JP2007520565A - Synthesis of dehydrophenylahistine and their analogs, and dehydrophenylahistine and their analogs - Google Patents

Abstract

Disclosed are compounds of structure (I), as well as methods of making such compounds. Here, the method comprises reacting diacyl diketopiperazine with a first aldehyde to form an intermediate compound, reacting the intermediate compound with a second aldehyde, and a compound species having the general structure. The first aldehyde and the second aldehyde are selected from the group consisting of oxazole carboxaldehyde, imidazole carboxaldehyde, benzaldehyde, imidazole carboxaldehyde derivatives, and benzaldehyde derivatives, Wherein R ₁ , R ₁ ′, R ₁ ″, R ₂ , R ₃ , R ₄ , R ₅ and R ₆ , X ₁ and X ₂ , Y, Z, Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently producing that may be) be defined in a manner consistent with the accompanying description. compositions and methods for treating vascular growth also It is shown.

Description

[Background of the invention]
[Field of the Invention]
The present invention relates to compounds and synthetic processes in the chemical and medical fields. More specifically, the present invention relates to compounds and methods for producing compounds useful in the treatment of cancer and fungal infections.

[Related applications]
This application is a continuation-in-part of US patent application Ser. No. 10 / 632,531, filed Aug. 1, 2003, and US Provisional Application No. 60 / 542,073 filed Feb. 4, 2004. And US Provisional Application No. 60 / 624,262 filed on November 1, 2004, all of which are dehydrophenylahistins and their analogs, and dehydrophenylahistins and their Each of which is incorporated herein by reference in its entirety.

[Short description of related technology]
A single universal cell mechanism is thought to control the regulation of eukaryotic cell cycle processes. For example, Hartwell, L .; H. See Hartwell, LH et al., Science (1989), 246: 629-34. It is also known that cancer or immune disorders can occur if an abnormality occurs in the cell cycle control mechanism. Thus, as is also known, anti-tumor agents and immunosuppressive agents can be one of the substances that regulate the cell cycle. Accordingly, new methods of producing eukaryotic cell cycle inhibitors are needed as antitumor and immune enhancing compounds and should be useful in human cancer treatment as chemotherapeutic and antitumor agents It is. For example, Rovage, M.M. See (Roberge, M. et al.), Cancer Res. (1994), 54, 6115-21.

Fungi, especially pathogenic fungi and their associated infections represent a further clinical challenge. With respect to existing antifungal agents, there are limitations in the development and / or discovery of efficacy and toxicity and pathogenic fungal strains that are currently available or resistant to drugs under development. For example, fungi that are pathogenic to humans include C.I. C. albicans, C.I. C. tropicalis, C.I. Kefir, C. kefyr. C. krusei and C.I. Candida species including C. galbrata, A. A. fumigatus and A. fumigatus. Aspergillus genus including A. Flavus, Cryptococcus neoformans, Blastomyces dermatitidis including Blastomyces dermatitidis, Pneumocystis Carini (Pneumocystis carinii), Coccidioides immitis, Basidiobolus ranarum, Conidiobolus spp., Histoplasma capsulatum, R. R. oryzae and R. oryzae Rhizopus genus including R. microsporus, Cunninghamella, Rhizomucor, Paracoccidioides brasiliensis, Pseudalle, boys, Pseudalle Rhinosporidium seeberi and Sporothrix schenckii (Kwon-Chung, KJ & Bennett, JE) 1992, Medical Mycology, Lea and Febiger, Malvern, Pennsylvania (Ma
lvern, PA)).

  Recently, tryprostatin A and B (which are diketopiperazines consisting of proline and isoprenylated tryptophan residues), as well as five other structurally related diketopiperazines, have undergone cell cycle progression in M phase. (Cui, C. et al., 1996, J Antibiotics, 49: 527-33, Cui, C. et al., 1996, J Antibiotics, 49: 534-40), these compounds also affect microtubule assembly (Usui, T. et al., 1998, Biochem J, 333: 543-48, Condon, M. (see Kondon, M. et al., 1998, J Antibiotics, 51: 801-04). In addition, it binds to the colchicine binding site (CLC site) of tubulin, a macromolecule composed of two 50 kDa subunits (α- and β-tubulin) and a major component of microtubules. By doing so, natural and synthetic compounds have been reported that inhibit mitosis and thus inhibit the eukaryotic cell cycle. For example, Iwasaki, S .; (Iwasaki, S.), 1993, Med Res Rev, 13: 183-198, Hamel, E .; (Hamel, E.), 1996, Med Res Rev, 16: 207-31, Weisenberg, R.A. C. See (Weisenberg, R.C. et al.), 1969, Biochemistry, 7: 4466-79. Microtubules are thought to be involved in several essential cellular functions such as axonal transport, cell motility and cell morphology determination. Accordingly, inhibitors of microtubule function can have a wide range of biological activities and are applicable for medical purposes and agricultural purposes. Also, CLC, steganacin (see Kupchan, SM et al., 1973, J Am Chem Soc, 95: 1335-36), podophyllotoxin (Saket, DL ( Sackett, DL), 1993, Pharmacol Ther, 59: 163-228), and combretastatin (Pettit, GR et al., 1995, J Med Chem, 38: 166- A colchicine (CLC) site ligand such as 67) can prove useful as a eukaryotic cell cycle inhibitor, and thus may be useful as a chemotherapeutic agent.

The diketopiperazine-type metabolites are mycotoxins (Horak, RM et al. (Horak RM
et al.), 1981, JCS Chem Comm, 1265-67, Ali, M. et al. (See Ali M. et al., 1898, Toxicology Letters, 48: 235-41) or secondary metabolites (Smedsgaard J et al., 1996, J Microbiol Meth, 25: 5-17), but with little regard to the specific structure of diketopiperazine-type metabolites or their derivatives, and in particular their anti-tumor activity in vivo. unknown. These compounds are not only isolated as mycotoxins, but the chemical synthesis of phenilahistine, a type of diketopiperazine-type metabolite, is described in Hayashi in J. Org. Chem., (2000) 65, 8402. (Hayashi et al.). In this technical field, dehydrophenylahistine, which is one of such diketopiperazine-type metabolite derivatives, is produced by dehydrogenation by the enzymatic action of its parent phenilahistine. As cancer development increases, animal cell-specific proliferation-inhibiting activity and substantially purified diketopiperazine-type metabolite derivative species with high antitumor activity and selectivity There is a particular need to produce it chemically. Therefore, there is a particular need for an efficient method for synthetically producing diketopiperazine-type metabolite derivatives that are substantially purified and structurally and biologically characterized.

  Similarly, PCT Publication No. WO / 0153290 (July 26, 2001) discloses dehydrophenilahis by exposing phenylahistine or certain phenylahistine analogues to a dehydrogenase obtained from Streptomyces albulus. A non-synthetic method for producing styne is described.

[Summary of Invention]
With respect to the compound species having the structure of the following formula (I), a compound and a synthetic production method of the compound are disclosed.

The disclosed compounds have the formula (I)
(Wherein R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, -CO-O-R _7, cyano, alkylthio, poly Alkyl halides including alkyl halides, carbonyl halides and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl. , Cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted Lumpur, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ are independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, Including substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl Alkyl halide, carbonyl halide and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroa Lumpur, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with an R ₅ group as defined above;
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with the above R ₅ group, an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond can be either a single bond or a double bond,
However, when R ₁ , R ₁ ′, R ₂ , R ₃ , R ₄ and R ₅ are each a hydrogen atom in a specific compound, X ₁ and X ₂ are each an oxygen atom, and R ₆ is 3 , 3-dimethylbutyl-1-ene or hydrogen atom does not apply)
It has the following structure.

This method
Reacting a diacyl diketopiperazine with a first aldehyde to form an intermediate compound;
Reacting the intermediate compound with a second aldehyde to yield the compound species having the general structure described above,
The first aldehyde and the second aldehyde are selected from the group consisting of oxazole carboxaldehyde, imidazole carboxaldehyde, benzaldehyde, imidazole carboxaldehyde derivatives, and benzaldehyde derivatives, thereby producing a compound.

The disclosed compounds have the structure of formula (I), wherein
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ are independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, Including substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl Alkyl halide, carbonyl halide and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroa Lumpur, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with an R ₅ group as defined above;
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with the above R ₅ group, an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond can be either a single bond or a double bond.

  In a preferred embodiment of the compounds and methods of the present invention, the imidazole carboxaldehyde is 5- (1,1-dimethyl-2-ethyl) imidazole-4-carboxaldehyde and the benzaldehyde is a single methoxy group. including. Further preferred embodiments of the compounds described herein include compounds having t-butyl, dimethoxy, chloro and methylthiophene groups, and methods for making such compounds, as well as Tables 2, 3 And the compounds described in Table 4 and methods of making such compounds.

  Similarly, methods and materials for treating neoplastic tissue or preventing infection by cancer or pathogenic fungi are disclosed. These methods and materials are particularly well suited for the treatment of mammalian subjects, more specifically humans, and include administering dehydrophenylahistine or analogs thereof to a subject. The method includes administering to the subject an antitumor or antifungal effective amount of dehydrophenylahistine or an analog thereof.

  Further embodiments relate to a method of treating a condition in an animal, the method being described herein in an amount effective to reduce vascular proliferation or in an amount effective to reduce vascular density. Administration of such compounds to animals may be included. Exemplary conditions include tumors (eg, cancer) and other conditions associated with or dependent on angiogenesis (eg, immune and non-immune inflammation, rheumatoid arthritis, rheumatoid arthritis, psoriasis, diabetic Retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, post-lens fibroproliferation, rubeosis, capillary growth in atherosclerotic plaques, osteoporosis, etc.). In some embodiments, the disease is not cancer.

  Another embodiment relates to a method of inducing vascular collapse in an animal. This method can include, for example, treating the animal with a therapeutically effective amount of a compound having formula (I) as described herein. The therapeutically effective amount of the compound can cause tubulin depolymerization in the vasculature.

  Preferably, the animal is a human. Preferably, the disease can be a tumor, diabetic retinopathy, age-related macular degeneration, and the like. In some aspects, the disease is not cancer, or cancer can be specifically excluded from the methods and uses described above. Preferably, the compound is KPU-02.

  A further embodiment is a pharmaceutical composition for treating or preventing vascular proliferation, wherein a pharmaceutically effective amount of a compound disclosed herein is a pharmaceutically acceptable carrier for the compound In conjunction with a pharmaceutical composition. Vascular proliferation can also be a symptom of diseases such as cancer, age-related macular degeneration and diabetic retinopathy.

  Some embodiments relate to methods of preferentially targeting tumor vasculature over non-tumor tissue vasculature. This method may include the step of administering to an animal, preferably a human, a compound having the structure of formula (I) as described herein. The non-tumor tissue can be, for example, skin, muscle, brain, kidney, heart, spleen, intestine, and the like. The tumor vasculature may be preferentially targeted over non-tumor tissue vasculature, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%.

  Another embodiment relates to a method of preferentially targeting tumor vasculature over non-tumor tissue vasculature, wherein the method preferentially targets tumor vasculature over non-tumor tissue vasculature Can be administered to animals.

  A further embodiment relates to the use of a compound having the structure of formula (I) in the preparation of a medicament for the treatment of a condition associated with or dependent on increased vasculature. In some aspects, the condition may be cancer, while certain types of cancer or all cancers may be specifically excluded. The situation can be any other condition associated with hypervascularization, associated with the vasculature, or dependent on the vasculature. Examples include immune and non-immune inflammation, rheumatoid arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, post-lens fibroproliferation, rubeosis, Capillary growth in osteosclerosis, osteoporosis, etc. are mentioned.

  The accompanying drawings are incorporated in and constitute a part of this specification and merely illustrate certain preferred embodiments of the invention. Together with the remainder of the specification, the accompanying drawings are intended to assist those skilled in the art in describing the preferred modes of making particular compounds of the invention.

Detailed Description of Preferred Embodiments
Each reference cited herein, including U.S. patents cited herein, is hereby fully incorporated by reference in its entirety, to the maximum extent permitted by law. It is considered to be done. US patent application Ser. No. 10 / 632,531, both filed Aug. 1, 2003, entitled “Synthesis of dehydrophenylahistine and analogs thereof, and dehydrophenylahistine and analogs thereof” PCT application PCTUS03 / 24232 is hereby incorporated by reference in its entirety.

  The present disclosure provides a method for the synthetic production of compounds, including novel compounds, including dehydrophenylahistine and dehydrophenylahistine analogs, and also provides a relatively high yield, pharmaceutically acceptable cell cycle inhibitor, Provided is a method for producing an antitumor agent and an antifungal agent, wherein the compound and / or derivative thereof is one of the active ingredients in these cell cycle inhibitors, antitumor agents and antifungal agents. It is. Other objectives include providing novel compounds that cannot be obtained by currently available non-synthetic methods. Similarly, it is an object to provide a method of treating cancer, particularly human cancer, comprising administering a member of a novel anti-tumor compound species in an amount effective to inhibit tumor growth. The invention also provides a method of preventing or treating pathogenic fungi in a subject, comprising administering to the subject an antifungal effective amount of a member of a novel antifungal compound species, for example Administering dehydrophenylahistine or an analog thereof in an amount and manner that provides the intended antifungal effect. In preferred embodiments of the compounds disclosed herein and methods of making and using such compounds, although not necessarily all of the embodiments of the invention, these objectives have been achieved.

  Disclosed herein are compounds and methods for preparing the compound species, which compounds have the following formula (I):

(Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ are independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, Including substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl Alkyl halide, carbonyl halide and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroa Lumpur, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R, R ₁ ′ and R ₁ ″ are either covalently bonded to each other or not covalently bonded to each other;
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with an R ₅ group as defined above;
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with the above R ₅ group, an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond may be either a single bond or a double bond).

This method
Reacting a diacyl diketopiperazine with a first aldehyde to form an intermediate compound;
Reacting the intermediate compound with a second aldehyde to produce the compound species having the general structure to produce a compound of formula (I),
The first aldehyde and the second aldehyde are selected from the group consisting of oxazole carboxaldehyde, imidazole carboxaldehyde, benzaldehyde, imidazole carboxaldehyde derivatives, and benzaldehyde derivatives, thereby forming the formula (I)
(Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ are independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, Including substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl Alkyl halide, carbonyl halide and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroa Lumpur, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom;
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with the above R ₅ group, an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
Dashed bonds form compounds that can be either single bonds or double bonds.

  Also provided are pharmaceutically acceptable salts and prodrug esters of compounds of formula (I) and formula (II), and methods for synthesizing such compounds by the methods disclosed herein are provided. .

The term “prodrug ester” refers specifically to a prodrug ester of a compound of formula (I) synthesized by the methods disclosed herein, eg, by hydrolysis in blood or within a tissue. , Refers to a chemical derivative of a compound that is rapidly transformed in vivo to yield a compound of the invention. The term “prodrug ester” refers to a derivative of a compound disclosed herein formed by adding any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of prodrug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and the art including (5-R-2-oxo-1,3-dioxolen-4-yl) methyl groups Other groups known in the art may be mentioned. Other examples of prodrug ester groups are described, for example, in T.W. Higuchi and V. Stella (T. Higuchi and V. Stella), “Pro-drugs as Novel Delivery Systems”, Vol. 14, ACS Symposium Series, American Chemical Society (1975), and “Drug Design” Bioreversible Carriers in Drug Design: Theory and Application ”, E. B. Edited by EB Roche, Pergamon Press: New York, 14-21 (1987), which provides examples of esters useful as prodrugs for compounds containing carboxyl groups.

  As used herein, the term “prodrug ester” also refers to a chemical derivative of a compound that is rapidly transformed in vivo to yield a compound of the invention, for example by hydrolysis in blood. The term “prodrug ester” refers to a derivative of a compound disclosed herein formed by adding any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of prodrug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and the art including (5-R-2-oxo-1,3-dioxolen-4-yl) methyl groups Other groups known in the art may be mentioned. Other examples of prodrug ester groups are described, for example, in T.W. Higuchi and V. Stella (T. Higuchi and V. Stella), “Pro-drugs as Novel Delivery Systems”, Vol. 14, ACS Symposium Series, American Chemical Society, (1975), and “Drugs” Bioreversible Carriers in Drug Design: Theory and Application ", E. B. Ed. E. B. Roche, Pergamon Press: New York, 14-21 (1987), which provides examples of esters useful as prodrugs for compounds containing carboxyl groups.

The term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt of a compound, particularly when referring to a pharmaceutically acceptable salt of a compound of formula (I) synthesized by the methods disclosed herein. Is an acceptable salt, preferably an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable salts include alkali metal salts (sodium or potassium), alkaline earth metal salts (calcium or magnesium), or ammonia or pharmaceutically acceptable organic amines such as C ₁ -C ₇ Ammonium salts derived from alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris (hydroxymethyl) -aminomethane. For compounds synthesized by the above method that are basic amines, preferred examples of pharmaceutically acceptable salts include pharmaceutically acceptable inorganic or organic acids such as hydrohalic acid, sulfuric acid, phosphoric acid. Or aliphatic or aromatic carboxylic acids or sulfonic acids such as acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid Acid addition salt.

As used herein, the term “pharmaceutically acceptable salt” also refers to any pharmaceutically acceptable salt of a compound, preferably an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable salts include alkali metal salts (sodium or potassium), alkaline earth metal salts (calcium or magnesium), or ammonia or pharmaceutically acceptable organic amines such as C ₁ -C ₇ Ammonium salts derived from alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris (hydroxymethyl) -aminomethane. For compounds that are basic amines, preferred examples of pharmaceutically acceptable salts include pharmaceutically acceptable inorganic or organic acids such as hydrohalic acid, sulfuric acid, phosphoric acid, or aliphatic or aromatic. Acid addition salts of carboxylic acids or sulfonic acids such as acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, p-toluenesulfonic acid or naphthalenesulfonic acid.

  Preferred pharmaceutical compositions disclosed herein include pharmaceutically acceptable salts and prodrug esters of the compounds of formula (I) synthesized by the methods disclosed herein. Thus, where the manufacture of a pharmaceutical formulation involves intimate mixing of a pharmaceutical excipient and an active ingredient in the form of a salt, a non-basic pharmaceutical excipient, i.e. an acidic excipient or It is preferred to use any neutral excipient.

In preferred embodiments of the compound methods disclosed herein, relatively strong planar quasi-tricyclic structures can be formed. In order to stabilize such a relatively strong planar quasi-tricyclic structure, R ₃ may preferably be selected to be hydrogen.

In other preferred embodiments of the compounds and methods described herein, n is 0 or 1, more preferably 1, and Z ₂ , Z ₃ and Z ₄ are each independently an oxygen atom. More preferably, at least one of Z ₂ , Z ₃ and Z ₄ is a carbon atom, and at least two of Z ₂ , Z ₃ and Z ₄ are carbon atoms. Most preferred. Z may be all carbon atoms at the same time.

  Still other preferred embodiments of the methods and compositions disclosed herein include the following formulas (Ia) and (Ib):

  Embedded image wherein the variable group is as defined herein, including a compound having the structure:

As used herein, the term “halogen atom” means any one of the radioactive stable atoms in the seventh column of the Periodic Table of Elements, ie fluorine, chlorine, bromine or iodine, and fluorine and chlorine Is preferred.

As used herein, the term “alkyl” refers to any unbranched or branched substituted or unsubstituted saturated hydrocarbon, a C ₁ -C ₆ unbranched saturated unsubstituted carbon. Hydrogen is preferred, with methyl, ethyl, isobutyl and tert-butyl being most preferred. Of the substituted saturated hydrocarbons, C ₁ -C ₆ mono and di and perhalogen substituted saturated hydrocarbons and amino substituted hydrocarbons are preferred and are perfluoromethyl, perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl. Is most preferred. The term “substituted” has its ordinary meaning as found in many current patents in the related art. For example, US Pat. Nos. 6,583,143, 6,509,331, 6,506,787, 6,500,825, 5,922,683, See 5,886,210, 5,874,443, and 6,350,759. Specifically, the definition of the term “substituted” is as broad as that provided in US Pat. No. 6,583,143, which patent is “substituted”. Is defined as any group such as alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, wherein at least one hydrogen atom is replaced by a substituent. The term “substituted” is also as broad as the definition provided in US Pat. No. 6,509,331, which includes the term “substituted alkyl” Alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, Keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocycle, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted Alkyl, -SO- aryl, -SO- heteroaryl, -SO ₂ - alkyl, -SO ₂ - substituted alkyl, -SO ₂ - aryl and -SO ₂ - is selected from the group consisting of heteroaryl, 1-5 It is defined to refer to an alkyl group having 1 substituent, preferably 1 to 3 substituents, preferably 1 to 10 carbon atoms. The other patents mentioned above also provide standard definitions for the term “substitution” that is well understood by those skilled in the art. The term “cycloalkyl” refers to any non-aromatic hydrocarbon ring having preferably 5 to 12 atoms constituting the ring. The term “acyl” refers to an alkyl or aryl group derived from an oxo acid, with an acetyl group being preferred.

As used herein, the term “alkenyl” means any unbranched or branched substituted or unsubstituted, unsaturated hydrocarbon, including polyunsaturated hydrocarbons, C ₁ -C _Six unbranched monounsaturated and diunsaturated unsubstituted hydrocarbons are preferred, and monounsaturated dihalogen substituted hydrocarbons are most preferred. A z-isoprenyl moiety is particularly preferred at the R ₁ and R ₄ positions of the compounds of structure (I). The term “cycloalkenyl” refers to any non-aromatic hydrocarbon ring having preferably 5 to 12 atoms constituting the ring.

  As used herein, the terms “aryl”, “substituted aryl”, “heteroaryl” and “substituted heteroaryl” are preferably 5, 6, or 7 atoms, most preferably comprising a ring. Refers to an aromatic hydrocarbon ring having 6 atoms. “Heteroaryl” and “substituted heteroaryl” refer to an aromatic hydrocarbon ring in which at least one heteroatom, for example an oxygen, sulfur or nitrogen atom, is present in the ring together with at least one carbon atom.

The term “alkoxy” refers to any unbranched or branched substituted or unsubstituted saturated or unsaturated ether, preferably a C ₁ -C ₆ unbranched saturated unsubstituted ether, preferably methoxy, Also preferred are dimethyl, diethyl, methyl-isobutyl and methyl-tert-butyl ether. The term “cycloalkoxy” refers to any non-aromatic hydrocarbon ring having 5 to 12 atoms, preferably constituting the ring.

  As used herein, the terms “purified”, “substantially purified” and “isolated” do not include other different compounds with which the compound is normally associated in its natural state. Refers to a compound, and thus a compound of the invention comprises at least 0.5%, 1%, 5%, 10% or 20%, most preferably at least 50% or 75% by weight of a given sample. Includes mass.

  Compounds of formula (I) are known in the art and can be chemically synthesized or prepared from available reagents. For example, the modification of diacyl diketopiperazine (diacetyl diketopiperazine) is described in WO 95/90, for example by Loughlin et al., 2000, Bioorg Med Chem Lett, 10:91, or by Broccini et al. 21832. Diacyl diketopiperazine (diacetyl diketopiperazine) can be prepared, for example, by diacetylation of inexpensive 2,5-piperazinedione (TCI catalog number G0100, 25 g) with sodium acetate and anhydrous sodium acetate. The diacetyl structure of the activated ketopiperazine can be replaced with other acyl groups to include carbamates such as Boc (t-butoxycarbonyl), Z (benzoyloxycarbonyl) and the like.

  Imidazole carboxaldehyde can be prepared according to the procedure disclosed in, for example, Hayashi et al., 2000, J Organic Chem, 65: 8402, as shown below.

The synthetic methods disclosed herein may preferably be carried out in DMF and under a deoxygenated atmosphere in the presence of cesium carbonate as a base. Inert atmospheres are described, for example, by Watanabe et al., 18th International Congress of Heterocyclic Chemistry, Yokohama, Japan (July 30, 2001) (18th International Congress of Heterocyclic Chemistry in Yokohama, Japan
(30 July 2001)), Abstract, page 225, to prevent possible oxidation of the active α-carbon atom of the diketopiperazine ring (see below) that can occur during treatment with cesium carbonate.

  Other embodiments of the synthetic methods include modifications of compounds used or involved in the synthesis of compounds represented by formula (I). Such derivatives include modifications to the phenyl ring, introduction of other aromatic ring systems, positions of the aromatic rings, changes to the imidazole ring system and / or further modifications to the 5-position of the imidazole ring. But you can. Examples of such modifications are described in Example 4, for example. The results of such modifications include compounds that can increase the nitrogen content of the phenyl ring and / or increase the solubility of the compound. Other modifications may incorporate derivatives of known tubulin inhibitors, thereby mimicking the activity of tubulin inhibitors. Other modifications can simplify the synthesis of β-ketoesters involved in the production of imidazole carboxaldehyde used in the methods disclosed herein.

Pharmaceutical Compositions The present invention is optionally and preferably manufactured according to the methods disclosed herein in a pharmaceutical composition comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration. Including a compound disclosed herein, having a pharmaceutically effective amount of the product disclosed above in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are known in the pharmaceutical arts, such as “Remington's Pharmaceutical Sciences”, Mack Publishing Co. (AR Gennaro). Ed. (1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

  Compositions of dehydrophenylahistine or dehydrophenylahistine analogs include tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for injectable administration, transdermal It may be formulated and used as patches for administration, subcutaneous deposits and the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as solid forms suitable as solutions or suspensions in liquid prior to injection or infusion, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and human serum albumin. In addition, if desired, injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances such as wetting agents and pH buffering agents. If desired, preparations that enhance absorption (eg, liposomes) may be utilized.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or media include fatty oils such as sesame oil, or other organic oils such as soybean oil, grapefruit oil or almond oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Can be mentioned. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the production of highly concentrated solutions.

  Pharmaceutical preparations for oral use are suitable for combining the active compounds with solid excipients, optionally milling the resulting mixture and, if desired, obtaining tablets or dragee cores. It may be obtained by processing the mixture of granules after adding the auxiliaries. Suitable excipients are in particular fillers such as sugars containing lactose, sucrose, mannitol or sorbitol, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose Cellulose preparations such as sodium and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The dragee core is provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which optionally include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. It may contain. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification of the active compound dose or to characterize different combinations of active compound doses. For this purpose, concentrated sugar solutions may be used, which optionally include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. It may contain. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification of the active compound dose or to characterize different combinations of active compound doses. Such formulations can be manufactured using methods known in the art (eg, US Pat. No. 5,733,888 (injectable composition), US Pat. No. 5,726,181). (Poorly water-soluble compounds), 5,707,641 (therapeutically active proteins or peptides), 5,667,809 (lipophilic agents), 5,576,012 (possible) Solubilized polymer active substance), 5,707,615 (antiviral formulation), 5,683,676 (particulate drug), 5,654,286 (topical formulation) 5,688,529 (oral suspension), 5,445,829 (sustained release formulation), 5,653,987 (liquid formulation), Nos. 641,515 (controlled release formulations) and 5,6 No. 1,845 refer to the (spherical formulation)).

In addition, a variety of pharmaceutical compositions known in the pharmaceutical art for applications including intraocular, intranasal and intraauricular delivery are disclosed herein. Pharmaceutical formulations include water-soluble forms such as eye drops, or gellan gum (Shedden et al., 2001, Clin Ther, 23 (3): 440-50) or hydrogels (Meyer et al. ( Mayer et al.), 1996, Ophthalmologica 210: 101-3) in aqueous ophthalmic solutions, ointments and ophthalmic suspensions of active compounds (eg microparticles, containing drugs suspended in a liquid carrier medium) Small polymer particles (Joshi, A., 1994 J
Ocul Pharmacol 10: 29-45), lipid soluble formulations (Alm et al., 1989 Prog Clin Biol Res 312: 447-58), and microspheres (Mordenti, 1999 Toxicol Sci 52: 101-6)), and ocular inserts. Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. The pharmaceutical compositions may also include drops and sprays that are often manufactured to stimulate nasal secretions in many ways to ensure the maintenance of normal ciliary action. As disclosed in “Remington's Pharmaceutical Sciences” (Mack Publishing, 18th edition) and as known to those skilled in the art, suitable formulations are most often and preferably, etc. It is tonic and slightly buffered to maintain a pH of 5.5 to 6.5, and in most cases and preferably includes an antimicrobial preservative and a suitable drug stabilizer. Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such otic formulations include glycerin and water.

  When used as a cell cycle inhibitor, tumor growth inhibitory or fungal growth inhibitory compound, the compound of formula (I) can be administered by either the oral or parenteral route. When administered orally, the compounds of formula (I) can be administered in capsules, tablets, granules, sprays, syrups or other forms. When administered parenterally, the compound of formula (I) is administered as an aqueous suspension, oily preparation, etc. or as an infusion, suppository, ointment, ointment, etc. In particular, it can be administered intraperitoneally, intravenously or intramuscularly. Similarly, a compound of formula (I) may be topically, rectally, or as deemed appropriate by those skilled in the art to optimally contact the compound with the tumor and thereby inhibit tumor growth. It may be administered vaginally. Local administration at the site of the tumor is also contemplated either before or after resection of the tumor, as well as controlled release formulations, depot formulations and infusion pump delivery.

Methods of Administration The invention also encompasses methods of making and administering the disclosed chemical compounds and the disclosed pharmaceutical compositions. Such disclosed methods include, in particular, (a) administration by oral route including administration in capsules, tablets, granules, sprays, syrups, or other forms, (b) as aqueous suspensions, oily preparations, etc. Or by parenteral route including subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal administration by infusion, suppository, ointment, ointment, etc., injection or infusion, (c) topical Administration, (d) rectal administration, or (e) vaginal administration, and (f) a controlled release formulation, depot, as deemed appropriate by one of ordinary skill in the art for optimal contact of the compound with living tissue. Administration by formulation and infusion pump delivery. As further examples of such modes of administration, and as further disclosure of modes of administration, various methods of administering the disclosed chemical compounds and pharmaceutical compositions, including modes of administration by intraocular, intranasal and intraauricular routes, are provided. Disclosed herein.

  The pharmaceutically effective amount of dehydrophenylahistine or dehydrophenylahistine analog composition required as a dose depends on the route of administration, the type of animal, including the human being treated, and the particular animal of interest. Depending on body characteristics. The dose can be tailored to achieve the desired effect, but will depend on factors such as weight, diet, co-medication, and other factors that one of ordinary skill in the medical arts will recognize.

  In carrying out the above methods, the products or compositions can be used alone or in combination with each other or in combination with other therapeutic or diagnostic agents. For example, as disclosed herein, the compounds disclosed herein may be CPTs that are other actives, specifically other chemotherapeutic agents such as biologics and certain chemotherapeutic agents. When used in combination with -11, taxoten (docataxel) and paclitaxel, it is effective in treating cancer. The compounds disclosed herein also include anti-vascular agents, anti-angiogenic agents, other VEGFs such as Erbuitux (Imclone / bristol-Myers) and Iressa (AstraZeneca). Inhibitors and biologics, more specifically at least one anti-VEGF antibody, particularly a monoclonal antibody against the VEGF receptor including DC101, other including a rat monoclonal antibody that inhibits mouse VEFG receptor 2 (flk-1) When used in combination with an active substance, it is effective in treating cancer. Such a combination can be utilized in vivo, usually in mammals, preferably in humans, in vivo, or in vitro. When such a combination is used in vivo, the compounds disclosed in the present invention may be used alone or in combination with other chemotherapeutic agents or other biologic products to use various dosage forms. Administered parenterally, intravenously, by infusion or injection, subcutaneously, intramuscularly, into the colon, rectally, vaginally, nasally or intraperitoneally into a mammal in various ways. It's okay. Such a method may also be applied to in vivo chemical activity testing.

  As will be readily apparent to those skilled in the art, the useful in vivo doses to be administered, and the particular mode of administration, include age, weight and species of mammal being treated, the particular compound used, and These compounds vary depending on the particular application for which they are used. The determination of effective dosage levels, that is, the dosage levels necessary to achieve the desired result, can be made by one skilled in the art using routine pharmacological methods. Usually, clinical application of the product to humans starts with a lower dosage level and increases the dosage level until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration for compositions identified by the methods of the invention using established pharmacological methods.

  In non-human animal studies, potential product applications begin at higher dose levels and reduce the dose until the desired effect is no longer achieved or adverse side effects disappear. The dosage can vary widely depending on the desired effects and the signs of treatment. Usually, the dosage can be about 10 microgram / kg to 100 mg / kg body weight, preferably about 100 microgram / kg to 10 mg / kg body weight. Alternatively, the dose may be calculated based on the patient's surface area, as will be appreciated by those skilled in the art. Administration may be oral administration every 3 days, every other day, daily, twice daily, or three times daily.

  The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, for example, Fingl et al., “The Pharmacological Basis of Therapeutics”, 1975. It should be noted that the attending physician will understand how and when to terminate, interrupt or adjust administration depending on toxicity or organ dysfunction. Conversely, the attending physician will also understand that the therapy is adjusted to a higher level if the clinical response is not appropriate (except for toxicity). The size of the dose administered in the management of the disorder of interest will vary depending on the severity of the condition to be treated and the route of administration. The severity of symptoms can be assessed, for example, in part by standard prognostic assessment methods. Furthermore, the dose, and possibly the dose frequency, will also vary according to the age, weight and response of the individual patient. A program similar to the above may be used in veterinary medicine.

  Depending on the specific conditions to be treated, such agents can be formulated and administered systemically or locally. Various techniques for formulation and administration can be found in “Remington's Pharmaceutical Sciences”, 18th Edition, Mack Publishing Co., Easton, Pennsylvania (1990). Suitable routes of administration include oral, rectal, transdermal, vaginal, transmucosal or enteral administration, intramuscular, subcutaneous, intraspinal injection, and intrathecal, directly into the ventricle, intravenously, by infusion, intraperitoneally, Parenteral delivery including intranasal or intraocular injection may be mentioned.

  For injection or infusion, the agent may be formulated in aqueous solutions, for example in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The use of a pharmaceutically acceptable carrier to formulate a compound disclosed herein for the practice of the invention in a dose suitable for systemic administration is within the scope of the invention. With proper choice of carrier and proper manufacturing practices, the compositions disclosed herein, particularly those formulated as solutions, may be administered parenterally, for example, by intravenous injection or infusion. The compounds of the present invention can be readily formulated into pharmaceutically suitable carriers for oral administration using pharmaceutically acceptable carriers known in the art. Such carriers allow the compound to be formulated into tablets, pills, capsules, solutions, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques known to those skilled in the art. For example, such agents may be administered as described above after encapsulating in liposomes. All molecules present in the aqueous solution at the time of liposome formation are entrapped within the aqueous interior. Both liposome contents are protected from the external microscopic environment and are efficiently delivered to the cytoplasm because the liposomes fuse with the cell membrane. Further, due to the hydrophobic nature of the liposomes, small organic molecules may be administered directly into the cell.

  Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredient, these pharmaceutical compositions contain suitable pharmaceutically acceptable ingredients, including excipients and auxiliaries that facilitate processing of the active compound into preparations that can be used pharmaceutically. Possible carriers may be included. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions. The pharmaceutical composition may be manufactured in a manner known per se, for example by conventional mixing, dissolving, granulating, dragee making, flotation, emulsification, encapsulation, incorporation or lyophilization process.

  The compounds disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound or a subset of compounds that share a particular chemical moiety can be determined by determining toxicity in vitro against a mammalian cell line, preferably a cell line such as a human cell line. Can be established. The results of such studies are often useful for predicting toxicity in mammals, and more specifically in animals such as humans. Alternatively, the toxicity of a particular compound in animal models such as mice, rats, rabbits, or monkeys can be determined using known methods. The effectiveness of a particular compound may be established using a number of art-recognized methods such as in vitro methods, animal models, or human clinical trials. In vitro models understood in the art exist for almost all types of symptoms including those alleviated by the compounds disclosed herein, including cancer, cardiovascular disease and various fungal infections. Similarly, acceptable animal models may be used to establish the effectiveness of chemicals for treating such conditions. When selecting a model to determine efficacy, one of ordinary skill in the art can be guided by current state of the art to select the appropriate model, dose, and route of administration, and dosing regimen. Of course, human clinical trials can also be used to determine the effectiveness of a compound in humans.

  When used as an anti-cancer agent or tumor growth inhibiting compound, the compounds disclosed herein can be administered by either the oral or parenteral route. When administered orally, the compounds disclosed herein can be administered in capsules, tablets, granules, sprays, syrups or other forms. When administered parenterally, the compounds disclosed herein can be administered as aqueous suspensions, oily preparations, etc. or as infusions, suppositories, ointments, ointments, etc., administered by injection or infusion. In that case, it can be administered subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally or the like. Similarly, a compound disclosed herein may be topically, rectally, rectal, if deemed appropriate by one of ordinary skill in the art to optimally contact the compound with the tumor and thereby inhibit tumor growth. Or it may be administered to the vagina. Local administration at the site of the tumor or other disease state is also contemplated either before or after tumor resection, or as part of a treatment understood in the art of the disease state. Controlled release formulations, depot formulations and infusion pump delivery are contemplated as well.

When used as an anti-cancer or anti-tumor agent, the amount of active ingredient is about 0.0007 mg / day to about 7,000 mg / day, more preferably about 0.07 mg / day to about 70 mg / day, preferably It may be administered orally or parenterally to a human patient once per day, or less preferably, from 2 to about 10 times per day. Alternatively and preferably, the compound may be preferably administered in a prescribed amount continuously, for example by intravenous infusion. Thus, for a patient weighing 70 kilograms, a preferred daily dose of an effective anti-tumor component is about 0.0007 mg / kg / day to about 35 mg / kg / day (1.0 mg / kg / day and 0.5 mg / kg). /
Day), more preferably from 0.007 mg / kg / day to about 0.050 mg / kg / day (including 0.035 mg / kg / day). Nevertheless, as will be appreciated by those skilled in the art, in certain circumstances, in order to effectively and aggressively treat especially advanced or fatal tumors, the above preferred dosage ranges are exceeded, Or in some cases, it may be necessary to administer the anti-tumor compound in much greater amounts.

  When used as an antifungal agent, the preferred amount of dehydrophenylahistine or an analog thereof effective for the treatment or prevention of a particular fungal pathogen is determined by standard clinical techniques, depending somewhat on the characteristics of the fungus and the degree of infection. Can be determined. In vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. Effective doses may be extrapolated from dose response curves derived from in vitro analysis, or preferably from animal models. The exact dosage level should be determined by the attending physician or other health care provider, the route of administration and the individual's age, weight, gender and general health, nature of the infection, severity and clinical stage Depends on known factors, including (or not using) combination therapy.

  Effective doses of dehydrophenillahistin or analogs thereof are usually in the range of about 0.01 to about 50 mg / kg, preferably about 0.1 to about 10 mg / kg (mammalian body weight) per day. It is administered in doses or multiple doses. In general, the compounds may be administered to a patient in need of such treatment at a daily dose range of about 1 to about 2,000 mg per patient.

  Known surfactants, excipients, lubricants, suspending agents, and pharmaceutically acceptable film-forming substances for formulating dosages containing the compounds disclosed herein as tumor growth inhibiting compounds In addition, a coating aid or the like may be used. Preferably, alcohols, esters, sulfated aliphatic alcohols and the like may be used as surfactants. Sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, metasilicic acid Magnesium aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, etc. may be used as excipients, magnesium stearate, talc, hardened oil etc. as lubricant Palm oil, olive oil, sesame oil, peanut oil, soybean may be used as a suspending or lubricant, cellulose acetate phthalate as a derivative of carbohydrates such as cellulose or sugar, or polyvinyl Acetate as a derivative Le - may be used methacrylic acid copolymer as the suspending agent may be used as suspending agents plasticizers such as phthalates. In addition to the preferred ingredients described above, sweeteners, fragrances, colorants, preservatives, and the like may be added to the compound dosage formulation, particularly when the compound is administered orally.

  The compositions disclosed herein in pharmaceutical compositions may also include a pharmaceutically acceptable carrier. Such compositions may be prepared for storage and subsequent administration. Acceptable carriers or diluents for therapeutic use are known in the pharmaceutical arts, eg, “Remington's Pharmaceutical Sciences”, Mack Publishing Co. (AR Gennaro, edited by AR Gennaro). 1985). For example, such compositions may be formulated and used as tablets, capsules or solutions for oral administration, suppositories for rectal or vaginal administration, sterile solutions or suspensions for injection administration. Injectables can be prepared in liquid forms or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or in conventional forms such as emulsions. Suitable excipients include, but are not limited to, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances such as wetting agents, pH buffering agents and the like. Absorption enhancing preparations (eg, liposomes) may be utilized if desired.

  The pharmaceutically effective amount of the composition required as a dose depends on the route of administration, the type of animal being treated, and the physical characteristics of the particular animal of interest. The dose can be tailored to achieve the desired effect, but depends on factors such as weight, diet, concomitant medications, and other factors recognized by those skilled in the medical arts.

  Products or compositions as described above can be used alone or in combination with each other or in combination with other therapeutic or diagnostic agents. Specifically, the compound products disclosed herein may be used alone or in combination with other chemotherapeutic agents or biologics (including antibodies) or for fungal infections for the treatment of cancer. For treatment, it may be used in combination with other anti-infective substances. These products or compositions can be utilized in vivo or in vitro. The useful dosage and the most useful mode of administration will vary depending on age, weight and the animal being treated, the particular compound used and the particular application using these composition (s). The size of the dose in the management or treatment for a particular disease will vary depending on the severity of the condition to be treated and the route of administration, and depending on the disease state and their severity, the composition will be formulated, It can be administered systemically or locally. Various techniques for formulation and administration can be found in “Remington's Pharmaceutical Sciences”, 18th Edition, Mack Publishing Co., Easton, Pennsylvania (PA) (1990).

  Known surfactants for formulating compounds of formula (I), preferably synthetically prepared by the methods disclosed herein, as cell cycle inhibitors, tumor growth inhibitors, or antifungal compounds , Excipients, lubricants, suspending agents, and pharmaceutically acceptable film-forming substances and coating aids may be used. Preferably, alcohols, esters, sulfated aliphatic alcohols and the like may be used as surfactants, and sucrose, glucose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicate, magnesium aluminate, metasilicate. Magnesium aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, etc. may be used as excipients, magnesium stearate, talc, hardened oil, etc. Coconut oil, olive oil, sesame oil, peanut oil, soybean, etc. may be used as a suspension or lubricant, cellulose acetate phthalate as a derivative of carbohydrates such as cellulose or sugar, or Vinegar as a derivative of polyvinyl Methyl - may be used methacrylic acid copolymer as the suspending agent may be used as suspending agents plasticizers such as phthalates. In addition to the preferred ingredients described above, sweeteners, fragrances, colorants, preservatives and the like may be added to the compound dosage formulations prepared by the above methods, particularly when the compound is administered orally.

  The cell cycle inhibitor, antitumor agent and antifungal agent that can be produced by the above method are about 0.001 mg / kg / day to about 10,000 mg / kg / day of active ingredient, more preferably about 0.1 mg / kg. / Day to about 100 mg / kg / day of active ingredient, preferably periodically once every 3 days, once every other day, once per day, twice per day, or less preferred However, it may be administered orally or parenterally to human patients from 2 to about 10 times per day. Alternatively and preferably, the compound produced by the above method can be preferably administered in a defined amount continuously, for example by intravenous infusion. Thus, for a patient weighing 70 kilograms, the preferred daily dose of active anti-tumor component is from about 0.07 mg / day to about 700 grams / day, more preferably from 7 mg / day to about 7 grams / day. Nevertheless, as will be appreciated by those skilled in the art, in certain circumstances, in order to effectively and aggressively treat especially advanced or fatal tumors, the above preferred dosage ranges are exceeded, Or, in some cases, it may be necessary to administer the antitumor compound produced by the above methods in much greater amounts.

When the cell cycle inhibitor produced by the above method is used as a biochemical test reagent, the compound produced by the method of the present invention can be dissolved in an organic solvent or a water-containing organic solvent, and any of various cultured cell systems. When applied directly, it inhibits cell cycle progression. Examples of the organic solvent that can be used include methanol, methyl sulfoxide and the like. The formulation can be, for example, a powder, granule or other solid inhibitor, or a liquid inhibitor prepared using an organic or hydrous organic solvent. Preferred concentrations of the compounds produced by the methods of the present invention for use as cell cycle inhibitors are generally in the range of about 1 to about 100 μg / ml, although the most appropriate usage will be understood by those skilled in the art As such, it will vary depending on the type of cultured cell line and the purpose of use. Similarly, for certain applications, it may be necessary or desirable for those skilled in the art to use amounts outside the above ranges.

  From a pharmaceutical perspective, certain embodiments provide a method for preventing or treating a fungal infection and / or pathogenic fungus in a subject, wherein the subject comprises dehydrophenylahistine or an analog thereof. Administration of the product, eg, administration of dehydrophenylahistine or an analog thereof in an amount and manner that provides the desired antifungal effect.

  Other embodiments include treatment or prevention of patients infected with pathogenic fungi such as those described above or below.

  Another embodiment is the use of one or more other antifungal agents, particularly agents other than dehydrophenilahistine or analogs thereof (eg, amphotericin B or analogs or derivatives thereof (14 (s) -hydroxyamphotericin) B methyl ester, amphotericin B hydrazide with 1-amino-4-methylpiperazine, and other derivatives), or other polyene macrolide antibiotics (eg, nystatin, candicidin, pimaricin and natamycin, Flucytosine, griseofulvin, echinocandin or aureobasidin (including naturally occurring analogs and semi-synthetic analogs), dihydrobenzo [a] naphthacenequinone, nucleoside peptide antifungals (including polyoxins and nikkomycin), naphthifine and other Squalene Infection by allylamines such as xidase inhibitors and pathogenic fungi that are resistant to azoles, imidazoles and triazoles such as clotrimazole, miconazole, ketoconazole, econazole, butconazole, oxyconazole, terconazole, itraconazole or fluconazole, etc. See, for example, Turner and Rodriguez, 1996, Current Pharmaceutical Design, 2: 209-224, for additional conventional antifungal agents and new agents under development. Another embodiment is when the patient is allergic to one or more other antifungal agents, or otherwise intolerant or unresponsive, or the use of other antifungal agents In those who are contraindicated in other cases, Including the treatment or prevention of patients infected with protogenic fungi, which include, among others, the antifungal agents disclosed above and the antifungal agents disclosed elsewhere herein. Can be mentioned.

  In the methods of treatment or prevention described above, dehydrophenylahistine or an analog thereof is administered to a subject in an effective antifungal amount.

  Other embodiments may be used in combination with the administration of one or more other antifungal agents, including, for example, any of the above-described agents or agent types (eg, preferably in a lipid or liposome formulation). Dehydrophenylahistine with amphotericin B, an azole or triazole such as fluconazole, in combination with treatment with eg aureobasidin, dihydrobenzo [a] naphthacenequinone, or echinocardin), and a different dehydrophenylahistine or analog thereof Or the treatment or prevention of patients infected with pathogenic fungi by administration of analogs thereof.

Dehydrophenylahistine or an analog thereof is administered before, after, other antifungal agents are administered
Or they may be administered simultaneously. In certain embodiments, combination therapy allows for the use of a reduced amount of one or both of the antifungal agents relative to the amount used when used alone.

  Yet another embodiment relates to the administration of dehydrophenylhistine or an analog thereof to a subject for the treatment or prevention of infection by pathogenic fungi, wherein the subject is e.g. genetic disease, diabetes Or induced by drugs or other in connection with chemotherapy or radiotherapy for HIV or other infections, cancer or other diseases, or treatment of tissue or organ transplantation or autoimmune disorders As a result of immune suppression, it is either immunosuppressed or immunocompromised. If the patient is being treated or will be treated with an immunosuppressive agent, eg, in connection with tissue or organ transplantation, dehydrophenylahistine or an analog thereof may be combined with the immunosuppressive agent (s). They may be co-administered to treat or prevent pathogenic fungal infections.

  Another aspect of the present invention is the administration of one or more anti-HIV therapeutics (eg, including an HIV protease inhibitor, reverse transcriptase inhibitor, or antiviral agent) together with an antifungal dehydrophenilahistine or Treatment or prevention of infection by pathogenic fungi in patients infected with or suspected of being infected by administration of the analog. Dehydrophenillahistin or an analog thereof may be administered before, after, or simultaneously with the administration of the anti-HIV agent (s).

  Another aspect of the present invention is the combination of administration of one or more other antibiotic compounds, particularly one or more antibacterial agents, preferably in an effective amount and regimen for treating or preventing bacterial infection. Treatment or prevention of patients infected with pathogenic fungi by administration of the fungus dehydrophenilahistine or analogs thereof. Similarly, dehydrophenilahistine or an analog thereof may be administered before, after, or simultaneously with the administration of the other agent (s).

  Pathogenic fungal infections that can be treated or prevented by the disclosed methods include, among others, aspergillosis (including invasive pulmonary aspergillosis), blastomiasis (significant or rapidly progressive infection and blastomies in the central nervous system) ), Candidiasis (including kidney stones, urinary tract obstruction, kidney transplantation or retrograde candidiasis in the urinary tract in patients with poorly controlled diabetes mellitus), coccidioidomycosis (not responding well to other chemotherapy) Chronic disease), cryptococcosis, histoplasmosis, mucormycosis (including craniofacial mucormycosis and mucor pneumonia), paracoccidioidomycosis and sporotrichosis. Administration of a composition comprising an antifungal amount of one or more dehydrophenillahistines or analogs thereof is such that the fungus is resistant to one or more other antifungal therapies or one or more other antifungals. It should be noted that the therapy may be particularly useful for treating or preventing pathogenic fungal infections in mammalian subjects, eg contraindicated as described above.

  Also provided is an antifungal pharmaceutical composition containing at least one antifungal dehydrophenylahistine or an analog thereof for use in practicing the disclosed methods. These pharmaceutical compositions may be packaged with appropriate package inserts, particularly with instructions and information, particularly regarding their antifungal use. Also provided is a pharmaceutical composition containing one or more dehydrophenylhistines or analogs thereof together with a second antifungal agent.

Methods of Treating Fungal Infections Certain embodiments disclosed herein include, for example, aspergillosis (including invasive pulmonary aspergillosis), blastosis (significant or rapidly progressive infection and central nervous system) ), Candidiasis (including kidney stones, urinary tract obstruction, renal transplantation or retrograde candidiasis in the urinary tract in patients with poorly controlled diabetes mellitus), coccidioidomycosis (sufficient for other chemotherapy) Treatment of pathogenic fungal infections including cryptococcosis, histoplasmosis, mucormycosis (including craniofacial mucormycosis and mucor pneumonia), paracoccidioidomycosis and sporotrichosis, including chronic diseases that do not respond to Relates to how to prevent. The method may include administering to a human subject at least one antifungal dehydrophenylahistine or analog thereof as described above to treat or prevent a fungal infection. In certain embodiments, the dehydrophenylahistine or analog thereof is amphotericin B, or one or more non-dehydrophenylahistine or analog antifungal agents such as imidazole or thiazole agonists as described above. It may be administered in combination with administration.

  Pathogenic fungal infections are local, for example caused by Candida, Trichophyton, Microsporum or Epidermophyton species, among other organisms Or it can be mucosal, e.g. caused by Candida albicans (e.g. phlegm and vaginal candidiasis). Infections include, for example, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Coccidioides, Paracoccidioides, and Paracoccidioides Or it may be systemic caused by a Blastomyces species. Infection may also include fungal mycomas, chromoblast mycosis, cryptococcal meningitis or phycomicosis.

  Further embodiments include Candida spp. (C. albicans, C. tropicalis, C. kefyr, C. krusei and C. galbrata ( C. galbrata), Aspergillus species (including A. fumigatus and A. Flavus), Cryptococcus neoformans, Blast myces ( Blastomyces species (including Blastomyces dermatitidis), Pneumocystis carinii, Coccidioides immitis, Basidiobolus ranarum, Conidiobolus, Conidiobolus ) Species, Histoplasma capsulatum, Rhizopus species (R. oryzae) and R. microsporus (R. mi) crosporus), Cunninghamella spp., Rhizomucor spp., Paracoccidioides brasiliensis, Pseudallescheria boydii, Rinosporidium sp. And a method of treating or preventing a pathogenic fungal infection selected from the group consisting of Sporothrix schenckii. Similarly, the above method requires a non-immunosuppressive antifungal dehydrophenilahistine or analog thereof so that a fungal infection is treated or prevented without inducing an adverse immunosuppressive effect. Administration to a patient may be included.

  Further embodiments include one or more antifungal agents referred to elsewhere herein (eg, one of amphotericin B, flucytosine, imidazole and triazole (eg, fluconazole, ketoconazole, itraconazole, and others). To the method of treating or preventing pathogenic fungal infections that are resistant to other antifungal therapies, including pathogenic fungal infections that are resistant to). The method comprises administering to a patient one or more antifungal dehydrophenillahistines or analogs thereof in an amount and dosage regimen such that a fungal infection resistant to another antifungal therapy in a subject is treated or prevented. Doing so may be included.

Further embodiments include one or more other antifungals in patients who are allergic, intolerant or not responsive to another antifungal therapy, or referred to elsewhere herein. Use of other antifungal agents, including agents (eg, including one of amphotericin B, flucytosine, imidazole, and triazole (including, for example, fluconazole, ketoconazole, itraconazole, and other above-described examples)) It relates to a method for treating or preventing pathogenic fungal infections in people who are contraindicated in some cases. The method may include administering to the patient one or more antifungal dehydrophenilahistine or an analog thereof in an amount such that a fungal infection is treated or prevented.

Packaged dehydrophenillahistin or analogs thereof Certain embodiments may include packaged dehydrophenylahistine or analogs thereof, preferably packaged non-immunosuppressive antifungal dehydrophenillahistin or analogs thereof. The term is packaged with instructions for administering dehydrophenylhistine or analog (s) as an antifungal agent without causing adverse immunosuppressive effects in a human subject, as described above. Is intended to include at least one dehydrophenylhistine or analog thereof. In some embodiments, the non-immunosuppressive antifungal dehydrphenylhistine or an analog thereof is a member of one of the above-mentioned preferred compound subsets. The dehydrophenylahistine or analog thereof can be simply packaged with the instructions, or another dehydrophenylahistine or analog thereof, rapamycin or another ingredient or additive, for example a component of the above pharmaceutical composition Can be packaged with one or more of the following: The package may contain one or more containers filled with one or more of the ingredients of the pharmaceutical composition. Optionally, such container (s) may be accompanied by a notice in the form prescribed by the government that regulates the manufacture, use or sale of a pharmaceutical or biological product, Indicates approval by the manufacturing, use or sales organization for human administration.

The following non-limiting examples are meant to describe the preferred methods using certain preferred embodiments. Variations in the details of the particular method used and variations in the exact chemical composition obtained will be apparent to those skilled in the art.
(Example)

A. Synthesis of Dehydrophenylahistine As shown in FIG. 1, dehydrophenylahistine was synthesized by condensation according to the following basic reaction scheme.

N, N′-Diacetyl-2,5-piperazinedione Complete 2,5-piperazinedione 1 [2,5-piperazinedione (Aldrich G640-6) in 100 mL acetic anhydride (Ac ₂ O), 25 2.0 g, 0.218 mol] was mixed with sodium acetate (NaOAc) (17.96 g, 0.218 mol). The mixture was heated at 110 ° C. for 8 hours using a double coil condenser in an Ar atmosphere. After removal of Ac ₂ O by evaporation, the residue is dissolved in AcOEt, washed with 10% citric acid, 10% NaHCO ₃ and saturated NaCl (3 times each), dried over Na ₂ SO ₄ , vacuum Concentrated in. The residue was triturated with ether to form a solid. This solid was recrystallized from EtOAc with ether-hexane to give 1,6.4 g (61%) of N, N′-diacetyl-2,5-piperazinedione.

1-acetyl-3-{(Z) -1- [5- (1,1-dimethyl-2-propenyl) -1H-4-imidazolyl] methylidene}]-2,5-piperazinedione 2
To a solution of 5- (1,1-dimethyl-2-propenyl) imidazole-4-carboxaldehyde (100 mg, 0.609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1.22 mmol). The solution was repeatedly evacuated to remove oxygen, flushed with Ar, followed by the addition of Cs ₂ CO ₃ (198 mg, 0.609 mmol) and the evacuation-pour process was repeated again. The resulting mixture was stirred at room temperature for 5 hours. After removal of the solvent by evaporation, the residue is dissolved in a mixture of EtOAc and 10% Na ₂ CO ₃ and the organic phase is washed once more with 10% Na ₂ CO ₃ and 3 times with saturated NaCl, and Na ₂ SO. Dried by ₄ and concentrated in vacuo. The residual oil was purified by column chromatography on silica using CHCl ₃ -MeOH (100: 0 to 50: 1) as the eluent to give 2, 60 mg (33%) of a pale yellow solid.

To a solution of 2 (30 mg, 0.099 mmol) in dehydrophenylhistine DMF (0.8 mL), benzaldehyde (51 μL, 0.496 mmol, 5 eq) was added and the solution was repeatedly evacuated over a short period of time, Oxygen was removed and Ar was flushed, followed by the addition of Cs ₂ CO ₃ (53 mg, 0.149 mmol, 1.5 eq) and the evacuation-pouring process was repeated again. The resulting mixture was heated at 80 ° C. for 2.5 hours. (The temperature must be increased gradually. Rapid heating increases the production of the E isomer at the benzylidene moiety.) After removing the solvent by evaporation, the residue is dissolved in EtOAc and washed with 2% water. Washed three times with saturated NaCl, dried over Na ₂ SO ₄ and concentrated in vacuo. Spots with bright yellow-green emission at 365 nm UV can be observed on TLC using CHCl ₃ -MeOH (10: 1). The purity of this crude product was 75% or more from HPLC analysis. The resulting residue was dissolved in 90% MeOH water and applied to a reverse phase HPLC column (YMC-Pack, ODS-AM, 20 × 250 mm) using a linear gradient from 70% MeOH to 74% MeOH in water. Eluting at a flow rate of 12 mL / min over 16 minutes, the desired fractions were collected and concentrated by evaporation to give 19.7 mg (60%) of yellow dehydrophenylahistine. The HPLC profile of the synthetic crude dehydrophenilahistine is shown in FIG.

  As shown in FIG. 4, in the purification of dehydrophenylahistine, the main peak was the desired Z-form compound of dehydrophenylahistine. The formation of the E isomer was observed as a minor component (about 10%), which eluted as a more polar peak than the Z isomer. As other minor peaks, reduced Z and E compounds in which the dimethylallyl moiety of dehydrophenylahistine was reduced were also observed. The formation of these reducing compounds was attributed to aldehyde 2 with reducing impurities, which were produced during reduction with DIBAL-H and were not separated in subsequent processes.

  These trace compounds can be removed by purification by preparative HPLC, and dehydrophenilahistine having a Z configuration at the benzylidene moiety can be obtained with a purity of 95% or more and a yield of 60% ( 20% yield over 2 steps). Compounds with an E configuration at the imidazole side chain of the diketopiperazine ring are not observed on this HPLC chart, suggesting that the first reaction from compounds 1-3 in FIG. 1 is Z-selective.

B. Chemical properties The dehydrophenylahistine compound is a pale yellow solid. Its structure is confirmed by standard NMR analysis.

Synthesis and physical properties of tBu-dehydrophenylahistine derivatives A structural derivative of dehydrophenylahistine was synthesized according to the following reaction scheme to produce tBu-dehydrophenylahistine. The synthesis by Route A (see FIG. 1) is similar in some respects to the synthesis of dehydrophenylahistine synthesized in Example 1.

Route A
N, N′-diacetyl-2,5-piperazinedione 1 was prepared in the same manner as Example 1.

1) 1-acetyl-3-{(Z) -1- [5-tert-butyl-1H-4-imidazolyl] methylidene}]-2,5-piperazinedione (16)

To a solution of 5-tert-butylimidazole-4-carboxaldehyde 15 (3.02 g, 19.8 mmol) in DMF (30 mL) was added compound 1 (5.89 g, 29.72 mmol) and the solution was The exhaust-pour process was repeated again by repeatedly evacuating for a short period of time to remove oxygen and flushing with Ar, followed by the addition of Cs ₂ CO ₃ (9.7 g, 29.72 mmol). The resulting mixture was stirred at room temperature for 5 hours. After removing the solvent by evaporation, the residue is dissolved in a mixture of EtOAc and 10% Na ₂ CO ₃ and the organic phase is dissolved in 10%.
Washed once more with% Na ₂ CO ₃ and 3 times with saturated NaCl, dried over Na ₂ SO ₄ and concentrated in vacuo. The residual oil was purified by column chromatography on silica using CHCl ₃ -MeOH (100: 0 to 50: 1) as eluent to give a pale yellow solid 16, 1.90 g (33%). It was. ¹ H NMR (270 MHz, CDCl ₃ ) δ 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) t-Bu-dehydrophenilahistine

1-acetyl-3-{(Z) -1- [5-tert-butyl-1H-4-imidazolyl] methylidene}]-2,5-piperazinedione (16) (11 mg, in DMF (1.0 mL)) 0.038 mmol) solution was added benzaldehyde (19 μL, 0.19 mmol, 5 eq) and the solution was repeatedly evacuated briefly to remove oxygen, flushed with Ar, followed by Cs ₂ CO ₃ ( 43 mg, 0.132 mmol, 3.5 eq) was added and the exhaust-pour process was repeated again. The resulting mixture was heated at 80 ° C. for 2.5 hours. After removal of the solvent by evaporation, the residue was dissolved in EtOAc, washed twice with water and three times with saturated NaCl, dried over Na ₂ SO ₄ and concentrated in vacuo. The resulting residue was dissolved in 90% MeOH water and applied to a reverse phase HPLC column (YMC-Pack, ODS-AM, 20 × 250 mm) using a linear gradient from 70% MeOH to 74% MeOH in water. Eluting at a flow rate of 12 mL / min over 16 minutes, collecting the desired fractions and concentrating by evaporation, yielded 6.4 mg (50%) of yellow tert-butyl-dehydrophenilahistine. ¹ H NMR (270 MHz, CDCl ₃ ) δ 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).

  The dehydrophenylahistine reaction to produce tBu-dehydrophenylahistine is the same as Example 1.

  The total yield of recovered tBu-dehydrophenilahistine was 16.5%.

Path B
N, N′-diacetyl-2,5-piperazinedione 1 was prepared in the same manner as Example 1.

1) 1-acetyl-3-[(Z) -benzylidene 1] -2,5-piperazinedione (17)

To a solution of benzaldehyde 4 (0.54 g, 5.05 mmol) in DMF (5 mL) was added compound 1 (2.0 g, 10.1 mmol) and the solution was repeatedly evacuated in a short time to remove oxygen. Then, Ar was flushed, followed by the addition of Cs ₂ CO ₃ (1.65 g, 5.05 mmol) and the exhaust-pouring process was repeated again. The resulting mixture was stirred at room temperature for 3.5 hours. After removal of the solvent by evaporation, the residue is dissolved in a mixture of EtOAc and 10% Na ₂ CO ₃ and the organic phase is washed once more with 10% Na ₂ CO ₃ and 3 times with saturated NaCl, and Na ₂ SO. Dried by ₄ and concentrated in vacuo. The remaining solid was recrystallized from MeOH-ether to give 17 off-white solids: yield 1.95 g (79%).

2) t-Bu-dehydrophenilahistine

To a solution of 1-acetyl-3-[(Z) -benzylidene 1] -2,5-piperazinedione (17) (48 mg, 0.197 mmol) in DMF (1.0 mL) was added 5-tert-butylimidazole- 4-Carboxaldehyde 15 (30 mg, 0.197 mmol) was added and the solution was repeatedly evacuated briefly to remove oxygen, flushed with Ar, followed by Cs ₂ CO ₃ (96 mg, 0.296 mmol). And the exhaust-pour process was repeated again. The resulting mixture was heated at 80 ° C. for 14 hours. After removal of the solvent by evaporation, the residue was dissolved in EtOAc, washed twice with water and three times with saturated NaCl, dried over Na ₂ SO ₄ and concentrated in vacuo. The resulting residue was dissolved in 90% MeOH water and applied to a reverse phase HPLC column (YMC-Pack, ODS-AM, 20 × 250 mm) using a linear gradient from 70% MeOH to 74% MeOH in water. Eluting at a flow rate of 12 mL / min over 16 minutes, collecting the desired fractions and concentrating by evaporation, yielded 0.8 mg (1.2%) of yellow tert-butyl-dehydrophenilahistine.

  The total yield of recovered tBu-dehydrophenilahistine was 0.9%.

  The HPLC profile of synthetic crude tBu-dehydrophenylhistine from Route A and Route B is shown in FIG.

  Two other tBu-dehydrophenylhistine derivatives were synthesized according to the route A method. In the synthesis of a further tBu-dehydrophenylhistine derivative, modification to benzaldehyde compound 4 was performed.

  FIG. 4 shows the HPLC profile (column: YMC-Pack ODS-AM (20 ×) of the synthetic dehydrophenylhistine of Example 1 (FIG. 2) and the tBu-dehydrophenillahistine compound produced by Route A exemplified above. 250 mm), gradient: methanol-water system for 20 minutes, followed by 100% methanol system for 10 minutes, 65% to 75%, flow rate: 12 mL / min, OD 230 nm).

  The order of introduction of aldehydes is related to yield and is therefore a synthetic aspect. An analog of dehydrophenillahistine was synthesized as a control or model where the dimethylallyl group was changed to a tert-butyl group with similar steric hindrance at the 5-position of the imidazole ring.

  The synthesis of this “tert-butyl (tBu) -dehydrophenylahistine” using “Route A” was as described above. In particular, the order of aldehyde introduction followed the dehydrophenylahistine synthesis exactly and showed a total yield of 16.5% tBu-dehydrophenylahistine. This yield was similar to the yield of dehydrophenilahistine (20%). The introduction of the first benzaldehyde 4 yields a 76% yield of intermediate compound 17 using the “route B” in which the order of aldehyde introduction is the reverse order of “route A” for dehydrophenylhistine synthesis. However, only a trace amount of the desired tBu-dehydroPLH was obtained with a total yield of 0.9%. This result shows that it can be difficult to introduce very bulky imidazole-4-carboxaldehyde 15 with a substituent having a quaternary carbon on the adjacent 5-position in the imidazole ring into the intermediate compound 17. And that the order of aldehyde introduction is an important aspect for obtaining high yields of dehydrophenylahistine or an analog of dehydrophenylahistine using the synthesis disclosed herein. Suggested.

  As shown in FIG. 4, from the HPLC analysis of the final crude product, formation of a very high content of tBu-dehydrophenylhistine and a small amount of by-product was observed in the crude sample of Path A (left). ). However, in the sample obtained using route B, a relatively smaller amount of the desired tBu-dehydrophenilahistine and some other by-products were observed (right).

Alternative, larger scale synthesis of dehydrophenylhistine and analogs 3-Z-benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] Synthesis of piperazine-2,5-dione [dehydrophenilahistine] (1)

Reagents: a) LDA, CH ₃ CHO, b) Tos-Cl, pyridine, c) DBU, d) NaOH, e) C ₂ Cl ₂ O ₂ , f) KOOCCH ₂ COOEt, BuLi, g) SO ₂ Cl ₂ , _{h) H 2 NCHO, H 2} O, i) LiAlH 4, j) MnO 2, k) 1,4- diacetyl - piperazine-2,5-dione, Cs ₂ CO _3, l) benzaldehyde, Cs ₂ CO ₃

  3-hydroxy-2,2-dimethyl-butyric acid methyl ester

  Add a solution of LDA (2M, 196 ml, 0.39 mol) in heptane / THF / ethylbenzene to a solution of methyl isobutyrate (45 ml, 0.39 mol) in THF (270 mL) at −60 ° C. under argon. The resulting mixture was stirred for 30 minutes. A solution of acetaldehyde (27 ml, 0.48 mol) in THF (45 ml) pre-cooled to −60 ° C. was added slowly and the resulting solution was stirred for an additional 30 minutes. Saturated ammonium chloride (50 ml) was added and the solution was warmed to room temperature. The reaction mixture was extracted with ethyl acetate and the extract was washed with HCl (2M), sodium bicarbonate followed by brine. The organic layer was dried over magnesium sulfate, filtered and evaporated to give a clear oil (52.6 g). Distillation at 76-82 ° C./30 mmHg gave pure 3-hydroxy-2,2-dimethylbutyric acid methyl ester (42.3 g, 74%). (Burk et al., J. Am. Chem. Soc., 117: 4423-4424 (1995)).

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

  2,2-Dimethyl-3- (toluene-4-sulfonyloxy) -butyric acid methyl ester

  To a cooled (0 ° C.) solution of 3-hydroxy-2,2-dimethylbutyric acid methyl ester (52.0 g, 0.36 mol) in pyridine (100 ml) was added p-toluenesulfonyl chloride (69.0 g, 0.36 mol). Was gradually added. The mixture was warmed to room temperature and stirred for 60 hours. The reaction was again cooled in ice and acidified by adding HCl (2M). The resulting solution is extracted with ethyl acetate and the extract is washed with HCl followed by brine, dried and evaporated to give an oil which forms a white precipitate upon standing. did. This mixture was dissolved in a minimal amount of ethyl acetate followed by addition of light oil to give a white precipitate that was collected and washed with an additional amount of light oil. The filtrate was evaporated to some extent to collect a second product of crystals, and in addition to the first product, 2,2-dimethyl-3- (toluene-4-sulfonyloxy) -butyric acid methyl ester (81. 2 g, 76%).

¹ H NMR (400 NMz, CDCl ₃ ) δ 1.12 (s, 3H); 1.13 (s, 3H); 1.24 (d, J = 6.4 Hz, 3H); 2.45 (s, 3H, -PhMe); 3.58 (s, 3H, -OMe); 4.94 (quartet, J = 6.4Hz, 1H, H3), 7.33 (d, J = 8.0Hz, 2H) 7.78 (d, J = 8.0 Hz, 2H).

  Additional crude 2,2-dimethyl-3- (toluene-4-sulfonyloxy) -butyric acid methyl ester (19.0 g, 18%) was obtained by evaporation of the final filtrate.

  2,2-Dimethyl-but-3-enoic acid methyl ester

  A solution of 2,2-dimethyl-3- (toluene-4-sulfonyloxy) -butyric acid methyl ester (18.06 g, 0.06 mol) in DBU (15 ml) was heated at 140-160 ° C. for 3.5 hours. The mixture was cooled to room temperature and then diluted with ether. The mixture was washed with HCl (1M), sodium bicarbonate, followed by brine. The ether layer was dried and evaporated to some extent to give a concentrated solution (10 g) of 2,2-dimethyl-but-3-enoic acid methyl ester. (Savu and Katzenellenbogen, J. Org. Chem, 46: 239-250 (1981)). Due to the volatility of the product (bp 102 ° C.) further evaporation was avoided. (Tsaconas et al., Aust. J. Chem., 53: 435-437 (2000)).

¹ H NMR (400 NMz, CDCl ₃ ) δ 1.31 (s, 6H); 3.68 (s, 3H); 5.06 (d, J = 17.1 Hz, 1H, —CH═CH ₂ ); 11 (d, J = 10.7Hz, 1H, -CH = CH 2); 6.03 (dd, J = 17.1,10.7Hz, 1H, -CH = CH 2).

  2,2-Dimethyl-but-3-enoic acid

  The ether solution (10 g) of 2,2-dimethyl-but-3-enoic acid methyl ester described above is diluted with ethanol (25 ml), sodium hydroxide (4 M, 22 ml) is added and the mixture is stirred overnight. did. The solution was evaporated to some extent to remove the ethanol and the resulting mixture was added to HCl (1M, 100 ml). The product was extracted with ethyl acetate and the extract was dried and evaporated to give 2,2-dimethyl-but-3-enoic acid (6.01 g, 88%, 2 steps). (Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.33 (s, 6H); 5.11 (d, J = 10.8 Hz, 1H, —CH═CH ₂ ); 5.15 (d, J = 17.2 Hz) , 1H, —CH═CH ₂ ); 6.05 (dd, J = 17.2, 10.8 Hz, 1H, —CH═CH ₂ ).

Monoethyl hydrogen malonate (Wierenga and Skulnick, “
Aliphatic and aromatic β-ketoesters from monoethyl malonate: Ethyl 2-Butyrylacetate (“Aliphatic and Aromatic β-keto Esters from Monoethyl Malonate: Ethyl 2-Butyrylacetate”), Organic Syntheses, Collective Volume, 7,213).

Potassium ethyl malonate (25.0 g, 0.15 mol) was suspended in water (15.6 ml) and cooled in an ice bath. After concentrated HCl (12.5 ml) was added dropwise over 30 minutes, the mixture was stirred for an additional 10 minutes. The precipitate was filtered and washed twice with ether. The filtrate was separated and the aqueous phase was extracted with ether. The combined ether solution was dried (MgSO ₄ ) and evaporated to give monoethyl hydrogen malonate (19.2 g, 99%) as an oil which was left under vacuum (or 50 ° C. / 1 mm for 1 hour).

  4,4-Dimethyl-3-oxo-hex (hex) -5-enoic acid ethyl ester

  Oxalyl chloride (3.83 ml, 43.9 mmol) was cooled to 2,2-dimethyl-but-3-enoic acid (5.0 g, 43.9 mmol) and DMF (1 drop) in anhydrous dichloromethane (25 ml) ( 0 ° C.). The mixture was stirred at 0 ° C. for 1 hour followed by 16 hours at room temperature. By fractional distillation (121 ° C./760 mmHg), 2,2-dimethyl-but-3-enoyl chloride (4.1 g, 71%) was obtained.

  Monoethyl hydrogen malonate (7.2 g, 0.05 mol) and bipyridyl (a few milligrams) were dissolved in THF (90 ml) and nitrogen was flushed through the system. After the solution was cooled to −70 ° C., BuLi (2.5 M in hexane, 37 ml, 0.09 mol) was added. After adding only about 10 ml of BuLi, the solution turned pink and required additional THF (15 ml) to allow magnetic stirring. The cooling bath was removed and the remaining BuLi was added to allow the temperature to reach −10 ° C., at which point the solution became colorless. The mixture was again cooled to −60 ° C. and a solution of 2,2-dimethyl-but-3-enoyl chloride (4.1 g, 0.03 mol) in THF (12 ml) was added dropwise. After the addition was complete, the mixture was warmed to 0 ° C. and stirred for 3 hours, then it was added to a 1: 1 mixture of ether / 1M HCl (260 ml) at 0 ° C. for an additional 1.5 hours. Stir. The organic layer was removed and washed with HCl (1M), sodium bicarbonate solution, brine, then dried and evaporated to 4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (5. 6 g, 98%). (Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)). Pure material was obtained by distillation (160 ° C./1 mmHg) using a Kugelrohr oven.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.26 (s, 6H); 1.27 (t, J = 6.9 Hz, 3H, —CH ₂ CH ₃ ); 3.51 (s, 2H); 18 (q, J = 6.
_{9Hz, 2H, -CH 2 CH 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-oxo-hex-5-enoic acid ethyl ester

  Sulfuryl chloride (0.84 ml, 10.4 mmol) was cooled to 4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (1.83 g, 9.93 mmol) in chloroform (7 ml). ° C) was added to the solution. The resulting mixture was warmed to room temperature and stirred for 30 minutes, after which it was heated under reflux for 2 hours. After cooling to room temperature, the reaction mixture was diluted with chloroform, followed by washing with sodium bicarbonate, water, followed by brine. The organic phase was dried and evaporated to give 2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (2.01 g, 93%) as a brown oil. (Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.28 (t, J = 7.0 Hz, 3H, —CH ₂ CH ₃ ); 1.33 (s, 3H); 1.34 (s, 3H); 24 (q, J = 7.0 Hz, 2H, —CH ₂ CH ₃ ); 5.19 (s, 1H); 5.28 (d, J = 16.9 Hz, 1H, —CH═CH ₂ ); 5 .29 (d, J = 10.9 Hz, 1H, —CH═CH ₂ ); 5.96 (dd, J = 16.9, 10.9 Hz, 1H, —CH═CH ₂ ).

_{LC / MS t R = 8.45 (} 219.3 [M (Cl 37) + H] +) min.

  This material was reacted without further purification.

  5- (1,1-Dimethyl-allyl) -3H-imidazole-4-carboxylic acid ethyl ester

2-Chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (19.4 g, 0.09 mol) and water (1.94 ml, 0.11 mol) in formamide (36.8 ml) The suspension was shaken briefly and then dispensed into 15 × 18 ml vials. The vial was sealed and heated at 150 ° C. for 5 hours. After cooling to room temperature, the contents of the vial were combined and extracted exhaustively with chloroform. The extract was dried and evaporated to give a concentrated formamide solution (14.7 g). This was added to a silica column (diameter 7 cm, height 11 cm) packed with 1% MeOH / 1% Et ₃ N in chloroform. Elution of the column with 2 L of this mixture followed by 2% MeOH / 1% Et ₃ N 2 L in chloroform gave 5- (1,1-dimethyl-allyl) -oxazole-4-carboxylic acid in the initial fraction. There was obtained a compound (1.23 g, 7%) which appeared to be the acid ethyl ester.

_{HPLC (214nm) t R = 8.68} (50.4%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.40 (t, J = 7.2 Hz, 3H, —CH ₂ CH ₃ ); 1.54 (s, 6H); 4.38 (t, J = 7.2 Hz) _{, 2H, -CH 2 CH 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] +) fraction.

  The desired 5- (1,1-dimethyl-allyl) -3H-imidazole-4-carboxylic acid ethyl ester was recovered from the later fraction (3.13 g, 17%). (Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)).

_{HPLC (214nm) t R = 5.52} (96.0%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.38 (t, J = 7.0 Hz, 3H); 1.57 (s, 6H); 4.35 (q, J = 7.0 Hz, 2H); 04-5.14 (m, 2H, —CH═CH ₂ ); 6.28 (dd, J = 18.0, 10.4 Hz, 1H, —CH═CH ₂ ); 7.52 (s, 1H) .

_{LC / MS t R = 5.30 (} 209.1 [M + H] +, 417.2 [2M + H] +) min.

  Additional 5- (1,1-dimethyl-allyl) -3H-imidazole-4-carboxylic acid ethyl ester was also recovered from the column (3.59 g, 19%), which was less pure but still sufficient for further reaction Met.

Another by-product isolated from a similar reaction (smaller scale) by further elution of the column with 5% MeOH / 1% Et ₃ N in chloroform is 5- (1,1-dimethyl-allyl)- It was a compound considered to be 3H-imidazole-4-carboxylic acid (0.
27 g, 9%).

_{HPLC (245nm) t R = 5.14} (68.9%) min.

¹ H NMR (400 MHz, CD ₃ OD) δ 1.45 (s, 6H); 4.97 (d, J = 10.6 Hz, 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] +) fraction.

  [5- (1,1-Dimethyl-allyl) -3H-imidazol-4-yl (yl)]-methanol

  A solution of 5- (1,1-dimethyl-allyl) -3H-imidazole-4-carboxylic acid ethyl ester (3.13 g, 15.0 mmol) in THF (60 ml) was added to lithium hydride in THF (40 ml). It was added dropwise to a suspension of aluminum (95% suspension, 1.00 g, 25.0 mmol) and the mixture was stirred at room temperature for 4 hours. Water was added until gas evolution ceased and the mixture was stirred for 10 minutes before being filtered through a sintered funnel. The precipitate is washed with THF followed by methanol, and the filtrate and washings are combined, evaporated and lyophilized to give [5- (1,1-dimethyl-allyl) -3H-imidazol-4-yl. ] -Methanol (2.56 g, 102%) was obtained. Residual water was removed by azeotroping with chloroform prior to further reaction. (See Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)).

_{HPLC (240nm) t R = 3.94} (56.8%) min.

¹ H NMR (400 MHz, CD ₃ OD) δ 1.43 (s, 6H); 4.57 (s, 2H); 5.01 (d, J = 10.5 Hz, 1H, —CH═CH ₂ ); 5 .03 (d, J = 17.7 Hz, 1H, —CH═CH ₂ ); 6.10 (dd, J = 17.7, 10.5 Hz, 1H, —CH═CH ₂ ); 7.46 (s , 1H).

_{LC / MS t R = 3.77 (} 167.3 [M + H] +) min.

  5- (1,1-Dimethyl-allyl) -3H-imidazole-4-carbaldehyde

  Manganese dioxide (20 g, 0.23 mol) was added to [5- (1,1-dimethyl-allyl) -3H-imidazol-4-yl] -methanol (2.56 g, 0.02 mol) in acetone (300 ml). Add to the solution and stir the resulting mixture at room temperature for 5 hours. The mixture was filtered through filter paper and the residue was washed with acetone. The filtrate and washings were combined and evaporated to give 5- (1,1-dimethyl-allyl) -3H-imidazole-4-carbaldehyde (1.82 g, 51%). (Hayashi et al., J. Org. Chem., 65: 8402-8405 (2000)).

_{HPLC (240nm) t R = 4.08} (91.5%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.56 (s, 6H); 5.16 (d, J = 10.6 Hz, 1H, —CH═CH ₂ ); 5.19 (d, J = 17.3 Hz) _{, 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-acetyl-3- [5 '-(1,1-dimethyl-allyl) -1H-imidazole-4'-Z-ylmethylene] -piperazine-2,5-dione

  To a solution of 5- (1,1-dimethyl-allyl) -3H-imidazole-4-carbaldehyde (1.78 g, 0.01 mol) in DMF (35 ml) was added 1,4-diacetyl-piperazine-2,5. -Dione (8.59 g, 0.04 mol) was added and the mixture was evacuated before flushing with argon. After the exhaust-pour process was repeated two more times, cesium carbonate (3.53 g, 0.01 mol) was added. After the evacuation-casting process was repeated three more times, the resulting mixture was heated at 45 ° C. for 5 hours. The reaction mixture was evaporated to some extent (heated under high vacuum) until small, and the resulting solution was added dropwise to ice water (50 ml). The yellow precipitate was collected, washed with water, and then lyophilized to give 1-acetyl-3- [5 ′-(1,1-dimethyl-allyl) -1H-imidazole-4′-ylmethylene] -piperazine -2,5-dione (1.18 g, 36%) was obtained. (Hayashi, personal communication (2001)).

_{HPLC (214nm) t R = 6.01} (72.6%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.53 (s, 6H); 2.64 (s, 3H); 4.47 (s, 2H); 5.19 (d, J = 17.3 Hz, 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) [1,4-diacetyl-piperazine-2,5 -Dione contaminating contaminants δ 2.59 (s, 6H); 4.60 (s, 4H) were observed about 2%. ]

_{LC / MS t R = 6.65 (} 303.3 [M + H] +, 605.5 [2M + H] +) min. (Note that different systems are used).

  3-Z-benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5-dione

  1-acetyl-3- [5 ′-(1,1-dimethyl-allyl) -1H-imidazol-4′-ylmethylene] -piperazine-2,5-dione (2.91 g, 9.D) in DMF (70 ml). Benzaldehyde (4.89 ml, 48.1 mmol) was added to the 62 mmol) solution and the solution was evacuated and flushed with argon. After the exhaust-pour process was repeated two more times, cesium carbonate (4.70 g, 14.4 mol) was added. After the evacuation-casting process was repeated three more times, the resulting mixture was heated under a temperature gradient as shown below.

After a total time of 5 hours, the reaction was cooled to room temperature and the mixture was added to ice cold water (500 ml). The precipitate was collected, washed with water (300 ml) and then lyophilized to give a yellow solid (2.80 g). This material was dissolved in chloroform (250 ml), filtered through filter paper and azeotroped to evaporate residual water. Residual yellow precipitate (2.70 g, HPLC (214 nm) t _R = 7.26 (93.6%) min) was dissolved to some extent in chloroform (20 ml) and the suspension was sonicated for 5 min. The solid was collected and air dried to give 3-Z-benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5- Dione (1.82 g, 54%) was obtained. (Hayashi, personal communication (2001)). m. p. 239-240 ° C (dec.).

_{HPLC (214nm) t R = 6.80} (1.92) min, 7.33 (95.01%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.53 (s, 6H); 5.18 (d, J = 17.6 Hz, 1H, —CH═CH ₂ ); 5.21 (d, J = 11.0 Hz) , 1H, —CH═CH ₂ ); 6.06 (dd, J = 17.6, 11.0 Hz, 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, H2 ″); 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) min.

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

  Additional 3-Z-benzylidene-6- [5 ″-(1,1-dimethylallyl) -1H-imidazole-4 ″ -Z-ylmethylene] -piperazine-2,5-dione (0. 76 g, 29%).

_{HPLC (214nm) t R = 7.29} (84.5%) min.

  3-E-Benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5-dione

Purification of a crude sample of the material synthesized as described above by preparative HPLC yielded the geometric isomer 3-E-benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5-dione (1.7 mg) was obtained.

_{HPLC (214nm) t R = 6.75} (87.79) minutes.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.52 (s, 6H); 5.19 (d, J = 20.8 Hz, 1 H, CH═CH ₂ ); 5.22 (d, J = 14.0 Hz, _{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 / z 349.5 [M + H] ⁺ , 390.4 [M + CH ₄ CN] ⁺ .

  Synthesis of 3-Z-benzylidene-6- (5 "-tert-butyl-1H-imidazole-4" -Z-ylmethylene) -piperazine-2,5-dione (2)

Reagents: g) SO ₂ Cl ₂ , h) H ₂ NCHO, H ₂ O, i) LiAlH ₄ , j) MnO ₂ , k) 1,4-diacetyl-piperazine-2,5-dione, Cs ₂ CO ₃ , l) Benzaldehyde, Cs ₂ CO ₃

  2-Chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester

Sulfuryl chloride (14.0 ml, 0.17 mol) was added to a cooled (0 ° C.) solution of ethyl pivaloyl acetate (27.17 g, 0.16 mol) in chloroform (100 ml). The resulting mixture was warmed to room temperature and stirred for 30 minutes, after which it was heated under reflux for 2.5 hours. After cooling to room temperature, the reaction mixture was diluted with chloroform, followed by washing with sodium bicarbonate, water, followed by brine.

  The organic phase was dried and evaporated to give 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (33.1 g, 102%) as a clear oil. (Durant et al., “Aminoalkylimidazoles and Process for their Production”, patent number GB1341375 (UK, 1973)).

_{HPLC (214nm) t R = 8.80} (92.9%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.27 (s, 9H); 1.29 (t, J = 7.2 Hz, 3H); 4.27 (q, J = 7.2 Hz, 2H); 22 (s, 1H).

¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.8, 26.3, 45.1, 54.5, 62.9, 165.1, 203.6.

  5-tert-butyl-3H-imidazole-4-carboxylic acid ethyl ester

After shaking a solution of 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (25.0 g, 0.12 mol) in formamide (47.5 ml) and water (2.5 ml), Distribute into 15 x 8 ml vials. All vials were sealed and subsequently heated at 150 ° C. for 3.5 hours. After the vial was cooled to room temperature, water (20 ml) was added and the mixture was extracted exhaustively with chloroform. Removal of chloroform gave a concentrated formamide solution (22.2 g) which was added to a flash silica column (diameter 6 cm, height 12 cm) packed with 1% MeOH / 1% Et ₃ N in chloroform. did. Elution of the column with 2.5 L of this mixture followed by 2% MeOH / 1% Et ₃ N 1 L in chloroform gives 5-tert-butyl-oxazole-4-carboxylic acid ethyl ester in the initial fraction. The expected product (6.3 g, 26%) was obtained.

HPLC (214nm) t _R = 8.77 min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.41 (t, J = 7.2 Hz, 3H); 1.43 (s, 9H); 4.40 (q, J = 7.2 Hz, 2H); 81 (s, 1H).

¹³ C NMR (100 MHz, CDCl ₃ ) δ 14.1, 28.8, 32.5, 61.3, 136.9, 149.9, 156.4, 158.3.

ESMS m / z 198.3 [M + H] ⁺ , 239.3 [M + CH ₄ CN] ⁺ .

_{LC / MS t R = 7.97 (} 198.1 [M + H] +) min.

  5-tert-Butyl-3H-imidazole-4-carboxylic acid ethyl ester was recovered from the later fraction (6.20 g, 26%). (Durant et al., “Aminoalkylimidazoles and Process for their Production”, patent number GB1341375 (UK, 1973)).

_{HPLC (214nm) t R = 5.41} (93.7%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.38 (t, J = 7.0 Hz, 3H); 1.47 (s, 9H); 4.36 (q, J = 7.2 Hz, 2H); 54 (s, 1H).

¹³ C NMR (100 MHz, CDCl ₃ ) δ 13.7, 28.8, 32.0, 59.8, 124.2, 133.3, 149.2, 162.6.

ESMS m / z 197.3 [M + H] ⁺ , 238.3 [M + CH ₄ CN] ⁺ .

Further elution of the column with 5% MeOH / 1% Et ₃ N 1L gave a compound that appeared to be 5-tert-butyl-3H-imidazole-4-carboxylic acid (0.50 g, 2%).

_{HPLC (245nm) t R = 4.68} (83.1%) min.

¹ H NMR (400 MHz, CD ₃ OD) δ 1.36 (s, 9H); 7.69 (s, 1H).

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.37 (s, 9H); 7.74 (s, 1H).

¹ H NMR (400 MHz, CD ₃ SO) δ 1.28 (s, 9H); 7.68 (s, 1H).

ESMS m / z 169.2 [M + H] ⁺ , 210.4 [M + CH ₄ CN] ⁺ .

  (5-tert-butyl-3H-imidazol-4-yl) -methanol

A solution of 5-tert-butyl-3-imidazole-4-carboxylic acid ethyl ester (3.30 g, 16.8 mmol) in THF (60 ml) was added to a suspension of lithium aluminum hydride in THF (40 ml) ( 95% suspension, 0.89 g, 22.2 mmol) and the mixture was stirred at room temperature for 3 hours. Water was added until gas evolution ceased and the mixture was stirred for 10 minutes before being filtered through a sintered funnel. The precipitate was washed with THF followed by methanol and the filtrate and washings were combined and evaporated. The residue was lyophilized overnight to give (5-tert-butyl-3H-imidazol-4-yl) -methanol (2.71 g, 105%) as a white solid. (Durant et al., “Aminoalkylimidazoles and Process for theirs”
Production "), Patent No. GB1341375 (UK, 1973)).

_{HPLC (240nm) t R = 3.70} (67.4%) min.

¹ H NMR (400 MHz, CD ₃ OD) δ 1.36 (s, 9H); 4.62 (s, 2H); 7.43 (s, 1H).

¹³ C NMR (100 MHz, CD ₃ OD) δ 31.1, 33.0, 57.9, 131.4, 133.9, 140.8.

_{LC / MS t R = 3.41 (} 155.2 [M + H] +) min.

  This material was used without further purification.

  5-tert-Butyl-3H-imidazole-4-carbaldehyde

  Manganese dioxide (30 g, 0.35 mol) was added to a heterogeneous solution of (5-tert-butyl-3H-imidazol-4-yl) -methanol (4.97 g, 0.03 mol) in acetone (700 ml). The resulting mixture was stirred at room temperature for 4 hours. The mixture was filtered through a celite pad and the pad was washed with acetone. The filtrate and washings were combined and evaporated. The residue was triturated with ether to give 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 51%) as a colorless solid. (Hayashi, personal communication (2000)).

_{HPLC (240nm) t R = 3.71} (89.3%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.48 (s, 9H); 7.67 (s, 1H); 10.06 (s, 1H).

_{LC / MS t R = 3.38 (} 153.2 [M + H] +) min.

  Additional 5-tert-butyl-3H-imidazole-4-carbaldehyde (1.88 g, 38%) was obtained by evaporation of the trituration-derived filtrate.

  1-acetyl-3- (5'-tert-butyl-1H-imidazole-4'-Z-ylmethylene) -piperazine-2,5-dione

  To a solution of 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 164.4 mmol) in DMF (50 ml) was added 1,4-diacetyl-piperazine-2,5-dione (6.50 g). , 32.8 mmol) was added and the solution was evacuated before flushing with argon. After the evacuation-pour process was repeated two more times, cesium carbonate (5.35 g, 16.4 mmol) was added. After the evacuation-casting process was repeated three more times, the resulting mixture was stirred at room temperature for 5 hours. The reaction mixture was evaporated to some extent until it was small (heated under high vacuum) and the resulting solution was added dropwise to water (100 ml). The yellow precipitate was collected and then lyophilized to give 1-acetyl-3- (5′-tert-butyl-1H-imidazole-4′-Z-ylmethylene) -piperazine-2,5-dione (2.24 g). 47%). (Hayashi, personal communication (2000)).

_{HPLC (214nm) t R = 5.54} (94.4%) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.47 (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).

¹³ C NMR (100 MHz, CDCl ₃ + CD ₃ OD) δ 27.3, 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 "-tert-butyl-1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5-dione

A solution of 1-acetyl-3- (5′-tert-butyl-1H-imidazole-4′-Z-ylmethylene) -piperazine-2,5-dione (2.43 g, 8.37 mmol) in DMF (55 ml). To was added benzaldehyde (4.26 ml, 41.9 mmol) and the solution was evacuated and flushed with nitrogen. After the exhaust-pour process was repeated two more times, cesium carbonate (4.09 g, 12.6 mol) was added. After the evacuation-casting process was repeated three more times, the resulting mixture was heated under a temperature gradient as shown below. After a total time of 5 hours, the reaction was cooled to room temperature and the mixture was added to ice cold water (400 ml). The precipitate was collected, washed with water, and lyophilized to give a yellow solid (2.57 g, HPLC (214 nm) t _R = 6.83 (83.1%) min). This material was dissolved in chloroform (100 ml) and the remaining water was evaporated azeotropically to yield a brown oil. This was dissolved in chloroform (20 ml) and cooled in ice. After 90 minutes, the yellow precipitate was collected and air dried to give 3-Z-benzylidene-6- (5 "-tert-butyl-1H-imidazole-4" -Z-ylmethylene) -piperazine-2,5- Dione (1.59 g, 56%) was obtained. (Hayashi, personal communication (2000)).

_{HPLC (214nm) t R = 6.38} (2.1%), 6.80 (95.2) min.

¹ H NMR (400 MHz, CDCl ₃ ) δ 1.46 (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) min.

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

Additional 3-Z-benzylidene-6- (5 "-tert-butyl-1H-imidazol-4" -Z-ylmethylene) -piperazine-2,5-dione (0.82 g, 29%) by evaporation of the chloroform solution was gotten. _{HPLC (214nm) t R = 6.82} (70.6%) min.

General experimental sodium bicarbonate refers to a 5% solution.

  Organic solvents were dried over sodium sulfate unless otherwise stated.

Analysis conditions NMR conditions
¹ H NMR (400 MHz) analysis was performed on a Varian Inova Unity 400 MHz NMR machine. Samples were measured in deuterated chloroform containing 0.1% TMS (unless otherwise noted). Chemical shift (ppm) refers to 3.30 ppm of CH ₃ OH for samples measured in TMS (0.00 ppm) or CD ₃ OD. The coupling constant is expressed in hertz (Hz).

Analytical HPLC condition system 6 conditions:
RP-HPLC was performed on a Rainin Microsorb-MV C18 (5 μm, 100 、) 50 × 4.6 mm column.
Buffer A: 0.1% aqueous TFA
Buffer B: 0.1% TFA in 90% MeCN water
Gradient: 0% to 100% buffer B over 11 minutes
Flow rate: 1.5 mL / min

LCMS conditions LCMS was performed on a Perkin-Elmer Sciex API-100 instrument. LC conditions:
Reversed phase HPLC analytical column: Monitor 5 μm C18 50 × 4.6 mm
Solvent A: 0.1% TFA in water
Solvent B: 0.085% TFA in 90% MeCN water
Gradient: 0% to 100% B over 11.0 minutes
Flow rate: 1.5 mL / min Wavelength: 214 nm
MS conditions:
Ion source: ion spray detection: flow rate for ion counting mass spectrometer: 300 μL / min (1.5 mL / min) after splitting from the column.

ESMS Conditions ESMS was performed on a Perkin Elmer / Sciex-API III LC / MS / MS using an electrospray inlet.
Solvent: 0.1% AcOH in 60% MeCN water
Flow rate: 25 μL / min Ion spray: 5,000 V
Orifice plate: 55V
Collection time: 2.30 minutes Scan range: 100-1,000 amu / z
Scan step size: 0.2 amu / z

Preparative RP-HPLC purification conditions Reversed phase HPLC purification was performed using Nebula on a Waters XterraMS column (19 x 50 mm, 5 μm, C18) using the following conditions: Solvent A: 0.1 % TFA water solvent B: 0.1% TFA in 90% MeCN water
Gradient: 5% to 95% B over 4 minutes
Flow rate: 20 mL / min Wavelength: 214 nm

Abbreviations are as follows: brs: broad singlet, BuLi: n-butyllithium, d: doublet, DBU: 1,8-diazabicyclo [5.4.0] undec-7-ene, ESMS: electrospray mass spectrometry , HCl: hydrochloric acid, HPLC: high performance liquid chromatography, LCMS: liquid chromatography mass spectrometry, LD: lithium diisopropylamide, M ⁺ : molecular ion, m: multiplet, MeCN: acetonitrile, M: mass spectrometry, MW: molecular weight, NMR: nuclear magnetic resonance, q: quartet, s: singlet, t: triplet, t _R : retention time, TFA: trifluoroacetic acid, THF: tetrahydrofuran

Detailed procedure for the synthesis of dehydrophenilahistine

  1-acetyl-3-{(Z) -1- [5- (1,1-dimethyl-2-propenyl) -1H-4-imidazolyl] methylidene}]-2,5-piperazinedione (2)

To a solution of 5- (1,1-dimethyl-2-propenyl) imidazole-4-carboxaldehyde (100 mg, 0.609 mmol) in DMF (2 mL) was added compound 1 (241 mg, 1.22 mmol). The solution was repeatedly evacuated to remove oxygen, flushed with Ar, followed by the addition of Cs ₂ CO ₃ (198 mg, 0.609 mmol) and the evacuation-pour process was repeated again. The removal of oxygen is preferred because such removal is believed to reduce the oxidation of the alpha carbon at the 6-position of the diketopiperazine ring. The resulting mixture was stirred at room temperature for 5 hours. After removal of the solvent by evaporation, the residue was dissolved in a mixture of EtOAc and 10% Na ₂ CO _3, the organic phase is washed three times with 10% Na ₂ CO ₃ once more, and saturated NaCl, Na ₂ CO Dried by ₃ and concentrated in vacuo. The residual oil was purified on silica by column chromatography using CHCl ₃ -MeOH (100: 0 to 50: 1) as eluent to give 2, 60 mg (33%) of a pale yellow solid.

To a solution of 2 (30 mg, 0.099 mmol) in dehydrophenylhistine DMF (0.8 mL), benzaldehyde (51 μL, 0.496 mmol, 5 eq) was added and the solution was repeatedly evacuated over a short period of time, Oxygen was removed and Ar was flushed, followed by the addition of Cs ₂ CO ₃ (53 mg, 0.149 mmol, 1.5 eq) and the evacuation-pouring process was repeated again. The resulting mixture was heated at 80 ° C. for 2.5 hours. (The temperature must be increased gradually. Rapid heating increases the production of the E isomer at the benzylidene moiety.) After removing the solvent by evaporation, the residue is dissolved in EtOAc and washed with 2% water. Washed three times with saturated NaCl, dried over Na ₂ SO ₄ and concentrated in vacuo. Spots with bright yellow-green emission at 365 nm UV can be observed on TLC using CHCl ₃ -MeOH (10: 1). The purity of this crude product was 75% or more from HPLC analysis. The resulting residue was dissolved in 90% MeOH water and applied to a reverse phase HPLC column (YMC-Pack, ODS-AM, 20 × 250 mm) using a linear gradient from 70% MeOH to 74% MeOH in water. Elution over a period of 16 minutes at a flow rate of 12 mL / min, the desired fractions were collected and concentrated by evaporation to give 19.7 mg (60%) of yellow dehydrophenylahistine, the yield for each step Not optimized for.

Biological properties of dehydrophenylahistine and dehydrophenylahistine analogs Biological evaluation The biological properties of the synthesized tBu-dehydrophenylahistine and dehydrophenylahistine were evaluated in both HT29 human colon cells and PC-3 prostate adenocarcinoma cells.

HT-29 (ATCC HTB-38), a human colorectal adenocarcinoma, was prepared from McCoy's complete medium (10% FBS, 1 mM Na pyruvate, 1 × NEAA, 2 mM L-glutamine and 100 IU / ml and 100 μg / ml Pen). / McCoy 5A medium with L-glutamine and 25 mM HEPES supplemented with / Strep, respectively. PC-3 (ATCC CRL-1435), ie human prostate adenocarcinoma, F12K complete medium (10% FBS, 2 mM glutamine, 1% HEPES, and F12K medium supplemented with 100 IU / ml and 100 μg / ml Pen / Strep, respectively) ) Maintained in. Cell lines were cultured in a 95% humidified incubator at 37 ° C., 5% CO ₂ .

For tumor cytotoxicity assays, HT-29 or PC-3 cells are seeded in Corning 3904 black-walled clear-bottom culture plates at 5,000 cells / well in 90 μl of complete medium. The plates were incubated overnight to allow the cells to establish and enter a logarithmic growth phase. A 20 mM stock solution of dehydrophenylahistine and tBu-dehydrophenylahistine was prepared in 100% DMSO and stored at −20 ° C. Ten-fold concentrated serial dilutions of the two compounds were prepared in appropriate culture media to a final concentration ranging from 2.0 × 10 −5 M to 2.0 × 10 ⁻¹⁰ M. A 10 μl volume of 10 × serial dilution was added to the test wells in triplicate and the plates were returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.

48 hours after drug exposure, 10 μl of 0.2 mg / ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in PBS without Mg ²⁺ , Ca ²⁺ was added to each well. The plate was then returned to the incubator for 3-4 hours. Plates were removed and resazurin fluorescence was measured using a 530 nm excitation and 590 nm emission filter in a Fusion fluorimeter (Packard Instruments). Background was determined using resazurin dye without cells and subtracted from the data for all experimental wells. Data was analyzed using Prism software (GraphPad Software). Data were normalized to the mean of cells treated with medium alone (100% cell growth) and EC ₅₀ values were determined using a standard sigmoidal dose response curve fitting algorithm.

  As shown in Table 1 below, tBu-dehydrophenillahistin shows about 4 times greater cytotoxic activity compared to dehydrophenilahistine.

  See also FIG. 41 for further data on HT-29, PC-3 and P-388 cells.

B. Structure and activity studies of dehydrophenylahistine derivatives The cytotoxic effects of phenylahistine, dehydrophenylahistine and various derivatives of dehydrophenylahistine were compared to P388 mouse leukemia cells, HT-29 human colon cells, and PC-3 prostate adenocarcinoma cells. Inspected with.

As explained above, HT-29, a human colorectal adenocarcinoma, was prepared using McCoy's complete medium (10% FBS, 1 mM Na pyruvate, 1 × NEAA, 2 mM L-glutamine and 100 IU / ml and 100 μg / ml). McCoy's 5A medium with L-glutamine and 25 mM HEPES, each supplemented with Pen / Strep. PC-3, a human prostate adenocarcinoma, was maintained in F12K complete medium (F12K medium supplemented with 10% FBS, 2 mM glutamine, 1% HEPES, and 100 IU / ml and 100 μg / ml Pen / Strep, respectively). Cell lines were cultured in a 95% humidified incubator at 37 ° C., 5% CO ₂ .

For tumor cytotoxicity assays, HT-29 or PC-3 cells are seeded in Corning 3904 black-walled clear bottom tissue culture plates at 5,000 cells / well in 90 μl complete medium and the plates are incubated overnight. The cells were established and entered the logarithmic growth phase. A 20 mM stock solution of dehydrophenylahistine and tBu-dehydrophenylahistine was prepared in 100% DMSO and stored at −20 ° C. Ten-fold concentrated serial dilutions of the two compounds were prepared in appropriate culture media to final concentrations ranging from 2.0 × 10 ⁻⁵ M to 2.0 × 10 ⁻¹⁰ M. A 10 μl volume of 10 × serial dilution was added to the test wells in triplicate and the plates were returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.

48 mg after drug exposure, 0.2 mg / ml resazurin in PBS without Mg ²⁺ , Ca ²⁺ (obtained from Sigma-Aldrich Chemical Co.)
10 μl was added to each well and the plate was returned to the incubator for 3-4 hours. Plates were removed and resazurin fluorescence was measured using a 530 nm excitation and 590 nm emission filter in a Fusion fluorimeter (Packard Instruments). Background was determined using resazurin dye without cells and subtracted from the data for all experimental wells. Data was analyzed using Prism software (GraphPad Software). Data were normalized to the mean of cells treated with medium alone (100% cell growth) and EC ₅₀ values were determined using a standard sigmoidal dose response curve fitting algorithm.

The EC ₅₀ and IC ₅₀ values for phenylahistine, dehydrophenylahistine and dehydrophenylahistine derivatives are summarized in Table 2 below.

Modifications to the phenyl ring have a significant effect on cytotoxic activity. Compared to the activity of tBu-dehydrophenilahistine (# 6), the activity of the methoxy group (KPU-9) in the meta position is IC ₅₀ 20.8 ± 3.3 nM in P388 cells, compared with other derivatives. The highest activity was shown in comparison. KPU-9 derivatives also showed cytotoxicity in HT-29 cells (EC ₅₀ 31 nM). Dehydrophenylahistine, tBu-dehydrophenylahistine (KPU-2) and KPU-9 derivatives were all cytotoxic in P388 cells.

C. Further structure and activity studies of dehydrophenylahistine derivatives The cytotoxic effects of phenylahistine, dehydrophenylahistine and various derivatives of dehydrophenylahistine were determined using the above methodology, using HT-29 human colon cells and PC-3 prostate gland Tested with cancer cells.

  Further cytotoxicity assays were performed as described above under this example using resazurin fluorescence as an indicator of cell viability. The results are shown below in Table 3.1.

Other dehydrophenylahistine analogs Modifications relating to the synthesis of dehydrophenylahistine derivatives Other derivatives of dehydrophenylahistine are synthesized using the techniques described above alone or in combination with other known organic synthesis techniques.

The modifications to the diacyl diketopiperazine and the first and second aldehydes involved in the synthesis method vary depending on the desired derivative to be produced. Derivatives with the following are synthesized:
A) modifying the phenyl ring and / or introducing other aromatic ring systems;
B) changing the position of the aromatic ring,
C) changing the imidazole aromatic ring system and / or D) modifying the 5-position on the imidazole ring.

  The figure below represents a region of dehydrophenylahistine compounds that are modified to produce derivatives of dehydrophenylahistine. Non-limiting examples of modifications are disclosed and will be understood by those skilled in the art based on this disclosure.

Developing the above modifications to dehydrophenylahistine compounds, the derivatives of the compounds have the following substituents (A) on the phenyl ring: —CF ₃ , —SO ₂ NH ₂ (—SO ₂ NR ₁ R ₂ ), _{-SO 3 H, -CONH 2 (-CONR} 1 R 2), - may contain such COOH. Other ring structures (C) may also include:

B. Examples of synthesized dehydrophenylahistine derivatives Further examples of synthesized dehydrophenylahistine derivatives are disclosed in Table 4.

C. Evaluation of Dehydrophenillahistine Derivative The evaluation of the above-described derivatives is evaluated according to the method described in Example 3. Further evaluation of derivatives includes specific effects such as inhibitory effects on cell proliferation, effects on specific cellular mechanisms (ie, microtubule function), determination of effects on cell cycle progression, evaluation of in vitro anti-tumor activity against cancer cell lines, etc. Expanded to activity. Some evaluation method protocols are shown below.

1) Cell growth inhibitory effect of dehydrophenilahistine and its analogs In each well of a 96-well microtiter plate, an EMEM culture medium (Nissei Seyak Co., Ltd.) having an antitumor effect on A-549 cells derived from human lung cancer A-549 derived from human lung cancer prepared to 10 ⁵ cells / ml in a culture medium obtained by adding 10% fetal calf serum to a company (Nissui Seiyaku Co., Ltd.) Add 100 μl of cells. The derivative methanol solution obtained from the above example is added to the top row of wells, the specimen is diluted by the semi-log dilution method and added, and the plate is placed in a carbon dioxide gas incubator at 37 ° C. for 48 hours. Incubate. After adding the resulting product to a 10 μl lot containing MTT reagent (3- (4,5-dimethyl-2-thiazole) -2,5-diphenyl-2H-tetrabromide (1 mg / ml PBS), Incubate for 6 hours in a carbon dioxide gas incubator at 37 ° C. Discard the culture medium and dissolve the crystals produced in the cells in 100 μl / well of dimethyl sulfoxide, followed by microscopic absorption of light at 595 nm. Measure with a plate reader Compare the light absorption of untreated cells with the light absorption of cells treated with a known concentration of the sample to calculate the concentration of the sample that inhibits 50% cell growth (IC ₅₀ ).

2) Cell cycle inhibitory activity of dehydrophenilahistine and its analogs Cell line A431 is derived from human lung cancer. Using EMEM culture medium containing 10% fetal bovine serum and 1% MEM non-essential amino acid solution (SIGMA M2025), A431 cells were incubated at 37 ° C. in an incubator saturated with 5% carbon dioxide gas and water vapor. Incubate with. A purified sample of dehydrophenylahistine obtained by the above method is added to cells in the logarithmic growth phase, and the progression of the cell cycle is analyzed by flow cytometry and microscopic observation.

  The effect on the cell cycle progression of HeLa cells is shown in FIG.

Structure-activity relationships of synthesized dehydrophenylahistine (dehydroPLH) derivatives 1) Summary of derivative synthesis Many (but not all) of the derivatives of dehydroPLH disclosed herein include one on the phenyl ring, Includes 2 or 3 modifications (Table 5 below). The derivative was synthesized by the method described above. As shown in Table 5, certain compounds showed more potent cytotoxic activity than dehydroPLH and tBu-dehydroPLH. The most potent compound showing an EC ₅₀ value of 3 nM was KPU-90. This value was 16 and 4 times higher than that of dehydroPLH and tBu-dehydroPLH, respectively. These derivatives have monosubstitution at the o or m position of the phenyl ring with halogen atoms such as fluorine and chlorine atoms or methyl, vinyl or methoxy groups. Derivatives with substitutions on heteroaryl structures such as naphthalene, thiophene and furan rings also elicited potent activity. KPU-35, 42, 69, 80 and 81 also showed higher activity than tBu-dehydroPLH.

2) Introduction of methoxy group to phenyl ring Colchicine recognizes the same binding site on β-tubulin as PLH. Colchicine has four characteristic methoxy groups on its A and B rings. A series of substitutions with single or multiple methoxy groups were performed. The results of cytotoxic activity are shown in Table 6.

  The results showed that substitution at the m or o position increased cytotoxic activity against HT-29 cells. KPU-9 and 16 showed high activity. Methoxy derivatives with triple substitution (KPU-11, 17 and 45) also showed activity. The structure of KPU-24 was confirmed by mass spectrometry.

3) Modification with electron withdrawing groups In order to study more extended structure-activity relationships on the phenyl ring, a series of different functional groups were introduced, including both electron withdrawing groups and electron donating groups. The results of cytotoxicity against HT-29 cells are shown in Table 7 and Table 8, respectively.

  Substitution at the o or m position effectively increased activity. These results were in good agreement with the methoxy group.

  The present disclosure is not bound or limited to any particular scientific theory. Nonetheless, those of ordinary skill in the art will appreciate that the results presented herein may be preferred to induce more potent activities with relatively small functional groups that do not significantly affect steric hindrance. Slightly larger groups such as ethoxy groups (compared to methoxy groups) or Br atoms (compared to Cl atoms) can affect steric hindrance, which is undesirable for interaction with tubulin binding sites, for example. It will be understood that this may be interpreted as implying. Furthermore, since the electrical properties of these substituents did not affect activity, these relatively small substituents do not interact directly with the β-tubulin binding site, but are suitable for dehydroPLH binding. It is suggested to restrict the conformation of. Alternatively, another possible hypothesis is that the introduction of hydrophilic hydroxyl groups that can form hydrogen bonds as hydrogen donors dramatically reduces activity, so that the hydrophobic property is on β-tubulin. It may be a more important factor at the binding site at the o or m position.

  As shown in Table 9, the effect of substituents on cytotoxic activity at the o-position may be ordered as in the case of the m-position as shown in Table 10. Compounds with effective functional groups that showed higher activity than tBu-dehydroPLH may also be further modified. In addition, since a shift in stereochemistry from Z to E was observed under irradiation with visible light, a substituent that decreases the electron density in the conjugated double bond may contribute to a decrease in the shift from Z to E by light. Yes, resulting in a more physicochemically more stable structure. Temperature can also cause this transition.

Modifications at two sites on the ring are powerful but may be desirable for the development of biologically stable compounds at the same time. The phenyl ring of phenylahistine is oxidized by cytochrome P-450. Thus, a double modification that reduces the electron density of the phenyl ring may be effective to avoid P-450 oxidation. Thus, combinations of small electron withdrawing groups such as fluorine atoms for elements that can increase activity such as -OMe, -Me, -Cl, -F and Br are more powerful and biologically stable. Drug compounds can be produced.

4) Substitution of phenyl ring to aryl heterocycle The phenyl ring may also be replaced by a heteroaryl group. The results of such replacement for cytotoxic activity are shown in Table 11. The aryl nitrogen atom forms a hydrogen bond with the NH group of the diketopiperazine ring and can restrict the conformation of the molecule between the pyridine and the diketopiperazine ring to a single plane, thus allowing the active conformation of dehydroPLH. The formation requires a certain level of dihedral angle formed by steric repulsion between the amide hydrogen atom of the diketopiperazine ring and the o-hydrogen atom of the phenyl ring (FIG. 6).

  Replacement of the phenyl ring with a smaller furan or thiophene ring (eg KPU-29 or KPU-42) showed activity. The phenyl ring can be changed to other aromatic structures while maintaining strong activity.

5) Metabolism of Phenylahistin In his recent study, (±) -phenylhistine was treated with rat liver microsomes or human liver P450. In the human case, at least seven metabolites were detected, two of which, namely P1 and P3, were major metabolites, representing more than 60% of the recovered metabolites.

Since there is no exoolefin structure in tBu-dehydroPLH, the synthesized derivatives of the present invention do not have oxidation like P1 and P4. However, oxidations such as P3 and P5 are formed during liver metabolism. Various derivatives that prevent such metabolism are effective in avoiding P450 oxidation on the phenyl ring. The imidazole ring can also be modified to avoid undesired oxidation.

6) Physicochemical stability of dehydro PLH Physicochemical stability is one of the undesirable problems of dehydro PLH. Phenylhistine does not have this problem because there is no additional olefin structure in the benzyl moiety. However, in dehydroPLH, the benzylidene moiety can be readily activated, probably with visible light, and the transition from Z to E frequently occurs due to the presence of longer conjugates of double bonds. This transition occurred even under standard room lighting. In the cytotoxicity assay, some compounds migrate to the E form during incubation, but this transition is equilibrated at a 1: 1 ratio in the case of dehydroPLH. This transition can be controlled. This transition from Z to E is also known for combretastatin A, the same type as tubulin inhibitors, and several studies have been reported to ameliorate this problem.

7) Prodrug synthesis The E form may also be used as a prodrug of dehydroPLH, or one or more of its analogs, including the analogs described herein. One of the undesirable properties of anti-tubulin drugs involves low selectivity between tumor and intact tissue, but these drugs belong to one of the molecular targeted therapies. This causes undesirable side effects. However, the negative side effects of anti-microtubule drugs can be reduced if the compound functions selectively only in tumor tissue. Since dehydroPLH (Z form) can be produced from its E isomer by irradiation with visible light, when E form is administered and light irradiation is carried out only at the tumor site, only the tumor is produced by light. Damaged by the body, adverse effects on intact tissue are reduced.

  The E form is bulky but can be chemically protected by the addition of a biodegradable acyl group, which is introduced into the diketopiperazine ring as a prodrug. This acyl group can be cleaved by a protease in the body. Therefore, the acylated E compound can be maintained before administration, and after administration, it can be changed to a true E form and transferred to a physiologically active Z form by local light irradiation.

  A synthetic scheme for this acyl E form of tBu-dehydroPLH is outlined in FIG.

Synthetic pharmaceutical formulation of dehydrophenylahistine 1) Formulation administered intravenously by infusion, injection, infusion, etc. 10 mg of the compound synthesized by the above method is aseptically added to each vial containing 5 g of powdered glucose. Add and seal. After filling with nitrogen, helium or other inert gas, store the vial in a cool dark place. Prior to use, the contents are dissolved in ethanol and added to 100 ml of 0.85% saline solution. The resulting solution would be suitable by those skilled in the art as a method of inhibiting the growth of cancerous tumors, from about 10 ml / day to about 1,000 ml / day in humans diagnosed with such tumors. Intravenously by infusion or by subcutaneous or intraperitoneal injection.

2) Formulation to be administered orally etc. A mixture obtained by thoroughly mixing 1 g of the compound synthesized by the above method, 98 g of lactose and 1 g of hydroxypropylcellulose is formed into granules by any conventional method. The granules are completely dried and sieved to obtain a granule preparation suitable for packaging in a bottle or by heat sealing. The resulting granule preparation is administered orally at about 100 ml / day to about 1,000 ml / day, depending on the symptoms, as would be appropriate by one skilled in the art of treating cancerous tumors in humans. Is done.

3) Formulations to be administered topically Administration of an effective amount of a compound to an individual can also be accomplished topically by administering the compound (s) directly to the affected area of the individual's skin. For this purpose, the compound to be administered or applied can be a pharmaceutically acceptable topical carrier such as a gel, ointment, lotion or cream (which can be water, glycerol, alcohol, propylene glycol, fatty alcohol). , Triglycerides, fatty acid esters, or carriers such as mineral oils). Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate in water (5%), or sodium lauryl sulfate (5%) in water. Other materials such as antioxidants, wetting agents, viscosity stabilizers, and similar agents may be added as needed. A transdermal penetration enhancer such as Azone may also be included. Further, it is anticipated that in certain situations, the compound may be placed in a device that is placed on, in or under the skin. Such devices include patches, implants, and injections that release compounds to the skin by either passive or active release mechanisms.

In Vitro Pharmacology of KPU-2, KPU-35 and t-Butylphenylhistine As an in vitro efficacy study conducted using KPU-2, KPU-35 and t-butylphenylhistine A) a panel of 6 tumor cell lines, B) studies in multidrug resistant tumor cells, and C) studies to determine the mechanism of action.

A) Study of KPU-2, KPU-35 and t-Butylphenylhistine in a panel of 6 tumor cell lines The following cell lines (source in parentheses) were used: HT29 (human colon tumor, ATCC, HTB) -38), PC3 (human prostate tumor, ATCC, CRL-1435), MDA-MB-231 (human breast tumor, ATCC, HTB-26), NCI-H292 (human non-small cell lung tumor, ATCC, CRL-1848) ), OVCAR-3 (human ovarian tumor, ATCC, HTB-161), B16-F10 (mouse melanoma, ATCC, CRL-6475) and CCD-27sk (normal human fibroblasts, ATCC, CRL-1475). Cells were maintained at subconfluent density in their respective culture media.

  Cytotoxicity assays were performed as described above in Example 4 using resazurin fluorescence as an indicator of cell viability.

The disclosed compounds are effective agents against a variety of different and distinct tumor cell lines. Specifically, for example, KPU-2 and KPU-35 are both HT-29 tumor cell lines with respect to both efficacy (active in the low nanomolar range) and potency (most responsive for maximum cytotoxic effects). Was the most effective. t-Butyl-phenilahistine showed its maximum efficacy against the PC-3 tumor cell line, while maximum potency was demonstrated against the HT-29 cell line. KPU-2 and KPU-35 were generally 10-40 times more potent than t-butyl-phenylhistine, whereas potency was similar for all three compounds in various tumor cell lines. HT-29, PC-3, MDA-MB-231 and NCI-H292 tumor cell lines all responded similarly to NPI compounds, whereas B16-F10 appeared to be somewhat less sensitive. . t-Butyl-phenilahistine showed a significant difference between normal fibroblasts and tumor cell lines at a rate of> 20 to> 100, except for the OVCAR-3 cell line.

B) Studies in drug resistant cell lines One of the major challenges in the use of chemotherapeutic agents in clinical oncology is the development of resistance to drug effects by tumor cells. There are several mechanisms for the development of resistance, each of which has a different effect on chemotherapeutic drugs. These mechanisms include increased expression of ATP-dependent efflux pumps such as PDR-glycoprotein encoded by MDR1 or multidrug resistance associated protein 1 encoded by MRP1. Reduced drug intake, altered drug targets, increased repair of drug-induced DNA damage, altered apoptotic pathways, and activation of cytochrome P450 enzymes are in addition to the mechanisms by which cancer cells become resistant to anticancer drugs. It is an example. Selected compounds were studied in three different cell lines showing two different resistance mechanisms: overexpression of P-glycoprotein and altered topoisomerase II activity.

1) Human uterine sarcoma tumor cell line pair: MES-SA (taxol sensitive) and MES-SA / Dx5 (taxol resistant)
This cell line expresses elevated mdr-1 mRNA and P-glycoprotein (efflux pump mechanism). Pretreatment with cyclosporin A (CsA) inhibits P-glycoprotein and restores activity in resistant cell lines for compounds resulting from increased resistance P-glycoprotein.

As can be seen from Table 13, KPU-2 and KPU-35 had similar efficacy in sensitive cell lines as sensitive systems, and the effectiveness of t-butyl-phenylhistine was only slightly reduced. Cyclosporine A (CsA) pretreatment did not change the effectiveness of the selected compounds. In contrast, taxol was virtually inactive in MES-SA / DX5-resistant cell lines, whereas this compound was very potent in sensitive cell lines. CsA treatment restored the sensitivity of the MES-SA / DX5 cell line to taxol. The MES-SA / DX5 cell line also showed reduced sensitivity to etoposide (60-fold), doxorubicin (34-fold) and mitozantrone (20-fold).

  These data show that the effects of KPU-2, KPU-35 and t-butyl-phenylhistine are not affected by the taxol-related resistance mechanism (p-glycoprotein) in this cell line, and taxol-derived cross-resistance is not , Indicating that this model does not occur for these selected compounds.

  See further data shown in FIG.

2) Human acute promyelocytic leukemia cell line pair: HL-60 (mitoxantrone sensitive) and HL-60 / MX-2 (mitoxantrone resistant)
This cell line is considered to have atypical drug resistance properties with altered topoisomerase II catalytic activity without overexpression of P-glycoprotein.

  As can be seen in Table 14, these results indicate that the efficacy of the selected novel compounds is very similar in sensitive and resistant HL-60 cell lines. In contrast, mitoxantrone loses 24 fold potency by certain factors in resistant HL-60 / MX-2 cell lines.

  Thus, KPU-2, KPU-35, and t-butyl-phenylhistine are not affected by the same resistance mechanism as mitoxantrone in this cell line, and in this model mitoxantrone-derived to these selected novel compounds. There is no cross resistance.

3) Human breast carcinoma cell line pair: MCF-7 (taxol sensitive) and MCF-7 / ADR (taxol resistant)
This study involves KPU-2 compared to taxol. KPU-2 showed similar efficacy in both sensitive and resistant members of this cell line pair. In contrast, taxol was virtually inactive in resistant cell lines, whereas there was low nanomolar efficacy in sensitive cell lines (Table 15).

  These studies confirm that taxol resistance does not transfer to KPU-2 in various human tumor cell lines.

C) Study of mechanism of action 1) Action on microtubule function To evaluate the effect of KPU-2 and t-butyl-phenhistine on tubulin compared to colchicine and taxol by staining with α-tubulin. Human umbilical vein endothelial cells (HuVEC from Cambrex) were used in this study.

  30 minutes exposure to KPU-2, t-butyl-phenylhistine or colchicine (all at 2 μM) resulted in a lack of intact microtubule structure and cell membranes in HuVEC cells compared to that observed in DMSO controls. Taxol did not induce microtubule depolymerization under these conditions, whereas microtubule depolymerization was indicated by blebbing (a clear indicator of apoptosis). Colchicine is a known microtubule depolymerizer, whereas taxol is a tubulin stabilizer. Similar results were obtained when CCD-27sk cells were exposed to KPU-2 or colchicine.

2) Induction of apoptosis Apoptosis and its dysregulation play an important role in oncology. Selective induction of the programmed cell death cycle in tumor cells is the goal of many chemotherapeutic drug discovery programs. This induction of apoptosis is characterized by characteristic cell membrane blebbing, DNA fragmentation, hyperphosphorylation of the anti-apoptotic factor Bcl-2, activation of the caspase cascade and cleavage of poly (ADP ribose) polymerase (PARP) It can be proved by various methods including:

  Characteristic signs of apoptotic cell death include cell membrane blebbing, nuclear disruption, cell contraction and condensation, and ultimately cell death very different from necrotic cell death. KPU-2 induced typical morphological changes associated with the early stages of apoptosis in human prostate tumor cells. A similar view was also evident in the treatment of HuVEC cells with KPU-2.

3) DNA fragmentation A feature of the final stage of apoptosis is DNA cleavage between nucleosomes that produces a unique ladder pattern that can be visualized by gel electrophoresis. Using this approach, the effect of KPU-2 on DNA laddering in Jurkat cells (human T-cell leukemia line) compared to halimide and dehydrophenylahistine (KPU-1) did. KPU-2 induced DNA laddering at a concentration of 1 nM, whereas harimid and KPU-1 were much less effective.

4) Activation of the caspase cascade Several enzymes in the caspase cascade including caspase-3, caspase-8 and caspase-9 are activated during apoptosis. The activity of caspase-3 was monitored in Jurkat cells after treatment with KPU-2, KPU-35 and t-butyl-phenylhistine.

The results show that caspase-3 was activated in a dose-dependent manner by treatment with all three compounds in a manner similar to Harimide. Caspase-3 activation occurred over a similar concentration range for cytotoxic IC ₅₀ in Jurkat cell lines (Table 16).

5) Cleavage of poly (ADP-ribose) polymerase (PARP) in Jurkat cells To assess the ability of these compounds to induce apoptosis in Jurkat cells, cleavage of poly (ADP-ribose) polymerase (PARP) Was monitored. PARP is a 116 kDa nuclear protein that is one of the major intracellular targets of caspase-3. Cleavage of PARP produces a stable product of 89 kDa, and this process can be easily monitored by Western blotting. Caspase cleavage of PARP is one of the attributes of apoptosis and therefore serves as an excellent marker for this process. KPU-2 at 100 nM induced PARP cleavage in Jurkat cells 10 hours after exposure of the cells to the compound. KPU-2 appeared to be more active than either Harimide or KPU-1.

6) Enhanced vascular permeability in HuVEC cells Compounds that depolymerize microtubules (eg combretastatin A-4-phosphate, ZD6126) have been shown to induce vascular collapse in tumors in vivo. ing. This vascular collapse occurs after rapid induction of vascular cell permeability first to the electrolyte and then to the immediate macromolecule. Increased permeability of HuVEC cells to fluorescently labeled dextran is used as a surrogate assay for vascular collapse.

  KPU-2, KPU-35 and t-butyl-phenylhistine all rapidly (within 1 hour) induced significant HuVEC monolayer permeability to the same extent as colchicine. Taxol, a microtubule stabilizer, was inactive in this assay (Figure 12).

7) Profile in a wide range of kinase screens KPU-2 was initially screened at a concentration of 10 μM in a panel of 60 different kinases. The ATP concentration was 10 μM. Four kinases were inhibited by more than 50% in the primary screen and the IC ₅₀ determined in the secondary screen is presented in Table 17. All IC ₅₀ values are in the low micromolar range, which indicates that inhibition of these kinases is not related to the low nanomolar activity observed with respect to tumor cell cytotoxicity.

In vivo pharmacology Preliminary studies with KPU-2 were performed in mice using MX-1 (chest) and HT-29 (colon) xenograft models and the P-388 mouse leukemia tumor model. Other tumor models selected based on activity in the in vitro tumor panel were the DU-145 (prostate), MCF-7 (chest), and A549 (lung) cell lines. A human pancreatic tumor (MiaPaCa-2) was also included. New compounds were studied as monotherapy and in combination with clinically used chemotherapeutic agents. The doses of selected novel compounds were determined by an acute tolerability test (maximum tolerated dose, MTD) and adjusted as needed during each study. The dose of chemotherapeutic agent used clinically was selected based on historical studies.

  KPU-2 was the first compound to be studied in these five tumor models. In accordance with the initial results of this study, all three compounds were compared in HT-29 human colon tumor, DU-145 human prostate and MCF-7 human breast tumor xenograft models.

All the above models use the subcutaneous xenograft implantation technique and are potentially subject to the selective effects of compounds on the subcutaneous vasculature resulting in expanded (or apparent) antitumor activity. Cheap. To prevent this possibility, two other tumor models have been incorporated into the study. One of these is the observation of lung metastases after intravenous injection of B16-F10 mouse melanoma tumor cells. Another model is transplantation of MDA-231 human breast tumor cells in mouse mammary fat pad. Although this latter model is a xenograft model, the subcutaneous vasculature plays no role.

Method 1) Xenograft model The animals used (exceptions are indicated for individual studies) are 5-6 weeks old female nude mice (nu / nu) (~ 20 g, Harlan). there were. Group sizes were 9-10 mice per group unless otherwise stated.

  The cell lines used for tumor transplantation were HT-29 human colon tumor, MCF-7 human breast tumor, A549 human non-small cell lung tumor, MiaPaCa-2 human pancreatic tumor, DU-145 human prostate tumor.

  Selected novel compounds were administered as monotherapy by the intraperitoneal (ip) route at the doses shown for individual studies. In combination studies, selected reference chemotherapy agents were injected 15-30 minutes prior to compound.

  The carriers used in these studies are DMSO 12.5%, Cremaphor 5% and peanut oil 82.5% for the selected novel compounds, and for Taxotere (1: 3) polysorbate 80: 13% ethanol, for paclitaxel (1: 1) cremaphor: ethanol, for CPT-11, contained 20 mg irinotecan hydrochloride, 45 mg sorbitol NF powder, and 0.9 mg lactic acid per mL of solution. . The pH was adjusted to 7.4 with NaOH or HCl. Saline dilutions are used to achieve the injection concentration used for the reference compound.

HT-29 Human Colon Tumor Model HT-29 tumor fragments collected from tumors growing subcutaneously (sc) in nude mouse hosts were transplanted subcutaneously into animals by trocar. When the tumor size reached 5 mm x 5 mm (about 10-17 days), the animals were matched to the treatment group and the control group. Starting from the first day, the mice were weighed twice a week, and a tumor measurement result was obtained using a caliper twice a week. Tumor measurement results were converted to estimated tumor weight (mg) using the formula (W ² × L) / 2. When the estimated tumor weight of the control group reached an average of 1,000 mg, the mice were weighed and sacrificed to remove the tumor. Tumors were weighed and the average tumor weight per group was calculated and tumor growth inhibition (TGI) was determined for each group (100% —change in average treated tumor weight / change in average control tumor weight × 100).

  In this model, selected new compounds were injected intraperitoneally every 3 days for 15 days [1, 4, 8, 11 and 15 (q3d × 5)] unless otherwise noted for individual studies. CPT-11 was administered intraperitoneally on days 1, 8 and 15 (qw × 3).

MCF-7 human breast tumor model female nude mice (approximately 20 g) were transplanted subcutaneously with 21-day release estrogen (0.25 mg) pellets 24 hours later, and MCF-7 tumor fragments (nude mice). Harvested from subcutaneous tumors in the host) were implanted subcutaneously. Subsequently, the study proceeded as described for the HT-29 model using taxotere as a standard chemotherapeutic agent.

In this model, new compounds were injected by intraperitoneal route daily from day 1 to day 5 (including day 1 and day 5 (qd × 5)) unless otherwise noted for individual studies. . Taxotere was administered intravenously on days 1, 3 and 5 (qod × 3).

A549 Human Lung Tumor Model A549 tumor fragments collected from tumors growing subcutaneously in nude mouse hosts were transplanted subcutaneously into animals by trocar. When the tumor size reached 5 mm x 5 mm (about 10-17 days), the animals were matched to the treatment group and the control group. The remaining studies proceeded as described for the HT-29 model using taxotere and CPT-11 as standard chemotherapeutic agents.

  In this model, unless otherwise noted for individual studies, test compounds were administered ip in a q3d × 5 dose schedule for CPT-11 combination or qd × 5 dose regimen for taxotere. It was administered by the internal route. CPT-11 was administered by the intraperitoneal route on a qw × 3 schedule. Taxotere was administered intravenously in a qod × 3 dose regimen.

MiaPaCa-2 Human Pancreatic Tumor Model MiaPaCa-2 tumor fragments collected from tumors growing subcutaneously in nude mouse hosts were transplanted subcutaneously into animals by trocar. When the tumor size reached 5 mm x 5 mm (about 10-17 days), the animals were matched to the treatment group and the control group. The remaining studies proceeded as described for the HT-29 model using gemcitabine as a standard chemotherapeutic agent.

  In this model, unless otherwise noted for individual studies, test compounds were administered every 3 days on days 1, 4, 7, 10 and 15 (q3d × 5), intraperitoneal route Administered. Gemcitabine was administered by the intraperitoneal route on days 1, 4, 7, and 10 (q3d × 4).

DU-145 Human Prostate Tumor Model A fragment of DU-145 tumor collected from a tumor growing subcutaneously in a male nude mouse host was transplanted subcutaneously into male mice by trocar. When tumors reached approximately 5 mm x 5 mm (about 13-17 days), animals were matched to treatment and control groups. The remaining studies proceeded as described for the HT-29 model using taxotere as a standard chemotherapeutic agent.

  In this model, test compounds were administered by the intraperitoneal route on day 1, day 3, day 5, day 8 and day 11 (q3d × 5) unless otherwise noted for individual studies. . Taxotere was administered intravenously on days 1, 3 and 5 (q2d × 3).

2) Non-subcutaneous transplanted tumor model The animals used were 5-6 week old female nude mice (nu / nu) (MDA-231 study) or B6D2F1 mice (B16-F10 study) (approximately 20 g, Harlan). . Group sizes were 10 mice per group unless otherwise stated.

  The cell lines used were MDA-MB-231 human breast tumor cells and B16-F10 mouse melanoma cells.

  NPI compounds were administered as a monotherapy by the intraperitoneal route at the doses indicated for individual studies. For combination studies, the selected reference chemotherapeutic agent was injected 15-30 minutes before the NPI compound.

2 × 10 ⁶ MDA-231 cells collected from MDA-231 human breast tumor in vitro cell culture were injected into the mammary fat pad of female nude mice. When the tumor size reached 5 mm x 5 mm (approximately 14-28 days), the animals were adapted to the treatment group and the control group. Subsequently, the study proceeded as described for the HT-29 model using paclitaxel as a standard chemotherapeutic agent.

  In this model, test compounds were administered by the intraperitoneal route on days 1, 4, 8, 11, and 15 (q3d × 5) unless otherwise noted for individual studies. . Paclitaxel was administered by intraperitoneal route from day 1 to day 5 (qd × 5).

B16-F10 metastatic mouse melanoma model On day 0, mice were subjected to B16-F10 cells (prepared from an in vitro cell culture of B16-F10 cells) by the iv route. On day 1, mice were randomly extracted into treatment and control groups and treatment was initiated. Starting from day 1, mice were weighed twice a week. All mice were sacrificed on day 16, lungs were removed, weighed, and surface colonies were counted. Results are expressed as mean colonies of treated mice / mean colonies of control mice (T / C) × 100%. Metastatic growth inhibition (MGI) is the number subtracted from 100%. Paclitaxel was the standard chemotherapeutic agent used in this study.

  In this model, test compounds were administered by intraperitoneal route from day 1 to day 5 (qd × 5) unless otherwise noted for individual studies. Paclitaxel was administered intravenously from day 1 to day 5 (qd × 5).

  If appropriate (n ≧ 3), the results are expressed as mean ± SEM. Unless otherwise stated, statistical analysis of studies in several groups was performed by Neuman-Keuls post-test using ANOVA. A one-sided t-test was also used based on the assumption that a compound or drug, or combination thereof, reduces tumor growth.

Results Study of HT-29 human colon tumor xenograft model In vivo evaluation of KPU-2 +/− CPT-11 in the HT-29 human colon tumor xenograft model This study was conducted in the HT-29 model for KPU-2 alone and in combination with the related chemotherapeutic agent CPT-11. Were evaluated for changes in dosage strength and dosing regimen.

  KPU-2 was administered at a dose of 7.5 mg / kg daily for 5 days (qd × 5) ip, 3.75 mg / kg at a dose of 7.5 mg / kg, twice daily for 5 days, 1 at a dose of 7.5 mg / kg. Administered ip every other day for 10 days (qod × 5) and at a dose of 7.5 mg / kg every other day for 15 days (q3d × 5) ip. The combination of NPI-2358 and CTP-11 by ip at q3d × 5 at a dose of 7.5 mg / kg produced a significantly greater effect than either compound alone, which persisted for the duration of the study (FIG. 13 ). These observations in the live part of the study were confirmed by the group mean final tumor weight at the time of dissection, in which only the combination group showed a statistically significantly lower tumor weight than the control. Furthermore, the difference between the average tumor weight of the combination therapy group and the average tumor weight of the CPT-11 monotherapy group was statistically significant (FIG. 14). Examination of the individual final tumor weights at the time of dissection reveals a greater effect in concurrent therapy (FIG. 14). The TGI for concurrent therapy was 78%, compared to 38.9% for CPT-11 alone. The TGI for the combination therapy group exceeds the 58% NCI criteria for positive results.

Standard Chemotherapy Study of KPU-2 +/- for 2.5 Human Tumor Xenograft Models This study consists of 5 different departments, each with its own protocol, timing, dosing regimen and reference compound Have. Each department is considered within the presentation of a specific tumor model.

  The purpose of the HT-29 sector study was to find a slightly higher dose of KPU-2 in the HT-29 human colon tumor xenograft model (10 mg / kg, ip, q3d × 5), where there is a significant difference between KPU-2 (7.5 mg / kg, ip, q3d × 5) and CPT-11 (100 mg / kg, ip, qw × 3) A synergistic effect was observed.

  As can be observed in FIG. 15, the combination of KPU-2 and CPT-11 in this model resulted in a significant synergistic effect in inhibiting tumor growth, which was observed in treatment day 29 in the combination therapy group. Until almost completely inhibited. The combination therapy maintained efficacy and the estimated tumor growth for this group was significantly lower than either single therapy group. Therefore, administration of KPU-2 and CPT-11 inhibits tumor growth and is an effective anti-tumor treatment.

  Observation of the live part of the study (estimated tumor growth, FIG. 15) is supported by measuring the weight of the tumor excised at dissection (FIG. 16). The tumor weight in the combination group was significantly lower than the control (p <0.01), as was the tumor weight for CPT-11 alone (p <0.05).

  When individual final tumor weights were considered (FIG. 16), the tumor size for the combination group was usually smaller than the other treatment or control groups. The TGI of the combination group was 65.8%, showing a positive effect by the NCI criteria, whereas the monotherapy did not reach the NCI criteria of TGI> 58%.

3. Study of the activity of KPU-2, KPU-35 and t-butyl-phenylhistine in the HT-29 human colon tumor xenograft study The results of this study are presented in FIG. All combination therapy groups showed significant synergistic effects between the new compound and CPT-11. Individual tumor weights demonstrate the effectiveness of concurrent therapy treatment (FIG. 18). In each case, the TGI of the combination group exceeded the positive effect NCI criteria, whereas the CGI-11 monotherapy TGI did not reach this level.

4). Summary of the effects of KPU-2, KPU-35 and t-butyl-phenylhistine combined with CPT-11 in the HT-29 human colon tumor xenograft model When combined with CPT-11, KPU-2 is a standard The effect of the chemotherapeutic agent CPT-11 was increased to a level well above the NCI criteria of TGI ≧ 58% for positive effects. The results obtained by the three studies are very similar to both survival observations (Figure 19) and the weight of the tumor excised at dissection (Figure 20).

Study in the DU-145 human prostate tumor xenograft model Two studies were completed using this model. The first study included KPU-2 alone and in combination with taxotere, and the second study compared KPU-2, KPU-35 and t-butyl-phenilahistine used alone and in combination with taxotere. .

1. Effect of KPU-2 in combination with taxotere in DU-145 human prostate tumor xenograft model As can be seen from the data obtained in the live part of this study (Figure 21), the most effective of DU-145 human prostate tumor The treatment was KPU-2 + Taxotere combination therapy. The treatment effect was most noticeable early in the study and appeared to decrease as the study progressed. From the 20th to 27th day of treatment, the combination therapy provided a clear TGI above the NCI criteria (TGI ≧ 58%), and the estimated tumor weight of the combination therapy was significantly greater than either single therapy It was low.

2. Activity of KPU-2, KPU-35 and t-butyl-phenylhistine alone or in combination with taxotere in DU-145 human prostate tumor xenograft model Obtained with KPU-2 in combination with taxotere in the above study Based on the data obtained, a second study was started comparing KPU-2, KPU-35 and t-butyl-phenylhistine, used alone and in combination with taxotere.

  Observations made during the live part of this study indicate that the combination of either KPU-2 or KPU-35 with taxotere significantly reduces tumor growth than taxotere alone (FIG. 22). Tumor growth was almost completely prevented by KPU-35 in combination with taxotere.

  The tumor weight excised at the time of dissection confirmed the observations made in the live section of the study. The combination of taxotere with either KPU-2 (FIG. 23) or KPU-35 (FIG. 24) was significantly more effective than taxotere alone in preventing tumor growth. In the case of KPU-35, 3 out of 10 mice showed signs of tumor shrinkage. Tumor growth inhibition index showed significant inhibition of tumor growth in KPU-2 (group average = 74.1%) and almost total inhibition in KPU-35 (group average = 92.5%). Taxotere alone did not reach the NCI established criteria for positive effects (TGI ≧ 58%).

5). Study in the MCF-7 human breast tumor xenograft model This study compared the effects of KPU-2, KPU-35 and t-butyl-phenylhistine in the MCF-7 human breast tumor xenograft model. Compound doses were administered on days 1, 2, 3, 4, and 7. Taxotere was administered on days 1, 3 and 7.

  The selected novel compounds have a statistically significant effect at an early stage when used in combination with taxotere in this model, apparently almost completely blocking estimated tumor growth (FIG. 25). Of the three compounds, KPU-2 appears to be the most effective, and t-butyl-phenylhistine also showed a significant potentiation of taxotere.

6). A549 Human Non-Small Cell Lung Tumor Xenograft Model Study Survival in this study (FIG. 26) indicates that the combination of KPU-2 (7.5 mg / kg, ip, qd × 5) with taxotere is a control It was shown to result in significant inhibition of tumor growth when compared to the group or any single therapy group. This was confirmed by the tumor weight at the time of dissection and the mean of the co-therapy group was significantly lower than the mean of taxotere alone or the control group (FIG. 27). The tumor weight of the co-therapy group forms a cluster of low tumor weights, showing a consistent effect.

  When calculating the tumor growth index, the co-treatment group showed a TGI of 74.4%, well above the NCI criteria for positive effects (TGI ≧ 58%) when compared to the control group. Taxotere alone showed 26.1% TGI.

7). Study in the MDA-231 human breast tumor orthotopic xenograft model This model involves the placement of human tumor tissue in the mouse mammary fat pad, which is a substitute for the natural environment. In this manner, the possibility of a positive effect due to a specific action in the subcutaneous vascular bed is avoided. This study compared the effects of KPU-2 (7.5 mg / kg, ip, q3d × 5) used alone and in combination with paclitaxel (16 mg / kg, ip, qd × 5).

  Entering the third week of the study, a significant inhibition of tumor growth, a very significant effect, was seen in the combination therapy group. This effect appeared to be more pronounced than Taxotere alone (FIG. 28).

8). Study in mouse melanoma B16F10 metastatic tumor model In this study, we used alone and paclitaxel for the number of metastases appearing on the lung surface 16 days after intravenous injection of B16F10 melanoma cells into mice. The effects of KPU-2, KPU-35 and t-butyl-phenylhistine in combination with the test were tested. This model is not a xenograft model, but does not include a high degree of vascularization into the tumor mass.

  In this model, the most effective treatment was KPU-2 alone (41.6% and 35.0% MGI, respectively) showing an average metastasis count approximately 10% lower than that in paclitaxel (Fig. 29). Although this study itself does not prove that combination therapy is more effective than monotherapy, KPU-2, KPU-35 and t-butyl-phenylhistine are most effective in highly vascularized tumors It shows that.

Assay for activity against pathogenic fungi Dehydrophenillahistin against pathogenic fungi for the above-mentioned known antifungal compounds for use in determining the AF / IS value of dehydrophenylahistine or analogs thereof Or the relative activity of its analogues is measured directly against fungal organisms, for example by adaptation of the microtiter plate of the NCCLS broth macrodilution method described in Diagn Micro and Infect Diseases, 21: 129-133, (1995) Is done. Antifungal activity can also be determined in all animal models of fungal infection. For example, a steroid-treated mouse model of mucor pneumonia may be used (Goldaill, LZ and Sugar (Goldaill, LZ & Sugar, AM) 1994 J Antimicrob Chemother 33: 369-372). . Illustratively, in such studies, a large number of animals are not administered dehydrophenylahistine or its analogs before, during or after infection with fungi, respectively, or various doses of dehydro Phenylhistine or an analog thereof (and / or in combination with one or more other fungal agents) is administered, or a positive control (eg, amphotericin B) is administered. Animals may be treated once every 24 hours with a selected dose of dehydrophenylhistine or an analog thereof, a positive control, or carrier alone. The process is continued for a predetermined number of days, for example up to 10 days. Animals are observed for a period of time after the treatment period (eg, a total of 3 weeks) and mortality is assessed daily. Models can include systemic, pulmonary, vaginal and other infection models with or without other treatments (eg, treatment with steroids) designed to mimic susceptible human subjects.

To further illustrate, one method of determining therapeutic efficacy in vivo (eg, ED ₅₀ expressed in dehydrophenylahistine or analogs thereof (mg) / subject (kg)) is a rodent model. It is a system. For example, mice are infected with a fungal pathogen (eg, 10 ⁶ C. Albicans cells / mouse), such as by intravenous infection, at approximately 10 times the 50% lethal dose of the pathogen. Immediately following the fungal infection, the dehydrophenylahistine compound is administered to the mice at a predetermined dosage volume. ED ₅₀ is calculated from the survival rate recorded on day 20 after infection by the method of Van der Waerden (Arch Exp Pathol Pharmakol 195: 389-412, 1940). In general, untreated control animals die from 7 to 13 days after infection.

In another exemplary embodiment, C. aeruginosa grown on Sabouraud dextrose agar (SDA) slope media at 28 ° C. for 48 hours. C. Albicans Wisconsin (C43) and C.I. C. tropicalis (C112) is suspended in physiological saline and adjusted to a transmittance of 46% at 550 nm with a spectrophotometer. The inoculum is further adjusted with a hemocytometer and the plate count is confirmed to be approximately 1 or 5 × 10 ⁷ CFU / ml. CF-1 mice are infected by injection of 1 or 5 × 10 ⁶ CFU into the tail vein. An antifungal agent in ethanol: water (10:90) is administered intravenously or subcutaneously 4 hours after infection and once a day for more than 3 or 4 days thereafter. Monitor survival daily. ED ₅₀ can be defined as the dose that allows for 50% survival of mice.

Assessment of antimicotic activity Benzimidazole and griseofulvin are anti-tubulin agents capable of binding to fungal microtubules. Once bound, these compounds interfere with cell division and intracellular transport in susceptible organisms, leading to cell death. Commercially, benzimidazoles are used as fungicides in veterinary medicine and plant disease control. Botrytis cinerea, Beauveria bassiana, Helminthosporium solani, Saccharomyces cerevisiae and Aspergillus sp. It is easily affected by the molecules. However, toxicity issues and increasing drug resistance have a negative impact on their use. Griseofulvin is used clinically to treat skin, hair and nail ringworm infections caused by Trichophyton spp., Microsporum spp. And Epidermophyton floccosum. The However, its antifungal range is limited to this type of fungal organism. Genotoxicity is also a major side effect. Terbinafine is an alternative first-line treatment but is more expensive. In addition, clinical resistance has recently been observed with Trichophyton rubrum, a major virulence factor in all dermatophyte infections.

  In Candida albicans, microtubule / microfiber formation is affected when cells are exposed to the microtubule inhibitors nocodazole and chloroprofam. These results further confirm the search for cytoskeletal inhibitors as effective anti-fungal agents. Accordingly, the antifungal activity of some of the compounds disclosed herein was evaluated.

Specifically, the disclosed compounds were evaluated together with commercially available microtubule inhibitors as well as approved antifungal agents. As test compounds and controls used in this study, (−)-phenylhistine, KPU-1, KPU-2, KPU-11 and KPU-17, KPU-35, t-butylphenylhistine, colchicine (three Candida species) (Candida) commercial microtubule inhibitors tested against isolates), Benomyl (commercial microtubule inhibitors tested against three Candida isolates), griseofulvin (six dermatophytes) Commercial microtubule inhibition and antibiotic control in tests against fungal isolates, amphotericin B (antibiotic control to test against three Candida isolates), itraconazole ( Antibiotic control to test against two Aspergillus isolates).

Microorganisms tested with these compounds include Candida albicans, Candida glabrata, Aspergillus fumigatus (Aspergillus
fumigatus), Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum. Various two isolates were tested except for Candida glabrata (one isolate).

The antifungal susceptibility test is performed by the National Committee for Clinical Laboratory Standards, M38-A “Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi; Approved Standards) ”. This includes testing in RPMI-1640 with glutamine and no bicarbonate, inoculum size 0.4-5 × 10 ⁴ and incubation at 30 or 35 ° C. for 48 hours. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that resulted in an 80% reduction in turbidity when compared to a control tube containing no drug. Drug concentrations were 0.03-16 μg / ml for study compounds and 0.015-8 μg / ml for itraconazole and griseofulvin.

  The minimum inhibitory concentration (MIC) at which the compound inhibited the growth of the target microorganism was evaluated according to a modified version of the NCCLS protocol. The minimum inhibitory concentration (MIC) is determined at the first 24 hour interval, where growth can be measured in a control tube containing no drug. The defined MIC was the lowest concentration that showed an 80% reduction in turbidity when compared to the growth control. The minimum lethal concentration (MLC) was determined by plating 0.1 μl from each MIC concentration and each concentration above the MIC. MLC was obtained at the first concentration showing no more than 5 colonies of fungal growth showing 99.95% death. When MICs were obtained, the minimum bactericidal concentration (MFC) was determined to evaluate the fungistatic / bactericidal properties of the compounds. This procedure involves diluting drug-treated cell samples (taken from test wells containing compounds at and above MIC concentrations) to compound concentrations significantly lower than the inhibitory concentration and agar samples. With deposition on the plate. A compound is scored as fungistatic if the cell is able to resume growth and is scored as fungicidal if the compound has killed the organism and cannot be regrown It was done.

  The compounds disclosed herein have been found to be effective against two Trichophyton species. T. rubrum is a fundamental virulence factor for human dermatophyte infection and may be a notable important organism in the development of clinical agents.

  The compounds KPU-2, KPU-11 and KPU-17, KPU-35 and t-butylphenylahistine are in efficacy with griseofulvin, the current standard drug used to treat dermatophyte infections It was equivalent or in some cases more powerful.

The compounds (-)-phenhisthistine and KPU-1 are not significantly more potent than the other compounds when tested against T. rubrum, and the sensitive T. mentagrophytes isolate When tested against fungi, it was weak but comparable to the others.

  In those cases where the MFC can be determined, the results indicate that these compounds are virtually fungistatic (see Table 19 and Table 20).

[Evaluation of vascular targeting activity]
Tumors and tumor conditions can be treated using the compounds disclosed herein. Occlusion of blood supply in a tumor by a vascular targeting agent (VTA) induces tumor regression. For example, the compounds disclosed herein, including NPU-02 and KPU-35, can be VTA. Many VTAs exhibit their vascular effects by interacting with colchicine binding sites on microtubules. This interaction induces the characteristic rapid collapse and occlusion of the established vasculature in the tumor, thus neglecting the integrity of existing blood vessels and leading to necrosis.

  Vascular collapse can occur, for example, within 30-60 minutes of exposure to VTA, changing the shape of immature and proliferating endothelial cells in the central part of the tumor, but changing the shape of quiescent and mature endothelial cells. Including not changing. This differential effect on vascular cells provides a rationale for selective effects on tumors due to the higher proportion of proliferating immature endothelial cells in tumor vessels relative to normal vessels. VTA can be classified into three overlapping ranges of activity: (1) strong vascular and cytotoxic effects, (2) strong vascular effects with weak cytotoxic effects, and (3) with weak vascular effects. Strong cytotoxic effect.

In vivo vascular targeting activity of KPU-02 and KPU-35 Animal models are essential to study new therapies that inhibit tumor-induced angiogenesis, target established tumor vasculature, and inhibit tumor growth. is there.

  A mouse syngeneic “pseudo orthotopic” breast cancer model was used to address these issues. Torres Filho et al., Microvascular Research (1995) 49, 212-226, which is hereby incorporated by reference in its entirety. In order to create a “pseudo orthotopic environment”, the cover glass of the dorsal subcutaneous (skin flap) tuft was removed and a small piece of mammary fat pad from a donor mouse was transplanted into the tuft. Tumor spheroids containing N202 mammary tumor cells transduced with histone (H2B) green fluorescent protein (GFP) were applied on the fat pad graft. The use of H2B-GFP transduced cells makes it possible to visualize tumor growth and to monitor mitosis and apoptosis.

  Fluorescence video microscopy allows a relatively non-invasive study of tumor microcirculation in conscious mice. This model provides data on the effects of a compound on tumor vasculature, tumor growth, mitosis and apoptosis and is useful for testing the activity of a compound, either alone or in combination with other therapeutic agents It is. Using this model, KPU-02 and KPU-35 have been shown to induce rapid vascular collapse and regression of established tumors leading to central necrosis after a single intravenous administration.

  On day 12 of tumor growth, the mice were given a 2-minute infusion of 5 mg / kg KPU-35, a 5-minute intravenous infusion of 10 mg / kg KPU-02, or vehicle (10% solutol in water (w / w) +2 Intravenous treatment by rapid infusion of (% DMSO). On day 13, a 5 minute infusion of 10 mg / kg KPU-02, KPU-35 or excipient was given. Treatment with KPU-02 or KUP-35 showed good tolerability. The mice were observed for an additional 2 days. Tumor area within and around the tumor, blood flow velocity and blood vessel density were visualized. Real-time observations were recorded at various time points using still pictures and video microscopes.

  This study demonstrates rapid collapse of the central vasculature after a single intravenous treatment with either KPU-02 or KPU-35. Changes in vascular function resulted in significant central tumor necrosis with no observed effect on the vasculature around the fat pad or skin (FIG. 30). These observations support the selectivity and specificity of KPU-02 and KPU-35, both of which can individually disrupt established tumor vasculature.

In vivo activity of KPU-02 in human tumor xenografts When KPU-02 is administered with CPT-11 (irinotecan), taxotere or paclitaxel, human colon (HT-29), breast (MCF-7, MDA-MB231) And in the lung (A549) tumor xenograft model, significant anti-tumor activity was seen (Table 21). The effect of KPU-02 in HT-29 was strong and reproducible in these studies, showing a dose-dependent effect, ie 7.5 mg / kg was statistically higher than 2.5 mg / kg (FIG. 32). , FIG. 33).

In vitro activity of KPU-02 and KPU-35 in HuVEC cells The above in vivo effect of KPU-02 and KPU-35 on tumor vasculature was supported by the in vitro effect of the same compounds in HuVEC cells. Human umbilical vein endothelial cells are considered a good in vitro model of tumor endothelium, which is considered “immature”. Tumor endothelium lacks supporting vascular wall cells and is increasingly dependent on the microtubule network for tumor vasculature integrity. Thus, disruption of the microtubule network causes vascular collapse.

KPU-02 induces rapid tubulin depolymerization in HuVEC cells.
Human umbilical vein endothelial cells (HuVEC, Cambrex CC2519A) were maintained at subconfluent density in EGM-2 (Cambrex) medium. Cells were cultured in an incubator at 37 ° C. in 5% CO ₂ and 95% humidified air. For tubulin staining assays, HuVEC cells were seeded on tissue culture compatible coverslips (Fisher) at a density of 3 × 10 ⁴ cells / ml in EGM-2. Plates were returned to the incubator for 2 days.

  Test compound stock (20 mM) solutions were prepared in 100% DMSO. A 400-fold dilution of the compound was prepared in 100% DMSO. A 5 μl volume of dilution was added to individual wells, resulting in a final concentration of 200 nM. The final concentration of DMSO was 0.25% in all samples. The plate was returned to the incubator for 30 minutes. HuVEC cells were treated with 200 nM KPU-02 or KPU-35 for 30 minutes.

Cells were rinsed in dPBS and then fixed in 10% (v / v) neutral buffered formalin for 10 minutes at room temperature. After fixation, α-tubulin was visualized by indirect immunofluorescence. Specifically, cells were permeabilized for 10 minutes in 0.2% (v / v) Triton X-100 / dPBS. After washing the cells, the coverslips are transferred to a humidified chamber and the coverslips are blocked in antibody buffer (2% (w / v) BSA / 0.1% (v / v) Tween 20 / dPBS) for 2 hours. did. Coverslips were incubated for 1 hour with 50 μl of 0.1 μg / ml mouse α-tubulin (Molecular Probes) in antibody buffer, then washed and darkened with 50 μl of 1 μg / ml goat anti-mouse FITC (Jackson ImmunoResearch Laboratories). Incubated for 1 hour. Finally, the cells were washed, treated with 2 μg / ml DAPI (Molecular Probes) for 10 minutes, rinsed with H ₂ O, and Vectashield (Vector
Labs) Mounted for microscopic examination using mounting medium. Cells were imaged using a 60 × oil immersion objective on an upright microscope (Olympus BX51). Images were captured digitally using a CCD camera and Magnafire 2.0 software (Olympus). Subsequent image processing was performed with Photoshop Elements 2.0 (Adobe) and Microsoft Powerpoint.

  FIG. 33 shows that KPU-02 and KPU-35 rapidly induce tubulin depolymerization in HuVEC cells.

KPU-02 induces volume-dependent monolayer permeability in HuVEC cells.
Human umbilical vein endothelial cells (HuVEC, Cambrex CC2519A) were maintained at subconfluent density in EGM-2 (Cambrex) medium. Cells in 5% CO ₂ in incubator
And cultured at 37 ° C. in 95% humidified air. For monolayer permeability assays, HuVEC cells were seeded at 1 × 10 ⁵ cells / ml in EGM-2 medium on a fibronectin-coated 3.0 μm Fluoroblock insert (Becton Dickinson) in a 24-well plate. Plates were returned to the incubator for 4 days to allow cells to reach confluence.

  Test compound stock solutions (20 mM) were prepared in 100% DMSO. Serial dilutions of 10-fold concentrated compounds were prepared in EGM-2. 10 μl volumes of serial dilutions were added in duplicate to the test inserts, resulting in final concentrations ranging from 2 μM to 2 nM. The final concentration of DMSO was 0.25% in all samples. Cells were treated with 2 nM-2 μM KPU-02 for 15 minutes.

  FITC-dextran (50 mg / ml) (38.2 kDa, Sigma) in dPBS was diluted 2.5-fold in EGM-2 and 10 μl of FITC-dextran was added to each insert. The final concentration of FITC-dextran was 1 mg / ml. The plate was returned to the incubator and after 30 minutes, the fluorescence in the lower chamber of the 24-well plate was read using a Fluoreximeter (Packard Bioscience) with λex = 485 nm and λem = 530 nm filters.

  FIG. 34 shows that KPU-02 is capable of inducing monolayer permeability in a dose-dependent manner. The results shown in FIG. 34 represent the mean ± SD of 3 independent experiments.

Blood flow in the P22 rat sarcoma model with ¹²⁵ I-IAP Tumor blood flow was assessed in the model using quantitative ¹²⁵ I-iodoantipyrine (IAP) technique in rats bearing P22 rat sarcoma. KPU-02 (15 mg / kg, IP) significantly and selectively reduced tumor blood flow to 23% of the excipient 1 hour after administration, and blood flow was significantly reduced after 24 hours. (59% of excipients). In contrast, blood flow in non-tumor tissues was affected to a much lesser extent at 1 hour (see FIG. 35).

  The reduction in blood flow at 24 hours after administration was more variable between tissues for KPU-02 compared to vehicle, as shown in FIG. Blood flow to the tumor was most affected. Other tissues showed a slight reduction in blood flow. Skeletal muscle blood flow appeared to increase 24 hours after administration.

  The effects of KPU-02 observed at 1 hour appear to last longer than effects previously reported for CA4P using the same technique and appear to be more selective for tumor blood flow It was.

In experiments using the P22 rat sarcoma model, KPU-02 7.5 and 15 mg / kg
It was demonstrated that IP (n = 2 per dose) resulted in dose-dependent tumor necrosis by 24 hours after administration, with the highest dose resulting in nearly complete necrosis of the tumor as shown in FIG. All tumors in KPU-02 treated rats showed signs of necrosis, whereas no tumors in vehicle treated rats. VTAs handled in the clinic (eg, CA4P, ZD6126, AVE8062) use IAP methodologies (or similar techniques) to show similar qualitative effects on tumor blood and use dce-MRI techniques. Used to demonstrate reduced blood flow in P22 rat sarcoma tumors and humans. Stevenson JP, Rosen M, Sun W, Gallagher M, Haller DG, Vaughn D, et al. “To patients with cancer Phase I trial of the antivascular agent combretastati: Phase I trial of the antivascular agent combretastati
n A4 phosphate on a 5-day schedule to patients with cancer: magnetic resonance imaging evidence for altered tumor blood flow ”, J Clin Oncol 2003; 21 (23): 4428-38, Evelhoch JL, LoRusso PM PM), He Z, DelProposto Z, Porin L, Corbett TH, et al., "Magnetic resonance imaging of mouse and human tumor responses to vascular targeting agent ZD6126. Magnetic resonance imaging measurements of the response of murine
and human tumors to the vascular-targeting agent ZD6126), Clin Cancer Res 2004; 10 (11): 3650-7, and Gadgeel SM, LoRusso PM, Ozniak AJ, Wheeler C (Wheeler C.), “A dose-escalation study of the novel vascular-targeting agent, ZD6126, in patients with solid tumors” Proc Am Soc Clin Oncol 2002; 21: Abstract 438, each of which is incorporated herein by reference in its entirety.

Combination therapy with microtubule targeting agents The view that VTA selectively damages the vasculature in the central part of the tumor relative to the periphery, which restores functionality is the chemotherapeutic drug for VEGF and EGF (Taxol, Support the use of these agents in combination with vinblastine and cisplatin), radiation therapy and angiogenesis inhibitors. The new VTA should not replace these therapies, but complement these therapies and provide higher antitumor activity.

Treatment of other conditions In addition to cancer, other diseases can be treated using the VTAs disclosed herein. Conditions include other tumors, retinopathy, and blood supply, preferably any other condition that remains viable and / or depends on blood supply from new vasculature to grow or Disease.

  Many conditions are associated with excessive or inappropriate vasculature. Examples of conditions associated with excess vasculature include inflammatory disorders such as immune and non-immune inflammation, rheumatoid arthritis, rheumatoid arthritis and psoriasis; diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular Disorders related to inadequate or inappropriate vascular invasion, such as degeneration, corneal graft rejection, post lens fibroproliferation, rubeosis, capillary growth in atherosclerotic plaques, and osteoporosis; and for example solid tumors, tumor metastases, To support hematogenous tumors (eg, leukemia), hemangiofibroma, capo sarcoma, benign tumors (eg, hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas), and tumor growth Cancer-related disorders, including other cancers that require angiogenesis. Further examples of vasculature-dependent diseases include, for example, Osler-Weber syndrome, myocardial neovascularization, plaque neovascularization, telangiectasia, hemophilic joints and wound granulation. Furthermore, excess vasculature is also associated with clinical problems as part of biological and mechanical grafts (tissue / organ grafts, stents, etc.). The compounds and compositions of the present invention can be used to target the vasculature, preferably preferentially to the disease vasculature over the non-diseased tissue vasculature, and thus the compounds and The composition can be used in the treatment of such conditions. Other diseases in which angiogenesis plays a role and the compounds and compositions of the invention can be used are known in the art.

Examples of retinopathy include age-related macular degeneration (ARMD), diabetic retinopathy, and the like. Pathological angiogenesis is a major contributor to a number of retinopathy that is a major cause of blindness in the developed world. Kahn and Hiller, Am J Ophthalmol (1974) 78, 58-67, which is hereby incorporated by reference in its entirety. For example, retinal and disc neovascularization is seen in 30-50% of patients with diabetic retinopathy for over 20 years. Yanko et al., Retina (2003) 23, 518-522 (This is
Which is hereby incorporated by reference in its entirety). Furthermore, subretinal neovascularization is a serious complication in approximately 10% of patients with macular degeneration. Ferris et al, Arch Ophthalmol (1984), 102, 1640-1642, which is hereby incorporated by reference in its entirety.

Vascular targeting agents (eg combretastatin A-4 (CA-4)) have been shown to cause neovascular disruption in non-neoplastic tissues. Gricks and others (Griggs
et al), Br J Cancer (2001) 84, 832-835, which is hereby incorporated by reference in its entirety. In addition, CA-4P has been shown to inhibit retinal neovascularization seen during proliferative retinopathy. Griggs et al, Am J Path (2002) 160, 1097-1103, which is hereby incorporated by reference in its entirety. Finally, it has been demonstrated that CA-4P phosphate inhibits the development of VEGF-induced retinal neovascularization, inhibits the development of choroidal neovascularization and / or causes its partial regression. Nambu et al. (Nambu et
al), Invest Ophthalmology & Visual Sci (2003) 44, 3650-3655, which is hereby incorporated by reference in its entirety. The compounds disclosed herein can be used to treat retinopathy. For example, the Grix (2001 and 2002) and Nambu methodologies are used to treat retinopathy. In addition, the compounds and compositions disclosed herein may reduce such retinopathy by applying the compound and / or composition to the target area in an amount effective to reduce vascular density and / or vascular proliferation. Can be used to treat.

[Structure-activity relationship]
The effect of activity from various modifications on the phenyl ring of tBu-dehydroPLH is shown by the data in FIG. Substitution with relatively hydrophobic groups and smaller functional groups at the m or o positions increased or maintained cytotoxic activity in HT-29 cells, whereas substitution at the p position decreased activity. It is clear. While not being bound by any particular theory, this data suggests rigorous recognition of the phenyl ring by tubulin.

  3D-QSAR (CoMFA) analysis (see FIG. 39) also supports the presence of sterically favorable regions at the m and o positions and the presence of sterically unfavorable regions at the p positions. X-ray crystallography (see Figure 40) shows that the conformation of the strong derivative is a certain amount between the phenyl ring and the quasi-tricyclic core (cor) template formed by the DKP and imidazole rings. This suggests that a dihedral angle is required. Thus, modification by appropriate conformational restriction of the phenyl ring can induce potent activity. Without being bound by any particular theory, the binding mode of the PLH derivative at the colchicine binding site of tubulin may differ from that of colchicine and its known homologues.

[In vitro action on microtubules]
Purification of microtubule proteins and tubulin Microtubule proteins (MTP) were prepared as previously described (Farrell KW and Wilson L. (1987). Tubulin-colchicine complex. The body differentially poisons opposite microtubule ends (Tubulin-colchicine complexes differentially poison opposite microtubule ends) Biochemistry 23 (16): 3741-8 (which is hereby incorporated by reference in its entirety) ))). An MTP preparation composed of 70% tubulin and 30% microtubule associated protein (MTP) consists of PEM100 (100 mM 1-4 piperazine diethanesulfonic acid (Pipes), 1 mM MgSO ₄ , 1 mM EGTA, pH 6.8) and Isolated from bovine brain by 3 cycles of warm polymerization and cold depolymerization in 1 mM GTP. MTP was frozen frozen in liquid nitrogen and stored at -70 ° C until use. Tubulin was purified from microtubule proteins by phosphocellulose chromatography (PC-tubulin) and stored in PEM (50 mM Pipes, 1 mM MgSO ₄ , 1 mM EGTA, pH 6.8). Protein concentration was determined by the Bradford assay (Sigma Chemicals, St. Louis, MO) using bovine serum albumin as a standard (Bradford, 1976).

Test Agent A stock solution of KPU-02 was prepared in DMSO at a concentration of 20 mM. A stock solution of Combrestatin A4 (National Cancer Institute, Bethesda, MD) (CA4) was prepared in DMSO at a concentration of 5 mM. Colchicine (Sigma Chemicals, St. Loius, MO) (CLC) was prepared at a concentration of 3 mM in water. All agents were shielded from ambient light using an amber-colored Eppendorf tube. Serial dilutions were made to the desired concentration in DMSO and / or PEM50.

Determination of steady state microtubule polymer mass MTP (2 mg / ml) was polymerized into microtubules in the presence of a series of drug concentrations in PEM100 containing 1 mM GTP and a final DMSO concentration of 0.5%. Samples were monitored by light scattering at 350 nm for 75 minutes at 37 ° C.

The polymerization reaction was centrifuged and the concentration of microtubule protein in the supernatant, a measure of soluble tubulin at steady state, and the pellet, a measure of microtubule polymer, was used to calculate the inhibition of polymerization. . After incubation, the polymerized microtubules were separated from unpolymerized MTP and sedimented by centrifugation (150,000 × g, 45 minutes, 37 ° C.). After removing the supernatant and depolymerizing the microtubule pellet in deionized H ₂ O (24 hours, 0 ° C.), the protein was determined by Bradford assay.

  The percent inhibition was calculated by the two methods and the values obtained from the two methods were compared. In one method, the ratio of microtubule protein in the pellet (drug to non-drug) was calculated. Another ratio of microtubule protein in the pellet to supernatant (drug to non-drug) was also calculated. The numbers were in close agreement and were used because the former values were subject to smaller variances and experimental perturbations.

Microtubule average length distribution Transmission electron microscopy was used to determine the average length distribution of microtubules in the absence or presence of the test agent. At 75 minutes and prior to settling, a 10 μl aliquot from the polymer mass experiment was fixed by dilution into 290 μl PEM100 buffered 0.2% glutaraldehyde. 30 μl of fixed sample was fixed on a formvar coated 150 ICG mesh electron microscope grid for 90 seconds. Excess sample was wiped off with Whatman filter paper. Cytochrome C 30 μl (1 mg / ml) was applied for 30 seconds to enhance protofilament resolution and promote negative staining. Uranyl acetate (1.5%) was applied for 20 seconds and the excess was wiped off. Jeol electron microscope 1200
Observation was performed at a magnification of 2000 times and 30,000 times in EX11. A Zeiss MOPIII was used to determine the microtubule length distribution and average length for at least 100 microtubules per sample.

CLC competition assay PC-tubulin (0.2 mg / ml) was incubated for 120 minutes at 37 ° C. with 1 mM GTP, 1% DMSO, 10 μM test agent and 7-25 μM [ ³ H] CLC in PEM50. Following measurement of [ ³ H] CLC binding, a DEAE-cellulose filter binding assay was performed as previously described (Wilson, 1970). This method relies on adsorption of tubulin onto filter paper impregnated with DEAE-cellulose. Whatman DE81 filter paper was pre-wetted with PEM50 prior to sample application. A total 100 μl reaction volume was applied to a 2.5 cm disc filter paper on parafilm on ice. The paper disc was replaced by 5 successive 50 mls of PEM50 and washed by immersion for 5 minutes / wash at 4 ° C. to remove all unbound colchicine. Subsequently, a paper disk with tubulin-conjugated colchicine attached thereto was attached to a Beckman Coulter.
Counted directly in scintillation vials containing 2 ml of Ready protein solution (Fullerton, CA). All discs were washed together. Negligible binding of unbound CLC to the paper disc was seen in the control, either in the presence or absence of tubulin.

K _i values were calculated by linear regression of double reciprocal plots of experimental data in Microsoft Excel. Determined K _m value of tubulin regarding CLC under the experimental conditions is first, x is an equal sections to -1 / K _m. K _{m app} , K _{m in} the presence of drug was determined experimentally. K _i was determined for competitive inhibition α = K _m (1+ [I] / K _i ) using the relationship K _{m app} = α K _m .

Fluorescence spectroscopy Fluorescence measurements were performed using a Perkin-Elmer LS50B fluorescence spectrophotometer. PC-tubulin (0.2 mg / ml) was incubated with 0-30 μM KPU-02 in PEM50, 2 mM GTP, 3% DMSO. The interaction between KPU-02 and tubulin is due to fluorescence of 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt (bis ANS, Molecular Probes, Eugene, OR). An excitation wavelength of 395 nm and an emission wavelength maximum of 487 nm were reported. The excitation band and emission band were 10 nm. This experiment was performed twice.

The bis-ANS fluorophore explores the hydrophobic surface of the protein, and changes in the intensity of the bis-ANS fluorescence signal are the result of changes in the solvent accessible surface area of the protein. Bis-ANS can be used to report binding if there are some conformational changes that alter the tubulin-bis-ANS interaction upon ligand binding.

  PC-tubulin (0.2 mg / ml) was incubated with 0-30 μM KPU-02 at 25 ° C. for 20 minutes. Subsequently, bis-ANS (25 μM) was added and the relative fluorescence intensity of the samples was measured within 15 minutes at 25 ° C. Collecting a buffer blank spectrum showed that KPU-02 + Bis-ANS produced negligible fluorescence in the experimental wavelength range.

K _d is the equation F = ((− F _max × L) / (K _d + L)) + F ₀ , where F is the fluorescence intensity of bis-ANS-tubulin in the presence of total ligand concentration L. And F _max is the bis-ANS fluorescence intensity of fully liganded tubulin and F ₀ is the bis-ANS fluorescence in the absence of drug) in Sigmaplot and Microsoft Excel. Determined by fitting experimental data. F _max was determined by plotting 1 / (F ₀ -F) against 1 / L and extrapolating to 1 / L = 0. The percentage of binding site B occupied by KPU-02 was determined using the following relationship: B = (F ₀ -F) / (F ₀ -F _max ). Assuming that the concentration of free ligand is a single binding site per tubulin dimer, Lfree = L−B [C] where [C] is the molar concentration of the ligand binding site. Determined.

Inhibition of microtubule polymerization by KPU-02 KPU-02, CA4 and CLC were assayed for their ability to alter the polymerization of MAP-rich tubulin (MTP) (2 mg / ml) in a cell-free system in vitro. Initially, inhibition of polymerization was assayed using tubulin without phosphocellulose purified microtubule-associated protein (data not shown). KPU-02 was a more potent inhibitor against MT built with glycerol and DMSO seeds when compared to MT built in the presence of MAP co-purifying with tubulin. Although microtubule polymers in the absence of stabilized MAP did not reach steady state for 2 hours, these assays indicated that KPU-02 interacted directly with purified tubulin and KPU-02 Has been demonstrated not to exert its main effect through MAP.

KPU-02 and CA4 inhibited MT polymerization more potently than CLC as measured by light scattering (FIG. 44) and sedimentation analysis (FIG. 45). MTP (2 mg / ml) was polymerized into microtubules in the presence of a range of drug concentrations and was able to reach a steady state when monitored by light scattering at 350 nm. FIG. 41 shows DMSO drug excipient (white diamonds), 1.25 μM (white squares), 2.5 μM (−) and 5 μM (white circles) NPI-2358 (a), CA4 (b) and CLC (c). 2 represents the turbidity spectrum of microtubule protein polymerization in the presence of. KPU-02 and CA4 inhibited MT polymerization with comparable efficacy. FIG. 45 represents the inhibition of microtubule polymerization in the absence or presence of a series of KPU-02 (open circles), CA4 (open squares) and colchicine (open diamonds) concentrations. Total polymer mass after 75 minutes build was determined by sedimentation. Error bars are standard deviation values from three experiments. The concentration at which polymerization was inhibited by 50% (IC ₅₀ ) is 2.4 ± 0.4 μM for KPU-02, 2.2 ± 0.3 μM for CA4, and 7.6 ± 2.4 μM for CLC (Table 1). 22). (The variance obtained by statistical analysis is reported as the standard deviation value unless otherwise stated). At concentrations above the IC ₅₀ for in vitro polymerization of MAP-rich tubulin, MTP exhibits aggregation kinetics, suggesting that KPU-02 and CA4 sequester proteins and prevent microtubule assembly.

  As shown in FIG. 44, all three of the tested agents resulted in a concentration dependent inhibition of the degree of microtubule polymerization at 1.25-5 μM. There are two important differences to note between the spectra. First, the initial rate of increase in absorbance over time decreases with increasing drug concentration (FIGS. 44A and 44B). The spectrum shows that there is a delay period for MT formation in the presence of KPU-02 and CA4. Drugs that significantly and rapidly reduce the soluble construction eligibility pool of tubulin reduce the initial rate of polymerization. In contrast, the initial rate of polymerization is unchanged at all concentrations of CLC (FIG. 44C). Second, MTP in the presence of KPU-02 or CA4 does not reach steady state at high concentrations (greater than 5 μM) as shown by absorbance values that increase linearly with time (FIGS. 44A and 44B). ). In contrast, MTP in the presence of CLC reaches steady state at high drug concentrations (FIG. 44C).

The amount of drug required to inhibit polymerization by 50% (IC ₅₀ ) was determined from an analysis of the linear relationship between the decrease in microtubule polymer precipitated by centrifugation and the increase in drug concentration ( FIG. 45). Error bars in FIG. 45 represent standard deviation values from at least 3 independent experiments.

Reduction of average microtubule length measured by transmission electron microscopy Transmission electron microscopy is used to describe polymers formed at steady state and to evaluate conclusions derived from light scattering spectra. -Performed by microtubule polymerization reaction. KPU-02, CA4 and CLC all reduced the length of microtubules formed in the steady state. MT gradually decreased with increasing drug concentration (FIGS. 46, 47 and 48). FIG. 46 represents a frequency histogram of average microtubule length in vitro at steady state in the presence of (A) KPU-02, (B) CA4 and (C) CLC. Zeiss MOPIII was used to determine microtubule length distribution and average length. At least 100 microtubules were counted per drug concentration. FIG. 47 represents the electron microscopy used to record microtubules in the absence or presence of test compound. At 75 minutes, a sample from the polymer mass experiment was fixed and stained and 2 in a Jeol electron microscope-1200 EX11.
Observation was performed at a magnification of 000 times. (A) Representative electron micrographs of MAP-rich microtubules formed in vitro at steady state in the presence of KPU-2, (b) CA4 and (C) CLC. Scale bar is 10 μM. FIG. 48 represents a graphical summary of MT length reduction at steady state in the presence of KPU-02, CA4 and colchicine. The black bar is 1.25 μM, and the shaded bar is 2.5 μM. In the presence of KPU-02 and CA4, MT exhibits aggregation kinetics such that MTP is detected by turbidity, and gradually decreases with increasing drug concentration to a drug concentration at which MT is no longer observed. Error bars are standard deviation values from measurements of at least 100 microtubules.

  KPU-02, CA4 and CLC did not affect MT nucleation. The myriad short microtubules formed in the polymerization reaction demonstrate that the presence of KPU-02, CA4 or CLC does not affect nucleation. When nucleation is affected, a few longer microtubules are observed in the drug-treated sample versus the control sample, as opposed to the myriad shorter microtubules.

  KPU-02 and CA4 were equally potent in reducing the average MT length. At the lowest drug concentration analyzed by electron microscopy, 1.25 μM, KPU-02 and CA4 decreased the mean MT length by approximately 70% and CLC decreased by 40% (FIG. 48).

At drug concentrations above IC ₅₀ for in vitro microtubule polymerization, microtubules are not observed by electron microscopy for KPU-02 and CA4. In contrast, microtubules were observed by electron microscopy for all assayed CLC concentrations. At drug concentrations above IC ₅₀ , microtubule proteins in the presence of KPU-02 and CA4 show aggregation kinetics characterized by a linear increase in light absorbance over time (FIGS. 44A and 44B). In the presence of CLC, the light scattering polymer reaches a steady state (FIG. 44C). Despite the observation that MTP with KPU-02 or CA4 increases absorbance at 350 nm over time, no drug-specific protein aggregates were observed.

Fluorescence spectroscopy Tubulin (0.2 mg / ml) was incubated with a series of concentrations of KPU-02 in PEM50, 2 mM GTP for 20 minutes at 25 ° C. KPU-02 quenched bisANS fluorescence in a concentration dependent manner (FIG. 49A). K _d = 10 ± 1.6 μM (standard error) for KPU-02 and tubulin as measured by non-linear regression analysis of bis-ANS fluorescence intensity at maximum emission (FIG. 49B). A double reciprocal plot of the binding data resulted in a dissociation constant of 6.4 μM, assuming a single binding site for KPU-02 per tubulin dimer (FIG. 49C). The two different K _d values obtained by the non-linear and linear regression analysis methods were close enough that the values were considered approximately equivalent. FIG. 49A represents the fluorescence emission spectrum of tubulin in the presence of increasing KPU-02. Drug binding results in quenching of bis-ANS fluorescence. FIG. 49B represents the fluorescence maximum at 487 nm suitable for obtaining the tubulin K _d for 10 μM KPU-02 (standard deviation 1.6 μM). The inset is unprocessed. FIG. 49C represents the double reciprocal transformation of the binding data assuming 1 mole of drug / tubulin dimer (mol).

Competitive inhibition of CLC binding KPU-02 and CA4 competitively inhibited CLC binding to tubulin (Figure 50). FIG. 50 shows the results of the inhibition assay, where phosphocellulose purified tubulin (0.2 mg / ml) was added in the absence of KPU-02 or CA4 (white diamonds) or 10 μM KPU-02 (open circles). Alternatively, it was incubated with various concentrations of [ ³ H] CLC in the presence of 10 μM CA4 (white squares). Tubulin-CLC K _m is 11 ±
The inhibition constants for KPU-02 and CA4 were 3.2 ± 1.7 μM and 2.4 ± 0.3 μM, respectively. Constants were calculated from 3 independent experiments. The colchicine-tubulin binding reaction is time and temperature dependent and the binding dissociation constant is K _d = 0.1-1 μM depending on the assay conditions (Wilson L and Meza I.). (1973), Mechanism of action of colchicine. The mechanism of action of colchicine.
properties of sea urchin sperm tail outer doublet tubulin) Journal of Cell Biology 58 (3): 709-19, which is hereby incorporated by reference in its entirety. Under the test conditions, the K _m of tubulin for CLC is 11 ± 4.4 μM. K _m can be regarded as the total K _d of tubulin for CLC, but due to the time dependence of CLC binding, K _m is greater than the value reported for K _d . The K _i for KPU-02 and CA4 were 3.2 ± 1.7 μM and 2.4 ± 0.3 μM, respectively. K _i is defined as the amount of drug required to inhibit CLC binding by 50% and K _i is based on the amount of radioactive CLC bound to tubulin. K _i is a measure of the ability of a drug to compete with CLC, and K _i is not a direct measure of drug-tubulin binding dissociation because of the way binding affinity is reported.

Results At all concentrations of CLC assayed, MAP rich tubulin reached steady state. In contrast, at higher KPU-02 or CA4 drug concentrations, MAP rich tubulin did not polymerize to steady state and microtubules were not observed by electron microscopy. KPU-02 and CA4 effectively reduced the concentration of available tubulin. This reduction in the pool of soluble tubulin increased the MT critical concentration and prevented polymerization. in
The stoichiometric amount of KPU-02 and CA4 required to reduce the vitro polymer mass is linked to the data that microtubule proteins do not reach steady state above that concentration, and KPU-02 and CA4 are soluble. It suggested that tubulin is bound and acts by a sequestering mechanism that prevents polymerization.

Observation by electron microscopy at steady state, ie MAP-rich microtubules formed in the presence of the test agent, was consistent with the proposed mechanism that KPU-02 and CA4 sequester tubulin. In the presence of KPU-02, CA4 and CLC, a concentration-dependent decrease in average microtubule length was seen. In the presence of KPU-02 and CA4, a drug concentration dependent decrease in the initial rate of polymerization was seen, indicating that these drugs reduce the available tubulin for elongation. This decrease in initial polymerization rate was not seen when using CLC due to its slow association with tubulin. Furthermore, microtubules were formed at CLC concentrations above the CLC IC ₅₀ for polymerization, but microtubules were not formed at KPU-02 or CA4 concentrations above the KPU-02 or CA4 IC ₅₀ for polymerization. Without being bound by any particular theory, the concentration of soluble tubulin bound by KPU-02 or CA4 must be below the critical concentration required for tubulin polymerization to proceed.

Binding studies showed that tubulin has a lower affinity for KPU-02 than CLC. Inhibition of CLC binding to tubulin by KPU-02 and CA4 occurs within a 20 minute incubation period, indicating that the association of KPU-02 and CA4 with tubulin approaches equilibrium relatively faster than CLC. (Data not shown). KPU-02 is a study that characterizes phenilahistin (harimid) (Kanoh K, Kohno S, Kataka J, Takahashi J and Uno I., ( 1999), (-)-Phenylahistine inhibits tubulin polymerization to arrest cells during mitosis ((-)-Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization). The Journal of Antibiotics 52 ( 2): In line with 134-141, which is incorporated herein by reference in its entirety, competitively inhibited CLC binding to tubulin at sites that overlap the CLC binding site. CA4, a structural analog of CLC, also competitively inhibited CLC binding, although not bound by any particular theory, despite sharing the tubulin binding region with CLC, KPU-02 and CA4 , It appears to interact with tubulin and inhibit microtubules by a mechanism different from CLC.

[In vivo action on microtubules]
Cell Culture Studies MCF7 human breast cancer cells (American Type Culture Collection, Manassas, Va.) Stably transfected with GFP-α-tubulin (Clontech, Palo Alto, Calif.) Were placed in 250 ml tissue culture flasks or 35 mm 6 well plates. The cells were cultured in Dulbecco's modified Eagle medium (Sigma) supplemented with 5% fetal bovine serum, 0.1% penicillin / streptomycin and non-essential amino acids (doubling time, 29 hours) at 37 ° C. with 5% CO ₂ . Cells are replaced with KPU-02, CA4 or CLC prepared as described in Example 14 by replacing the original medium with an equal volume of medium containing the required concentration of test agent or DMSO excipient. Incubation was continued for 20 hours at 37 ° C.

Progression of mitosis The fraction of cells at mitosis (mitotic index) at a given drug concentration was determined in the breast cancer cell line MCF7. Cells were plated at a density of 3 × 10 ⁴ cells / ml in 6-well plates. After 24 hours, cells were incubated for 20 hours over a series of concentration ranges (1 nM to 1 μM) in the absence or absence of drug. The medium is collected and the cells are rinsed with versene (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH ₂ PO ₄ , 8.1 mM Na ₂ PO ₄ and 0.5 mM EDTA), detached with trypsin, and the medium. Was added back to ensure that the dividing and difficult to bind dividing cells were included in the analysis. Cells were fixed with 10% formalin in PBS overnight at 37 ° C., permeabilized in methanol for 10 minutes, and stained with 4,6-diamidino-phenylindole (DAPI) to visualize nuclei. Stained cells were spread on coverslips in Vectashielded mounting medium (Burlingame, CA) and sealed on slides with nail enamel. The splitting index was determined using fluorescence microscopy. The results are the mean and standard deviation of 4-7 experiments, in which 300 cells were counted for each concentration. The IC ₅₀ was the drug concentration that experimentally induced 50% of the maximum mitotic accumulation at 20 hours.

Immunofluorescence microscopy Cells were prepared as for mitotic progression except cells were seeded onto coverslips treated with poly-L-lysine (50 μg / ml, Sigma). On the day of staining, cells were rinsed in PBS and fixed overnight at 37 ° C. with 10% formalin. Cells were rinsed in PBS, permeabilized in methanol at −20 ° C. and hydrated with PBS. Coverslips were treated with 20% normal goat serum in PBS / BSA (1%) for 1 hour at room temperature. Cells were incubated for 1 hour at room temperature in DM1A / DM1B, a mouse monoclonal cocktail of anti-α- and β-tubulin diluted in PBS / BSA, and then stained with FITC-conjugated secondary antibody and DAPI. Cover slips were mounted using Prolong antifade medium (Molecular Probes, Eugene OR).

Preparation of cells for analysis of microtubule dynamics Cells were prepared as for mitotic progression, except that in order to promote cell spreading, cells were treated with poly-L-lysine (50 μg / ml, Sigma ) For 2 hours followed by laminin and fibronectin (10 μg / ml, Sigma) on glass coverslips pretreated for 1 hour at 37 ° C. Cells were serum starved by incubating with drugs or DMSO for 20 hours. Prior to analysis, coverslips were transferred to recording medium (culture medium lacking phenol red and sodium bicarbonate, buffered with 25 mM HEPES and supplemented with 3.5 g / L sucrose). To prevent photobleaching, the cells were sealed in a double-cover glass sealed chamber immediately after adding Oxylase (30 μl / ml, Oxyrase Inc, Mansfield, OH) to the recording medium.

Time-lapse microscopy and image acquisition Microtubules were prepared using Plan apochromat 1.4N. A. Observations were made using a Nikon Eclipse E800 fluorescence microscope with a x100 objective. The stage was sealed in a Pyrex box and maintained at 36 ± 1 ° C. by a forced air heating system. Thirty images of each cell were obtained using a Photometrics CoolSNAP HQ digital camera (Tucson, AZ) driven by Metamorph software (Universal Imaging, Media, PA) at 10 MHz, 300 ms exposure time, amplification factor 2 and luminance. Acquired at 4 second intervals, using 2 × 2 binning to enhance.

Analysis of microtubule dynamics The location of the positive end of the microtubule over time was tracked using the Metamorph Track Points application exported to Microsoft Excel and analyzed using Real Time Measurement software. Individual microtubule lengths were graphed as a function of time. Individual growth and shortening rates were determined by linear regression. A change of 0.5 μm or more between two points was considered a growth or shortening event, and a change of less than 0.5 μm between two points was considered to be a period of attenuated kinetics or interruption. . For each condition, at least 25 microtubules were analyzed. Results are the mean and standard deviation of at least 3 independent experiments.

  The time-based catastrophe frequency for each microtubule was calculated by dividing the number of catastrophes per microtubule by the time spent during growth or decay. The time-based rescue frequency per microtubule was calculated by dividing the total number of rescues per microtubule by the time spent shortening. Distance-based catastrophe and rescue frequency were similarly calculated by dividing the number of transitions by the length that was grown or shortened, respectively. Visible microtubules for less than 2 minutes were included in the frequency analysis. The kinetics per microtubule were calculated as the growth and shortened length divided by the total microtubule life. Microtubules visible for more than 0.3 minutes were included in the kinetic analysis.

Cell cycle progression arrested in early metaphase The concentration range for KPU-02, CA4 and CLC that cells accumulate during mitosis was determined. After 20 hours, 60-70% of the cells were studied for MT depolymerizers such as vinca alkaloids and 2-methoxyestradiol, and MT stabilizers such as taxol, epothilone B and discodermolide (Jordan MA ) (2002), Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Current Medicinal Chemistry-Anti-Cancer Agents 2: 1-17 This was inhibited in the previous metaphase as compared to 30-40% of the cells in metaphase in its entirety (incorporated herein by reference) Drug concentration required for 50% maximal mitotic arrest ( IC ₅₀₎ was evaluated in the drug 1 nM to 1 [mu] M (Figure 51). Figure 51, KPU-02, CA4 and log regarding mitotic progression inhibition by CLC [ Product] represents a response curve MCF7 cells were cultured in the presence of NPI-2358 (open circles), CA4 (open squares) and colchicine (open diamonds). Plated at a density of 3 × 10 ⁴ cells / ml in. After 24 hours, cells were incubated for 20 hours over a range of concentrations (1 nM to 1 μM) in the absence or presence of drug. Cells were fixed and stained with DAPI to visualize nuclei, fluorescence microscopy was used to determine mitotic index, the results are the mean and standard deviation of 3 or 4 experiments, In the experiment, 300 cells were counted for each drug concentration, the mitotic inhibition IC ₅₀ for KPU-02 was 17.4 ± 1.2 nM, and the mitotic inhibition IC _{50 for} CA4 was 5.4 ± 0.7 nM. so Ri, antimitotic IC ₅₀ for CLC was 23.8 ± 3.1 nM (Table 23).

  Most MT targeting agents block mitosis at the transition from metaphase to late phase. Mitotic arrest at the transition from metaphase to late phase is associated with suppression of MT kinetics. While not being bound by any particular theory, the earlier pre-medium blockade, when compared to other depolymerized drugs that stabilize MT kinetics at low concentrations (eg, vinblastine) with MT polymer depletion, A different mechanism of action for 02 is suggested.

Spindle and interphase array MT depolymerization KPU-02, CA4 and CLC were observed to be potent microtubule depolymerizers in MCF7 cells. Spindle microtubules are more susceptible to depolymerization and / or inhibition of polymerization than interphase array microtubules, but both microtubule populations were affected (FIG. 52). FIG. 52 represents an immunofluorescence microscopy image of MCF7 cells. Interphase arrays are more stable to depolymerization by KPU-02, CA4 and CLC than spindles. Cells were prepared and seeded as for mitotic progression and treated with mitotic inhibition IC ₅₀ for 20 hours for each drug. Cells were incubated in DM1A / DM1B, a mouse monoclonal cocktail of anti-α- and β-tubulin, and then stained with FITC-conjugated secondary antibody and DAPI. a to d, control (a), KPU-02 (b), tubulin in CA4 (c) and CLC (d) treated cells, and e to h, control (a), KPU-02 (f), CA4 (G) and CLC (h) DNA in cells treated. Thin arrows indicate spindle polymer and mitotic chromosomes, thick arrows indicate interphase arrays and nuclei.

With IC ₂₅ on mitotic arrest, KPU-02 dramatically changed the spindle morphology. 53A-53C represent immunofluorescence microscopy images of MCF7 cells treated with KPU-02 (A), CA4 (B) and CLC (C) for 20 hours. Spindle destruction with increasing drug concentration. 1-4, control (1), the concentration of IC ₂₅ about antimitotic (2), IC ₅₀ relates antimitotic (3)
And α- and β-tubulin at twice the IC ₅₀ for mitotic inhibition (4); 5-8, corresponding images of DNA for adjacent panels. In IC ₂₅ with respect to KPU-02 or CA4 normal bipolar spindle was present (FIGS. 53A and FIG. 53B). Compound treated cells had unipolar or bipolar spindles with unassociated chromosomes. In contrast, for CLC, a normal bipolar spindle persisted with IC ₂₅ (FIG. 53C). In IC ₅₀ for KPU-02, 75% of the mitotic cells, and contain asters or focus of tubulin, the remaining cells had no detectable mitogenic polymer. In the presence of CLC, half of the cells were bipolar with unassociated chromosomes and the other half were unipolar. There was almost no detectable MT polymer in mitotic cells treated with KPU-02, CA4 or CLC at a concentration of IC ₅₀ doubling with respect to mitotic arrest.

The intermicrotubule array was more resistant to depolymerization than the spindle for all of the compounds tested (FIG. 52). However, polymer qualitative reduction was observed in a dose-dependent manner for all three compounds (FIGS. 54A-54C). 54A-54C represent immunofluorescence microscopy images of MCF7 cells treated with KPU-02 (a), CA4 (b) and CLC (c) for 20 hours. Interphase MT depolymerization with increasing drug concentration. 1-4, α- and β-tubulin in control (1), concentration of IC ₂₅ for anti-mitotic activity (2), IC ₅₀ (3) for anti-mitotic activity and 2 × IC ₅₀ (4) for anti-mitotic activity; 8. Corresponding image of DNA for adjacent panels. Presumably, MAP-rich tubulin is sequestered in in vitro polymer mass assays, while tubulin is sequestered in these interphase cells despite the presence of intracellular stabilized MAP.

Lack of suppression or regulation of MT dynamic instability in living MCF7 cells KPU-02 as well as CA4 are MTs at concentrations that give 25% (Table 24) or 50% (Table 25) of maximum division inhibition in MCF7 cells. Has no measurable effect on dynamic instability. While not being bound by any particular theory, these data suggest that the antiproliferative mechanism of action of KPU-02 (and CA4) is primarily due to inhibition of MT polymerization rather than suppression of microtubule dynamics. To do.

  The above examples are described merely to facilitate an understanding of the present invention. Thus, it will be appreciated by those skilled in the art that the disclosed methods and compounds include, and in other cases, provide additional derivatives of dehydrophenylahistine.

  Those skilled in the art will readily appreciate that the present invention is well adapted, for example, to obtain the stated objects and advantages, as well as other specific details. The methods and procedures described herein are representative of presently preferred embodiments, are exemplary, and are not intended as limitations on the claims of the invention. Modifications and other uses herein will occur to those skilled in the art and are encompassed within the spirit of the invention.

  It will be readily apparent to those skilled in the art that various alternatives and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

  As mentioned above, all patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are hereby incorporated by reference to the extent permitted by law, and thus each individual patent and publication is specifically and independently shown to be incorporated by reference. Can be treated.

  The invention, which is appropriately described herein by way of example, may be practiced in the absence of any element (s), limitation (s) not specifically disclosed herein. . The terms and expressions that have been used are used as descriptive terms and not as limiting terms, and in the use of such terms and expressions, are the equivalents of the features shown and described, or portions thereof. There is no intention to exclude. It will be understood that various modifications are possible within the scope of the claims of the present invention. Thus, although the present invention has been specifically disclosed with preferred embodiments and optional features, modifications and changes to the concepts disclosed herein may be made by those skilled in the art, and such modifications and changes are not It should be understood that this is considered to be within the scope of the claims of the present invention.

  In certain figures, compounds are identified using alternative symbolic representations. The complete chart for converting these alternative symbolic representations is as follows:

Diacyl diketopiperazine 1 is reacted with imidazole carboxaldehyde 2 to obtain intermediate compound 3, which is reacted with benzaldehyde 4 to produce dehydrophenylahistine to produce dehydrophenylahistine. It is a figure which shows the reaction scheme to be made. It is a figure showing the HPLC profile of synthetic crude dehydrophenillahistine. Diacyl diketopiperazine 1 is reacted with benzaldehyde 4 to give intermediate compound 17, which is reacted with imidazole carboxaldehyde 15 to produce dehydrophenylahistine, resulting in dehydrophenylahistine. It is a figure which shows the reaction scheme to be made. FIG. 3 is a diagram representing an HPLC profile of crude synthetic tBu-dehydrophenylahistine generated from Route A and Route B. FIG. 2 shows two modification methods for dehydroPLH for potent cytotoxic activity. FIG. 6 represents the putative active conformation of dehydroPLH at the phenyl moiety. It is a figure showing the cytochrome P450 metabolism of phenilahistine. It is a figure which shows the ZE rearrangement of tBu-dehydroPLH. It is a figure showing the synthetic image and prodrug image of acyl-E-tBu-dehydroPLH. It is a figure showing the temperature gradient of 3-Z-benzylidene-6- [5 "-(1,1-dimethylallyl) -1H-imidazole-4" -Z-ylmethylene] -piperazine-2,5-dione. It is a figure showing the temperature gradient of 3-Z-benzylidene-6- (5 "-tert-butyl-1H-imidazole-4" -Z-ylmethylene) -piperazine-2,5-dione. FIG. 6 represents the effect of KPU-2, KPU-35 and t-butyl-phenhistine compared to colchicine and taxol on HuVEC monolayer permeability to FITC-dextran. FIG. 10 depicts the effect of KPU-2 alone and in combination with CPT-11 on estimated tumor growth in an HT-29 human colon tumor xenograft model. FIG. 10 depicts the effect of KPU-2 alone and in combination with CPT-11 on the weight of tumor excised at dissection in individual mice in an HT-29 human colon tumor xenograft model. FIG. 5 depicts the effect of KPU-2 alone and in combination with CPT-11 on estimated tumor growth in the HT-29 human colon tumor xenograft model. FIG. 10 depicts the effect of KPU-2 alone and in combination with CPT-11 on the weight of tumor excised at dissection in individual mice in an HT-29 human colon tumor xenograft model. FIG. 5 depicts the effect of KPU-2 alone and in combination with CPT-11 on estimated tumor growth in the HT-29 human colon tumor xenograft model. FIG. 5 depicts the effect of KPU-35 alone and in combination with CPT-11 on estimated tumor growth in the HT-29 human colon tumor xenograft model. FIG. 5 depicts the effect of t-butyl-phenilahistine alone and in combination with CPT-11 on estimated tumor growth in an HT-29 human colon tumor xenograft model. FIG. 10 depicts the effect of KPU-2 alone and in combination with CPT-11 on the weight of tumor excised at dissection in individual mice in an HT-29 human colon tumor xenograft model. FIG. 5 depicts the effect of KPU-35 alone and in combination with CPT-11 on the weight of tumor excised at dissection in individual mice in an HT-29 human colon tumor xenograft model. FIG. 6 depicts the effect of t-butyl-phenilahistin alone and in combination with CPT-11 on the weight of tumor excised at dissection in individual mice in an HT-29 human colon tumor xenograft model. FIG. 6 depicts the effect of KPU-2 alone and in combination with CPT-11 on tumor growth in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 6 depicts the effect of KPU-2 alone and in combination with CPT-11 on tumor growth in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 6 depicts the effect of KPU-2 alone and in combination with CPT-11 on tumor growth in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 3 depicts the effect of KPU-2 alone and in combination with CPT-11 on final tumor weight in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 3 depicts the effect of KPU-2 alone and in combination with CPT-11 on final tumor weight in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 3 depicts the effect of KPU-2 alone and in combination with CPT-11 on final tumor weight in an HT-29 human colon tumor xenograft model (comparison of three studies). FIG. 4 depicts the effect of KPU-2 alone or in combination with taxotere on estimated tumor growth in a DU-145 human prostate tumor xenograft model. FIG. 6 represents the effect of KPU-2 alone and in combination with taxotere on estimated tumor growth based on observations made in the living part of the DU-145 human prostate tumor xenograft model. FIG. 6 represents the effect of KPU-35 alone and in combination with taxotere on estimated tumor growth based on observations made in the living part of the DU-145 human prostate tumor xenograft model. FIG. 7 depicts the effect of t-butyl-phenilahistine alone and in combination with taxotere on estimated tumor growth based on observations made in the living part of the DU-145 human prostate tumor xenograft model. FIG. 4 depicts the effect of KPU-2 alone and in combination with taxotere on individual tumor weights excised at dissection in a DU-145 human prostate tumor xenograft model. FIG. 4 represents the effect of KPU-35 alone and in combination with taxotere on individual tumor weights excised at dissection in a DU-145 human prostate tumor xenograft model. FIG. 6 represents the effect of KPU-2 alone and in combination with taxotere in the MCF-7 human breast tumor xenograft model. FIG. 5 represents the effect of KPU-35 alone and in combination with taxotere in the MCF-7 human breast tumor xenograft model. FIG. 5 depicts the effect of t-butyl-phenilahistine alone and in combination with taxotere in the MCF-7 human breast tumor xenograft model. FIG. 6 depicts the effect of KPU-2 alone and in combination with taxotere on estimated tumor growth in an A549 human lung tumor xenograft model. FIG. 6 depicts the effect of KPU-2 alone and in combination with taxotere on tumor weight excised at dissection in an A549 human lung tumor xenograft model. FIG. 5 represents the effect of KPU-2 alone and in combination with paclitaxel on the estimated tumor weight in an MDA-231 human breast tumor model transplanted into mouse mammary fat pad. FIG. 7 represents the effect of KPU-2 alone and in combination with paclitaxel in a mouse melanoma B16F10 metastatic tumor model. FIG. 5 represents the effect of KPU-35 alone and in combination with paclitaxel in a mouse melanoma B16F10 metastatic tumor model. FIG. 5 represents the effect of t-butyl-phenylhistine alone and in combination with paclitaxel in a mouse melanoma B16F10 metastatic tumor model. FIG. 31 represents the effect of KPU-35 and KPU-02 on tumor vasculature in the dorsal skinfold chamber of FIG. FIG. 6 depicts the effect of KPU-02 in combination with CPT-11 on estimated tumor weight in an HT-29 human colon tumor xenograft model. FIG. 6 depicts the effect of KPU-02 in combination with CPT-11 on excised tumor weight in an HT-29 human colon tumor xenograft model. FIG. 6 depicts the effect of KPU-02 in combination with CPT-11 on excised tumor weight in an HT-29 human colon tumor xenograft model. FIG. 3 represents rapid tubulin depolymerization in HuVEC cells induced by KPU-02 and KPU-35. FIG. 6 represents the effect of KPU-02 on monolayer permeability in HuVEC cells. FIG. 2 represents the effect of KPU-02 on tumor blood flow in a p22 rat sarcoma model using the 125I-IAP technique. FIG. 2 represents the effect of KPU-02 on tumor blood flow in a p22 rat sarcoma model using the 125I-IAP technique. FIG. 5 represents the effect of KPU-02 15 mg / kg IP (expressed as% vehicle control) on blood flow in various tissues 1 and 24 hours after dosing. FIG. 6 is a graph showing the effect of tumor necrosis induced by KPU-02 7.5 mg / kg IP and 15.0 mg / kg IP in a P22 rat sarcoma model. FIG. 6 lists the activity of various tBu-dehydro-PLH derivatives in HT-29 cells. FIG. 38 is a continuation of FIG. FIG. 38 is a continuation of FIG. FIG. 38 is a continuation of FIG. FIG. 38 is a continuation of FIG. It is a figure showing 3D QSAR (CoMFA) analysis of a tBu-dehydro-PLH derivative. It is a figure showing the X ray crystal analysis of a tBu-dehydro-PLH derivative. FIG. 3 represents the biological activity of various phenylhistine derivatives compared to colchicine. It is a figure showing the effect with respect to cell cycle progression of HeLa cells by tBu-dehydro-PLH (KPU-2) and KPU-35. FIG. 5 represents the effect of dehydro-PLH and tBu-dehydro-PLH (KPU-2) on drug sensitivity and drug resistant tumor cell lines when compared to paclitaxel. FIG. 2 is a diagram showing a turbidity spectrum of microtubule protein polymerization in the presence of DMSO drug excipient (white rhombus), 1.25 μM (white square), 2.5 μM (−) and 5 μM (white circle) KPU-02. . FIG. 3 is a diagram showing a turbidity spectrum of microtubule protein polymerization in the presence of DMSO drug excipient (white rhombus), 1.25 μM (white square), 2.5 μM (−) and 5 μM (white circle) CA4. FIG. 5 represents a turbidity spectrum of microtubule protein polymerization in the presence of DMSO drug excipient (white rhombus), 1.25 μM (white square), 2.5 μM (−) and 5 μM (white circle) CLC. FIG. 2 represents inhibition of MT in the absence or presence of a series of KPU-02 (open circles), CA4 (open squares) and colchicine (open diamonds) concentrations. It is a figure showing the frequency histogram of the average microtubule length in vitro in the steady state in presence of KPU-02. It is a figure showing the frequency histogram of the average microtubule length in vitro in the steady state in presence of CA4. FIG. 5 is a diagram showing a frequency histogram of average microtubule length in vitro in a steady state in the presence of CLC. It is a figure showing the electron micrograph of the MAP rich microtubule formed in vitro in the steady state in presence of KPU-02. It is a figure showing the electron micrograph of the MAP rich microtubule formed in vitro in the steady state in presence of CA4. FIG. 2 is an electron micrograph of a MAP-rich microtubule formed in vitro in a steady state in the presence of CLC. It is a figure showing the graph outline | summary of MT length reduction | decrease in the steady state in presence of KPU-02, CA4, and colchicine. FIG. 3 is a diagram showing a fluorescence emission spectrum of tubulin in the presence of increasing KPU-02. FIG. 6 represents the fit to the fluorescence emission maximum at 487 nm to obtain the tubulin K _d for KPU-02. The inset shows the unprocessed state. It is a figure showing the double reciprocal conversion of combined data. Colchicine to tubulin using various concentrations of [ ³ H] CLC in the absence of KPU-02 or CA4 (open diamonds) or in the presence of 10 μM KPU-02 (open circles) or 10 μM CA4 (open squares) FIG. 6 represents the results of a graphical expression of a binding competition inhibition assay. The figure showing the log [compound] response curve of mitotic progression inhibition by KPU-02, CA4 and CLC in MCF7 cells cultured in the presence of KPU-02 (white circle), CA4 (white square) and colchicine (white rhombus) It is. It is a figure showing the immunofluorescence microscope image of MCF7 cell. ad: tubulin in control (a) tubulin in control, (b) KPU-02, (c) CA4 and (d) CLC treated cells; e-h: DNA in control (e) DNA in control, ( f) KPU-02, (g) CA4 and (h) CLC treated cells. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed with KPU-02. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed by CA4. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed by CLC. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed with KPU-02. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed by CA4. It is a figure showing the immunofluorescence microscope image of the MCF7 cell processed by CLC.

Claims (41)

A compound having the structure of the following formula (I) and inducing vascular collapse:

Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ each independently represent a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R, R ₁ ′ and R ₁ ″ are either covalently bonded to each other or not covalently bonded to each other;
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with an R ₅ group as defined above;
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with R ₅ , an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond can be either a single bond or a double bond). The compound of claim 1, wherein the first aldehyde is imidazole carboxaldehyde.   The compound of claim 1, wherein the second aldehyde is benzaldehyde. The compound according to claim 1, wherein R ₂ , R ₃ , R ₅ and R ₆ are each a hydrogen atom. The compound according to claim _1, wherein X ₁ and X ₂ are each an oxygen atom. The compound of claim ₁ , wherein R ₄ is saturated C ₁ -C ₁₂ alkyl. The saturated C ₁ -C ₁₂ alkyl is a tertiary butyl group, A compound according to claim 6. The compound of claim 1, wherein R ₁ comprises substituted phenyl.   The compound according to claim 8, wherein the substituted phenyl group is methoxybenzene.   The compound of claim 1, wherein the first aldehyde is benzaldehyde.   2. The compound of claim 1, wherein the second aldehyde is imidazole carboxaldehyde.   The compound according to claim 1, wherein n is 0 or 1.   The compound according to claim 1, wherein n is 1. n is 1, Z, respectively Z _1, Z _2, Z ₃ and Z ₄ is a carbon atom A compound according to claim 1.   A method of treating a condition in an animal comprising administering to the animal the compound of claim 1 in an amount effective to reduce vascular growth or an amount effective to reduce vascular density.   Said conditions include immune and non-immune inflammation, rheumatoid arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, post-lens fibroproliferation, rubeosis, 16. The method of claim 15, wherein the method is selected from the group consisting of capillary growth in atherosclerotic plaques, as well as osteoporosis.   16. The method of claim 15, wherein the condition is a neoplastic condition.   The method of claim 17, wherein the neoplastic condition is cancer.   16. The method of claim 15, wherein the condition is not cancer.   17. A method according to claim 15 or 16, wherein the condition is retinopathy.   21. The method of claim 20, wherein the retinopathy is diabetic retinopathy.   21. The method of claim 20, wherein the retinopathy is age-related macular degeneration.   The method of claim 15, wherein the animal is a human.   The method of claim 15, wherein the compound is KPU-02.   16. The method of claim 15, wherein the condition is a condition associated with hypervascularization. A method of inducing vascular collapse in an animal comprising treating said animal with a therapeutically effective amount of a compound having formula (I), said therapeutically effective amount of said compound in said vasculature A method that causes tubulin depolymerization, wherein the compound has the following structure:

Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl , Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl Alkyl halide, carbonyl halide and carbonyl-CCO-R ₇ (wherein R ₇ is a hydrogen atom, a halogen atom, saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cyclo Alkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Roariru, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ each independently represent a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R, R ₁ ′ and R ₁ ″ are either covalently bonded to each other or not covalently bonded to each other;
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with R ₅ as defined above,
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with R ₅ , an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of the n number of Z (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond can be either a single bond or a double bond.   27. The method of claim 26, wherein the animal is a human.   27. The method of claim 26, wherein the human has a disease selected from the group consisting of a tumor, diabetic retinopathy and age-related macular degeneration.   27. The method of claim 26, wherein the disease is not cancer.   27. The method of claim 26, wherein the compound is KPU-02.   A pharmaceutical composition for treating or preventing vascular proliferation, comprising a pharmaceutically effective amount of a compound according to claim 1 together with a pharmaceutically acceptable carrier for the compound. object.   32. The pharmaceutical composition of claim 31, wherein the vascular proliferation is a symptom of a disease selected from the group consisting of cancer, age-related macular degeneration and diabetic retinopathy. A method of preferentially targeting tumor vasculature over non-tumor tissue vasculature,
Administering a compound having the structure of formula (I) to an animal:
Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ each independently represent a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R, R ₁ ′ and R ₁ ″ are either covalently bonded to each other or not covalently bonded to each other;
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Selected from the group consisting of substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl groups and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each unsubstituted or substituted with R ₅ as defined above,
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with R ₅ , an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of n (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
The dashed bond can be either a single bond or a double bond.   34. The method of claim 33, wherein the non-tumor tissue is selected from the group consisting of skin, muscle, brain, kidney, heart, spleen and intestine.   The tumor vasculature is preferentially targeted about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% over non-tumor tissue vasculature. Item 34. The method according to Item 33.   34. The method of claim 33, wherein the animal is a human.   A method of preferentially targeting tumor vasculature over non-tumor tissue vasculature, comprising administering to an animal an agent that preferentially targets tumor vasculature over non-tumor tissue vasculature. Use of a compound having the structure of formula (I) in the preparation of a medicament for the treatment of a condition associated with or dependent on increased vasculature:
Where
R ₁ , R ₄ and R ₆ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R ₁ ′ and R ₁ ″ each independently represent a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl Substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azide, substituted nitro, phenyl, and substituted phenyl, hydroxy, carboxy, —CO—O—R ₇ , cyano, alkylthio, polyhalogenated alkyl. halogenated alkyl including, halogenated carbonyl, and carbonyl -CCO-R ₇ (wherein, R ₇ is a hydrogen atom, a halogen atom, and saturated C ₁ -C ₂₄ alkyl, unsaturated C ₁ -C ₂₄ alkenyl, cycloalkyl, Cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and is selected from the group consisting of to) selected from substituted phenyl group,
R, R ₁ ′ and R ₁ ″ are either covalently bonded to each other or not covalently bonded to each other;
R ₂ , R ₃ and R ₅ are each independently a hydrogen atom, a halogen atom, and saturated C ₁ -C ₁₂ alkyl, unsaturated C ₁ -C ₁₂ alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, amino,
Selected from the group consisting of substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
X ₁ and X ₂ are independently selected from the group consisting of oxygen, nitrogen and sulfur atoms, each unsubstituted or substituted with R ₅ as defined above,
Y is selected from the group consisting of a nitrogen atom, a nitrogen atom substituted with R ₅ , an oxygen atom, a sulfur atom, a sulfur oxide atom, a methylene group and a substituted methylene group;
n is an integer of 0, 1 or 2;
each of the n Z (if not 0) and Z ₁ , Z ₂ , Z ₃ and Z ₄ are each independently selected from carbon, sulfur, nitrogen or oxygen atoms;
The dashed bond can be either a single bond or a double bond.   40. Use according to claim 38, wherein the condition is cancer.   40. Use according to claim 38, wherein the condition is not cancer.   Said conditions include immune and non-immune inflammation, rheumatoid arthritis, rheumatoid arthritis, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, post-lens fibroproliferation, rubeosis, 39. Use according to claim 38, selected from the group consisting of capillary growth in atherosclerotic plaques, as well as osteoporosis.

Images