaction is carried out 5 hours altogether, make reactant be cooled to room temperature, and reaction mixture is added (500ml) in the frozen water.Collect the precipitation that produces, water (300ml) washs freeze-drying then, obtains yellow solid (2.80g).This material is dissolved in the chloroform (250ml), removes residual water with azeotropic with filter paper filtering and evaporation.With residual yellow mercury oxide (2.70g, HPLC (214nm) t _R=7.26 (93.6%) min.) be partially dissolved in chloroform (20ml), gained suspension is used supersound process 5 minutes, collect the gained solid subsequently and use dry air, obtain 3-Z-benzylidene-6-[5 "-(1; the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene] piperazine-2,5-diketone (1.82g, 54%) (Hayashi, Personal Communication (2001)), m.p.239-240 ° (dec.).

HPLC(2?14nm)t _R＝6.80(1.92)min，7.33(95.01％)。

¹H?NMR(400MHz，CDCl ₃)δ1.53(s，6H)；5.18(d，J＝17.6Hz，1H，-CH＝CH ₂)；5.21(d，J＝11.0Hz，1H，-CH＝CH ₂)；6.06(dd，J＝17.6，11.0Hz，1H，-CH＝CH ₂)；6.99(s，1H，-C-C＝CH)；7.00(s，1H，-C-C＝CH)；7.30-7.50(m，5×ArH)；7.60(s，H ₂″)；8.07(bs，NH)；9.31(bs，NH)；12.30(bs，NH)。

LC/MS t _R=6.22 (349.3[M+H] ⁺, E isomer), 6.73 (349.5[M+H] ⁺, 697.4[2M+H] ⁺, Z isomer) and min.

ESMS?m/z?349.5[M+H] ⁺，390.3[M+CH ₄CN] ⁺。

The evaporation of gained chloroformic solution is obtained more 3-Z-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene] piperazine-2,5-diketone (0.76g, 29%).

HPLC(214nm)t _R＝7.29(84.5％)min。

3-E-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene] piperazine-2, the 5-diketone

The crude samples of as above synthetic material with preparation HPLC chromatogram purification, is obtained geometrical isomer 3-E-benzylidene-6-[5 "-(1, the 1-dimethyl-allyl)-1H-imidazoles-4 "-the Z-methylene] piperazine-2,5-diketone (1.7mg).

HPLC(214nm)t _R＝6.75(87.79)min。

¹H?NMR(400MHz，CDCl ₃)δ1.52(s，6H)；5.19(d，J＝20.8Hz，1H，CH＝CH ₂)；5.22(d，J＝14.0Hz，1H，CH＝CH ₂)；6.05(dd，J＝18.0，10.4Hz，1H，CH＝CH ₂)；6.33(s，1H，C-C＝CH)；6.90-7.65(m，7H)。

ESMS?m/z349.5[M+H] ⁺，390.4[M+CH ₄CN] ⁺。

3-Z-benzylidene-6-(5 "-tertiary butyl-1H-imidazoles-4 "-the Z-methylene) piperazine-2,5-diketone (2) synthetic

Reagent: g) SO ₂Cl ₂H) H ₂NCHO, H ₂O; I) LiAlH ₄J) MnO ₂K) 1,4-diacetyl piperazine-2,5-diketone, Cs ₂CO ₃L) phenyl aldehyde, Cs ₂CO ₃

2-chloro-4,4-dimethyl-3-oxygen-Valeric acid ethylester

(14.0ml, (27.17g is in chloroform 0.16mol) (100ml) solution 0.17mol) to add the tri-methyl ethyl acetoacetate that cools off (0 °) with SULPHURYL CHLORIDE.Make the gained mixture be warming up to room temperature and stirred 30 minutes, subsequently it is heated 2.5h under reflux temperature.After being cooled to room temperature, the gained reaction mixture is diluted with chloroform, use sodium bicarbonate, water and salt water washing subsequently in order.

The dry also evaporation of organic phase is obtained clarifying buttery 2-chloro-4,4-dimethyl-3-oxygen-Valeric acid ethylester (33.1g, 102%).(Durant et al., " Aminoalkylimidazoles and Process for theirProduction. " patent No. GB1341375 (Great Britain, 1973)).

HPLC(214nm)t _R＝8.80(92.9％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.27(s，9H)；1.29(t，J＝7.2Hz，3H)；4.27(q，J＝7.2Hz，2H)；5.22(s，1H)。

¹³C?NMR(100MHz，CDCl ₃)δ13.8，26.3，45.1，54.5，62.9，165.1，203.6。

The 5-tertiary butyl-3H-imidazoles-4-carboxylic acid, ethyl ester

With 2-chloro-4, (25.0g, (47.5ml) solution of methane amide 0.12mol) and water (2.5ml) shake up 4-dimethyl-3-oxy pentanoic acid ethyl ester, in the bottle of the 15 * 8ml that packs into then.With the whole sealings of bottle and at 150 ° of heating 3.5h.Make bottle be cooled to room temperature, to wherein adding entry (20ml) and the gained mixture thoroughly being extracted with chloroform.Remove chloroform and obtain spissated formamide soln (22.2g), with its adding 1%MeOH/1%Et ₃In the quick silicon-dioxide chromatography post (6cm diameter, 12cm height) that the chloroformic solution of N soaks.With this mixture of 2.5L described chromatography column is carried out wash-out, use the 2%MeOH/1%Et of 1L then ₃The chloroformic solution wash-out of N in the part than wash-out morning, obtains suspecting being the product of the 5-tertiary butyl-oxazoles-4-carboxylic acid, ethyl ester (6.3g, 26%).

HPLC(214nm)t _R＝8.77min。

¹H?NMR(400MHz，CDCl ₃)δ1.41(t，J＝7.2Hz，3H)；1.43(s，9H)；4.40(q，J＝7.2Hz，2H)；7.81(s，1H)。

¹³C?NMR(100MHz，CDCl ₃)δ14.1，28.8，32.5，61.3，136.9，149.9，156.4，158.3。

ESMSm/z198.3[M+H] ⁺，239.3[M+CH ₄CN] ⁺。

LC/MS?t _R＝7.97(198.1[M+H] ⁺)min。

From the part of back wash-out, reclaim and obtain the 5-tertiary butyl-3H-imidazoles-4-carboxylic acid, ethyl ester (6.20g, 26%).(Durant et al., " Aminoalkylimidiazoles and Process for theirProduction. " patent No. GB 1341375 (Great Britain, 1973)).

HPLC(214nm)t _R＝5.41(93.7％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.38(t，J＝7.0Hz，3H)；1.47(s，9H)；4.36(q，J＝7.2Hz，2H)；7.54(s，1H)。

¹³C?NMR(100MHz，CDCl ₃)δ13.7，28.8，32.0，59.8，124.2，133.3，149.2，162.6。

ESMSm/z197.3[M+H] ⁺，238.3[M+CH ₄CN] ⁺。

Use 1L5%MeOH/1%Et ₃N obtains suspecting to the further wash-out of described chromatography column and is the compound of the 5-tertiary butyl-3H-imidazoles-4-carboxylic acid (0.50g, 2%).

HPLC(245nm)t _R＝4.68(83.1％)min。

¹H?NMR(400MHz，CD ₃OD)δ1.36(s，9H)；7.69(s，1H)。

¹H?NMR(400MHz，CDCl ₃)δ1.37(s，9H)；7.74(s，1H)。

¹H?NMR(400MHz，CD ₃SO)δ1.28(s，9H)；7.68(s，1H)。

ESMSm/z169.2[M+H] ⁺，210.4[M+CH ₄CN] ⁺。

(the 5-tertiary butyl-3H-imidazol-4 yl)-methyl alcohol

With the 5-tertiary butyl-3-imidazoles-4-carboxylic acid, ethyl ester (3.30g, 16.8mmol) THF (60ml) drips of solution add lithium aluminum hydride (95% suspension, 0.89g, 22.2mmol) be suspended in the suspension of gained among the THF (40ml), and the gained mixture at room temperature stirred 3h.Add water till no longer producing gas, reaction mixture was stirred 10 minutes, filter with sinter funnel subsequently.Precipitation is successively with THF and methanol wash, with filtrate and washings merging and evaporation.The resistates freeze-drying is spent the night, and obtains (the 5-tertiary butyl-3H-imidazol-4 yl) methyl alcohol (2.71g, 105%).(Durant et al., " Aminoalkylimidazoles and Processfor their Production. " patent No. GB1341375 (Great Britain, 1973)).

HPLC(240nm)t _R＝3.70(67.4％)min。

¹H?NMR(400MHz，CD ₃OD)δ1.36(s，9H)；4.62(s，2H)；7.43(s，1H)。

¹³C?NMR(100MHz，CD ₃OD)δ31.1，33.0，57.9，131.4，133.9，140.8。

LC/MS?t _R＝3.41(155.2[M+H] ⁺)min。

This material need not to be further purified before using.

The 5-tertiary butyl-3H-imidazoles-4-formaldehyde

(30g, (4.97g in multi-phase solution 0.03mol), at room temperature stirs 4h with the gained mixture 0.35mol) to add (the 5-tertiary butyl-3H-imidazol-4 yl) methyl alcohol that is dissolved in acetone (700ml) with Manganse Dioxide.Reaction mixture is filtered with Celite pad (liner), and with this pad washing with acetone.With filtrate and washings merging and evaporation.Resistates grinds with ether, obtains the 5-tertiary butyl-3H-imidazoles-4-formaldehyde (2.50g, 51%) of colorless solid.(Hayashi，Personal?Communication(2000))。

HPLC(240nm)t _R＝3.71(89.3％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.48(s，9H)；7.67(s，1H)；10.06(s，1H)。

LC/MS?t _R＝3.38(153.2[M+H] ⁺)min。

The filtered liquid that grinds the back gained is evaporated, obtain the extra 5-tertiary butyl-3H-imidazoles-4-formaldehyde (1.88g, 38%).

1-ethanoyl-3-(5 '-tertiary butyl-1H-imidazoles-4 '-Z-methylene) piperazine-2, the 5-diketone

To the 5-tertiary butyl-3H-imidazoles-4-formaldehyde (2.50g adds 1 in DMF 164.4mol) (50ml) solution, 4-diacetyl piperazine-2, (6.50g 32.8mmol), carries out emptying to described solution to the 5-diketone, charges into argon gas subsequently.Emptying-gas replenishment process is repeated twice again, add subsequently cesium carbonate (5.35g, 16.4mmol).With emptying-gas replenishment process triplicate again, subsequently the gained mixture is at room temperature stirred 5h.Reaction mixture is partly evaporated (under vacuum, heating) up to the residue a little volume, the gained drips of solution is added in the entry (100ml).Collect the yellow mercury oxide freeze-drying then that produces, obtain 1-ethanoyl-3-[5 '-tertiary butyl-1H-imidazoles-4 '-Z-methylene] piperazine-2,5-diketone (2.24g, 47%).(Hayashi，Personal?Communication(2000))。

HPLC(214nm)t _R＝5.54(94.4％)min。

¹H?NMR(400MHz，CDCl ₃)δ1.47(s，9H)；2.65(s，3H)，4.47(s，2H)；7.19(s，1H)；7.57(s，1H)，9.26(s，1H)，12.14(s，1H)。

¹³C?NMR(100MHz，CDCl ₃+CD ₃OD)δ27.3，30.8，32.1，46.5，110.0，123.2，131.4，133.2，141.7，160.7，162.8，173.0

Lc/Ms?t _R＝5.16(291.2[M+H] ⁺，581.6[2M+H] ⁺)min。

3-Z-benzylidene-6-[5 "-tertiary butyl-1H-imidazoles-4 "-the Z-methylene] piperazine-2, the 5-diketone

To 1-ethanoyl-3-[5 '-tertiary butyl-1H-imidazoles-4 '-Z-methylene] piperazine-2, (2.43g, (4.26ml 41.9mmol), carries out emptying to described solution to the 5-diketone, charges into nitrogen subsequently to add phenyl aldehyde in DMF 8.37mmol) (55ml) solution.Emptying-gas replenishment process is repeated twice again, add subsequently cesium carbonate (4.09g, 12.6mmol).With emptying-gas replenishment process triplicate again, subsequently reaction mixture is heated under following thermograde.After reaction is carried out 5 hours altogether, make reactant be cooled to room temperature, and reaction mixture is added in the frozen water (400ml).Collect the precipitation that produces, wash freeze-drying then with water, obtain yellow solid (2.57g, HPLC (214nm) t _R=6.83 (83.1%) min.).This material is dissolved in the chloroform (100ml), and evaporation is removed residual water with azeotropic, obtains the oily matter of brown.It is dissolved in chloroform (20ml) also with ice-cooled.Collect the yellow mercury oxide that produces after 90 minutes, dry air obtains 3-Z-benzylidene-6-[5 "-tertiary butyl-1H-imidazoles-4 "-the Z-methylene] piperazine-2,5-diketone (1.59g, 56%) (Hayashi, Personal Communication (2000)).

HPLC(214nm)t _R＝6.38(2.1％)，6.80(95.2)min。

¹H?NMR(400MHz，CDCl ₃)δ1.46(s，9H)；7.01(s，1H，-C-C＝CH)；7.03(s，1H，-C-C＝CH)；7.30-7.50(m，5H，Ar)；7.60(s，1H)；8.09(bs，NH)；9.51(bs，NH)；12.40(bs，NH)。

LC/MS t _R=5.84 (337.4[M+H] ⁺, E isomer), 6.25 (337.4[M+H] ⁺, 673.4[2M+H] ⁺, Z isomer) and min.

ESMS?m/z?337.3[M+H] ⁺，378.1[M+CH ₄CN] ⁺。

Evaporate described chloroformic solution and obtain extra 3-Z-benzylidene-6-[5 "-tertiary butyl-1H-imidazoles-4 "-the Z-methylene] piperazine-2,5-diketone (0.82g, 29%).HPLC(214nm)t _R＝6.82(70.6％)min。

Common experimental conditions

Sodium bicarbonate refers to 5% solution.

Unless specifically stated otherwise, the organic solvent dried over sodium sulfate.

Analysis condition

The NMR condition

1H NMR (400MHz) analyzes and carries out on Varian Inova Unity 400MHz NMR equipment.Sample is carried (unless specifically stated otherwise) with the deuterochloroform that contains 0.1%TMS.Chemical shift (ppm) is a benchmark or to CD with relative TMS (0.00ppm) ₃The sample that OD carries is with CH ₃OH (3.30ppm) is a benchmark.Coupling (coupling) constant is represented with hertz (Hz).

Analyze the HPLC condition

System 6 conditions:

Rainin Microsorb-MV C18 (5 μ m, ) carry out RP-HPLC on 50 * 4.6mm post.

Buffer A: 0.1% moisture TFA

Buffer B: the 0.1%TFA that is dissolved in 90% moisture MeCN

Gradient: 0.100% buffer B in 11 minutes

Flow rate: 1.5mL/ minute

The LCMS condition

On Perkin-Elmer Sciex API-100 device, carry out LCMS.

The LC condition:

Reversed-phase HPLC is analyzed

Post: Monitor 5 μ m C1850 * 4.6mm

The aqueous solution of solvent orange 2 A: 0.1%TFA

Solvent B: the 0.085%TFA that is dissolved in 90% moisture MeCN

Gradient: 0-100% buffer B in 11.0 minutes

Flow rate: 1.5mL/ minute

Wavelength: 214nm

The MS condition:

Ion source: ion shower

Detector: ion counter

Arrive mass spectral flow rate: separate (1.5mL/ minute) back 300 μ L/ minute ESMS condition from post

On Perkin Elmer/Sciex-API III LC/MS/MS, carry out ESMS with the electrospray input unit.

Solvent: the 0.1%AcOH that is dissolved in 60% moisture MeCN

Flow rate: 25 μ L/ minutes

Ion shower: 5000V

Orifice plate (orifice plate): 55V

Acquisition time (acquisition time): 2.30 minutes

Sweep limit: 100-1000amu/z

Scanning step: 0.2amu/z

Preparation RP-HPLC purification condition

Employing Nebula Waters XterraMS post (19 * 50mm, 5 μ m C18) carry out the reversed-phase HPLC purifying under the following conditions:

Solution A: 0.1% moisture TFA

Solution B: the 0.1%TFA that is dissolved in 90% moisture MeCN

Gradient: 5-95%B in 4 minutes

Flow rate: 20mL/ minute

Wavelength: 214nm

It is as follows respectively to write a Chinese character in simplified form implication: brs: wide is unimodal; BuLi: n-Butyl Lithium; D: bimodal; DBU:1,8-diazabicyclo [5.4.0] 11 carbon-7-alkene; ESMS: electronic spraying mass spectrum; HCl: hydrochloric acid; HPLC: high performance liquid chromatography; LCMS: liquid chromatography mass; LD: lithium diisopropylamine; M ⁺: molion; M: multimodal; MeCN: acetonitrile; M: mass spectrum; MW: molecular weight; NMR: nucleus magnetic resonance; Q: quartet; S: unimodal; : three peaks; t _R: retention time; TFA: trifluoroacetic acid; THF: tetrahydrofuran (THF)

The concrete steps of synthetic dehydrophenylahistin

1-ethanoyl-3-{ (Z)-1-[5-(1,1-dimethyl-2-propenyl)-1H-4-imidazolyl] methylene radical }-2, the 5-piperazine Piperazine diketone (2)

To 5-(1,1-dimethyl-2-propenyl) imidazoles-4-formaldehyde (100mg, add in DMF 0.609mmol) (2mL) solution compound 1 (241mg, 1.22mmol), at short notice gained solution is carried out the repetition emptying to remove oxygen and to charge into Ar, add Cs subsequently ₂CO ₃(198mg 0.609mmol), and repeats the process of emptying-inflation once more.Preferably remove oxygen, because be considered to reduce the oxidation of the alpha-carbon of 6 on described piperazinedione ring like this.The gained mixture is at room temperature stirred 5h.Except that after desolvating, described resistates is dissolved in EtOAc and 10%Na by evaporation ₂CO ₃Mixture in, the gained organic phase is used 10%Na once more ₂CO ₃Wash and wash three times, use Na with saturated NaCl ₂CO ₃Drying, and under vacuum, concentrate.Residual oily matter with CHCl ₃-MeOH (100: 0 to 50: 1) is eluent silicon-dioxide column chromatography purification, obtains 60mg (33%) faint yellow solid 2.

Dehydrophenylahistin

(30mg, (51 μ L, 0.496mol 5eq), carry out the repetition emptying to remove oxygen and to charge into Ar to gained solution at short notice, add Cs subsequently to add phenyl aldehyde in DMF 0.099mmol) (0.8mL) solution to compound 2 ₂CO ₃(53mg, 0.149mmol 1.5eq), and repeat the process of emptying-inflation once more.(temperature must slowly promote at 80 ℃ of heating 2.5h with the gained mixture.Intensification can cause the increase at benzylidene position E-isomer growing amount rapidly)., except that after desolvating the gained resistates is dissolved among the EtOAc by evaporation, washes twice and wash three times, use Na with saturated NaCl ₂SO ₄Drying, and under vacuum, concentrate.On TLC, use CHCl ₃-MeOH (10: 1) can be observed bright orange green emitting point under 365nmUV.HPLC analyzes the purity of learning this crude product and surpasses 75%.The gained resistates is dissolved in 90% the MeOH aqueous solution and adds reversed-phase HPLC post (YMC-Pack, ODS-AM, in 20 * 250mm), and the MeOH aqueous solution of using from 70% to 74% linear gradient about 16 minutes with 12mL/ minute flow rate wash-out, collect required part and, obtain the yellow dehydrophenylahistin of 19.7mg (60%) (yield in each step not being optimized) although have by evaporation concentration.

Embodiment 4

The biological characteristics of dehydrophenylahistin and dehydrophenylahistin analogue

A. Biological assessment

In HT29 people's clone cell and PC-3 prostate cancer cell, the biological characteristics of synthetic tBu-dehydrophenylahistin and dehydrophenylahistin is estimated.

HT-29 (ATCC HTB-38) human colon cancer cell is maintained the full substratum of McCoy ' s (added McCoy ' the s 5A substratum that contains L-glutamine and 25mM HEPES of 10%FBS, the 1mM Sodium.alpha.-ketopropionate, 1 * NEAA, 2mM L-glutamine and be respectively 100IU/ml and Pen/Strep) in.PC-3 (ATCC CRL-1435) human benign prostatic cancer cells is maintained the full substratum of F12K (added the F12K substratum of 10%FBS; The 2mM glutamine; 1%HEPES; Be respectively 100IU/ml and

Pen/Strep) in.With clone at 37 ℃, 5%CO ₂, humidity 95% thermostat container in cultivate.

Tumour cell is carried out oxicity analysis, with HT-29 or PC-3 cell with 5,000 cells/well ( In the full substratum) be seeded in Coming 3904 black wall clear bottom (black-walledclear-bottom) tissue culturing plates, and this culture plate overnight incubation is made the cell growth and enters logarithmic phase.Dehydrophenylahistin and the tBu-dehydrophenylahistin stock solution of preparation 20mM in 100%DMSO, and be stored in-20 ℃.The spissated serial dilution of 10X of the described two kinds of compounds of preparation in suitable substratum, reaching the ultimate density scope is 20 * 10 ^-5M to 20 * 10 ^-10M.Will The 10X serial dilution of volume adds (totally three times) in the described test hole, and described culture plate was put back in the thermostat container 48 hours.Final DMSO concentration in all samples is 0.25%.

Be exposed to medicine after 48 hours, in every hole, add Be dissolved in no Mg ²⁺, Ca ²⁺The 0.2mg/ml resazurin (resazurin) (obtaining) of PBS from Sigma-Aldrich Chemical Co., described culture plate was put back in the thermostat container 3-4 hour.Remove culture plate, and in Fusion photofluorometer (PackardInstruments), excite and 590nm emission spectral filter detects the fluorescence of resazurin with 530nm.Measure background with not celliferous resazurin dyestuff, it is deducted from the data of all test holess.With Prism software (GraphPad Software) analytical data.Above-mentioned data are carried out normalization method to the mean value with the cell (growth of 100% cell) of matrix treatments only, and determine EC with sigmoidal (S type) the dose response curve fitting algorithm of standard ₅₀Value.

Shown in following table 1, compare the tBu-dehydrophenylahistin with dehydrophenylahistin and demonstrate big approximately 4 times cellular cytoxicity activity.

The cytotoxic effect of table 1 dehydrophenylahistin and derivative

Also referring to the other data in HT-29, PC-3 and P-388 cell shown in Figure 41.

B. The structure of dehydrophenylahistin derivative and activity research

In mouse leukemia cell, HT-29 people's clone cell and the PC-3 prostate cancer cell of P388, checked the cytotoxic effect of phenyl ahistins, dehydrophenylahistin and various dehydrophenylahistin derivatives.

As mentioned above, the HT-29 human colon cancer cell is maintained the full substratum of McCoy ' s (added McCoy ' the s 5A substratum that contains L-glutamine and 25mM HEPES of 10%FBS, 1mM Sodium.alpha.-ketopropionate, 1X NEAA, 2mM L-glutamine and be respectively 100IU/ml and Pen/Strep) in.PC-3 human benign prostatic cancer cells is maintained the full substratum of F12K (added the F12K substratum of 10%FBS; The 2mM glutamine; 1%HEPES; Be respectively 100IU/ml and Pen/strep) in.With clone at 37 ℃, 5%CO ₂, humidity 95% thermostat container in cultivate.

Cytotoxicity is analyzed, with HT-29 or PC-3 cell with 5,000 cells/well (in the full substratum of 90 μ l) is seeded in Coming 3904 black wall clear bottom (black-walledclear-bottom) tissue culturing plates, and this culture plate overnight incubation is made the cell growth and enters logarithmic phase.Dehydrophenylahistin and the tBu-dehydrophenylahistin stock solution of preparation 20mM in 100%DMSO, and be stored in-20 ℃.The spissated serial dilution of 10X of the described two kinds of compounds of preparation in suitable substratum, reaching the ultimate density scope is 20 * 10 ^-5M to 20 * 10 ^-10M.The 10X serial dilution of 10 μ l volumes is added (totally three times) in the described test hole, and described culture plate was put back in the thermostat container 48 hours.Final DMSO concentration in all samples is 0.25%.

Be exposed to medicine after 48 hours, in every hole, add Be dissolved in no Mg ²⁺, Ca ²⁺The 0.2mg/ml resazurin (obtaining) of PBS from Sigma-Aldrich Chemical Co., described culture plate was put back in the thermostat container 3-4 hour.Remove culture plate, and in Fusion photofluorometer (PackardInstruments), excite and 590nm emission spectral filter detects the fluorescence of resazurin with 530nm.Measure background with not celliferous resazurin dyestuff, it is deducted from the data of all test holess.With Prism software (GraphPad Software) analytical data.Above-mentioned data are carried out normalization method to the mean value with the cell (growth of 100% cell) of matrix treatments only, and determine EC with the sigmoidal dose response curve fitting algorithm of standard ₅₀Value.

Summed up the EC of phenyl ahistins, dehydrophenylahistin and dehydrophenylahistin derivative in the following table 2 ₅₀And IC ₅₀Value.

Table 2. Phenyl ahistins or dehydrophenylahistin And the SAR of dehydrophenylahistin derivative research

Modification pair cell toxicity activity to phenyl ring has remarkable influence.Compare with the activity of tBu-dehydrophenylahistin (#6), the compound that a position has methoxy group (KPU-9) shows the activity all higher than other derivative, its IC in the P388 cell ₅₀Value is 20.8 ± 3.3nM.Described KPU-9 derivative also shows cytotoxicity (EC in the HT-29 cell ₅₀31nM).Dehydrophenylahistin, tBu-dehydrophenylahistin (KPU-2) and KPU-9 derivative all show cytotoxicity in the P388 cell.

C. The structure of other dehydrophenylahistin derivative and activity research

Use above-mentioned method, in HT-29 people's clone cell and PC-3 prostate cancer cell, the cytotoxic effect of phenyl ahistins, dehydrophenylahistin and various other dehydrophenylahistin derivatives is checked.

Table 3. Phenyl ahistins or dehydrophenylahistin and other dehydrophenylahistin derivative SAR research

Application is adopted above-mentioned method in this embodiment as the resazurin fluorescence of cell viability indicator, carries out other cytotoxicity analysis.Described result is presented in the following table 3.1.

Table 3.1 Phenyl ahistins, dehydrophenylahistin and other dehydrophenylahistin spread out Biological research

Embodiment 5

Other dehydrophenylahistin analogue

A. Dehydrophenylahistin derivative synthetic is improved

Use separately aforementioned techniques or in conjunction with synthetic other the dehydrophenylahistin derivative of other known organic synthesis technology.

According to target derivative to be prepared, can change the diacyl piperazinedione and described first and second aldehyde that adopt in the described synthetic method.The synthetic derivative is:

A) other aromatic ring is modified and/or introduced to described phenyl ring,

B) position of the described aromatic nucleus of change,

C) change described imidazoles aromatic ring, and/or

D) 5 on the described imidazole ring are modified.

Figure below has shown the zone of modifying for preparation dehydrophenylahistin derivative on the dehydrophenylahistin compound.Herein disclosed is the example of the indefiniteness of modification, and those skilled in the art can fully understand on the basis of the disclosure.

A1) structure with known anti-tubulin compound serves as that alkyl, halogen, alkoxyl group, ethanoyl, sulfanilamide (SN), amino, hydroxyl, nitro or the like are modified to described phenyl ring in the basis.

2) introduce other aromatic ring

Or the like

The position of the described aromatic nucleus of B

C becomes other ring system

Or the like

D further modifies 5 of described imidazole ring

Or the like

The above-mentioned modification to the dehydrophenylahistin compound is expanded, and the derivative of described compound can comprise following substituting group :-CF on its phenyl ring (A) ₃,-SO ₂NH ₂(SO ₂NR ₁R ₂) ,-SO ₃H ,-CONH ₂(CONR ₁R ₂) ,-COOH or the like.Other ring system (C) also can comprise following groups:

B. The example of synthetic dehydrophenylahistin derivative

Other example of synthetic dehydrophenylahistin derivative is disclosed in the table 4.

Table 4. Other synthetic dehydrophenylahistin derivative

C. The evaluation of dehydrophenylahistin derivative

According to the method described in the embodiment 3 said derivative is estimated.Other evaluation to described derivative extends to the evaluation given activity, for example measure the restraining effect of on cell proliferation, to the effect of the effect of specific cells mechanism (being the microtubule function), cell cycle process, estimate anti tumor activity in vitro to cancer cell system or the like.Provided the scheme of some evaluation methods below.

1) The cell inhibitory effect effect of dehydrophenylahistin and analogue thereof

In each hole of 96-hole titer plate, add 100 μ l and in substratum, be prepared to 10 ⁵The A-549 cell that is derived from people's lung cancer of cell/ml, wherein said substratum is by at EMEM substratum (Nissui Seiyaku Co., Ltd.) foetal calf serum of adding 10% obtains in, and wherein said substratum has antitumor action to the A-549 cell that is derived from people's lung cancer.The methanol solution of the derivative that will obtain by above-mentioned listed example adds to be gone up in row's the hole most, uses the half-log method sample is diluted and to add, and described titer plate was hatched 48 hours in 37 ℃ in the carbon dioxide thermostat container.With the gained culture add a plurality of contain 10 μ lMTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-xenyl-2H-tetrabromide) (1mg/mlPBS) in, in the carbon dioxide thermostat container, hatched 6 hours subsequently in 37 ℃.Discard substratum and with the dissolution of crystals that produces in the described cell in the dimethyl sulfoxide (DMSO) in 100 μ l/ holes.Use microplate reader (microplate reader) to detect the photoabsorption of 595nm subsequently.By of the photoabsorption of comparison untreated cell, can calculate the concentration (IC of the described sample that suppresses 50% cell proliferation with the cell of crossing with the concentration known sample preparation ₅₀).

2) The cell cycle of dehydrophenylahistin and analogue thereof suppresses active

Cell strain A431 is derived from people's lung cancer.In 37 ℃ the A431 cell is hatched in the thermostat container saturated with the EMEM substratum that contains 10% foetal calf serum and 1%MEM non-essential amino acid solution (SIGMA M2025) with 5% carbon dioxide and water vapor.The purification sample of the dehydrophenylahistin that will obtain by aforesaid method adds and is in the above-mentioned cell of logarithmic phase, and by the flow cytometry and the process in microscopic analysis of cells cycle.

Embodiment 6

The structure-activity relation of synthetic dehydrophenylahistin (dehydrogenation PLH) derivative

1) general introduction during derivative synthesizes

Many in the dehydrogenation PLH derivative disclosed herein (but not being whole) have one, two or three modification group (seeing following Fig. 5) on its phenyl ring.Described derivative is by the aforesaid method synthetic.As shown in table 5, some compound exhibits goes out than dehydrogenation PLH and the higher cellular cytoxicity activity of tBu-dehydrogenation PLH.Active the highest, EC ₅₀Value is KPU-90 for the compound of 3nM.Above-mentioned value is higher 16 times and 4 times than the numerical value of dehydrogenation PLH and tBu-dehydrogenation PLH respectively.These derivatives have single replacement in an ortho position or a position of described phenyl ring, and substituting group is halogen atom (as fluorine and chlorine atom) or methyl, vinyl or methoxy group.On heteroaryl structure, have substituent derivative and also have effective activity such as naphthalene, thiophene and furan nucleus.KPU-35,42,69,80 and 81 also shows the activity that is higher than tBu-dehydrogenation PLH.

Table 5. Synthetic effective dehydrogenation PLH derivative

2) on described phenyl ring, introduce methoxy group

Colchicine can be discerned binding site identical on β-tubulin with PLH.Colchicine has 4 feature methoxy group on its A and B ring.Carried out a series of mono methoxies or Polymethoxylated replacement, the result is as shown in table 6 for its cellular cytoxicity activity.

Table 6. Methoxyl group replaces the effect to HT-29 cell proliferation

The above results proves in a position or adjacent replaces the cellular cytoxicity activity rising that makes the HT-29 cell.KPU-9 and 16 demonstrates high reactivity.Have trisubstituted methoxy derivatives (KPU-11,17 and 45) and also demonstrate activity.The structure of KPU-24 is determined by mass spectroscopy.

3) modify with electron withdrawing group

For the structure-activity relation on the described widely phenyl ring of research, introduced a series of different functional groups, comprise electron withdrawing group and donor residues.To the cytotoxicity result of HT-29 cell respectively shown in table 7 and 8.

At the ortho position or the effective enhanced activity of the replacement of a position.These results are very consistent with the situation of methoxyl group.

Table 7. electron-withdrawing group is to the effect of HT-29 cell proliferation

Table 8. Electron-donating group is to the effect of HT-29 cell proliferation

The disclosure is not defined or is limited to any concrete scientific theory.Yet, be to be understood that those skilled in the art can admit the conclusion that this paper presents and think that promptly less relatively functional group (that causes is sterically hindered less) can help showing stronger activity, be unfavorable for sterically hindered with for example tubulin binding site reaction and can cause such as the big slightly group of oxyethyl group (when comparing) or Br atom (when comparing) with the Cl atom with methoxyl group.In addition because these substituent characteristic electrons do not influence activity, therefore think these less relatively groups not with the binding site direct reaction of described β-tubulin, but limited the conformation that is suitable for above-mentioned bonded dehydrogenation PLH.Perhaps, as another possible hypothesis, because introduce hydrophilic oh group (it can be used as hydrogen donor and forms hydrogen bond) activity is significantly reduced, so hydrophobic property may be prior factor at the binding site to ortho position on described β-tubulin or contraposition.

As shown in table 9, can put in order the active effect of ortho-substituent pair cell toxicity, and as shown in table 10 to the situation of meta-substituent.Also can further modify compound with effective functional group (it demonstrates the high reactivity more than tBu-dehydrogenation PLH).And because the stereochemistry from Z to E that observes under radiation of visible light changes, the substituting group that therefore can reduce the electron density of described conjugated double bond can reduce the Z that caused by the light transformation to E, thus stable structure more on the generation physical chemistry.Temperature also can influence this transformation.

Modify the compound that can help developing effective and bio-stable in two parts of described ring.The phenyl ring of described phenyl ahistins can be by the cytochrome P-450 oxidation.Therefore, the double modification that can reduce the electron density of described phenyl ring can be avoided the oxidation of P-450 effectively.Therefore, with described little electron-withdrawing group (as fluorine atom) but with the unit of enhanced activity (as-OMe ,-Me ,-Cl ,-F and Br) combine, can produce the medical compounds of more effective and bio-stable.

Table 9. The summary that the ortho position is modified

Table 10. Between the position summary of modifying

4) with the described phenyl ring of aryl-heterocyclic substituted

Described phenyl ring also can be substituted by heteroaryl groups.The cellular cytoxicity activity result that this class is replaced is as shown in table 11.Because the nitrogen-atoms of described aromatic ring can form hydrogen bond with the NH group of described piperazinedione ring, and the molecular conformation that limits described pyridine and piperazinedione interannular is the monoplane structure, so the activity conformation of dehydrogenation PLH needs between the ortho-hydrogens atom of the hydrogen atom of acid amides of described piperazine ring and described phenyl ring to form by steric exclusion a certain size interfacial angle.

Table 11. Replace effect with heteroaryl ring to HT-29 cell proliferation

Substitute described phenyl ring (for example KPU-29 or 42) with less furans or thiphene ring and can show activity.Keeping to change described phenyl ring into other aromatic structure under the stronger active condition.

5) metabolism of phenyl ahistins

In recent research, (±)-phenyl ahistins is handled with rat liver microsome or people's liver P450s.In people's situation, detect seven kinds of meta-bolitess at least, and wherein two kinds (they being P1 and P3) be main meta-bolites, what account for described recovery meta-bolites surpasses 60%.

Since outside in tBu-dehydrogenation PLH, not having-and alkene structures, so current synthetic derivative does not resemble the oxidised form of P1 and P4.But, in the liver metabolism process, form oxidised form such as P3 and P5.The various metabolic derivatives of this class that stop can be avoided P450 oxidation on described phenyl ring effectively.Also can modify to avoid disadvantageous oxidation described imidazole ring.

6) physical and chemical stability of dehydrogenation PLH

The physical and chemical stability of dehydrogenation PLH is one of disadvantageous problem.In the phenyl ahistins,, therefore there is not this class problem because do not have other alkene structures at described benzyl moiety.Yet in dehydrogenation PLH, described benzylidene part can be easy to be activated (may by visible-light activated), and because there is the long conjugation of two keys, therefore the transformation of Z to E often takes place.This transformation even under normal indoor light, also can take place.In described cytotoxicity analysis, some compounds change the E-type in the process of hatching, although this transformation may reach the balance of 1: 1 ratio to dehydrogenation PLH.This transformation is controlled.Knownly in combretastatin A4 (similar type of tubulin inhibitor), also there be the transformation of this Z, and reported that some improve the research of this problem to E.

7) prodrug is synthetic

The E-type also can be used as the prodrug of dehydrogenation PLH or one or more its analogues (comprising those analogues as herein described).Although anti-tubulin medicine belongs to one of molecular targeted methods of treatment, one of unfavorable characteristic of these medicines comprises its lower selectivity to tumour and health tissues.This has caused disadvantageous side effect.Yet,, can reduce the adverse side effect of anti-microtubule medicine if described compound is only optionally brought into play function in tumor tissues.Because described dehydrogenation PLH (Z-type) can be produced by radiation of visible light by its E-isomer, therefore can be with the administration of described E-type and only knub position is carried out rayed, have only tumour to be damaged like this, and reduced counter productive health tissues by the Z-type that light produces.

By introducing large volume but biodegradable carboxyl groups can carry out chemoproection to described E-type, and described carboxyl groups can be used as prodrug and introduces on the described piperazinedione ring.This class carboxyl groups can be by intravital proteasome degradation.Therefore, keep the E-compound of acylations before administration, it becomes real E-type after the administration, it can be changed into bioactive Z-type by the local light irradiation.

The synthetic route of the tBu-dehydrogenation PLH of this class acyl group-E-type as shown in Figure 9.

Embodiment 7

The pharmaceutical preparation of synthetic dehydrophenylahistin

1) is used for the preparation of intravenous drip, injection, infusion or similar type administration

The usefulness the inventive method synthetic compound of aseptic adding 10mg and sealing in each bottle that contains 5g glucose powder.After charging into nitrogen, helium or other rare gas element, described bottle is kept at the dark place of low temperature.Use is preceding with described composition dissolve with ethanol, and it is added in 0.85% normal saline solution of 100ml.As the method that suppresses to make a definite diagnosis the intravital tumor growth of people of suffering from cancerous tumour, with gained solution to carry out intravenous administration in about 10ml/ days to about 1000ml/ days, its administering mode can be those skilled in the art's mode of thinking fit, as instillation or subcutaneous injection or peritoneal injection.

2) The preparation that is used for oral administration or similar type

By any conventional method, will make particle with the mixture that the inventive method synthetic compound, 98g lactose and 1g hydroxypropylcellulose thorough mixing obtain by 1g.With gained particle thorough drying and sieve, obtain being suitable for being packaged in the bottle or the granular preparation of heat-seal.According to symptom, the mode that the gained granular preparation can be thought fit by technician under people's cancerous tumour treatment field is to carry out oral administration in about 100ml/ days to about 1000ml/ days.

3) The preparation that is used for topical

Also can be by the direct described compound of administration in the zone of catching an illness of individual's skin being realized the described compound of the described individual effective dose of topical.For this purpose, described compound carries out administration or application with the form of the pharmaceutical composition that comprises the acceptable topical carrier of medicine, example gel, ointment, washing lotion or emulsifiable paste,, wherein said topical carrier includes but not limited to the carrier such as water, glycerine, alcohol, propylene glycol, Fatty Alcohol(C12-C14 and C12-C18), triglyceride level, fatty acid ester or mineral oil.Other topical carrier comprises the aqueous solution (5%) of kerosene, palmitinic acid isopropyl esters, polyoxyethylene glycol, ethanol (95%), polyoxyethylene mono-laurate or the aqueous solution (5%) of sodium laurylsulfonate.Can add other material if desired, for example antioxidant, wetting agent, viscosity stabilizer and similar agents.Also can comprise transdermal penetration toughener such as Azone.In addition, in some example, described compound can be arranged in be arranged on the skin, the device under skin or the skin.This class device comprises paster, implant and the injected material that can discharge described compound by passive or active releasing mechanism in skin.

Embodiment 8

The external pharmacology of KPU-2, KPU-35 and tert-butyl-phenyl ahistins

The vitro efficacy research of carrying out with KPU-2, KPU-35 and tert-butyl-phenyl ahistins comprises: the A) group of six kinds of tumor cell lines, B) study in the tumour cell of anti-multiple medicine, and C) determine the mechanism of effect.

A) In the group of six kinds of tumor cell lines to KPU-2, KPU-35 and tert-butyl-phenyl A Xi The research of this fourth

Use following clone (being the source in the bracket): (people clones tumour to HT29; ATCC; HTB-38), PC3 (human prostate tumour; ATCC; CRL-1435), MDA-MB-231 (human breast tumour; ATCC; HTB-26), NCI-H292 (people's non-small cell lung tumor; ATCC; CRL-1848), OVCAR-3 (people's ovarian tumor; ATCC; HTB-161), B16-F10 (mouse melanoma; ATCC; CRL-6475) and CCD-27sk (normal people inoblast; ATCC; CRL-1475).Cell is maintained inferior (subconfluent) density that merges in its culture medium separately.

According to above carrying out cytotoxicity analysis described in the embodiment 4, use the index of resazurin fluorescence as cell viability.

Described disclosed compound is the potent agent of the tumor cell line of anti-various differences and significant difference.Especially, for example KPU-2 and KPU-35 in intensity (in low nmole scope, activity being arranged) and to render a service that (the strongest to effect aspect the maximum cell toxic action) go up described HT-29 tumor cell line all be the most effective; The tert-butyl-phenyl ahistins demonstrates maximum intensity to described PC-3 tumor cell line, and described HT-29 clone is demonstrated maximum effectiveness; KPU-2 and KPU-35 intensity are usually than the high 10-40 of tertiary butyl ahistins times, and the effectiveness of all three kinds of compounds in described different tumor cell lines is suitable; Described HT-29, PC-3, MDA-MB-231 and NCI-H292 tumor cell line to the response class of NPI compound seemingly, described B16-F10 then shows to a certain degree insensitive.The tertiary butyl-phenyl ahistins shows tangible difference between normal fibroblast and described tumor cell line, except that to the OVCAR-3 clone, proportional range from 20 to 100.

Table 12. The work in the screening of tumour culture plate of KPU-2, KPU-35 and tert-butyl-phenyl ahistins The property

B) The research of drug-resistant cell system

A main challenge using chemotherapy agents in Clinical Oncology is the resistance of tumour cell to described drug effect development.Chemical sproof development has multiple mechanism, and wherein each all has different effects to chemotherapeutics.These mechanism comprise that the expression of the efflux pump (efflux pump) that ATP relies on rises, and described efflux pump is the P-glycoprotein of MDR1 coding or the multiple drug resistance associated protein 1 of MRP1 coding for example.Ingestion of medicines reduces, drug targets changes, acceleration reparation, the change of apoptosis pathway and the activation of cytochrome P 450 enzymes of drug-induced dna damage are cancer cell produces chemical sproof mechanism to cancer therapy drug other examples.In three kinds of different clones of two kinds of different resistance mechanism of performance (crossing of P-glycoprotein expressed and the active change of topoisomerase II), selected compounds is studied.

1) People's sarcoma of uterus tumor cell line is right: MES-SA (taxol sensitivity) and MES-SA DX are (anti- Taxol)

Mdr-1 mRNA and P-glycoprotein (overboard pump mechanism) that this expression of cell lines raises.Cause chemical sproof compound to raising, with S-Neoral-A (CsA) pre-treatment P-glycoprotein capable of blocking and recover the activity of those compounds in described drug-resistant cell is by P-glycoprotein.

As shown in table 13, KPU-2 has identical intensity with KPU-35 in the described sensitivity of described drug-resistant cell system's neutralization system, descend and the intensity of tert-butyl-phenyl ahistins is just slight.The intensity of S-Neoral-A (CsA) pre-treatment can not change selected compounds.Different with it, taxol is actually non-activity in described MES-SA DX drug-resistant cell system, and this compound is very potent in described sensitive cell line.CsA handles and has recovered the susceptibility of described MES-SA DX clone to taxol.Described MES-SA DX clone also demonstrates the susceptibility of reduction to etoposide (etoposide) (60 times), Zorubicin (34 times) and mitoxantrone (mitoxantrone) (20 times).

These data show, the effect of KPU-2, KPU-35 and tert-butyl-phenyl ahistins is not subjected to the influence of the relevant resistance mechanism of this described taxol (p-glycoprotein) in described clone, shows that simultaneously there is not cross resistance in taxol to these selected compounds in this model.

Table 13. KPU-2, KPU-35, tert-butyl-phenyl ahistins and taxol are at the MES-SA Japanese yew Activity in people's sarcoma of uterus tumor cell line of alcohol sensitivity and the anti-taxol of MES-SA DX5

Also referring to the other data shown in Figure 43.

2) The acute promyelocyte leukemia cell of people is right: HL-60 (mitoxantrone-sensitivity) and HL-60/MX-2 (anti-mitoxantrone)

This clone is considered to have atypical resistance, has the topoisomerase II catalytic activity of change and does not have cross expressing of P-glycoprotein.

As shown in table 14, these results show that the intensity of selected new compound is very similar in described sensitivity and resistance HL-60 clone.Different with it, mitoxantrone is renderd a service in resistance HL-60/MX-2 clone and has been descended 24 times.

Therefore, KPU-2, KPU-35 and tert-butyl-phenyl ahistins are insensitive to the resistance mechanism of mitoxantrone in this clone, show that simultaneously mitoxantrone does not have cross resistance to these selected new compounds in this model.

Table 14. KPU-2, KPU-35, tert-butyl-phenyl ahistins and mitoxantrone are anxious HL-60 people The property promyelocyte leukemia tumour responsive and drug-resistant cell be the activity of centering

3) MCF-7 is right: MCF-7 (taxol sensitivity) and MCF-7/ADR (anti-taxol)

This research compares KPU-2 and taxol.KPU-2 proof has similar intensity in right sensitivity of this clone and resistance member.Different with it, taxol non-activity in fact in described drug-resistant cell system, and in described sensitive cell line, have low nmole intensity (table 15).

These researchs are confirmed can not be transferred to KPU-2 to the resistance of taxol in the different people tumour cell.

Table 15. KPU-2 and taxol are the work of centering in MCF-7 human breast carcinoma sensitivity and drug-resistant cell The property

C) The research of the mechanism of action

1). to the effect of microtubule function

End user's huve cell in this research (from the HuVEC of Cambrex), by α-tubulin is dyeed, contrast colchicine and taxol have been estimated KPU-2 and the tert-butyl-phenyl ahistins effect to tubulin.

In described HuVEC cell, be exposed to KPU-2, tert-butyl-phenyl ahistins or colchicine (being 2 μ M) and induced microtubule depolymerization effect relative (show as and lack complete micro-tubular structure) and cytolemma to blister (blebbing) (apoptotic distinguishing mark) in 30 minutes, and taxol can not be induced the microtubule depolymerization effect under these conditions with observed result in the DMSO contrast.Colchicine is a known microtubule depolymerization reagent and taxol is the tubulin stablizer.The CCD-27sk cellular exposure be can be observed similar result in KPU-2 or colchicine.

2). cell death inducing

Apoptosis and insufficiency of accommodation thereof play an important role in oncology; The necrocytosis of selective induction sequencing circulation is the target of many chemotherapeutics development projects in tumour cell.By this inducing action of the provable pair cell apoptosis of different methods, aforesaid method comprises that distinctive cytolemma is blistered, dna break, the peroxophosphoric acidization of anti-apoptosis factor Bc1-2, the activation of caspase (apoptotic proteins enzyme) cascade reaction and the degraded of many (ADP ribose) polysaccharases (PARP).

Obviously different with the death of non-viable non-apoptotic cell, the characteristic signal of apoptotic cell death comprises that cytolemma is blistered, nuclear decomposition, cellular contraction and cohesion and final necrocytosis.KPU-2 induces the representative configuration relevant with the apoptosis commitment to change in the human prostate tumour cell.Can know the discovery similar phenomenon when handling the HuVEC cell with KPU-2.

3) .DNA fracture

Apoptotic late stage feature is a dna break between nucleosome, and it causes the tangible trapezoidal pattern that can be manifested by gel electrophoresis.This method is used for studying Jurkat cell (HTL system) compares the effect of KPU-2 with dehydrophenylahistin (KPU-1) dna ladder shapeization (laddering) with halimide.KPU-2 is in trapezoidalization of 1nM concentration inducing DNA, and the intensity of halimide and KPU-1 is much lower.

4) activation of .Caspase cascade reaction

In apoptotic process, comprise in the caspase cascade reaction that Caspase-3 ,-8 and-9 plurality of enzymes are activated.In the Jurkat cell, after handling, the Caspase-3 activity is monitored with KPU-2, KPU-35 and tert-butyl-phenyl ahistins.

The result shows, by to handle with all three kinds of compounds with the similar mode of halimide caspase-3 is activated in dose-dependent mode.In described Jurkat clone, with the Cytotoxic IC50 similar concentrations scope of expression in the activation (table 16) of described caspase-3 takes place.

Table 16. KPU-2, KPU-35 and the cell toxicant of tert-butyl-phenyl ahistins in the Jurkat cell The property

5) degraded of many (ADP ribose) polysaccharases (PARP) in the .Jurkat cell

In order to assess these compounds apoptosis-induced ability in the Jurkat cell, the degraded of many (ADP ribose) polysaccharases (PARP) is monitored.PARP is the nucleoprotein of 116kDa, and it is one of target in the main cell of Caspase-3.The degraded of PARP produces stable 89kDa product, and this process can be at an easy rate by the western marking (blotting) monitoring.PARP is one of apoptotic sign by the caspase degraded, and itself can be used as the good sign of this process.In described compound after 10 hours, the KPU-2 of 100nM can induce the degraded of PARP in the Jurkat cell with described cellular exposure.KPU-2 demonstrates than halimide or the higher activity of KPU-1.

6). enhanced vascular permeability in the HuVEC cell

(for example combretastatin A-4-phosphoric acid ester ZD6126) once demonstrated the interior blood vessel atrophy of induced tumor in vivo to the compound of depolymerization microtubule.Before the blood vessel atrophy, the perviousness of rapid induction of vascular cell (being at first) to developing macromole after the ionogen rapidly.With the HuVEC cell the infiltrative of fluorescently-labeled dextran strengthened as the indirect analysis to the blood vessel atrophy.

KPU-2, KPU-35 and tert-butyl-phenyl ahistins all rapid (in 1 hour) are induced tangible HuVEC mono-layer osmotic, and its degree and colchicine are similar.Microtubule stabilizer taxol non-activity (Figure 12) in this analysis.

7). the overview of kinases examination on a large scale

At first, in one group of 60 kinds of different kinases, KPU-2 is carried out examination with the concentration of 10 μ M; The concentration of ATP is 10 μ M.Four kinds of kinases are suppressed and surpass 50% in elementary examination, and the IC50 value of determining in the secondary examination is shown in table 17.All IC50 values all belong to low micro-molar range, show these kinase whose inhibition with active irrelevant to the observed low nmole of cytotoxicity.

Table 17. KPU-2 resists selected kinase whose activity

Kinases	IC50(μM)
		CDK1/Cyclin B (people)	10.1
C-RAF (people)	8.9
		JNK3 (rat)	6.8
Lyn (mouse)	11.1

Embodiment 9

Pharmacology in the body

In mouse, use MX-1 (breast) and HT-29 (clone) xenograft models and P-388 mouse leukemia tumor model that KPU-2 has been carried out preliminary study.According to activity, other tumor model of choosing in described external lesion list is DU-145 (prostate gland), MCF-7 (breast) and A549 (lung) clone.Also comprise human pancreas's tumour (MiaPaCa-2).Described new compound is studied as independent therapeutical agent or with the chemotherapy agents combination of clinical use.(maximum tolerated dose MTD) is determined the dosage of selected new compound, and can be adjusted in each research if desired by the acute tolerance test.The dosage of the chemotherapy agents of described clinical use is to select on the basis of former studies.

KPU-2 is first compound of studying in these five tumor models.According to the PRELIMINARY RESULTS of this research, all three kinds of compounds are cloned in tumour, DU-145 human prostate and the MCF-7 human breast tumor xenogeneic graft model HT-29 people compare.

Above-mentioned model all adopts the implantation technique of subcutaneous heterograft, and is subjected to compound produces amplification (or obvious) to the influence of the selectively acting of subcutaneous vascular system anti-tumor activity potentially.For fear of this possibility, two kinds of other tumor models have also been adopted in this research.One of them is the metastatic tumor of observation lung behind intravenous injection B16-F10 mouse melanoma tumour cell.Alternate model is to transplant MDA-231 human breast tumour cell in the buccal pad of mouse breast.Although back one model is an xenograft models, described subcutaneous vascular system is also inoperative.

Method

1). xenograft models

The animal of using is (exception statement in each research): 5-6 age in week (～20g, a female nude mice (nu/nu) Harlan); Unless specifically stated otherwise, the group size is every group of 9～10 mouse.

The cell that tumour transplatation is used is: HT-29 people clones tumour; MCF-7 human breast tumour; A549 people's non-small cell lung tumor; MiaPaCa-2 human pancreas tumour; DU-145 human prostate tumour.

Selected new compound is carried out administration by the intraperitoneal approach with the given dosage of each research as the single therapy agent; For combination research, injection in 15～30 minutes is selected with reference to chemotherapy agents before the described compound of administration.

The carrier that uses in these researchs is: 12.5%DMSO, 5%Cremaphor and 82.5% peanut oil are used for selected new compound; (1: 3) polysorbate80: 13% ethanol is used for taxotere; (1: 1) Cremaphor: ethanol is used for Paclitaxel; The every mL solution of CPT-11 is contained irinotecan hydrochloride, 45mg sorbyl alcohol NF powder and the 0.9mg lactic acid of 20mg, use NaOH or HCl that its pH value is transferred to 7.4.Dilute the injection concentration that obtains described reference compound with physiological saline.

HT-29 people clones tumor model

The HT-29 tumor fragment subcutaneous (s.c.) that to take from the subcutaneous growing tumors of nude mice host with trocar is implanted in the animal body.When tumor size reaches 5mm * 5mm (about 10-17 days), described animal is divided into treatment group and control group.Since first day, one week of mouse weighed twice, and used capplipers gauge (caliper) to measure tumor size twice weekly.With formula (W ²* L)/2 described tumor size is converted into the weight mg that estimates tumour.When the estimation tumor weight mean value of control group reaches 1000mg, mouse is weighed, puts to death and tumor resection.Tumour weighed and calculate every group average tumor weight, and determine every group inhibition rate of tumor growth (TGI) (100% deducts variation * 100 of the variation/control group exemplary embodiment lock of treatment group exemplary embodiment lock).

Unless independent research is had special declaration, in this model, selected new compound was once carried out peritoneal injection in per three days 15 days [the 1st, 4,8,11 and 15 day (q3dx5)]; At the 1st, 8 and 15 day (qwx3) with the administration of CPT-11 intraperitoneal.

MF-7 human breast tumor model

Subcutaneous transplantation MCF-7 tumor fragment (taking from nude mice host's Subcutaneous tumor) 24 hours before, to female nude mice (～20g) subcutaneous transplantation discharged the pill of oestrogenic hormon (0.25mg) in 21 days.Described in the HT-29 model, study then, use taxotere as the standard chemotherapy agents.

Unless independent research is had special declaration, in this model, new compound is injected by the intraperitoneal approach at 1-5 days every day, comprise (qdx5); At the 1st, 3 and 5 day (qodx3) with the administration of taxotere intraperitoneal.

A549 human lung cancer model

To take from the A549 tumor fragment subcutaneous transplantation of the subcutaneous growing tumors of nude mice host goes in the animal body with trocar.When tumor size reaches 5mm * 5mm (about 10-17 days), described animal is divided into treatment group and control group.The remaining part of research carries out according to described in the HT-29 model, uses taxotere and CPT-11 as the standard chemotherapy agents.

Unless independent research is had special declaration, in this model, uses the CPT-11 combination, or with the qdx5 dosage described test compounds is passed through the intraperitoneal administration with the taxotere combination with the q3dx5 dosage; By the intraperitoneal approach with qwx3 scheme administration CPT-11; With the qodx3 dosage with the administration of taxotere intraperitoneal.

MiaPaCa-2 human pancreas tumor model

To take from the MiaPaCa-2 tumor fragment subcutaneous transplantation of the subcutaneous growing tumors of nude mice host goes in the animal body with trocar.When tumor size reaches 5mm * 5mm (about 10-17 days), described animal is divided into treatment group and control group.The remaining part of research carries out according to described in the HT-29 model, uses gemcitabine (gemcitabine) as the standard chemotherapy agents.

Unless independent research is had special declaration, in this model, test compounds was once passed through the intraperitoneal administration in per three days at the 1st, 4,7,10 and 15 day (q3dx5); At the 1st, 4,7 and 10 day (q3dx4) gemcitabine is passed through the intraperitoneal administration.

DU-145 human prostate tumor model

To take from the DU-145 tumor fragment subcutaneous transplantation of the subcutaneous growing tumors of nude mice host goes in the male mice with trocar.When tumor size reaches～during 5mm * 5mm (about 13-17 days), described animal divided into treatment group and control group.The remaining part of research carries out according to described in the HT-29 model, uses taxotere as the standard chemotherapy agents.

Unless independent research is had special declaration, in this model, test compounds is passed through the intraperitoneal administration at the 1st, 3,5,8 and 11 day (q3dx5); At the 1st, 3 and 5 day (q2dx3) with the administration of taxotere intraperitoneal.

2). non-subcutaneous transplantation tumor model

Used animal is: 5-6 age in week (～20g, female nude mice (nu/nu) Harlan) (MDA-231 research) or B6D2F1 (B16-F10 research) mouse; Unless specifically stated otherwise, the size of group are every group of 10 mouse.

Used cell is: MDA-MB-231 human breast tumour and B16-F10 mouse melanoma cell.

The NPI compound is carried out administration by the intraperitoneal approach with the given dosage of each research as the single therapy agent; For combination research, injection in 15～30 minutes is selected with reference to chemotherapy agents before with the NPI compound administration.

MDA-231 human breast tumour

2 * 10 of cell in vitro cultivation is taken from injection in the breast buccal pad of female nude mice ⁶The MDA-231 cell.When tumor size reaches 5mm * 5mm (about 14-28 days), described animal is divided into treatment group and control group.The remaining part of research carries out according to described in the HT-29 model, uses Paclitaxel as the standard chemotherapy agents.

Unless independent research is had special declaration, in this model, test compounds is passed through the intraperitoneal administration at the 1st, 4,8,11 and 15 day (q3dx5); At 1-5 days (qdx5) Paclitaxel is passed through the intraperitoneal administration.

B16-F10 transitivity mouse melanoma model

Accept B16-F10 cell (taking from B16-F10 cells in vitro cell cultures) the 0th day mouse by the iv approach.At first day mouse is assigned randomly in treatment group and the control group, and begin treatment.Since first day, mouse is weighed weekly twice.At the 16th day all mouse are put to death, excise lung, weigh and the surface clone is counted.The result is expressed as average clone's number (T/C) * 100% of average clone's number/control group mice of treatment group mouse.Transforming growth inhibiting rate (MGI) is the above-mentioned numerical value of deduction from 100%.The standard chemotherapy agents that uses in this research is a Paclitaxel.

Unless independent research is had special declaration, in this model, described test compounds is passed through the intraperitoneal administration at 1-5 days (qdx5); At 1-5 days (qdx5) with the administration of Paclitaxel intraperitoneal.

In the time of suitably (n 〉=3), the result is expressed as mean value ± SEM.Unless specifically stated otherwise, check (post test) is carried out statistical analysis to result of study (using the researchs of carrying out of organizing) more behind use ANOVA and the Neuman-Keuls.Can reduce this hypothesis of tumor growth based on described compound or medicine or described combination, also use single tail t check (one-tailed t-test).

The result

Clone tumour xenogenesis skin HT-29 people and suppress research in the model

1.KPU-2+/-CPT-11 clones interior evaluating in the tumor xenogeneic graft model HT-29 people

Dose intensity when this research is used in combination to independent use KPU-2 or with relevant chemotherapeutics CPT-11 in the HT-29 model and the variation of dosage are assessed.

With 7.5mg/kg ip once a day totally five days (qdx5), totally five days, per two days one time 7.5mg/kgip of twice on the one 3.75mg/kg ip totally 10 days (qodx5) and per three days one time 7.5mg/kg ip the dosage of totally 15 days (q3dx5) with the KPU-2 administration.CTP-11 is combined dosed administration with 7.5mg/kg ip q3dx5 can produce effect when obviously using separately greater than arbitrary compound with NPI-2358, it all remains (Figure 13) in whole research process.The average final tumor weight of the group of these observed results that the live body of this research partly obtains during by postmortem confirms, wherein has only to compare with control group in conjunction with group to show the significant lower tumor weight of statistics.In addition, the average tumor weight differential of described combined treatment group and CPT-11 treatment group separately is statistics significant (Figure 14).After when postmortem, the final tumor weight of individuality being tested, can know the bigger effect (Figure 14) of seeing combined therapy.The TGI of combined therapy be 78% and relatively separately CPT-11 be 38.9%.The TGI of combined treatment group has surpassed the NCI standard (58%) of positive effect.

2.KPU-2+/-standard chemotherapeutics is to five kinds of people's tumor xenogeneic graft Study of model

This research is made up of five parts, and every part has its scheme, arrangement of time, dosage and reference compound separately.Each part all is interpreted as being included in the description of concrete tumor model.

The purpose of the HT-29 of this research part is that research is cloned to use in the tumor xenogeneic graft model HT-29 people and compared the effect of the KPU-2 of high dosage slightly with used dosage in the above-mentioned research, wherein observes the obvious synergistic effect between KPU-2 (7.5mg/kg ip q3dx5) and CPT-11 (100mg/kg ip qwx3).

As shown in Figure 15, KPU-2 and CPT-11 are combined in this model the obvious synergistic effect when suppressing tumor growth, and suppressed fully basically at the 29th a treatment day tumor growth this moment in described combined treatment group.Combined treatment has lasting effectiveness, and the tumor growth of estimating in this group is starkly lower than arbitrary single therapy group.Therefore, KPU-2 and CPT-11 administration can be suppressed tumor growth, be the effective antitumour therapy.

The weight measurement of the tumour of excising during postmortem (Figure 16) supported this research live body part observed result (tumor growth of estimation, Figure 15).Tumor weight in conjunction with group is starkly lower than control group (p＜0.01), equally also is starkly lower than the tumor weight (p＜0.05) of independent use CPT-11 group.

When considering individual final tumor weight (Figure 16), in conjunction with the tumor size of organizing generally less than other treatment group or control group.TGI in conjunction with group is 65.8%, shows positive effect according to the NCI standard, and separately treatment does not reach TGI〉58% NCI standard.

3. HT-29 people is cloned KPU-2, KPU-35 and the tertiary butyl in the tumor xenogeneic graft research The active research of phenyl ahistins

The result of this research is shown in Figure 17 and table 18.All show the obvious synergistic effect between the described new compound of combined treatment group and the CPT-11.The individual tumors attestation of weight validity of combined treatment (Figure 18).Under every kind of situation, all surpassed the NCI standard of positive effect in conjunction with the TGI of group, and CPT-11 separately the TGI of treatment do not reach this level.

4. KPU-2, KPU-35 and tert-butyl-phenyl ahistins and CPT-11 are combined in HT-29 people's gram The summary of the effect in the grand tumor xenogeneic graft model

When combining with CPT-11, KPU-2 is strengthened to the effect of CPT-11 (chemotherapy agents of standard) level of the positive effect NGI standard of super TGI far away 〉=58%.The result that above-mentioned three institutes get is suitable substantially aspect the weight (Figure 20) of the tumour of vivo observation result (Figure 19) and postmortem excision.

Research in the DU-145 human prostate tumor xenogeneic graft model

Finished two researchs with this model: first research comprises independent use KPU-2 and it is used in combination with taxotere; Second research will be used KPU-2, KPU-35 and tert-butyl-phenyl ahistins separately and be used in combination taxotere and compare.

1.KPU-2 be combined in work in the DU-145 human prostate tumor xenogeneic graft model with taxotere With

Can find from the data (Figure 21) of this research live body part gained, be that KPU-2 and taxotere combination are treated to the most effective methods of treatment of DU-145 human prostate tumour.Effect in the described treatment of the initial stage of this research is the most remarkable, but along with the carrying out of research constantly reduces.From 20-27 days of treatment, described combined treatment demonstrated the tangible TGI above NCI standard (TGI 〉=58%) really, and the estimation tumor weight of described combined treatment also is starkly lower than each independent methods of treatment.

2. independent KPU-2, KPU-35 and tert-butyl-phenyl ahistins or itself and taxotere are combined in Activity in the DU-145 human prostate xenograft models

Based on the data that combine with taxotere by KPU-2 in the above-mentioned research and obtain, carried out second research, independent use KPU-2, KPU-35 and tert-butyl-phenyl ahistins and the difference that is used in combination taxotere have been compared in this research.

The observed result of this research live body part shows no matter be KPU-2 or KUP-35, and it combines reduction effect to tumor growth all much larger than independent use taxotere (Figure 22) with taxotere.KPU-35 combines with taxotere and has hindered described growth of tumor basically fully.

The tumor weight that excises during postmortem has confirmed the observed result in this research live body stage.Be combined in when hindering tumor growth KPU-2 (Figure 23) or KPU-35 (Figure 24) and taxotere all obviously effective than independent use taxotere.When using KPU-35, three signs that demonstrate the tumour atrophy in ten mouse.Tumor growth suppresses index and shows that KPU-2 has significant tumor growth restraining effect (group on average=74.1%), and KPU-35 has then hindered tumor growth (organizing average=92.5%) basically fully.Independent taxotere does not then reach the positive effect standard (TGA 〉=58%) that NCI sets up.

5. the research in MCF-7 human breast tumor xenogeneic graft model

KPU-2, KPU-35 and the effect of tert-butyl-phenyl ahistins in MCF-7 human breast tumor xenogeneic graft model have been compared in this research.The the 1st, 2,3,4 and 7 day with given dose with described compound administration; At the 1st, 3 and 7 day with the taxotere administration.

In this model, when selected compounds is used in combination with taxotere, have onset early, statistics acts on significantly, obviously hindered estimated tumor growth (Figure 25) basically fully.In above-mentioned three kinds of compounds, KPU-2 is shown as the most effective, and the tert-butyl-phenyl ahistins also demonstrates obvious strong effect of crossing taxotere.

6. the research in A549 people's non-small cell lung tumor xenogeneic graft model

The observed result in this research live body stage (Figure 26) shows, compares with arbitrary single therapy group with control group, and (7.5mg/kg ip qdx5) has produced significant inhibition with being used in combination of taxotere to tumor growth to KPU-2.The tumor weight that postmortem obtains has confirmed The above results, and wherein the mean value of combined treatment group is significantly less than the group of independent use taxotere or the mean value of control group (Figure 27).The tumor weight value of described combined treatment group forms one group low tumor weight, shows the persistence of described effect.

When calculating the tumor growth index, compare described combined treatment group with control group and have 74.4% TGI, far surpass the NCI standard (TGI 〉=58%) of positive effect.The use taxotere has 26.1% TGI separately.

7. the research in MDA-231 human breast tumour coordination (orthotopic) xenograft models

This model comprises to be implanted people's tumor tissues in the mouse breast buccal pad (replacer of natural surroundings).Avoided in this way by to the specific action of veins beneath the skin tissue and the positive effect that produces.This research will be used KPU-2 separately, and (7.5mg/kg ip, (16mg/kg ip, effect qdx5) contrasts with being used in combination Paclitaxel in effect q3dx5).

When this research carried out for three weeks, in the combined treatment group, there be significant inhibition the (very significantly effect) in tumor growth.Much obvious (Figure 28) are compared in this effect with independent use taxotere.

8. the research in mouse melanoma B16 F10 metastatic tumo(u)r model

This research has been checked the intravenous injection of B16 F10 melanoma cell was gone into mouse after 16 days, uses KPU-2, KPU-35 and tert-butyl-phenyl ahistins separately and is used in combination the effect of Paclitaxel to the metastatic tumor number of lung surface appearance.This model is not an xenograft models; Yet it does not carry out the height vascularization in described tumor mass.

In this model, the most effective methods of treatment is to use KPU-2 (Figure 29) separately, and its mean transferred knurl number lacks 10% (MGI is respectively 41.6% and 35.0%) than independent use Paclitaxel approximately.Although this research itself can not description taken in conjunction treatment more effective than single therapy, it shows that really KPU-2, KPU-35 and tert-butyl-phenyl ahistins are the most effective in the tumour of height vascularization.

Embodiment 10

The analysis of enantiopathy substance fungi activity

Directly, use it for the AF/IS value of measuring described dehydrophenylahistin or its analogue to the antipathogen fungi relative reactivity of fungal organism bulk measurement (for example passing through the titer plate adaptation of NCCLS meat soup Macrodilution (macrodilution) method described in the Diagn Micro and Infect Diseases21:129-133 (1995)) dehydrophenylahistin or the above-mentioned relatively known antifungal compound of its analogue.Also can in the full animal model of fungi infestation, measure anti-mycotic activity.For example, can use pulmonary mucormycosis mouse model (Goldaill, the L.Z.﹠amp of steroid therapy; Sugar, A.M.1994 J Antimicrob Chemother 33:369-372).In this class research in the mode of explanation, before with fungi infestation, at that time or subsequently begin to a large amount of animals respectively administration do not have dehydrophenylahistin or its analogue (and/or in conjunction with one or more other anti-mycotic agents) or the positive control (for example amphotericin B) of dehydrophenylahistin or its analogue, various dosage.Can per 24 hours once, with dehydrophenylahistin or its analogue, the positive control of selected dosage or only animal is treated with carrier.The fate (as nearly 10 days) that continued treatment is predetermined.Behind the treatment stage animal is carried out the observation (for example 3 weeks) altogether of for some time, assess mortality ratio every day.Model can be adopted as simulation and suspect that infected human individual designs, and comprises or do not comprise other treatment (for example using steroid therapy), systemic infection, pulmonary infection, vaginal infection and other infection model.

In order to further specify, a kind ofly be used to measure cylinder therapeutic effect (ED ₅₀, for example be expressed as mg dehydrophenylahistin or its analogue/kg individuality) method be the rodent model system.For example, mouse is infected with the pathogenic agent fungi, for example with about 10 times to the pathogenic agent (10 of 50% lethal dose ⁶C.albicans cell/mouse) to infecting by intravenous injection.After the fungi infestation at once with the dehydrophenylahistin compound with predetermined dosage to the mouse administration.Calculate described ED by Van derWaerden (Arch Exp Pathol Pharmakol 195:389-412,1940) method from the survival rate that infects the 20th day record in back ₅₀Value.Usually, untreated animal death in 7-13 days after infection.

In another illustrative embodiment, to go up in C. albicans Wisconsin (C43) and the C.tropicalis (C112) of 28 ℃ of growth 48h at Sabouraud (Saab Luo Shi) agar glucose (SDA) slant culture (slant) and be suspended in the salt solution, and on spectrophotometer, the transmitance at 550nm place will be adjusted to 46%.Described inoculum is confirmed to reach about 1 or 5 * 10 with the further adjusting of hematimeter and with the plate count device ⁷CFU/ml.By with 1 or 5 * 10 ⁶CFU is injected into its afterbody blood vessel the CF-1 mouse is infected.Infect back 4h and reach 3 or 4 days thereafter once a day, will be dissolved in ethanol: the anti-mycotic agent of water (10: 90) carries out intravenously or subcutaneous administration.Monitor survival volume every day.ED ₅₀Be defined as the dosage that makes the survival of 50% mouse.

Embodiment 11

Assessment Antimicotic (antifungal) activity

Benzoglyoxaline and grisovin be can with the anti-tubulin reagent of fungi microtubule bonded.In case combination, these compounds hinder cell fission and the interior transportation of cell of responsive organism, cause necrocytosis.Commercial, with benzoglyoxaline as the mycocide in the control of veterinary medicine and crop disease.A large amount of fungal species comprise that grey mold (Botrytis cinerea), muscardine (Beauveria bassiana), potato cause of disease bacterium Helminthosporium sp. (Helminthosporium solani), yeast saccharomyces cerevisiae (Saccharomycescerevisiae) and Aspergillus are all to these molecule sensitivities.Yet toxicity problem and resistance rise its application have been caused negative impact.The tinea that grisovin is used for the treatment of the skin, hair and the nail that are caused by trichophyton, microsporum and acrothesium floccosum (Epidermophyton floccosum) clinically infects.Yet its antimycotic scope also is confined to this class fungal organism.Genotoxicity also is an important side effect.Another selectable first-line treatment (first-line treatment) Terbinafine (Terbinafine) is then expensive.In addition, in trichophyton (main pathogens that all dermophytes infect), observe clinical resistance recently.

In Candida albicans, when cellular exposure during in microtubule inhibitor R 17934 (nocodazole) and Y 3 (chloropropham), microtubule/microfilament forms and is affected.The further confirmation of these results can be with the cytoskeleton inhibitor as effective antifungal reagent.Therefore, assessed the antifungal activity of several compounds disclosed herein.

Especially, the microtubule inhibitor of disclosed compound and commercial distribution and generally acknowledged anti-mycotic agent are put together estimate.Test compounds and contrast used in this research are: (-)-phenyl ahistins, KPU-1, KPU-2, KPU-11 and KPU-17, KPU-35, the tert-butyl-phenyl ahistins, colchicine (at the microtubule inhibitor of the commercial distribution of 3 kinds of candidiasis strain isolateds (isolates) tests), F-1991 (Benomyl) (at the microtubule inhibitor of the commercial distribution of 3 kinds of candidiasis strain isolateds test), grisovin (being used for microtubule inhibitor and microbiotic contrast) at the commercial distribution of 6 kinds of dermophyte strain isolated tests, amphotericin B (being used for microbiotic contrast) at 3 kinds of candidiasis strain isolated tests, itraconazole (being used for microbiotic contrast) at 2 kinds of Aspergillus strain isolated tests.

The microorganism that is used to test these compounds comprises: Candida albicans, Candida glabrata, Aspergillus fumigatus, trichophyton, Trichophyton mentagrophytes (Trichophyton mentagrophytes), acrothesium floccosum.Except that Candida glabrata (strain isolated), each species uses two kinds of strain isolateds to test.

Method (M38-A " the Reference Method for BrothDilution Antifungal Susceptibility Testing of Conidium-Forming FilamentousFungi that provides according to the stdn council of U.S. clinical labororatory (National Committee for ClinicalLaboratoryStandards); Approved Standard ") finished antimycotic sensitivity tests.It is included in contain glutamine and not among the RPMI-1640 of potassium-containing hydrogen salt, 0.4-5 * 10 ⁴The inoculum size and 30 or 35 ℃ of conditions of hatching 48 hours under test.Minimum inhibitory concentration (MIC) is not defined as with there being medicine control tube (tube) and compares, and can make opacity reduce by 80% minimum concentration.To the compound medicine concentration of being studied is 0.03-16 μ g/ml, is 0.015-8 μ g/ml to itraconazole and grisovin.

Estimated minimum inhibitory concentration (MIC) according to the revision of NCCLS scheme, compound can stop the growth of target microorganism when this concentration.When can record growth in no medicine control tube the time, first measures minimum inhibition concentration (MIC) after 24 hours at interval.Described MIC is defined as to compare with growth control and demonstrates opacity and reduce by 80% minimum concentration.Determine minimum lethal concentration (MLC) by the concentration plating 0.1 μ l that is higher than described MIC from described MIC concentration and each.With first demonstrate 5 or still less the concentration of fungi clonal growth (representing 99.95% killing rate) be called MLC.When obtaining MIC, determine that minimum fungicidal concentration (MFC) is to estimate the fungi inhibition/fungicide performance of described compound.This process need is diluted to compound concentration with the cell sample (taking from the test hole that contains the compound that is equal to or higher than described MIC) of pharmacological agent and is starkly lower than described inhibition concentration, and places them on the agar plate.If described cell can continued growth, then described compound is designated as fungi and suppresses, and if there is no regrow, then be designated as fungicidal (because described compound has killed microorganism).

Compound exhibits disclosed herein goes out two kinds of trichophyton species effective.Trichophyton is the main pathogens of fell fungi infestation, and is the common-denominator target microorganism of exploitation clinical reagent.

KPU-2, KPU-11 and KPU-17, KPU-35﹠amp; Quite, effectiveness is higher than grisovin (the current standard drug reagent that is used for the treatment of the dermophyte infection) to the tert-butyl-phenyl ahistins in some cases on rendeing a service.

When testing at trichophyton, compound (-)-phenyl ahistins and KPU-1 render a service and are starkly lower than other compound, though and render a service during at the Trichophyton mentagrophytes strain isolated of sensitivity more weak more approaching.

In those examples, in the time can measuring MFC, the result shows that these compounds have the characteristic (referring to table 19 and 20) that suppresses fungi.

Embodiment 12

The blood-vessels target activity rating

Available compounds for treating tumour disclosed by the invention and neoplastic disease state.With the blood supply in the inaccessible tumour of blood-vessels target reagent (VTAs), induced the degeneration of this tumour.Compound disclosed by the invention (comprising NPU-02 and KPU-35) for example can be used as VTAs.Numerous VTAs by with microtubule on the colchicine binding site interact and to show its blood vessel function.The quick atrophy of characteristic and the obturation of the vascular system of having set up in this tumour induced in this interaction, therefore damaged the integrity of existing blood vessel, thereby caused necrosis.

For example, be exposed in VTA 30-60 minute, the blood vessel atrophy can take place, and relate to prematurity in the tumour middle body and propagation rather than the alteration of form of static and sophisticated endotheliocyte.Because with respect to normal blood vessels, in tumor vessel, exist the prematurity endotheliocyte of the propagation of higher percent, so the selectively acting that this difference to vascular cell act as tumour provides theoretical basis.VTAs can be divided into three kinds of eclipsed field of activities: (1) is blood vessel and cytotoxic effect effectively, and (2) have effective blood vessel function of faint cytotoxic effect, and (3) have the effective cell toxic action of weak vessel effect.

KPU-02 and KPU-35 blood-vessels target activity in vivo

Suppress tumor vascular system that tumor promotion vasculogenesis, target set up and the new therapy that suppresses tumor growth for research, animal model is necessary.

Mouse isogenic " false coordination " breast cancer model is used to study these problems.People such as Torres Filho, Microvascular Research (1995) 49,212-226 are incorporated herein by reference its full content at this.In order to create " false coordination environment ", remove the cover glass of the flap chamber (skinflap chamber) at back, and will be implanted into that this is indoor from the fritter breast buccal pad of donor mouse.On the top of buccal pad graft, use the tumour globe, this tumour globe comprises the N202 breast tumor cell with histone (H2B)-green fluorescent protein (GFP) transduction.The application of the cell of H2B-GFP transduction allows tumor growth and monitors mitotic division and apoptosis.

Fluorescence videomicroscopy technology allows relative Noninvasive research is carried out in the tumour microcirculation in the conscious mouse.This model can provide about the data of compound to tumor vascular system, tumor growth, mitotic division and apoptotic effect, this model be used for detection compound independent or with other therapeutical agent bonded activity.Utilize this model, after the single intravenous injection administration, KPU-02 and KPU-35 have shown and have induced blood vessel atrophy fast that this causes both central necrotic, and the degeneration of the tumour of having set up.

At tumor growth in the time of the 12nd day, with the KPU-35 venoclysis of 5mg/kg 2 minutes, with the KPU-02 venoclysis of 10mg/kg 5 minutes, or carrier group annotated and (contains 10%solutol (w/w)+2%DMSO) in the water and handle mouse.In the time of the 13rd day, KPU-02, the KPU-35 of usefulness 10mg/kg or vehicle infusion administration 5 minutes.The processing of KPU-02 or KPU-35 is by well tolerable.Observed mouse in addition two days.Observation in the tumour and tumour around tumour area, rate of blood flow and vessel density.On a plurality of time points, write down the Real Time Observation data with static images and videomicroscopy technology.

This studies confirm that after single KPU-02 or KPU-35 intravenous injection processing, the rapid atrophy of central vein system.The change of vascular function causes significant center neoplasm necrosis, and does not observe the effect (Figure 30) to the vascular system around buccal pad or the skin.Selectivity and the specificity of KPU-02 and KPU-35 supported in these observations, and KPU-02 and KPU-35 all can destroy the tumor vascular system of having set up individually.

The activity in vivo of KPU-02 in people's tumor xenogeneic graft

When KPU-02 and CPT-11 (Rinotecan), taxotere or Paclitaxel administration, at human colon tumor (HT-29), breast tumor (MCF-7; MDA-MB231) and in the tumor xenogeneic graft model of lung tumor (A549), see notable antitumor activity (table 21).In three research, the effect of KPU-02 in this HT-29 model is intensive, reproducible, and show dose dependency effect, and promptly 7.5mg/kg is higher than 2.5mg/kg (Figure 32,33) statistically.

KPU-02 and the KPU-35 external activity in the HuVEC cell

Above-mentioned KPU-02 and KPU-35 are supported by the interaction in vitro of same compound in the HuVEC cell effect in the body of tumor vascular system.Human umbilical vein endothelial cells is considered to the external model of good tumor endothelial, is considered to " immature ".Tumor endothelial lacks the cells of vascular wall of supporting and especially depends on the microtubule net of integrating tumor vascular system.Therefore, the destruction of tumour microtubule net causes the blood vessel atrophy.

KPU-02 induces tubulin depolymerization fast in the HuVEC cell

With Human umbilical vein endothelial cells (HuVECs; Cambrex CC2519A) merging density with the Asia maintains in EGM-2 (Cambrex) substratum.With this cell at 37 ℃, 5%CO ₂With cultivate in the incubator of 95% damp atmosphere.For the tubulin staining analysis, with the HuVEC cell with among the EGM-2 3 * 10 ⁴The density of cells/well is seeded on the compatible cover glass of tissue culture (Fisher).This plate was put back in this incubator 2 days.

The stock solution of preparation test compounds in 100%DMSO.The spissated diluent of 400X of the described compound of preparation in 100%DMSO.5 these diluents of μ l volumetrical are added in each hole, obtain the final concentration of 200nM.The final concentration of DMSO is 0.25% in all samples.Described culture plate was put back in the described incubator 30 minutes.KPU-02 or KPU-35 with 200nM handled the HuVEC cell 30 minutes.

At room temperature, in the formaldehyde of 10% (v/v) neutral buffered, fix 10 minutes before, described cell washes in dPBS.After fixing, by indirect immunofluorescence observation α-tubulin.Particularly, with the penetratingization processing 10 minutes in 0.2% (v/v) triton X-100/dPBS of described cell.Before described cover glass being transferred to moist chamber, wash this cell, this cover glass sealed 2 hours in antibody damping fluid (2% (w/v) BSA/0.1% (v/v) Tween20/dPBS).In washing and use in the dark before the 1 μ g/ml mountain sheep anti mouse FITC (Jackson immune Research laboratory) of 50 μ l hatches 1 hour, described cover glass is hatched with 0.1 μ g/ml mouse α-tubulin (Molecular Probes) in the antibody damping fluid of 50 μ l.At last, at H ₂Described cell is washed in flushing and with before the sealing of Vectashield (VectorLabs) mounting medium among the O, and with 2 μ g/ml DAPI (Molecular Probes) processing 10 minutes.On vertical microscope (Olympus BX51), use the 60x oil-immersion objective with this cell imaging.Catch this image with CCD photographic camera and Magnafire 2.0 softwares (Olympus) digitizing.In Photoshop Elements 2.0 (Adobe) and Microsoft Powerpoint, carry out post processing of image.

Figure 33 shows KPU-02 and KPU-35 rapid induction tubulin depolymerization in the HuVEC cell.

KPU-02 is inductive dose dependency mono-layer osmotic in the HuVEC cell

With Human umbilical vein endothelial cells (HuVECs; Cambrex CC2519A) merging density with the Asia maintains in EGM-2 (Cambrex) substratum.With this cell at 37 ℃, 5%CO ₂With cultivate in the incubator of 95% damp atmosphere.For the mono-layer osmotic analysis, with the HuVEC cell with 1 * 10 ⁵The density of cell/ml is seeded in the EGM-2 substratum on the 3.0 μ m Fluoroblok inserts (Becton Dickinson) of the fibronectin bag quilt in 24 orifice plates.This culture plate is put back in the described incubator 4 days to allow this cell to reach fusion.

The stock solution of preparation test compounds in 100%DMSO.The spissated serial dilutions of l0X of the described compound of preparation in EGM-2.This serial dilutions of 10 μ l volumetrical is added in the double test insert, obtain the final concentration of 2 μ M-2nM.The final concentration of DMSO is 0.25% in all samples.KPU-02 with 2nM-2 μ M handled described cell 15 minutes.

With dPBS (38.2kDa; Sigma) (50mg/ml) 2.5 times of dilutions in EGM-2 of the FITC-Dextran in (fluorescein isothiocyanate-dextran) add the FITC-Dextran of 10 μ l in every insert.The final concentration of FITC-Dextran is 1mg/ml.Described plate is put back in the described incubator, after 30 minutes, with having λ _Ex=485nm and λ _EmThe Fusion photofluorometer of the strainer of=530nm (Packard Bioscience) is measured the fluorescence of the low chamber in described 24 orifice plates.

Figure 34 has shown that KPU-02 can induce mono-layer osmotic in the dose-dependently mode.Result displayed is represented the mean value ± S.D. of three independent experiments among Figure 34.

Have ¹²⁵Volume of blood flow in the P22 rat sarcoma model of I-IAP

In rat, with quantitative with P22 rat sarcoma ¹²⁵Tumor blood flow amount in I-iodantipyrine (IAP) the technical evaluation model.1 hour the time, (15mg/kg IP) is reduced to 23% of carrier administration with the tumor blood flow amount to KPU-02 significantly and optionally after administration; After 24 hours volume of blood flow still keep remarkable minimizing (for the carrier administration 59%).On the contrary, in the time of 1 hour, the volume of blood flow in the nonneoplastic tissue is affected (seeing Figure 35) with much smaller degree influence.

As shown in figure 36, compare with carrier, for KPU-02, the minimizing of volume of blood flow is variable between tissue 24 hours the time after administration.Volume of blood flow to tumour is the most affected.Other tissue shows that less volume of blood flow reduces.24 hours the time, increasing appears in the volume of blood flow of skeletal muscle after administration.

Compare with the CA4P of the previous same technology of reporting of employing, the effect of the KPU-02 that observed in the time of 1 hour has shown for the longer persistence of tumor blood flow amount and bigger selectivity.

In the experiment of adopting P22 rat sarcoma model, as shown in figure 37, after administration 24 hours, 7.5 and the KPU-02 of 15mg/kg IP (the every dosage of n=2) administration produced the dose-dependently neoplasm necrosis, the highest dosage causes almost total necrosis of tumour.In the rat that described KPU-02 handles, all tumours show downright bad sign, yet tumour does not show this sign in the rat of vehicle treated.Enter clinical VTAs (for example CA4P, ZD6126, AVE8062) and shown similar qualitative effect tumor blood, wherein adopt IAP methodology (or similar technology) to confirm the minimizing of volume of blood flow in the P22 rat sarcoma tumour, adopt the dce-MRI technology to confirm the minimizing of blood in human body in flow.Referring to Stevenson JP, Rosen M, Sun W, Gallagher M, Haller DG, Vaughn D, Deng the people, " " Phase I trial of the antivascular agent combretastatin A4phosphate on a 5-day schedule to patients with cancer:magnetic resonanceimaging evidence for altered tumor blood flow, " (anti-angiogenic dose of phosphoric acid combretastatin A4 tested the I phase of 5 day time suffering from the cancer patients: the nuclear magnetic resonance image of the tumor blood flow amount of change is learned evidence) J Clin Oncol 2003; 21 (23): 4428-38; EvelhochJL, LoRusso PM, He Z, DelProposto Z, Polin L, Corbett TH waits the people, " Magneticresonanceimaging measurements of the response of murine and humantumors to the vascular-targeting agentZD6126, (nuclear magnetic resonance image learn to measure mouse with the reaction of the tumour people) " Clin CancerRes 2004 to blood-vessels target reagent ZD6126; 10 (11): 3650-7; And Gadgeel SM, LoRusso PM, Wozniak AJ, Wheeler C. " Adose-escalation study of the novel vascular-targeting agent; ZD6126; inpatients with solid tumors, " (in suffering from the patient of solid tumor, dosage-increase Journal of Sex Research of new blood-vessels target reagent ZD6126) Proc Am Soc Clin Oncol 2002; 21:abstract438; Be incorporated herein by reference at this full content each.

Combination therapy with the microtubule targeting agent

The investigator finds with respect to outer peripheral portion, VTAs optionally damages the vascular system in the tumour middle body, this has recovered functional, and this discovery has been supported these reagent and chemotherapy (taxol, vinealeucoblastine(VLB) and cis-platinum), radiation and direct angiogenesis inhibitor combined utilization at VEGF and EGF.New VTAs replenishes rather than alternative these therapies, and bigger anti-tumor activity is provided.

The treatment of other morbid state

Except cancer, also available VTAs disclosed by the invention treats other disease.Morbid state comprise other vegetation, retinopathy and any other depend on blood supply, be preferably the blood supply that depends on from neovasculature so that keep survival and/or the morbid state or the disease of propagation.

The numerous disease state is with too much or unsuitable vascular system is relevant.The example of the morbid state relevant with too much vascular system comprises diseases associated with inflammation, for example immunity and non-immunity inflammation, rheumatoid arthritis, beaevais' disease and psoriasis; The illness relevant with unsuitable or inappropriate vascular invasion for example is blood capillary proliferation and the osteoporosis in diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, Terry's sign disease, RI, the atherosclerotic plaque; And the illness relevant with cancer, for example comprise solid tumor, tumor metastasis, as leukemic haematogenous tumour, hemangiofibroma, Kaposi sarcoma, as angiomatous innocent tumour, acoustic tumor, neurofibroma, trachoma and botryomycosis hominis, and other needs vascularization to keep the cancer of tumor growth.The other example of vascular system-dependence disease comprises, for example, and Osler-Webber syndrome; The blood vessel of cardiac muscle takes place; The spot neovascularization; Telangiectasis; Bleeder's joint and wound granulation form.In addition, too much vascular system is learned and mechanical graft (tissue/organ graft, support or the like) as part biological, and is also relevant with clinical problem.Compound of the present invention and composition can be used for the target vascular system, and with respect to the vascular system of non-diseased tissue, preferably vascular system of preferential target diseased tissue, so described compound and composition can be used for treating this morbid state.Wherein to work and can adopt other disease of compound of the present invention and composition be conventionally known to one of skill in the art in vascularization.

The example of retinopathy comprises the macular degeneration relevant with the age (ARMD), diabetic retinopathy or the like.Pathologic vessels is the principal element that produces numerous retinal diseasess, and this numerous retinal diseases is principal element blind in the developed country altogether.Kahn and Hiller Am JOphthalmol (1974) 78,58-67 are incorporated herein by reference in its entirety.For example, suffering from of 30-50% retinal neovascularization formation and the formation of optic disk new vessel take place among the patient of 20 years diabetic retinopathies of surpassing.People Retina (2003) 23,518-522 such as Yanko are incorporated herein by reference in its entirety.In addition, in about 10% the patient who suffers from macular degeneration, new vessel formation is severe complications under the retina.People such as Ferris, Arch Ophthalmol (1984), 102,1640-1642 is incorporated herein by reference in its entirety.

Blood-vessels target reagent such as combretastatin A-4 (CA-4) has shown that the neovascularity that causes in the non-tumprigenicity tissue destroys.People such as Griggs, Br J Cancer (2001) 84,832-835 are incorporated herein by reference in its entirety.In addition, CA-4P has shown that being suppressed at the retinal neovascularization that takes place in the proliferating retinopathy process forms.People such as Griggs, Am J Path (2002) 160,1097-1103 are incorporated herein by reference in its entirety.At last, phosphoric acid CA-4P has shown the development that suppresses the formation of VEGF inductive retinal neovascularization and has suppressed the development of choroidal neovascularization and/or cause that the part of choroidal neovascularization disappears.People InvestOphthalmology ﹠amp such as Nambu; Visual Sci (2003) 44,3650-3655 are incorporated herein by reference in its entirety.Compound disclosed by the invention can be used for treating retinopathy.For example, Griggs (2001 and 2002) and the methodology of Nambu are used for the treatment of retinopathy.In addition, compound disclosed by the invention and composition can reduce vessel density and/or blood vessel hyperplasia by being applied to target region with treatment compound of significant quantity and/or composition, are used for the treatment of this retinopathy.

Effect when table 21 KPU-02 and chemotherapeutic associating in people's tumor xenogeneic graft model

Embodiment 13

Structure-activity relation

Data presentation described in Figure 38 come from the active result of the multiple modification on the phenyl ring of the tertiary butyl-dehydrogenation PLH.It is evident that in a position or adjacent is hydrophobic relatively and the replacement of minor feature base increases or kept cytotoxic activity in the HY-29 cell, and reduced activity in the replacement of contraposition.Be not bound by any particular theory, this data sheet is understood the strictness identification of tubulin to phenyl ring.

3D-QSAR (CoMFA) analyzes (seeing Figure 39) and has also supported to have three-dimensional range of profitability in a position and ortho position, and has three-dimensional unfavorable zone in contraposition.X-ray crystal analysis (seeing Figure 40) points out that the conformation of effective derivative need have a certain size interfacial angle between phenyl ring and the vacation of being made up of DKP and imidazole ring three ring templates (pseudo-tricyclic cor template).Therefore, the modification that utilizes appropriate phenyl ring conformational restriction to carry out may produce effective activity.Be not bound by any particular theory, the PLH derivative is different from colchicine and known analogue thereof at the binding pattern of the colchicine binding site of tubulin.

Embodiment 14

In external effect to microtubule

The purifying of tubulin and tubulin

Prepare tubulin (MTP) (referring to Farrell KW and Wilson L. (1987) Tubulin-colchicine complexes differentially poison opposite microtubuleends (tubulin-relative microtubule end of colchicine mixture otherness ground infringement) .Biochemistry 23 (16): 3741-8 is incorporated herein by reference in its entirety) as previously mentioned.By at PEM100 (100mM 1-4 piperazine two ethyl sulfonic acids (Pipes), 1mM MgSO ₄, 1mM EGTA pH6.8) and carry out three times thermopolymerization and cold depolymerization circulation among the 1mM GTP, isolates the MTP preparation of being made up of the microtubule-associated protein (MAPs) of 70% tubulin and 30% from the ox brain.MTP drip-freezes in liquid nitrogen and preserves until use in-70 ℃.By phosphocellulose chromatography (PC-tubulin) purifying tubulin from tubulin, and in PEM50, preserve (50mM Pipes, 1mMMgSO ₄, 1mM EGTA, pH6.8).Use bovine serum albumin as standard substance (Bradford, 1976), (Sigma Chemicals, St.Louis MO) determine protein concentration by the Bradford analysis.

Test agent

In DMSO with the stock solution of the prepared at concentrations KPU-02 of 20mM.In DMSO, examine not his spit of fland A4 (National Cancer Institute (National Cancer Institute), Bethesda, MD) stock solution of (CA4) with the prepared at concentrations of 5mM.In water with prepared at concentrations colchicine (Sigma Chemicals, St.Louis, MO) stock solution of (CLC) of 3mM.All reagent are managed isolation environment light with amber Eppendorf.Carry out serial dilution in DMSO and/or among the PEM50, make needed concentration.

Determining of the microtubule polymerization physique amount of stable state

Containing 1mM GTP and DMSO final concentration is when having the medicine of a series of concentration among 0.5% the PEM100, MTP (2mg/ml) to be aggregated into microtubule.Under 37 ℃, monitored sample 75 minutes with scattering of light at 350nm.

Polyreaction is centrifugal, the tubulin concentration in the supernatant liquor, when stable state the mensuration of solubility tubulin, and throw out, the mensuration of microtubule polymerization body is used for calculating the polymeric restraining effect.After hatching, by the centrifugal also microtubule (150,000xg, 45 minutes, 37 ℃) of precipitation polymerization that from unpolymerized MTP, separates.Remove supernatant liquor, before carrying out protein determination, at deionization H via the Bradford analysis ₂Among the O with the depolymerization of described microtubule throw out (24 hours, 0 ℃).

Calculate with two kinds of methods and to suppress per-cents, and the numerical value that will derive from these two kinds of methods compares.In one approach, calculate the ratio of the tubulin in the described throw out when not containing medicine of the tubulin in the described throw out when containing medicine.In other method, the tubulin when also calculating the tubulin in the described throw out when containing medicine the comparison of the tubulin in the described supernatant liquor not being contained medicine in the described throw out is to the ratio of the ratio of the tubulin in the described supernatant liquor.Described numerical value is near consistent, because the value of front has less difference and experimental interference, so use the value of front.

The mean length of microtubule distributes

Transmission electronic microscope technology is used to determine that the mean length of microtubule when not existing or having test agent distributes.In the time of 75 minutes and before the precipitation, by being fixed in PEM-100 buffered 0.2% glutaraldehyde that will be diluted in 290 μ l from 10 μ l equal portions of described polymer quality experiment.The fixed sample of 30 microlitres was placed 90 seconds on the 150ICG order electron microscope grid of polyvinyl formal bag quilt.Too much sample siphons away with Whatman filter paper.The cytochrome C (1mg/ml) 30 seconds of using 30 microlitres is to strengthen the precursor dissolving and negative staining is carried out easily.Use uranyl acetate (1.5%) 20 second, and siphon away too much part.In Jeol electron microscope-1200EX11,, observe grid during the 000X enlargement ratio in 2000X and 30.Zeiss MOPIII is used for determining the microtubule length distribution and the mean length of at least 100 microtubules of each sample.

The CLC competitive analysis

Under 37 ℃, in PEM50, use 1mM GTP, 1%DMSO, 10 μ M test agent and 7-25 μ M[ ³H] CLC hatched PC-tubulin (0.2mg/ml) 120 minutes.Measure [ ³H] the CLC combination, carrying out as previously described subsequently, the DEAE Mierocrystalline cellulose filters binding analysis (Wilson, 1970).This method depends on the absorption of tubulin to the filter paper of usefulness DEAE Mierocrystalline cellulose dipping.Before last sample, Whatman DE81 filter paper PEM50 pre-wetted.The reaction volume of 100 total μ l is applied to 2.5cm filter paper disk on Parafilm on ice.Under 4 ℃, this filter paper disk washs by immersing among the 50ml PEM50 that changes continuously for 5 times, to remove all unconjugated colchicine with 5 minutes speed of each washing.Then, containing 2ml BecKman Coulter ReadyProtein solution (Fullerton, the described filter paper disk that has the tubulin bonded colchicine of adhering to of direct census in scintillation vial CA).Whole filter paper disks is together washed.The unconjugated CLC that occurs in contrast can ignore the combination of this filter paper disk, no matter when existing or not having tubulin.

The linear regression calculating K of double reciprocal plot by described experimental data in Microsoft Excel _iValue.Utilize the x intercept to equal-1/K _m, determine that at first tubulin is to the K of CLC under experiment condition _mValue.Determine K in the presence of medicine with experimental technique _{M app}, K _mUse K _{M app}=α K _mRelation and competitive inhibition α=K _m(1+[I]/K _i) determine K _i

Fluorescence spectroscopy

Carry out fluorescence measurement with Perkin-Elmer LS50B spectrophotofluorometer.In PEM50,2mM GTP, 3%DMSO and 0-30 μ MKPU-02, hatch PC-tubulin (0.2mg/ml).Utilize the excitation wavelength of 395nm and the emission wavelength of 487nm, by 4,4 '-hexichol amido-1,1 '-dinaphthyl-5,5 '-disulfonic acid, di-potassium (bis-ANS; Molecular Probes, Eugene, OR) interaction of fluorescence report KPU-02 and tubulin.Excite and emission bandpass (band passes) for 10nm.Carry out twice this experiment.

Described bis-ANS fluorophor is surveyed proteic hydrophobic surface, and this bis-ANS fluorescence signal intensity change be this proteic solvent can and the result that changes of surface area.If part in conjunction with the time exist some conformational change, this conformational change changes the interaction of this tubulin-two-ANS, then bis-ANS can be used for reporting combination.

In the time of 25 ℃, hatched PC-tubulin (0.2mg/ml) 20 minutes with the KPU-02 of 0-30 μ M.Add bis-ANS (25 μ M) then, and in 25 ℃, the relative intensity of fluorescence of measure sample in 15 minutes.Collect the blank spectrum of damping fluid (Buffer blank spectra), and demonstrate KPU-02 and add bis-ANS and in the wavelength region of described experiment, produced insignificant fluorescence.

By experimental data being imported among Sigmaplot and the Microsoft Excel and being adopted equation F=((F _Max* L)/(K _d+ L))+F ₀Determine K _d, wherein F is the fluorescence intensity of the bis-ANS-tubulin in the presence of total ligand concentration L, F _MaxBe the bis-ANS fluorescence intensity of complete coordinate tubulin, and F ₀Be the bis-ANS fluorescence that does not exist under the condition of medicine.By with 1/ (F ₀-F) 1/L is drawn and is extrapolated to 1/L=0 determine F _MaxDetermine the part of the binding site B that occupies by KPU-02 in order to following relation: B=(F ₀-F)/(F ₀-F _Max).Use Lfree=L-B[C] determine the free ligand concentration, suppose that wherein each tubulin dimer has single binding site, [C] is the volumetric molar concentration of ligand-binding site point.

KPU-02 is to the inhibition of tubulin polymerization

In external cell free system, the ability that KPU-02, CA4 and CLC is changed tubulin (MTP) polymerization (2mg/ml) of being rich in MAP is analyzed.At first, with the inhibition (data not shown) of phosphorylated cotton tubulin analysis purifying, no microtubule-associated protein to polymerization.With respect to the MAPs of tubulin copurification in the presence of the MTs that assembles, KPI-02 is to the more effective inhibitor of MTs with glycerine and DMSO assembling.Though when not having stabilization MAP and existing, the microtubule polymerization body did not reach stable state in 2 hours, these are analyzed and confirm that KPU-02 is directly with the tubulin interaction of purifying and do not bring into play its basic role by MAP.

When measuring by scattering of light (Figure 44) and sedimentation analysis (Figure 45), KPU-02 and CA4 suppress the MT polymerization more consumingly than CLC.When having the medicine of a series of concentration, MTP (2mg/ml) is polymerized to microtubule, and by the scattering of light monitoring at 350nm, it has reached stable state.Figure 41 has shown the NPI-2358 (a) at DMSO pharmaceutical carrier (◇), 1.25 μ M (), 2.5 μ M (-) and 5M (zero), under the existence of CA4 (b) and CLC (c), and the turbidity spectrum of tubulin polymerization effect.KPU-02 and CA4 suppress the MT polymerization and have suitable usefulness.Figure 45 has shown when lacking or having a series of KPU-02 (zero), CA4 () and colchicine (◇) concentration, the inhibition of microtubule polymerization.By precipitating to determine the total polymerization physique amount of assembling after 75 minutes.Error bar is the standard deviation value from three experiments.Concentration (the IC that suppresses 50% polymerization ₅₀) be 2.4 ± 0.4 μ M to KPU-02, CA4 is that 2.2 ± 0.3 μ M and CLC are 7.6 ± 2.4 μ M (table 22).(unless refer else, the discrepancy report that obtains by statistical study is a standard deviation value).For polymerization in the body of the tubulin that is rich in MAP, be higher than IC at drug level ₅₀The time, MTP has shown kinetics of aggregation, this shows that KPU-02 and CA4 isolate albumen to stop the microtubule assembling.

Table 22 microtubule polymerization inhibition concentration

Compound	The average IC of polymer quality 50±sd(μM)	n
			KPU-02	2.4±0.4	4
CA4	2.2±0.3	3
			CLC	7.6±2.4	3

As shown in figure 44, three kinds of all described test agent produce the concentration dependent restraining effect of microtubule polymerization degree in the scope of 1.25-5 μ M.In described spectrum, note 2 important differences.At first, light absorption ratio initial growth rate in time reduces (Figure 44 A and 44B) with the increase of drug level.Described spectrum shows that the formation of MT exists the lag phase in the presence of KPU-02 and CA4.The medicine that also reduces the tubulin pond soluble, that the assembling ability is arranged significantly fast reduces the initial speed of polymeric.On the contrary, the initial speed of polymeric does not all have to change (Figure 44 C) in all CLC concentration.Secondly, as in time and shown in the linear light absorption value that increases, in the presence of KPU-02 or CA4, MTP does not reach stable state (Figure 44 A and 44B) when high drug level (more than the 5 μ M).On the contrary, in the presence of CLC, MTP reaches stable state (Figure 44 C) when high drug level.

By to analyzing with the linear relationship that the increase of drug level reduces, determine to suppress the needed medication amount (IC of 50% polymerization through the microtubule polymerization body of centrifugation ₅₀) (Figure 45).The error bar of Figure 45 is represented the standard deviation value from least three independent experiments.

Measure the minimizing of average microtubule length by transmission electronic microscope technology

The described polymer that reagent-microtubule polymerization reaction carrying out transmission electronic microscope technology is formed when describing stable state is also estimated by the conclusion that draws in the light scattering spectrum.KPU-02, CA4 and CLC all reduce the microtubule length that forms when stable state.MTs shorten gradually with the increase of drug level (Figure 46,47 and 48).Figure 46 shown (A) KPU-02, (B) CA4 and (C) CLC in the presence of, the frequency histogram of external average microtubule length when stable state.Described Zeiss MOPIII is used for determining the length distribution and the mean length of microtubule.Each drug level is counted 100 microtubules at least.Figure 47 has shown that Electron Microscopy is used to write down and has not existed or the microtubule when having test compounds.In the time of 75 minutes, the sample of the physique of auto-polymerization in the future amount experiment is fixed, dyeing, and in Jeol electron microscope-1200EX11, observe with the 2000x enlargement ratio.(A) KPU-02, (b) CA4 and (C) CLC in the presence of, during stable state at the exemplary electronic microscope figure of the microtubule that is rich in MAP of external formation.The scale bar is 10 μ M.Figure 48 has shown in the presence of KPU-02, CA4 and colchicine, the graphic summary that MT length reduces when stable state.The black square is 1.25 μ M, and the shade square is the medicine of 2.5 μ M.In the presence of KPU-02 and CA4, MTs shortens gradually with the increase of drug level, is under this concentration until drug concentrations, detects MTP by turbidity and shows kinetics of aggregation, and do not observe MTs.Error bar is from the standard deviation value that at least 100 microtubules are measured.

KPU-02, CA4 and CLC do not influence the MT nucleation.Numerous, short microtubule proof KPU-02, CA4 that forms in described polyreaction and the existence of CLC do not influence nucleation.If influence nucleation,, in the sample of drug treating, observe the long microtubule opposite, less with numerous short microtubules then with respect to control sample.

Aspect minimizing MT mean length, KPU-02 and CA4 have comparable validity.When the lowest concentration of drug 1.25 μ M that analyze by Electron Microscopy, KPU-02 and CA4 have reduced the MT mean length about 70%, and CLC has reduced about 40% (Figure 48).

Microtubule polymerization for external is higher than IC at drug level ₅₀The time, for KPU-02 and CA4, do not observe microtubule by Electron Microscopy.On the contrary, for all concentration of the CLC that is measured, observe microtubule by Electron Microscopy.Be higher than IC in concentration ₅₀The time, in the presence of KPU-02 and CA4, tubulin has shown kinetics of aggregation, it is characterized in that light absorption ratio in time and linear increase (Figure 44 A and 44B), and in the presence of CLC, the scattering of light polymer reaches stable state (Figure 44 C).MTP increases in time at 350nm in the presence of KPU-02 or CA4 although observe, and does not observe the protein aggregate of drug specificity.

Fluorescence spectroscopy

In the time of 25 ℃, in PEM50 and 2mM GTP, tubulin (0.2mg/ml) was hatched 20 minutes with the KPU-02 of a series of concentration.KPU-02 is with the fluorescence (Figure 49 A) of concentration dependent mode quencher bis-ANS.For by the fluorescence intensity of bis-ANS when the emission maximum attitude being carried out the KPU-02 and tubulin that nonlinear regression analysis measures Kd=10 ± 1.6 μ M (standard error) (Figure 49 B).Suppose that each tubulin dimer has the single binding site to KPU-02, the dissociation constant that the double reciprocal plot of described binding data produces is 6.4 μ M (Figure 49 C).By non-linear two different K that obtain with the linear regression analysis method _dValue is fully approaching, and thinks this numerical approximation equivalence.Figure 49 A has shown in the presence of ever-increasing KPU-02, the fluorescence emission spectrum of tubulin.Medicine is in conjunction with causing the bis-ANS Quenching of fluorescence.Figure 49 B has shown the fluorescent emission maximum value at 487nm, and match is to obtain the K of tubulin to KPU-02 _dValue, 10 μ M, standard deviation are 1.6 μ M.Illustration is a residue.Figure 49 C has shown the conversion two reciprocal of described binding data when supposing one mole of medicine/mole tubulin dimer.

CLC bonded competitive inhibition

KPU-02 and CA4 suppress CLC competitively in conjunction with tubulin (Figure 50).Figure 50 has shown the result of inhibition analysis, wherein do not have (◇) or having 10 μ M KPU-02 (zero) or during 10 μ MCA4 (), with multiple concentration [ ³H] CLC hatches the tubulin (0.2mg/ml) of phosphorylated cotton purifying.The Km of tubulin-CLC is 11 ± 4.4 μ M, and to suppress constant be 3.2 ± 1.7 μ M for KPU-02, is 2.4 ± 0.3 μ M for CA4.Computational constant from three independent experiments.Described colchicine-tubulin association reaction is the time and temperature dependent, and this bonded dissociation constant is K _d=0.1-1 μ M, this depends on the condition (Wilson L and Meza I) (1973) of described analysis.The mechanism of action of colchicine (mechanism of action of colchicine).Colchicinebinding properties of sea urchin sperm tail outer doublet tubulin (the colchicine binding characteristics of two tubulins in the sea urchin sperm tail outside) Journal of Cell Biology 58 (3): 709-19 is incorporated herein by reference in its entirety.Under test conditions, for CLC, the K of tubulin _mBe 11 ± 4.4 μ M.Can think K _mBe the K of tubulin substantially to CLC _dValue, still, since CLC bonded time-dependent manner, K _mCompare K _dThe value of being reported is big.Described K _iValue is 3.2 ± 1.7 μ M for KPU-02, is 2.4 ± 0.3 μ M for CA4.This K _iBe defined as and suppress 50% CLC in conjunction with required medication amount, and based on the amount in conjunction with the radioactivity CLC of tubulin.This K _iBe described medicine and the measurement CLC competitive capacity; It is not that medicine-tubulin is in conjunction with dissociated direct measurement, because report avidity in the described method.

The result

In all concentration of the CLC that is analyzed, the tubulin that is rich in MAP reaches stable state.On the contrary, in the KPU-02 or CA4 of higher drug concentration, the tubulin that is rich in MAP is not polymerized to stable state, and does not observe microtubule by electron microscope.KPU-02 and CA4 reduce the concentration of available tubulin effectively.This minimizing in solubility tubulin pond makes the threshold concentration of MT increase, and stops polymerization.Combine with the data that tubulin when those concentration are above does not reach stable state at required KPU-02 of external minimizing polymer quality and the chemistry amount of CA4, show that KPU-02 and CA4 work by isolation mechanism, in this isolation mechanism, the solubility tubulin is combined, stops polymerization.

By electron microscope observation, the microtubule that is rich in MAP that forms in the presence of described test agent is consistent with the mechanism that is proposed (being that KPU-02 and CA4 isolate tubulin) when described stable state.In the presence of KPU-02, CA4 and CLC, the mean length of tubulin exists concentration dependent to reduce.In the presence of KPU-02 and CA4, initial rate of polymerization exists the drug level dependency and reduces, and this shows that these medicines reduce the tubulin that can be used for prolonging.Because itself and tubulin slow fixation, so, do not see the reduction of initial rate of polymerization for CLC.In addition, for polymerization, surpass its IC in CLC concentration ₅₀Form microtubule during value, but surpass its IC at KPU-02 or CA4 concentration ₅₀Do not form microtubule during value.Be not bound by any particular theory, must be by the concentration of KPU-02 or CA4 bonded solubility tubulin to proceed below the required threshold concentration of tubulin polymerization.

In conjunction with studies show that with respect to CLC, tubulin has lower avidity to KPU-02.KPU-02 and CA4 took place in 20 minutes incubation period suppress CLC in conjunction with tubulin, this shows that KPU-02 and CA4 reach equilibrium ratio CLC faster (data not shown) with combining of tubulin.With described CLC-binding site eclipsed site on, KPU-02 competitive inhibition CLC is in conjunction with tubulin, this and consistent (the Kanoh K of research that characterizes phenyl A Xi Si spit of fland (halimide), Kohno S, Kataka J, Takahashi J and UnoI. (1999) (-)-Phenylahistin arrests cells inmitosis by inhibiting tubulin polymerization ((-)-phenyl A Xi Si spit of fland suppresses cell mitogen by suppressing the tubulin polymerization) The Journal of Antibiotics 52 (2): 134-141 is incorporated herein by reference in its entirety).Also competitive inhibition CLC combination of CA4 (analog of CLC).Be not subjected to the constraint of any particular theory, although it is evident that the shared tubulin land with CLC, KPU-02 and CA4 are by coming to interact with tubulin and suppress microtubule with the distinct mechanism of CLC.

Embodiment 15

Act in the body to microtubule

Cell culture studies

In the time of 37 ℃, at 5%CO ₂In, will be with GFP-α-tubulin (Clontech, Palo Alto, Canada) the MCF7 human breast cancer cell (American Type Culture Collection of stable transfection, the Manassas, VA) cultivate in the 250ml tissue culture flasks or the 35-mm-6 orifice plate in Dulbecco ' s improvement Eagle ' s substratum in, 5% foetal calf serum, 0.1% penicillin/streptomycin and non-essential amino acid (Sigma) (time doubles, 29 hours) have been added in this cultivation.Replace initial substratum by substratum,, and under 37 ℃, continued to hatch 20 hours with KPU-02, CA4 or the CLC incubated cell of method preparation as described in example 14 above with the equal volume that contains the test agent of desired concn or DMSO carrier.

The mitotic division process

Under given drug level, measure mitotic cell part (mitotic index) among the thymic carcinoma clone MCF7.With 3 * 10 ⁴The density of cell/ml with cell inoculation in 6 orifice plates.After 24 hours, when not having or existing the medicine of a series of concentration (1nM-1 μ M), cell was hatched 20 hours.Collect substratum and use versene (137mM NaCl, 2.7mM KCl, 1.5mMKH ₂PO ₄, 8.1mM Na ₂PO ₄With 0.5mM EDTA) flushing, separate with trypsinase, and be put back in the described substratum, with guarantee to suspend and adhere to not good mitotic cell and be included in the described analysis.Under 37 ℃, with 10% formaldehyde among the PBS cell fixation is spent the night, penetratingization processing is 10 minutes in methyl alcohol, and with 4,6-diamidino-Phenylindole (DAPI) dyes to manifest nucleus.Painted cell is coated on the Vectashield mounting medium, and (Burlingame CA) on the cover glass in, and is sealed on the wave carrier piece with nail varnish.Fluorescence microscopy is used for determining mitotic index.The result is the mean value and the standard deviation of 4-7 experiment, in this experiment, for 300 cells of every kind of concentration counting.IC50 is the drug level of inducing 50% maximum mitotic division to gather in the time of 20 hours experimentally.

The immunofluorescence microscopy technology

Except with cell inoculation PLL (50 μ g/ml, Sigma) on the cover glass of Chu Liing outside, adopt that method prepares cell described in the mitotic division process.Dyeing same day, flushing cell and in the time of 37 ℃ fixedly spends the night in 10% formaldehyde in PBS.Cell is washed in PBS,, and use the PBS hydration in-20 ℃ of penetratingization processing in methyl alcohol.At room temperature, handled cover glass 1 hour with 20% normal goats serum among the PBS/BSA (1%).At room temperature, hatched usefulness FITC-link coupled second antibody and DAPI dyeing then 1 hour among the mouse mono-clonal mixture of anti-α-tubulin that cell is diluted and β-tubulin, the DM1A/DM1B in PBS/BSA.With Prolong antidamping medium (Molecular Probes, Eugene OR) sealing cover glass.

Be used for the preparation of the cell of microtubule dynamic analysis

Adopt the method in the mitotic division process to prepare cell, just in order to promote the cell expansion, with cell inoculation on the glass cover slide, this glass cover slide is through PLL (50 μ g/ml, sigma) pre-treatment is 2 hours, (10 μ g/ml, sigma) pre-treatment is 1 hour through ln and fibronectin in the time of 37 ℃ subsequently.Also removed serum deprivation in 20 hours with medicine or DMSO incubated cell.Before analysis, cover glass is transferred in the record substratum (do not contain phenol red and sodium bicarbonate, cushion and add the substratum of 3.5g/L sucrose with 25mM HEPES).In order to prevent photobleaching, before cell being sealed in the chamber of double-canopy slide-seal, (Mansfield OH) adds in this record substratum for 30 μ l/ml, Oxyrase Inc. with Oxyrase immediately.

Microscopy and IMAQ at a slow speed

With Nikon Eclipse E800 fluorescence microscope microtubule with the apochromatic 1.4N.A.x100 object lens in plane.The described stage is encapsulated in the Pyrex box and by forcing the air heating system to maintain 36 ± 1 ℃.Application is by Metamorph software (Universal Imaging, Media, PA) drive, having 300ms time shutter, gain (gain) is that 2 and 2 * 2 combination of pixels (binning) are to strengthen the photometer CoolSNAP HQ digital camera (Tucson of brightness, AZ), when 10MHz, obtain 30 images of each cell with the interval of 4-s.

The microtubule analysis of dynamics

Employing exports the Metamorph Track Points application software of Microsoft Excel to, follows the tracks of the positive end position in time of microtubule, and analyzes with Real Time Measurement (measuring in real time) software.The length of each microtubule is traced the function for the time.Determine individual growth speed and shorten speed by linear regression.During the variation between 2 〉=0.5 μ m, be considered to take place to increase or shorten.During the variation between 2＜0.5 μ m, be considered to kinetics decay or termination period.For every kind of condition, analyze 25 microtubules at least.The result is the mean value and the standard deviation of at least three independent experiments.

By with the catastrophe number (the number ofcatastrophes) of every microtubule divided by increase or decay in time of consuming, calculate the time-based catastrophe frequency of every microtubule.By with total redemption number (the total number of rescues) of every microtubule divided by shortening the time that is consumed, calculate the time-based redemption frequency of every microtubule.Similarly, by with turnover number divided by the length that increases or the length of shortening, calculate based on the catastrophe frequency of distance respectively and save frequency.The microtubule of visible time≤2 minute is included in the described frequency analysis.With the dynamics calculation of every microtubule is to increase length and shorten length divided by the entire life by this microtubule.The microtubule of visible time 〉=0.3 minute is included in the described frequency analysis.

The cell cycle progress of when the prometaphase, blocking.

Determined the KPU-02 of cells accumulation, CA4 and CLC concentration range at m period.After 20 hours, the cell of 60-70% is suppressed at the prometaphase, and about such as the MT depolymerizing substance of vincaleucoblastine and 2-methoxyestradiol with such as taxol, in the MT Study of Stabilizers of epothilone B (epothilone B) and discodermolide, the cell that 30-40% is arranged is mid-term (JordanMA (2002) Mechanism of action of antitumor drugs that interact withmicrotubules and tubulin (with the mechanism of action of microtubule and the interactional antitumor drug of tubulin) .Current Medicinal Chemistry-Anti-Cancer Agents 2:1-17 is incorporated herein by reference in its entirety).The medicine of 1nM-1 μ M has been assessed 50% maximum mitotic block (IC ₅₀) required drug level (Figure 51).Figure 51 has shown by KPU-02, CA4 and CLC the inhibiting logarithm of mitotic division process [medicine] response curve.In the presence of NPI-2358 (zero), CA4 () and colchicine (◇), cultivate the MCF7 cell.In order to estimate mitotic index, with 3 * 10 ⁴The density of cell/ml with the MCF7 cell inoculation on 6 orifice plates.After 24 hours, when not having or existing the medicine of a series of concentration (1nM-1 μ M), cell was hatched 20 hours.Fixed cell, and with DAPI dyeing to manifest nucleus.The fluorescence Electron Microscopy is used for determining mitotic index.The result is the mean value and the standard deviation of three or four experiments, in this experiment, for 300 cells of every kind of drug level counting.Mitotic block IC ₅₀Value is that 17.4 ± 1.2nM, CA4 are that 5.4 ± 0.7nM and CLC are 23.8 ± 3.1nM (table 23) for KPU-02.

The restraining effect of table 23 pair mitotic division process

Compound	The average IC of polymer quality 50±sd(nM)	n
			KPU-02	17.4±1.2	4
CA4	5.4±0.7	3
			CLC	23.8±3.1	4

Great majority are blocked mitotic division in mid-term with the MT targeting agent in the transformation in later stage.It is relevant with the dynamic (dynamical) inhibition of MT to block mitotic division in mid-term in the transformation in later stage.Do not fettered by any specific theory, more Zao prometaphase prevent with the polymeric loss of MT shown jointly with respect to other such as vinealeucoblastine(VLB) stablize the dynamic (dynamical) MT depolymerization of MT medicine when the lower concentration, KPU-02 has the distinct mechanism of action.

The unzipping of the MTs that mitotic spindle and interkinesis arrange

Observing KPU-02, CA4 and CLC is effective microtubule depolymerization thing in the MCF7 cell.Though compare with the microtubule that the interkinesis arranges, the mitotic spindle microtubule suppresses more responsive, two kinds of microtubule group all effecteds (Figure 52) to depolymerization and/or polymeric.Figure 52 has shown the immunofluorescence micro-image of MCF7 cell.Compare with mitotic spindle, the interkinesis arranges more stable to the unzipping of KPU-02, CA4 and CLC.The preparation cell, and adopt method described in the mitotic division process to inoculate, for every kind of medicine, with the IC of described mitotic block ₅₀Value was handled cell 20 hours.Incubated cell in the monoclonal mixture of the mouse of anti-α-tubulin and β-tubulin, DM1A/DM1B dyes with FITC-link coupled second antibody and DAPI subsequently.A-d is the tubulin in contrast (a), in the cell handled of KPU-02 (b), CA4 (c) and CLC (d), and e-h is the DNA in the contrast (e), in the cell of KPU-02 (f), CA4 (g) and CLC (h) processing.Narrow arrow is pointed out mitotic spindle polymer and mitotic chromosome, and the arrow of broad is pointed out intermitotic arrangement and nucleus.

IC at mitotic block ₂₅During value, KPU-02 changes the morphology of spindle body significantly.Figure 53 A-C has shown the immunofluorescence electron micrograph image of handling 20 hours MCF7 cell with KPU-02 (A), CA4 (B) and CLC (C).Mitotic spindle destroys with the increase of drug level.1-4 is in contrast (1), mitotic block IC ₂₅Concentration (2), mitotic block IC ₅₀Concentration (3) and the mitotic block IC of twice ₅₀Concentration (4) in α-tubulin and β-tubulin; 5-8 is the corresponding D NA image in the contiguous illustration.For KPU-02 or CA4, at IC ₂₅The time do not have a normal bipolar spindle body (Figure 53 A and B).The cell of compound treatment contains unipolar or has the not chromosomal bipolar spindle body of set.On the contrary, for CLC, at IC ₂₅The time still have a normal bipolar spindle body (Figure 53 C).For KPU-02, at IC ₅₀The time, 75% mitotic cell contains the celestial body or the aggregate of tubulin, and remaining cell do not contain can detected mitotic division polymer.In the presence of CLC, the cell of half is to have not gather chromosomal Beale's ganglion cells, remaining half be unipolar.In concentration is the mitotic block IC of twice ₅₀The time, in the mitotic cell of handling with KPU-02, CA4 or CLC, almost detect polymer less than MT.

For the compound of all detections, to compare with mitotic spindle, the interkinesis of microtubule is arranged more anti-depolymerization (Figure 52).But,, observed polymer with the qualitative minimizing of dose-dependent manner (Figure 54 A-C) for three kinds of all compounds.Figure 54 A-C has shown the immunofluorescence electron micrograph image of handling 20 hours MCF7 cell with KPU-02 (a), CA4 (b) and CLC (c).Interkinesis MT unzipping is with the increase of drug level.1-4 is in contrast (1), mitotic block IC ₂₅Concentration (2), mitotic block IC ₅₀Concentration (3) and the mitotic block IC of twice ₅₀Concentration (4) in α-tubulin and β-tubulin; 5-8 is the corresponding D NA image in the contiguous illustration.Stablize MAPs although exist in the cell, tubulin may be isolated in these intermitotic cells, and this is isolated as the tubulin that is rich in MAP in external polymer mass analysis.

In the MCF7 cell of living, lack inhibition or adjusting to the MT dynamic instability

In the MCF7 cell, when the concentration of the maximum mitotic block that influences 25% (table 24) or 50% (table 25), KPU-02 and CA4 do not have measurable effect to the MT dynamic instability.Be not subjected to the constraint of any particular theory, these data show that the antiproliferative mechanism of action of KPU-02 (and CA4) mainly is because to MT polymeric restraining effect, rather than microtubule is suppressed dynamically.

The purpose that the foregoing description is provided is just in order to help to understand the present invention.Therefore, those skilled in the art are appreciated that the derivative that method disclosed by the invention and compound comprise maybe can provide other dehydrophenylahistin.

Those skilled in the art should be readily appreciated that the present invention is applicable to acquisition target for example as herein described and advantage and other natural characteristics very much.Method described herein and step are just represented embodiment preferred, and are exemplary, are not meant to limit the scope of the invention.Those skilled in the art include in spirit of the present invention its change of doing and other application.

To those skilled in the art apparently, can under the condition that does not depart from the scope of the invention and spirit, make various substitutions and modifications to the present invention.

As noted above, all patents mentioned in this specification sheets and publication are the degree for the technician who shows the technical field of the invention.This paper is incorporated herein by reference above-mentioned all patents and publication in allowed by law maximum range, should be considered as like this that independently patent and publication have illustrated especially and separately and be introduced into as a reference to each.

The invention that this paper illustrative is described can realize not possessing under not disclosed especially key element of any this paper or the restricted condition.Used term and phraseology all are used for illustration purpose and unrestricted purpose, and show without any hint, use this class term and express to mean eliminating its shown or the technical characterictic of description or equivalent of its part.Admittedly, can carry out various modifications within the scope of the invention.Therefore, though should be appreciated that the present invention is undertaken specifically disclosed by embodiment preferred and selectable technical characterictic, those skilled in the art can use the modification or the conversion of notion disclosed herein, and this class is revised or conversion is interpreted as belonging to scope of the present invention.

Claims (17)

1. Use of a compound of general formula (I) in the preparation of a drug that preferentially targets the vasculature of a tumor relative to the vasculature of a non-tumor tissue, wherein the compound has the following structure: in R ₁ is selected from hydrogen atom, halogen atom, C ₁ -C ₆ straight chain alkyl, C ₁ -C ₆ straight chain alkenyl excluding C ₁ alkenyl, C ₁ -C ₆ straight chain alkoxy, hydroxyl, C ₁ -C ₆ straight-chain alkylthio and halogenated C ₁ -C ₆ straight-chain alkyl, wherein the alkenyl is a monounsaturated or diunsaturated unsubstituted hydrocarbon group; Each R ₁ ' and R ₁ "is independently selected from hydrogen atom, halogen atom, C ₁ -C ₆ straight chain alkyl, C ₁ -C ₆ straight chain alkenyl excluding C ₁ alkenyl, C ₁ -C ₆ straight-chain alkoxy, hydroxyl, C ₁ -C ₆ straight-chain alkylthio and halogenated C ₁ -C ₆ straight-chain alkyl, wherein the alkenyl is monounsaturated or diunsaturated unsubstituted hydrocarbon group; R ₄ is tert-butyl; _R6 is hydrogen; _R2 and _R3 are both hydrogen; _X and _X are oxygen; Y is -NH; n is an integer equal to 1; each Z and each Z ₁ , Z ₂ , Z ₃ and Z ₄ are independently selected from carbon atoms; and Bonds indicated by dashed lines are either single or double bonds. 2. The use according to claim 1, wherein the non-tumor tissue is selected from the group consisting of skin, muscle, brain, kidney, heart, spleen and intestine. 3. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 10% over the vasculature of non-tumor tissue. 4. The use as claimed in claim 1, wherein n is equal to one. 5. The use as claimed in claim 1, wherein n is equal to 1 and each of Z, _Z1 , _Z2 , _Z3 and _Z4 is a carbon atom. 6. purposes as claimed in claim 1, wherein said compound has following structure:

7. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 20% relative to the vasculature of non-tumor tissue. 8. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 30% relative to the vasculature of non-tumor tissue. 9. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 40% over the vasculature of non-tumor tissue. 10. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 50% over the vasculature of non-tumor tissue. 11. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 60% over the vasculature of non-tumor tissue. 12. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 70% over the vasculature of non-tumor tissue. 13. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 80% over the vasculature of non-tumor tissue. 14. The use of claim 1, wherein the vasculature of the tumor is preferentially targeted by 90% over the vasculature of non-tumor tissue. 15. The use according to claim 1, wherein the halogenated C ₁ -C ₆ linear alkyl group is a polyhalogenated C ₁ -C ₆ linear alkyl group. 16. The use according to claim 1, wherein: R ₁ is selected from halogen atom, C ₁ -C ₆ straight chain alkyl, C ₁ -C ₆ straight chain alkenyl excluding C ₁ alkenyl, C ₁ -C ₆ straight chain alkoxy, hydroxyl, C ₁ - C ₆ straight chain alkylthio and halogenated C ₁ -C ₆ straight chain alkyl, wherein the alkenyl is a monounsaturated or double unsaturated unsubstituted hydrocarbon group; Each R ₁ ' and R ₁ "is independently selected from hydrogen atom, halogen atom, C ₁ -C ₆ straight chain alkyl, C ₁ -C ₆ straight chain alkenyl excluding C ₁ alkenyl, C ₁ -C ₆ straight-chain alkoxy, hydroxyl, C ₁ -C ₆ straight-chain alkylthio and halogenated C ₁ -C ₆ straight-chain alkyl, wherein the alkenyl is monounsaturated or diunsaturated unsubstituted Hydrocarbyl. 17. The use of claim 1, wherein: R ₁ is selected from a hydrogen atom, a halogen atom, a C ₁ -C ₆ straight chain alkyl group, a C ₁ -C ₆ straight chain alkoxy group, a hydroxyl group and a halogenated C ₁ -C ₆ straight chain alkyl group; Each of R ₁ ′ and R ₁ ″ is independently selected from a hydrogen atom, a halogen atom, a C ₁ -C ₆ linear alkyl group, a hydroxyl group, and a halogenated C ₁ -C ₆ linear alkyl group.

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