EP1926724A1 - Analogues de deshydrophenylahistines et utilisation therapeutique de ceux-ci - Google Patents
Analogues de deshydrophenylahistines et utilisation therapeutique de ceux-ciInfo
- Publication number
- EP1926724A1 EP1926724A1 EP06803950A EP06803950A EP1926724A1 EP 1926724 A1 EP1926724 A1 EP 1926724A1 EP 06803950 A EP06803950 A EP 06803950A EP 06803950 A EP06803950 A EP 06803950A EP 1926724 A1 EP1926724 A1 EP 1926724A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- substituted
- group
- tumor
- compound
- heteroaryl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
Definitions
- the present invention relates to compounds and methods of synthetic preparation in the fields of chemistry and medicine. More specifically, the present invention relates to compounds and procedures for making compounds useful in the treatment of cancer and the treatment of fungal infections.
- Fungi especially pathogenic fungi and related infections, represent an increasing clinical challenge.
- Existing antifungal agents are of limited efficacy and toxicity, and the development and/or discovery of strains of pathogenic fungi that are resistant to drugs currently available or under development.
- fungi that are pathogenic in humans include among others Candida spp. including C. albicans, C. tropicalis, C. kefyr, C. krusei and C, galbrata; Aspergillus spp. including A. fumigatus and A. flavus; Cryptococcus . neoformans; Blastomyces spp.
- Blastomyces dermatitidis including Blastomyces dermatitidis; Pneumocystis carinii; Coccidioides immitis; Basidiobolus ranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp. including R. oryzae and R, microsporus; Cunninghamella spp.; Rhizomucor spp.; Paracoccidioides brasiliensis; Pseudallescheria boydii; Rhinosporidium seeberi; and Sporothrix schenckii (Kwon-Chung, KJ. & Bennett, J.E. 1992 Medical Mycology, Lea and Febiger, Malvern, PA).
- tryprostatins A and B which are diketopiperazines consisting of proline and isoprenylated tryptophan residues
- five other structurally-related diketopiperazines inhibited cell cycle progression in the M phase, see Cui, C. et al, 1996 J Antibiotics 49:527-33; Cui, C. et al. 1996 J Antibiotics 49:534-40, and that these compounds also affect the microtubule assembly, see Usui, T. et al. 1998 Biochem J 333:543-48; Kondon, M. et al. 1998 J Antibiotics 51:801-04.
- CLC-site colchicine binding-site
- tubulin which is a macromolecule that consists of two 50 kDa subunits ( ⁇ - and ⁇ -tubulin) and is the major constituent of microtubules.
- CLC-site colchicine binding-site
- tubulin which is a macromolecule that consists of two 50 kDa subunits ( ⁇ - and ⁇ -tubulin) and is the major constituent of microtubules.
- Microtubules are thought to be involved in several essential cell functions, such as axonal transport, cell motility and determination of cell morphology.
- inhibitors of microtubule function may have broad biological activity, and be applicable to medicinal and agrochemical purposes.
- colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan, S.M. et al, 1973 JAm Chem Soc 95:1335-36, podophyllotoxin, see Sackett, D.L., 1993 Pharmacol Ther 59:163-228, and combretastatins, see Pettit, G.R. et al, 1995 J Med Chem 38:166-67, may prove to be valuable as eukaryotic cell cycle inhibitors and, thus, may be useful as chemotherapeutic agents.
- CLC colchicine
- diketopiperazine-type metabolites have been isolated from various fungi as mycotoxins, see Horak R.M. et al, 1981 JCS Chem Comm 1265-67; AIi M, et al, 1898 Toxicology Letters 48:235-41, or as secondary metabolites, see Smedsgaard J. et al, 1996 J Microbiol Meth 25:5-17, little is known about the specific structure of the diketopiperazine-type metabolites or their derivatives and their antitumor activity, particularly in vivo.
- R 2 and R 3 are each separately selected from the group consisting of a hydrogen atom; a halogen atom; mono-substituted; poly-substituted or unsubstituted, straight or branched chain variants of the following residues: Ci-Cj 2 alkyl, C 1 -C] 2 alkenyl, acyl, and alkoxy; and mono-substituted, poly- substituted or unsubstituted variants of the following residues: cycloalkyl, cycloalkoxy, aryl, heteroaryl, amino, nitro, and sulfonyl; or R 2 is a bond to Ar;
- R 4 and R 6 are each separately selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: C]-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted or unsubstituted valiants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl, amino, aminocarbonyl, amide,
- X] and X 2 are separately selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom substituted with a R 5 group;
- R 5 is selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C] 2 alkyl, unsaturated Ci-C] 2 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
- Y is selected from the group consisting of a nitrogen atom substituted with R 5 , an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group, and a substituted methylene group; n is O, 1, 2, 3, or 4; and
- u-xi ⁇ i ⁇ bstituted, straight or branched chain variants of the following r «es idues: C1-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, s*xy ⁇ * ⁇ lalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alks ⁇ ioxy, and alkylthio; mono-substituted, poly-substituted or
- Y is selected from the group consisting of an oxygen atom, a sulfur atom, and an* cr>:xidized sulfur atom.
- R 4 is a mono-substituted; poly-substituted or u ⁇ n ⁇ s; ⁇ ubstituted, straight or branched chain variant of C 1 -C 12 alkyl or C 1 -C 12 alkenyl.
- R 4 is selected from the group consisting of 3,3- dimethyl ⁇ ropyi-1-enLe or tert-butyl.
- Xi and X 2 are oxygen.
- Y is CD.
- n is 0.
- Ar is selected from the group cons- is' — ting of: with one or more subtituents selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: Q-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, ary
- R 2 is a bond to Ar, and the compound has the structure:
- Ri, R 4 , and R 6 are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Cj-C 24 alkyl, unsaturated Ci-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, - CO-O-R 7 , cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R7, wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated CpC 24 alkyl, unsaturated Cp C 24 alkenyl, cycloalkyl, cycl
- Ri 1 and Ri" are each independently selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C]-C 24 alkyl, unsaturated C]-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, - CO-O-R 7 , cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R 7 , wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated CpC 24 alkyl, unsaturated Cp C 24 alkenyl, cycloalkyl, cycloalkenyl, alk
- R, Ri' and Ri are either covalently bound to one another or are not covalently bound to one another;
- R 2 and R 3 are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated CpCi 2 alkyl, unsaturated Cp C 12 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups; m is an integer equal to zero, one or two; Z, for each separate m, if non-zero, and Zi, Z 2 , Z 3 and Z 4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom; and the dashed bonds may be either single or double bonds.
- Another embodiment includes a method for treating a condition in an animal, comprising administering to the animal a compound of formula II in an amount that is effective to reduce vascular proliferation or in an amount that is effective to reduce vascular density.
- the condition is selected from the group consisting of immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, and osteoporosis.
- said condition is a neoplastic condition.
- said neoplastic condition is cancer.
- the cancer is selected from the group consisting of one or more of colon cancer, breast cancer, lung cancer, pancreas cancer, prostate cancer, and melanoma.
- the condition is not cancer.
- said condition is a retinopathy.
- said retinopathy is diabetic retinopathy.
- said retinopathy an age-related macular degeneration.
- said animal is a human.
- ⁇ the condition is a condition associated with hypervascularization.
- Another embodiment includes a method of inducing vascular collapse in an animal, comprising treating said animal with a therapeutically effective amount of a compound of formula II, wherein said therapeutically effective amount of said compound causes tubulin depolymerization in said vasculature.
- said animal is a human.
- said human has a disease selected from the group consisting of a tumor, a diabetic retinopathy, and an age-related macular degeneration.
- the disease is not cancer.
- the tumor is selected from the group consisting of one or more of a colon tumor, a breast tumor, a lung tumor, a pancreas tumor, and a prostate tumor.
- Another embodiment includes a method of preferentially targeting tumor vasculature over non-tumor tissue vasculature, comprising administering to an animal a compound of formula II.
- the non-tumor tissue is selected from the group consisting of skin, muscle, brain, kidney, heart, spleen, and gut.
- the tumor vasculature is preferentially targeted over non-tumor tissue vasculature by about 10%, 20%, 30%, 40 « %, 50%, 60%, 70%, 80% and 90%.
- the animal is a human.
- Another embodiment includes a pharmaceutical composition comprising a compound of formula II together with a pharmaceutically acceptable carrier.
- Another embodiment includes a method for treating a tumor in an animal, comprising irradiating the tumor with radiation and administering to the animal a compound of Formula (II).
- the method comprises administering to the subject a composition comprising an effective antitumor or antifungal amount of a dehydrophenylahistin or its analog.
- neoplasms such as cancers
- other conditions associated with or which rely upon vascularization including for example, immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like.
- the disease is not cancer.
- inventions relate to methods of inducing vascular collapse in an animal.
- the methods can include treating said animal with a therapeutically effective amount of a compound of the Formula (I) or (II) as described herein, for example, The therapeutically effective amount of said compound can cause tubulin depolymerization in the vasculature.
- the animal can be a human.
- the disease can be a tumor, a diabetic retinopathy, an age-related macular degeneration, and the like.
- the disease is not cancer or cancer can be specifically excluded from the methods and uses.
- Still further embodiments relate to pharmaceutical compositions for treating or preventing vascular proliferation comprising a pharmaceutically effective amount of a compound disclosed herein together with a pharmaceutically acceptable carrier therefor.
- the vascular proliferation can be a symptom of a disease, for example, cancer, age-related macular degeneration and diabetic retinopathy.
- Some embodiments relate to methods of preferentially targeting tumor vasculature over non-tumor tissue vasculature.
- the methods can include the step of administering to an animal, preferably a human, a compound having the structure of Formula (I) or (II) as described herein.
- the non-tumor tissue can be, for example, skin, muscle, brain, kidney, heart, spleen, gut, and the like.
- the tumor vasculature can be preferentially targeted over non-tumor tissue vasculature, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%.
- Other embodiments relate to methods of preferentially targeting tumor vasculature over non-tumor tissue vasculature, which methods can include administering to an animal an agent that preferentially targets tumor vasculature over non-tumor tissue vasculature.
- the condition can be any other that is associated with hypervascularization, associated with vasculature or which relies upon vasculature.
- Examples include immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, - ⁇ x * -fcinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibropla ⁇ ia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like,
- Figure 1 illustrates a reaction scheme for producing dehydrcrrp. Hub: — ⁇ ⁇ nylahistins by reacting a diacyldiketopiperazine 1 with an iniidazolecarboxaldeheyde H2 ⁇ to yield an intermediate compound 3 which is reacted with a benzaldehyde 4 tzzo— produce a dehydrophenylahistin.
- Figure 2 depicts the HPLC profile of the tic crude dehydrophenylahistin.
- Figure 3 illustrates a reaction scheme for producing dehydio ⁇ p ⁇ _enylahistins by reacting a diacyldiketopiperazine 1 with a benzaldehyde 4 to yield aim L intermediate compound 17 which is reacted with an imidazolecarboxaldeheyde 15 i ⁇ c produce a dehydrophenylahistin,
- Figure 4 depicts the HPLC profiles of the crude s ⁇ y L"thetic tBu- dehyrophenylahistin produced from Route A and from Route B.
- Figure 5 illustrates two modification strategies for dehydroF"L E-I for potent cytotoxic activity
- Figure 6 depicts the putative active conformation of dehyc-lr :> PLH at the phenyl moiety
- Figure 7 depicts Cytochrome P450 metabolism of phenylahist:.-:ni.
- Figure 8 illustrates the Z-E migration of tBu-dehydroPLH
- Figure 9 depicts the synthesis and prodrug image o_rf scyl-E-tBu- dehydroPLH.
- Figure 10 depicts the temperature gradient of 3-Z-Benzylidene-6-[5"-(l , 1 - iethylallyl)-lH-imidazol-4"-Z-ylmethylene]-piperazine-2,5-dione.
- Figure .11 depicts the temperature gradient of 3-Z-benzylidene-6-(5"-tert- yl-lH-imidazol-4"-Z-ylmethylene)-piperazine-2,5-dione.
- Figure 12 depicts the effect of KPU-2, KPU-35 and t-butyl-phenylahistin omparison to colchicine and taxol on Hu VEC monolayer permeability to FITC-Dextran.
- Figure 13 depicts the effect of KPU-2 alone and in combination with CPT- >n estimated tumor growth in the HT-29 Human Colon Tumor Xenograft model.
- Figure 14 depicts the effect of KPU-2 alone and in combination with CPT- m the weight of tumors excised at autopsy in individual mice in the HT-29 Human Colon nor Xenograft model
- Figure 15 depicts the effect of KPU-2 alone and in combination with CPT- >n estimated tumor growth in the HT-29 Human Colon Tumor Xenograft model
- Figure 16 depicts the effect of KPU-2 alone and in combination with CPT- >n the weight of tumors excised at autopsy in individual mice in the HT-29 Human Colon ior Xenograft model.
- Figures 17A-C depict the effects of: A. KPU-2, B. KPU-35 and C. t-butyl- iylahistin alone and in combination with CPT-I l on estimated tumor growth in the HT- uman colon tumor xenograft model.
- Figures 18A-C depict the effects of A. KPU-2, B. KPU-35 and C. t-butyl- iylahistin alone and in combination with CPT-I l on the weight of tumors excised at psy in individual mice in the HT-29 Human Colon Tumor Xenograft model.
- Figures 19A-C depict the effects of KPU-2 alone and in combination with -11 on tumor growth in the HT-29 human colon tumor xenograft model: comparison of ; studies.
- Figures 20A-C depict the effects of KPU-2 alone and in combination with -11 on final tumor weights in the HT-29 human colon tumor xenograft model: parison of three studies.
- Figure 21 depicts the effects of KPU-2 alone or in combination with Taxotere on estimated tumor growth in the DU- 145 Human Prostate Tumor Xenograft Model.
- Figures 22A-C depict the effects of A. KPU-2, B. KPU-35 and C. t-butyl- phenylahistin alone and in combination with Taxotere on the estimated tumor growth based on observations made during the in-life portion of the DU- 145 Human Prostate Tumor Xenograft Model.
- Figure 23 depicts the effects of KPU-2 alone and in combination with Taxotere on the individual excised tumor weights at autopsy in the DU- 145 Human Prostate Tumor Xenograft Model.
- Figure 24 depicts the effects of KPU-35 alone and in combination with Taxotere on the individual excised tumor weights at autopsy in the DU- 145 Human Prostate Tumor Xenograft Model.
- Figures 25A-C depict the effects of A. KPU-2, B. KPU-35 and C. t-butyl- phenylahistin alone and in combination with Taxotere in MCF-7 Human Breast Tumor Xenograft model.
- Figure 26 depicts the effects of KPU-2 alone and in combination with Taxotere on estimated tumor growth in the A549 Human Lung Tumor Xenograft model.
- Figure 27 depicts the effects of KPU-2 alone and in combination with Taxotere on the excised tumor weights at autopsy in the A549 Human Lung Tumor Xenograft model.
- Figure 28 depicts the effects of KPU-2 alone and in combination with Paclitaxel on estimated tumor weight in the murine mammary fat pad implanted MDA-231 Human Breast Tumor model.
- Figures 29A-C depict effects of A. KPU-2, B. KPU-35 and C. t-butyl- phenylahistin alone and in combination with Paclitaxel in the Murine Melanoma B 16. FlO Metastatic Tumor Model.
- Figure 30 depicts effects of KPU-35 and KPU-02 on tumor vasculature in the dorsal skinfold chamber of Figure 30.
- Figure 31 depicts effect of KPU-02 in combination with CPT-Il on the estimated tumor weight in the HT-29 human colon tumor xenograft model.
- Figures 32A-B depict the effect of KPU-02 in combination with CPT-11 on the excised tumor weight in the HT-29 human colon tumor xenograft model
- Figure 33 depicts rapid tubulin depolymerization in HuVEC cells induced by KPU-02 and KPU-35.
- Figure 34 depicts effect of KPU-02 on monolayer permeability in HuVEC cells.
- Figures 35A-B depict the effect of KPU-02 on tumor (A) and tissue (B) blood flow in the P22 rat sarcoma model using the 125I-IAP technique.
- Figure 36 depicts the effect of KPU-02 15 mg/kg IP (expressed as % vehicle control) on blood flow in different tissues 1 and 24 hours post-dose.
- Figure 37 depicts the tumor necrosis induced by KPU-02 7.5 and 15.0 mg/kg IP in the P22 rat sarcoma model
- Figure 38 lists the activity of various tBu-dehydro-PLH derivatives at HT- 29 cells.
- Figure 39 depicts 3D QSAR (CoMFA) analysis of tBu-dehydro-PLH derivatives.
- Figure 40 depicts X-ray crystallographic analysis of tBu-dehydro-PLH derivatives.
- Figure 41 depicts the biologically activity of various phenylahistin derivatives compared to colchicine.
- Figure 42 depicts the effect on cell cycle progression of HeLa cells by tBu- dehydro-PLH (KPU-2) and KPU-35.
- Figure 43 depicts the effect of dehydro-PLH and tBu-dehydro-PLH (KPU- 2) on drug-sensitive and drug-resistant tumor cell lines as compared to paclitaxel.
- Figure 44A depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 ⁇ M (o), 2.5 ⁇ M (— ), and 5 ⁇ M (o) KPU-02.
- Figure 44B depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 ⁇ M ( ⁇ ), 2.5 ⁇ M (— ), and 5 ⁇ M (o) CA4.
- Figure 44C depicts turbidity spectra of microtubule protein polymerization in the presence of DMSO drug vehicle (0), 1.25 ⁇ M ( ⁇ ), 2.5 ⁇ M (— ), and 5 ⁇ M (o) CLC.
- Figure 45 depicts inhibition of MT in the absence or presence of a range of KPU-02 (o),CA-4 ( ⁇ ), and colchicine (0) concentrations.
- Figure 46A depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of KPU-02.
- Figure 46B depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of CA4.
- Figure 46C depicts frequency histograms of mean microtubule lengths in vitro at steady state in the presence of CLC.
- Figure 47A depicts electron micrographs of MAP-rich microtubules formed in vitro at steady state in the presence of KPU-02.
- Figure 47B depicts electron micrographs of MAP-rich microtubules formed in vitro at steady state in the presence of CA4.
- Figure 47C depicts electron micrographs of MAP-rich microtubules formed in vitro at steady state in the presence of CLC.
- Figure 48 depicts a graphical summary of MT length decrease at steady state in the presence of KPU-02, CA4, and colchicine.
- Figure 49A depicts fluorescence emission spectra of tubulin in the presence of increasing KPU-02.
- Figure 49B depicts a fit to fluorescence emission maxima at 487 nm to obtain the Ka of tubulin for KPU-02.
- the inset depicts residuals.
- Figure 49C depicts double reciprocal transformation of the binding data.
- Figure 50 depicts the graphical results of a competitive inhibition assay of colchicine binding to tubulin with various concentrations of [ 3 H]CLC in the absence (0), or presence of 10 ⁇ M KPU-02 (o) or 10 ⁇ M CA4 ( ⁇ ).
- Figure 51 depicts log [compound] response curves for mitotic progression inhibition by KPU-02, CA4, and CLC in MCF7 cells cultured in the presence of KPU-02 (o), CA4 ( ⁇ ), and colchicine (0).
- Figure 52 depicts immunofluorescence microscopy images of MCF7 cells, a-d: 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.
- Figure 53A depicts immunofluorescence microscopy images of MCF7 cells treated with KPU-02
- Figure 53B depicts immunofluorescence microscopy images of MCF7 cells treated with CA4.
- Figure 53C depicts immunofluorescence microscopy images of MCF7 cells treated with CLC.
- Figure 54A depicts immunofluorescence microscopy images of MCF7 cells treated with KPU-02
- Figure 54B depicts immunofluorescence microscopy images of MCF7 cells treated with CA4.
- Figure 54C depicts immunofluorescence microscopy images of MCF7 cells treated with CLC.
- Figure 55 shows that NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI- 2506 induce tubulin depolymerization within 30 minutes in HuVECs.
- the disclosure provides methods for the synthetic preparation of compounds, including novel compounds, including dehydrophenylahistin and dehydrophenylahistin analogs, and provides methods for producing pharmaceutically acceptable cell cycle inhibitors, antitumor agents and antifungal agents in relatively high yield, wherein said compounds and/or their derivatives are among the active ingredients in these cell cycle inhibitors, antitumor agents and antifungal agents.
- Other objects include providing novel compounds not obtainable by currently available, non-synthetic methods, It is also an object to provide a method of treating cancer, particularly human cancer, comprising the step of administering an effective tumor-growth inhibiting amount of a member of a class of new anti-tumor compounds.
- This invention also provides a method for preventing or treating a pathogenic fungus in a subject which involves administering to the subject an effective anti-fungal amount of a member of a class of new anti-fungal compounds, e.g., administering a dehydrophenylahistin or its analog in an amount and manner which provides the intended antifungal effect.
- an effective anti-fungal amount of a member of a class of new anti-fungal compounds e.g., administering a dehydrophenylahistin or its analog in an amount and manner which provides the intended antifungal effect.
- Ri, R 4 , and R 6 are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C 24 alkyl, unsaturated Ci-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R 7 , cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R 7 , wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated Ci-C 24 alkyl, unsaturated Ci-C 24 alkenyl, cycloalkyl, cyclo
- Ri' and R]" are independently selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C 24 alkyl, unsaturated Ci-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R 7 , cyano, alkylthio, halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-R 7 , wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated C]-C 24 alkyl, unsaturated C)-C 24 alkenyl, cycloalkyl, cycloalkenyl
- R, Ri 1 and Ri are either covalently bound to one another or are not covalently bound to one another;
- R 2 , R_3, and R 5 are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-Ci 2 alkyl, unsaturated Ci-Ci 2 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
- X J and X 2 are separately selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom, each either unsubstituted or substituted with a R 5 group, as defined above;
- Y is selected from the group consisting of a nitrogen atom substituted with R 5 , an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group, and a substituted methylene group;
- n is an integer equal to zero, one or two;
- Z for each separate n, if non-zero, san*d- Z 1 , Z 2 , Z 3 and Z 4 are each separately selected from a carbon atom, a sulfur atom, a n ⁇ tr-o-gem atom or an oxygen atom;
- the dashed bonds may be either single or ⁇ Lo»uifc>l-e bonds.
- the method comprises a method of producLnag; compounds of Formula (I) by the steps of:
- said first aldehyde and said second aldehxycters are selected from the group consisting of an oxazolecarboxaldeyhyde, imidazolecaLr ⁇ b « ⁇ »:s:a_ldehyde, a benzaldehyde, imidazolecarboxaldehyde derivatives, and benzaldehyde «d e ⁇ 5vatives, thereby forming a compound of Formula (I) wherein
- Ri, R 4 , and R 6 are each separately selected! fiom the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C 24 alkcjyl, unsaturated Ci-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substHi- ⁇ ec ⁇ l aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substitu_tes «d Mi ⁇ tro, phenyl, and substituted phenyl groups, hydroxy, carboxy, -CO-O-R 7 , cyano, alkyLtli io_, Ihalogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyl -CCO-Rj 1 wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated Ci -"C 2
- R]' and Ri" are independently is selectes ⁇ l f ⁇ r «oi ⁇ i the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-C 24 a-l ⁇ cyll,. "unsaturated C]-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, substLtnrtecd aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substittate di n ⁇ tro, phenyl, and substituted phenyl groups,.
- halogenated alkyl including polyhalogenated alkyl, halogenated carbonyl, and carbonyL — C CO-R 7 , wherein R 7 is selected from a hydrogen atom, a halogen atom, and saturated Cj-C 24 alkyl, unsaturated Cj-C 24 alkenyl, cycloalkyl, cycloalkenyl, alkoxy, cyclpalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, azido, substituted nitro, phenyl, and substituted phenyl groups;
- R 2 , R 3 , and R 5 are each separately selected from the group consisting of a hydrogen atom, a halogen atom, and saturated C]-Ci 2 alkyl, unsaturated C 1 -C 12 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
- Xi and X 2 are separately selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom, and
- Y is selected from the group consisting of a nitrogen atom, a substituted nitrogen atom with a R 5 group from above, an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group and a substituted methylene group;
- Z for each separate n, if non-zero, and Zj, Z 2 , Z 3 and Z 4 are each separately selected from a carbon atom, a sulfur atom, a nitrogen atom or an oxygen atom;
- the dashed bonds may be either single or double bonds.
- R 2 and R 3 are each separately selected from the group consisting of a hydrogen atom; a halogen atom; mono-substituted; poly-substituted or unsubstituted, straight or branched chain variants of the following residues: C J -C J2 alkyl, Ci-Ci 2 alkenyl, acyl, and alkoxy; and mono-substituted, poly- substituted or unsubstituted variants of the following residues: cycloalkyl, cycloalkoxy, aryl, heteroaryl, amino, nitro, and sulfonyl; or R 2 is a bond to Ar;
- R 4 and R 6 are each separately selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: Ci-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl, amino, aminocarbonyl, amide, aminocarbon
- Xi and X 2 are separately selected from the group consisting of an oxygen atom, a sulfur atom, and a nitrogen atom substituted with a R 5 group;
- R 5 is selected from the group consisting of a hydrogen atom, a halogen atom, and saturated Ci-Ci 2 alkyl, unsaturated Ci-Ci 2 alkenyl, acyl, cycloalkyl, alkoxy, cycloalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, nitro, and substituted nitro groups, sulfonyl and substituted sulfonyl groups;
- Y is selected from the group consisting of a nitrogen atom substituted with R 5 , an oxygen atom, a sulfur atom, a oxidized sulfur atom, a methylene group, and a substituted methylene group; n is O, 1, 2, 3, or 4; and
- Ar is a cyclic or polycyclic aryl or heteroaryl ring system comprising between one and three rings, wherein: each ring in said system is separately a 5, 6, 7, or 8 membered ring; each ring in said system separately comprises 0, 1, 2, 3, or 4 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen; and each ring in said system is optionally substituted with one or more subtituents selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: Ci-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly-sub
- R 4 is not tert-butyl.
- Y is selected from the group consisting of an oxygen atom, a sulfur atom, and an oxidized sulfur atom.
- R 4 is a mono-substituted; poly-substituted or unsubstituted, straight or branched chain variant of Ci-Ci 2 alkyl or Ci-Ci 2 alkenyl.
- R 4 is selected from the group consisting of 3,3- dimethyl ⁇ ropyl-1-ene or tert-butyl
- Xi and X 2 are oxygen.
- Y is N and R 5 is H or Y is O and R 5 is absent.
- n is 0.
- Ar is selected from the group consisting of:
- one or more subtituents selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly-substituted or unsubstituted, straight or branched chain variants of the following residues: Cj-C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono- substituted, poly-substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl, amino, aminocarbonyl, amide, aminocarbony
- n 0, R 2 is a bond to Ar, and the compound has the structure:
- phenyl ring in the structure is optionally substituted with one or more subtituents selected from the group consisting of hydrogen; halogen; hydroxyl; mono-substituted, poly- substituted or unsubstituted, straight or branched chain variants of the following residues: Ci- C 24 alkyl, C 2 -C 24 alkenyl, C 2 -C 24 alkynyl, alkoxy, acyl, arylalkyl, heteroarylalkyl, alkyloxycarbonyloxy, ester, arylalkoxy, alkoxy, and alkylthio; mono-substituted, poly- substituted or unsubstituted variants of the following residues: acyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, cycloalkoxy, aryl, heteroaryl, aryloxy, arylcarbonyl, heterocycloalkyl, carbonyl,
- R 4 is not 3,3-dimethylpropyl-l-ene or hydrogen.
- Ri, Ri', R 2 , R 3 , R 4 and R 5 are each a hydrogen atom and Xi and X 2 are each an oxygen atom, then R 4 is not 3,3-dimethylpropyl-l- ene or a hydrogen atom
- pro-drug ester especially when referring to a pro-drug ester of the compound of Formula (I) or (II) synthesized by the methods disclosed herein, refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood or inside tissues.
- pro-drug ester refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions.
- pro-drug ester groups examples include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5-R-2-oxo-l,3- dioxolen-4-yl)methyl group.
- Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and “Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups).
- pro-drug ester also refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood.
- pro-drug ester refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, mdanyl and methoxymethyl, as well as other such groups known in the art, including a (5-R-2-oxo-l,3-dioxolen-4-yl)methyl group.
- pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups).
- pharmaceutically acceptable salt especially when referring to a pharmaceutically acceptable salt of the compound of Formula (I) or (II) synthesized by the methods disclosed herein, refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound.
- Preferred examples of pharmaceutically acceptable salt are the alkali metal salts (sodium or potassium), the alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from eutically acceptable organic amines, for example Ci -C 7 allcj ⁇ /KL-s ⁇ i- ⁇ ai — inncinne, ylamine, triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminoj ⁇ «- ⁇ g- TQr-- M-ha-uane.
- the preferred examples of pharmaceutically acceptable salts are acid a_ ⁇ pharmaceutically acceptable inorganic or organic acids, for example, hy ⁇ cdi phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for -e:r: succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfcoi lfonic or naphthalenesulfonic acid.
- a_ ⁇ pharmaceutically acceptable inorganic or organic acids for example, hy ⁇ cdi phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for -e:r: succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfcoi lfonic or naphthalenesulfonic acid.
- R 3 may preferably be chose ⁇ cmi t_x-» b>e n.
- n is equal to zero or one, more preferable one, and Z 2 , Z 3 , and Z 4 , and each separately selected from an oxygen atom, a nitrogen atom, and a carbon atom, more preferable at one least one of Z 2 , Z 3 , and Z 4 being a carbon atom, and most preferable at least two of Z 2 , Z 3 , and Z 4 being a carbon atom, All Z's may simultaneous be carbon atoms.
- variable groups are as defined herein.
- halogen atom means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, i.e., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.
- alkyl means any unbranched or branched, substituted or unsubstituted, saturated hydrocarbon, with Ci-C 6 unbranched, saturated, unsubstituted hydrocarbons being preferred, with methyl, ethyl, iosbutyl, and tert-butyl being most preferred.
- substituted, saturated hydrocarbons C]-C 6 mono- and di- and per-halogen substituted saturated hydrocarbons and amino-substituted hydrocarbons are preferred, with perfluromethyl, perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl being the most preferred.
- substituted has its ordinary meaning, as found in numerous contemporary patents from the related art. See, for example, U.S. Patent Nos. 6,583,143, 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443; and 6,350,759. Specifically, the definition of substituted is as broad as that provided in U.S. Patent No. 6,583,143, which defines the term substituted as any groups such as alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, wherein at least one hydrogen atom is replaced with a substituent.
- substituted is also as broad as the definition provided in U.S. Patent No. 6,509,331, which defines the term “substituted alkyl” such that it refers to an alkyl group, preferably of from 1 to 10 carbon atoms, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of 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, heterocyclic, heterocyclooxy,
- cycloalkyl refers to any non- aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
- acyl refers to alkyl or aryl groups derived from an oxoacid, with an acetyl group being preferred,
- alkenyl means any unbranched or branched, substituted or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, with Ci-C 6 unbranched, mono-unsaturated and di -unsaturated, unsubstituted hydrocarbons being preferred, and mono-unsaturated, di-halogen substituted hydrocarbons being most preferred.
- a z- isoprenyl moiety is particularly preferred.
- cycloalkenyl refers to any non- aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
- aryl refers to aromatic hydrocarbon rings, preferably having five, six, or seven atoms, and most preferably having six atoms comprising the ring.
- heteroaryl refers to aromatic hydrocarbon rings in which at least one heteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring along with at least one carbon atom.
- alkoxy refers to any unbranched, or branched, substituted or unsubstituted, saturated or unsaturated ether, with Ci-C 6 unbranched, saturated, unsubstituted ethers being preferred, with methoxy being preferred, and also with dimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers also being preferred.
- cycloalkoxy refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.
- purified refers to the compound being free of other, dissimilar compounds with which the compound is normally associated in its natural state, so that the compound of the invention comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample,
- the compound of Formula (I) or (II) may be chemically synthesized or produced from reagents known and available in the art.
- diacyldiketopiperazine diacetyldiketopiperazine
- the diacyldiketopiperazine may be prepared, for example, by diacetylation of inexpensive 2,5-piperazinedione (TCI Cat. No. GOlOO, 25 g) with sodium acetate and sodium anhydride.
- TCI Cat. No. GOlOO, 25 g inexpensive 2,5-piperazinedione
- the diacetyl structure of the activated deketopiperazine can be replaced with other acyl groups, to include carbamates such as Boc (t-butoxycarbonyl), Z (benzoyloxy carbony 1) .
- the imidazolecarboxaldehyde may be prepared, for example, according the procedure disclosed in Hayashi et al, 2000 J Organic Chem 65: 8402 as depicted below:
- imidazolecarboxaldehyde derivative is an imidazole-4-carboxaldehyde 15 derivative which can be produced from, for example, a commercially available beta-ketoester 18 (TCI Cat, No. P 1031, 25mL) by the following route:
- the synthetic method disclosed herein may be preferably performed in the presence of cesium carbonate as a base in DMF and in a deoxygenated atmosphere.
- the inert atmosphere circumvents the probable oxidation of activated ⁇ -carbon atoms of the diketopiperazine ring during the treatment with cesium carbonate (see below) as reported, for example, by Watanabe et al, 18 lh International Congress of Heterocyclic Chemistry in Yokohama, Japan (30 July 2001), ' Abstract, page 225.
- Other embodiments of the synthetic method involve modifications to the compounds used in or otherwise involved in the synthesis of compounds represented by Formula (I).
- Such derivatives may include modifications to the phenyl ring, introduction of other aromatic ring systems, position of the aromatic ring, alterations to the imidazole ring system and/or further modifications to the 5-position on the imidazole ring. Examples of such modifications are discussed, for example, in Example 7.
- the result of such modifications includes increased nitrogen content of the phenyl ring and/or the compound which may increase compound solubility.
- Other modifications may incorporate derivatives of known tubulin inhibitors, thereby mimicking the activity of the tubulin inhibitors.
- Other modifications may simplify the synthesis of the ⁇ -ketoester involved in the production of the imidazolecarboxaldehyde used in the methods disclosed herein.
- the present invention also encompasses the compounds disclosed herein, optionally and preferably produced by the methods disclosed herein, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration, which have a pharmaceutically effective amount of the products disclosed above in a pharmaceutically acceptable carrier or diluent.
- Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack. Publishing Co. (A.R. Gennaro edit. 1985).
- Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
- sodium benzoate, ascorbic acid and esters of p- hydroxy benzoic acid may be added as preservatives.
- antioxidants and suspending agents may be used.
- the dehydrophenylahistin or dehydrophenylahistin analog compositions may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; patches for transdermal administration, and sub-dermal deposits and the like.
- injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension 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, human serum albumin and the like.
- the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
- absorption enhancing preparations for example, liposomes
- compositions 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 vehicles include fatty oils such as sesame oil, or other organic oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- compositions for oral use may be obtained by -combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
- suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
- PVP polyvinylpyrrolidone
- disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- Dragee cores are provided with suitable coatings.
- suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- Such formulations can be made using methods known in the art (see, for example, U.S.
- Patent Nos..5,733, 888 injectable compositions
- 5,726,181 poorly water soluble compounds
- 5,707,641 therapeutically active proteins or peptides
- 5,667,809 lipophilic agents
- 5,576,012 solubilizing polymeric agents
- 5,707,615 anti-viral formulations
- 5,683,676 porate medicaments
- 5,654,286 topical formulations
- 5,688,529 oral suspensions
- 5,445,829 extended release formulations
- 5,653,987 liquid formulations
- 5,641,515 controlled release formulations
- 5,601,845 spheroid formulations
- compositions well known in the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery.
- Pharmaceutical formulations include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., 2001 Clin Ther 23(3):440-50) or hydrogels (Mayer et al., 1996 Ophthalmologica 210:101-3); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug- containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A., 1994 J Ocul Pharmacol 10:29-45), lipid-soluble formulations (Aim et al., 1989 Prog CHn 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. Pharmaceutical compositions may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action. As disclosed in Remington's Pharmaceutical Sciences (Mack Publishing, 18 th Edition), and well-known to those skilled in the art, suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and appropriate drug stabilizers. Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.
- the compound of Formula (I) can be administered by either oral or a non-oral pathways.
- it can be administered in capsule, tablet, granule, spray, syrup, or other such form.
- it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection or infusion, subcutaneously, intreperitoneally, intravenously, intramuscularly, or the like.
- the compound may be administered topically, rectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the growth of the tumor.
- Local administration at the site of the tumor is also contemplated, either before or after tumor resection, as are controlled release formulations, depot formulations, and infusion pump delivery.
- the present invention also encompasses methods for making and for administering the disclosed chemical compounds and the disclosed pharmaceutical compositions.
- Such disclosed methods include, among-others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via injection or infusion, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or.
- the pharmaceutically effective amount of the dehydrophenylahistin or dehydrophenylahistin analog composition required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration.
- the dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
- the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
- the compounds disclosed herein are effective in the treatment of cancer when used in combination with other actives, specifically other chemotherapeutics, for example biologies and the specific chemotherapeutics CPT-I l, Taxotene (docataxel) and paclitaxel.
- the compounds disclosed herein are also effective in the treatment of cancer when used in combination with other actives, including anti-vascular agents, anti-angiogenenic agents, such as Erbuitux (Imclone/bristol-Myers) and Iressa (AstraZeneca), other VEGF inhibitors and biologies, more specifically, at least one anti- VEGF antibodies, especially monoclonal antibodies to the VEGF receptor, including DClOl, a rat monoclonal antibody, which blocks the mouse VEGF receptor 2 (flk-1).
- actives including anti-vascular agents, anti-angiogenenic agents, such as Erbuitux (Imclone/bristol-Myers) and Iressa (AstraZeneca), other VEGF inhibitors and biologies, more specifically, at least one anti- VEGF antibodies, especially monoclonal antibodies to the VEGF receptor, including DClOl, a rat monoclonal antibody, which blocks the mouse VEGF receptor 2 (f
- the disclosed compounds in employing them in vivo, may be administered to the mammal in a variety of ways, including parenterally, intravenously, via infusion or injection, subcutaneously, intramuscularly, colonically, rectally, vaginally, nasally or intraperitoneally, employing a variety of dosage forms. Such methods may also be applied to testing chemical activity in vivo,
- the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed.
- the determination of effective dosage levels can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.
- dosages may range broadly, depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 microgram/kg and 100 mg/kg body weight, preferably between about 100 microgram/kg and 10 mg/kg body weight. Alternatively dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art. Administration may be oral on an every third day, every other day, daily, twice daily, or thrice daily basis.
- 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, in The Pharmacological Basis of Therapeutics, 1975. It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
- the magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
- Suitable administration routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, via infusion, intraperitoneal, intranasal, or intraocular injections.
- the agents may be formulated in aqueous solutions, for example, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
- physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art.
- Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions disclosed herein, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection or infusion.
- the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
- Such carriers enable the compounds to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
- Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill, in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro- environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.
- these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
- suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
- the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
- the pharmaceutical compositions may be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
- Compounds disclosed herein can be evaluated for efficacy and toxicity using known methods.
- the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
- the toxicity of particular compounds in an animal model such as mice, rats, rabbits, or monkeys, may be determined using known methods.
- the efficacy of a particular compound may be established using several art recognized methods, such as in vitro methods, animal models, or human clinical trials.
- the compound When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or other such form. When administered non-orally, it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection or infusion, subcutaneously, intreperitoneally, intravenously, intramuscularly, intradermally, or the like. Similarly, it may be administered topically, rectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the growth of the tumor.
- Local administration at the site of the tumor or other disease condition is also contemplated, either before or after tumor resection, or as part of an art-recognized treatment of the disease condition.
- Controlled release formulations, depot formulations, and infusion pump delivery are similarly contemplated.
- an anti-cancer agent or an anti-tumor agent When used as an anti-cancer agent or an anti-tumor agent, may be orally or non-orally administered to a human patient in the amount of about .0007 mg/day to about 7,000 mg/day of the active ingredient, and more preferably about 0.07 mg/day to about 70 mg/day of the active ingredient at, preferably, one time per day or, less preferably, over two to about ten times per day.
- the compound may preferably be administered in the stated amounts continuously by, for example, an intravenous drip.
- the preferred daily dose of the active anti-tumor ingredient would be about 0.0007 mg/kg/day to about 35 mg/kg/day including 1.0 mg/kg/day and 0.5 mg/kg/day, and more preferable, from 0.007 mg/kg/day to about 0.050 mg/kg/day, including 0.035 mg/kg/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the anti-tumor compound in amounts that excess, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors,
- the preferable amount of the dehydrophenylahistin or its analog effective in the treatment or prevention of a particular fungal pathogen will depend in part on the characteristics of the fungus and the extent of infection, and can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed 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 precise dosage level should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the infection; the use (or not) of concomitant therapies.
- the effective dose of the dehydrophenylahistin or its analog will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, per day, administered in single or multiple doses.
- the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
- ком ⁇ онентs to formulate the dosage including the compounds disclosed herein as a tumor-growth-inhibiting compound, known surface active agents, excipients, smoothing agents, suspension agents and pharmaceutically acceptable film-forming substances and coating assistants, and the like may be used.
- alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a
- compositions disclosed herein in a pharmaceutical compositions may also comprise a pharmaceutically acceptable carrier. Such compositions may be prepared for storage and for subsequent administration. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 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 injectable administration. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions.
- Suitable excipients include, but are not limited to, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like.
- the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like.
- absorption enhancing preparations for example, liposomes
- the pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration.
- the dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
- the products or compositions, as described above, may be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents.
- the compounds can be administered or used in combination with treatments such as chemotherapy, radiation, and biologic therapies.
- the compounds can be administered or used with a chemotherapeutic agent.
- chemotherapeutics include Alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds, immunotherapeutics (antibodies, T-cells, epitopes), BRMs, and the like.
- Examples include, Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol), Docetaxel; topoisomerase " inhibibitors epipodophyllotoxins (Etoposide (VP-16), Teniposide (VM-26)), Camptothecin, nitrogen mustards (cyclophosphamide), Nitrosoureas, Carmustine, lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinoniycin (Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin, Cerubidine), Doxorubicin (Adriamycin), Idarubicin (Idamycin), Anthracenediones (Mitoxantrone), Bleomycin (Blenoxane), Plicamycin (Mithramycin, Antifolates (Methotrexate (Folex, Mexate)),
- the two major anticancer drugs in this category are 6-mercaptopurine and 6-thioguanine, Chlorodeoxyadenosine and Pentostatin, Pentostatin (2'-deoxycoformycin), pyrimidine antagonists, fluoropyrimidines (5-fluorouracil(Adrucil), 5- fluorodeoxyuridine (FdUrd) (Floxuridine)), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine, L-ASP ARAGINASE, Hydroxyurea, glucocorticoids, antiestrogens, tamoxifen, nonsteroidal antiandrogens, flutamide, aromatase inhibitors Anastrozole(Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine, Methotrexate, Cytoxan, Cytarabine, L- Asparaginase, Steroids: Prednisone and Dexamethasone.
- proteasome inhibitors such as bortezomib can be used in combination with the instant compounds, for example.
- biologies can include agents such as TRAIL antibodies to TRAIL, integrins such as alpha- V-beta-3 ( ⁇ V ⁇ 3) and / or other cytokine/growth factors that are involved in angiogenesis, VEGF, EGF, FGF and PDGF, immunotherapeutics, such as proteasome inhibitors, T cells, T cells vaccines, and the like.
- the compounds can be conjugated to or delivered with an antibody.
- Radiation therapy includes, but is not limited to, treatment with X-ray radiation and proton beam therapy. The above-described combination methods can be used to treat a variety of conditions, including cancer and neoplastic diseases, inflammation, and microbial infections.
- compositions can be utilized in vivo or in vitro.
- the useful dosages and the most useful modes of administration will vaiy depending upon the age, weight and animal treated, the particular compounds employed, and the specific use for which these composition or compositions are employed.
- the magnitude of a dose in the management or treatment for a particular disorder will vary with the severity of the condition to be treated and to the route of administration, and depending on the disease conditions and their severity, the- compositions may be formulated-and administered either systemically or locally.
- a variety of techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
- alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyiacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents.
- the cell cycle inhibitors, the antitumor agents, and the antifungal agents that may be produced by the method may be orally or non-orally administered to a human patient in the amount of about 0.001 mg/kg/day to about 10,000 mg/kg/day of the active ingredient, and more preferably about 0.1 mg/kg/day to about 100 mg/kg/day of the active ingredient at, preferably, once every three days on a cyclic basis, once every other day, one time per day, twice per day, or less preferably, over two to about ten times per day.
- the compound produced by the method may preferably be administered in the stated amounts continuously by, for example, an intravenous drip.
- the preferred daily dose of the active anti-tumor ingredient would be about 0.07 -mg/day to about 700 grams/day, and more preferable, 7 mg/day to about 7 grams/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the anti-tumor compound produced by the method in amounts that excess, or even far exceed, the above- stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors.
- the compound produced by methods of the invention inhibits the progression of the cell cycle when it is dissolved in an organic solvent or hydrous organic solvent and it is directly applied to any of various cultured cell systems.
- Usable organic solvents include, for example, methanol, methylsulfoxide, and the like.
- the formulation can, for example, be a powder, granular or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or a hydrous organic solvent.
- a preferred concentration of the compound produced by the method of the invention for use as a cell cycle inhibitor is generally in the range of about 1 to about 100 ⁇ g/ml, the most appropriate use amount varies depending on the type of cultured cell system and the purpose of use, as will be appreciated by persons of ordinary skill in the art. Also, in certain applications it may be necessary or preferred to persons of ordinary skill in the art to use an amount outside the foregoing range.
- certain embodiments provide methods for preventing or treating fungal infections and/or a pathogenic fungus in a subject, involve administering to the subject a composition including a dehydrophenylahistin or its analog, for example, administering the dehydrophenylahistin or its analog in an amount and manner which provides the intended antifungal effect.
- Other embodiments include the treatment or prevention of infection in a patient by a pathogenic fungus such as those listed above or referred to below.
- Another embodiment relates to the treatment or prevention of infection in a patient by a pathogenic fungus which is resistant to one or more other antifungal agents, especially an agent other than dehydrophenylahistin or its analog, including e.g.
- amphotericin B or analogs or derivatives thereof including 14(s)-hydroxyamphotericin B methyl ester, the hydrazide of ""amphotericin B with l-amino-4-methylpiperazine, and other derivatives
- polyene macrolide antibiotics including, e.g., nystatin, candicidin, pimaricin and natamycin; flucytosine; griseofulvin; echinocandins or aureobasidins, including naturally occurring and semi-synthetic analogs; dihydrobenzo[a]napthacenequinones; nucleoside peptide antifungals including the polyoxins and nikkomycins; allylamines such as naftifine and other squalene epoxidase inhibitors; and azoles, imidazoles and triazoles such as, e.g., clotrimazole, miconazole, ketoconazo
- Another embodiment involves, the treatment or prevention of infection in a patient by a pathogenic fungus in cases in which the patient is allergic to, otherwise intolerant of, or nonresponsive to one or more other antifungal agents or in whom the use of other antifungal agents is otherwise contra-indicated.
- Those other antifungal agents include, among others, those antifungal agents disclosed above and elsewhere herein.
- a dehydrophenylahistin or its analog is administered to the subject in an effective antifungal amount.
- Other embodiments relate to the treatment or prevention of infection by a pathogenic fungus in a patient by administration of a dehydrophenylahistin or its analog, in conjunction with the administration of one or more other antifungal agents, including for example, any of the previously mentioned agents or types of agents (e.g. in combination with treatment with amphotericin B, preferably in a lipid or liposome formulation; an azole or triazole such as fluconazole, for example; an aureobasidin; dihydrobenzofalnapthacenequinone; or an echinocardin) as well as with a different dehydrophenylahistin or its analog.
- agents or types of agents e.g. in combination with treatment with amphotericin B, preferably in a lipid or liposome formulation
- an azole or triazole such as fluconazole, for example; an aureobasidin; dihydrobenzofalnapthacenequinone; or an
- the dehydrophenylahistin or its analog may be administered before, after or at the same time the other antifungal agent is administered, In certain embodiments, the combination therapy will permit the use of reduced amounts of one or both antifungal components, relative to the amount used if used alone.
- Still other embodiments relate to administration of a dehydrophenylahistin or its analog to a subject for the treatment or prevention of infection by a pathogenic fungus, where the subject is immunosuppressed or immunocompromised, e.g. as the result of genetic disorder, disease such as diabetes or HIV or other infection, chemotherapy or radiation treatment for cancer or other disease, or drug- or otherwise induced immunosuppression in connection with tissue or organ transplantation or the treatment of an autoimmune disorder.
- a dehydrophenylahistin or its analog to a subject for the treatment or prevention of infection by a pathogenic fungus, where the subject is immunosuppressed or immunocompromised, e.g. as the result of genetic disorder, disease such as diabetes or HIV or other infection, chemotherapy or radiation treatment for cancer or other disease, or drug- or otherwise induced immunosuppression in connection with tissue or organ transplantation or the treatment of an autoimmune disorder.
- a dehydrophenylahistin or its analog may be co-administered with the immunosuppressive agent(s) to treat or prevent a pathogenic fungal infection.
- Another aspect of this invention is the treatment or prevention of infection by a pathogenic fungus in a patient infected, or suspected of being infected, with HIV, by administration of an antifungal dehydrophenylahistin or its analog, in conjunction with the administration of one or more anti-HIV therapeutics (including e.g. HIV protease inhibitors, reverse transcriptase inhibitors or anti-viral agents).
- an antifungal dehydrophenylahistin or its analog in conjunction with the administration of one or more anti-HIV therapeutics (including e.g. HIV protease inhibitors, reverse transcriptase inhibitors or anti-viral agents).
- the dehydrophenylahistin or its analog may be administered before, after or at the same time as administration of the anti-HIV agent(s). '
- Another aspect of this invention is the treatment or prevention of infection by a pathogenic fungus in a patient by administration of an antifungal dehydrophenylahistin or its analog, in conjunction with the administration of one or . more other antibiotic compounds, especially one or more antibacterial agents, preferably in an effective amount and regiment to treat or prevent bacterial infection.
- the dehydrophenylahistin or its analog may be administered before, after or at the same time as administration of the other agent(s).
- Pathogenic fungal infections which may be treated or prevented by the disclosed methods include, among others, Aspergillosis, including invasive pulmonary aspergillosis; Blastomycosis, including profound or rapidly progressive infections and blastomycosis in the central nervous system; Candidiasis, including retrograde candidiasis of the urinary tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplantation or poorly controlled diabetes mellitus; Coccidioidomycosis, including chronic disease which does not respond well to other chemotherapy; Cryptococcosis; Histopolasmosis; Mucormycosis ⁇ including e.g.
- compositions comprising an antifungal amount of one or more dehydrophenylahistin or its analogs may be particularly useful for treating or preventing a pathogenic fungal infection in a mammalian subject where the fungus is resistant to one or more other antifungal therapies, or where the use of one or more other antifungal therapies is contraindicated, e.g., as mentioned above.
- Antifungal pharmaceutical compositions containing at least one antifungal dehydrophenylahistin or its analog are also provided for use in practicing the disclosed methods. Those pharmaceutical compositions may be packaged together with an appropriate package insert containing, inter alia, directions and information relating to their antifungal use. Pharmaceutical compositions are also provided which contain one or more dehydrophenylahistin or its analog together with a second antifungal agent.
- Certain embodiments disclosed herein relate to methods for treating or preventing a pathogenic fungal infection, including for example Aspergillosis, including invasive pulmonary aspergillosis; Blastomycosis, including profound or rapidly progressive infections and blastomycosis in the central nervous system; Candidiasis, including retrograde candidiasis of the urinary tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplantaion or poorly controlled diabetes mellitus; Coccidioidomycosis, including chronic disease which does not respond well to other chemotherapy; Cryptococcosis; Histopolasmosis; Mucormycosis, including e.g.
- Aspergillosis including invasive pulmonary aspergillosis
- Blastomycosis including profound or rapidly progressive infections and blastomycosis in the central nervous system
- Candidiasis including retrograde candidiasis of the urinary tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplant
- the methods may involve administering at least one antifungal dehydrophenylahistin or its analog, as described above, to a human subject such that the fungal infection is treated or prevented.
- the dehydrophenylahistin or its analog may be administered in conjunction with administration of one or more non-dehydrophenylahistin or its analog antifungal agents such as amphotericin B, or an imidazole or triazole agent such as those mentioned previously.
- the pathogenic fungal infection may be topical, e.g., caused by, among other organisms, species of Candida, Trichophyton, Microsporum or Epiderinophyton or mucosal, e.g., caused by Candida albicans (e.g. thrush and vaginal candidiasis).
- the infection may be systemic, e.g., caused by Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastomyces spp.
- the infection may also involve eumycotic mycetoma, chromoblastomycosis, cryptococcal meningitits or phycomycosis.
- Further embodiments relate to methods for treating or preventing a pathogenic fungal infection selected from the group consisting of Candida spp. including C. albicans, C. tropicalis, C, kefyr, C. krusei and C. galbrata; Aspergillus spp. including A, fumigatus and A. flavus; Cryptococcus neoibrmans; Blastomyces spp.
- Blastomyces dermatitidis including Blastomyces dermatitidis; Pneumocystis carinii; Coccidioides immitis; Basidiobolus ranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp. including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizoniucor spp.; Paracoccidioides brasiliensis; Pseudallescheria boydii; Rhinosporidium seeberi; and Sporothrix schenckii.
- the method may involve administering a non-immunosuppressive antifungal dehydrophenylahistin or its analog to a patient in need thereof such that the fungal infection is treated or prevented without inducing an untoward immunosuppressive effect.
- Further embodiments relate to methods for treating or preventing a pathogenic fungal infection which is resistant to other antifungal therapy, including pathogenic fungal infections which are resistant to one or more antifungal agents mentioned elsewhere herein such as amphotericin B, flucytosine, one of the imidazoles or triazoles (including e.g. fluconazole, ketoconazole, itraconazole and the other previously mentioned examples).
- the methods may involve administering to the patient one or more antifungal dehydrophenylahistin or its analog, in an amount and dosing regimen such that a fungal infection resistant to another antifungal therapy in the subject is treated or prevented.
- Further embodiments relate to methods for treating or preventing a pathogenic fungal infection in a patient who is allergic to, intolerant of or not responsive to another antifungal therapy or in whom the use of other antifungal agents is otherwise contra- indicated, including one or more other antifungal agents mentioned elsewhere herein such as amphotericin B, flucytosine, one of the imidazoles or triazoles (including e.g. fluconazole, ketoconazole, itraconazole and the other previously mentioned examples).
- the methods may involve administering to such patient one or more antifungal -dehydrophenylahistin or its- analog, in an amount such that a fungal infection is treated or prevented.
- Certain embodiments relate to packaged dehydrophenylahistin or its analogs, preferably packaged nonimmunosuppressive antifungal dehydrophenylahistin or its analogs, which term is intended to include at least one dehydrophenylahistin or its analog, as described above, packaged with instructions for administering the dehydrophenylahistin or its analog(s) as an antifungal agent without causing a untoward immunosuppressive effects within a human subject.
- the non-immunosuppressive antifungal dehydrophenylahistin or its analog is a member of one of the preferred subsets of compounds described above.
- the dehydrophenylahistin or its analog can be packaged alone with the instructions or can be packaged with another dehydrophenylahistin or its analog, raparnycin or another ingredient or additive, e.g., one or more of the ingredients of the pharmaceutical compositions.
- the package can contain one or more containers filled with one or more of the ingredients of the phan-naceutical compositions.
- Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
- N,N'-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1. I) l-Acetyl-3- ⁇ (Z)-l-r5-tert-butyl-lH-4-imidazolyllmethylidene)l-2.5-piperazinedione (16)
- N,N'-diacethyl-2,5-pi ⁇ erazinedione 1 was prepared as in Example 1. 1) l-Acetyl-3-[YZ)-benzylidenel ⁇ l-2.5-piperazinedione (17)
- Figure 4 illustrates the similarities of the HPLC profiles (Column: YMC- Pack ODS-AM (20 x 250mm); Gradient: 65% to 75% in a methanol-water system for 20 min, then 10 min in a 100% methanol system; Flow rate: 12mL/min; O. D. 230 nm) from the synthesized dehydrophenylahistin of Example 1 (Fig 2) and the above exemplified tBu- dehydrophenylahistin compound produced by Route A.
- 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) l,4-diacetyl-piperazine-2,5-dione, Cs 2 CO 3 ; 1) benzaldehyde, Cs 2 CO 3
- Ethyl potassium malonate (25.0 g, 0.15 mol) was suspended in water (15.6 ml) and cooled in an ice bath. Concentrated HCl (12.5 ml) was added dropwise over 30 min, then the mixture was stirred for a further 10 min. The precipitate was filtered, then washed twice with ether. The filtrate was separated and the aqueous phase was extracted with ether. The combined ethereal solutions were dried (MgSO 4 ) and evaporated to afford, as an oil, nionoethyl hydrogen malonate (19.2 g, 99%) which was dried under vacuum overnight (or 50°/l mm for 1 h) prior to use.
- Oxalyl chloride (3.83 ml, 43.9 mmol) was added dropwise to a cooled (0°) solution of 2,2-dimethyl-but-3-enoic acid (5.0 g, 43.9 mmol) and DMF (1 drop) in anhydrous dichloromethane (25 ml). The mixture was stirred for 1 h at 0°, then for 16 h at room temperature. Fractional distillation (121°/760 mmHg) afforded 2,2-dimethyl-but-3-enoyl chloride (4. I g, 71%).
- Reagents g) SO 2 Cl 2 ; h) H 2 NCHO, H 2 O; I)LiAlH 4 ; j) MnO 2 ; k) 1,4- diacetyl-pi ⁇ erazine-2,5-dione, Cs 2 CO 3 ; 1) benzaldehyde, Cs 2 CO 3
- Sodium bicarbonate refers to a 5% solution.
- RP-HPLC was done on a Rainin Microsorb-MV Cl 8 (5 ⁇ m, 100A) 50 x 4.6 mm column.
- Buffer A 0.1% aqueous TFA
- Buffer B 0.1% TFA in 90% aqueous MeCN
- Ion Source Ionspray
- ESMS was done on a Perkin Elmer/Sciex-API III LC/MS/MS using an electrospray inlet.
- Orifice plate 55 V
- Scan range 100-1000 amu/z
- Reverse phase HPLC purification was carried out using Nebula with the Waters XterraMS column (19x50 mm, 5 ⁇ m, C18) using the following conditions:
- KPU-202-244 was prepared from compound 4 according to the procedure described for the synthesis of KPU-201.
- KPU-206 3-((Z)-l-f5-(tert- Butyl)-1H -4-oxazolyllmethylidene ⁇ -6-[(Z)-1-(3- chlorophenyl)methIidenel-2,5-piperazinedione:
- KPU-210 3- ⁇ (Z)-l-r5-(tert- Butyl)-1H -4-oxazolyllmethylidene ⁇ -6-[(Z)-1-(3- ethoxyphenyl)methIidene1-2,5-piperazinedione:
- KPU-221 3- ⁇ (ZVl-r5-(tert- Butyl)-1H -4-oxazolyllmethylidene ⁇ -6-[(Z)-1-(3- vinylphenyI)methlidenel-2,5-piperazinedione:
- KPU-224 3- ⁇ (Z)-l-r5-(tert- Butyl)-1H -4-oxazolyllmethylidene ⁇ -6-[(Z)-1-(2- methoxyphenyI)methIidenel-2,5-piperazinedione:
- KPU-240 3-((Z)-l-r5-(tert- Butyl)-1H -4-oxazolyllmethylidene ⁇ -6-[(Z)-1- (phenylsulfonyI-2- indoIvDmethIidene]-2,5-piperazinedione:
- Method B with silica gel column chromatography As the same method mentioned in A, compound 6 (3.28 g, 11.26 mmol) and benzaldehyde (1,72 mL, 16.9 mmol, 1.5 equiv.) were reacted in anhydrous DMF (110 mL) in the presence of Cs 2 CO 3 (7.33g, 0.34 mmol, 2 equiv.) for 3 h at 85 0 C in dark place.
- Ht-29 ATCC HTB-38
- McCoy's complete medium McCoy's 5A medium with L-glutamine and 25mM HEPES supplemented with 10% FBS, ImM Na pyruvate, IX NEAA, 2mM L- glutamine, and Pen/Strep at lOOIU/ml and lOO ⁇ g/ml, respectively.
- PC-3 (ATCC CRL- 1435), a human prostate adenocarcinoma, was maintained in F12K complete medium (F12K medium supplemented with 10% FBS; 2mM Glutamine; 1% HEPES; and Pen/Strep at 100IU/ml and lOO ⁇ g/ml, respectively).
- F12K complete medium F12K medium supplemented with 10% FBS; 2mM Glutamine; 1% HEPES; and Pen/Strep at 100IU/ml and lOO ⁇ g/ml, respectively.
- Cell lines were cultured at 37 0 C, 5% CO 2 in a 95% humidified incubator.
- HT-29 or PC-3 cells were seeded at 5,000 cells/well in 90 ⁇ l complete media into a Corning 3904 black- walled, clear-bottom tissue culture plate and the plate were incubated overnight to allow cells to establish and enter log phase growth.
- 20 mM stock solutions of dehydrophenylahistin and tBu-dehydrophenylahistin were prepared in 100% DMSO and stored at -20 °C.
- 1OX concentrated serial dilutions of the two compounds were prepared in appropriate culture medium for final concentrations ranging from 2.0 x 10 "5 M to 2.0 x 10 "10 M.
- Ten ⁇ l volumes of the 1OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
- tBu-dehydrophenylahistin demonstrates about a 4-times greater cytotoxic activity in comparison with dehydrophenylahistin.
- Table 1 Cytotoxic Effect of dehydrophenylahistin and derivative.
- HT-29 a human colorectal adenocarcinoma was maintained in McCoy's complete medium (McCoy's 5A medium with L-glutamine and 25mM HEPES supplemented with 10% FBS, ImM Na pyruvate, IX NEAA, 2mM L- glutamine, and Pen/Strep at 100IU/ml and lOO ⁇ g/ml, respectively).
- PC-3 a human prostate adenocarcinoma, was maintained in F12K complete medium (F12K medium supplemented with 10% FBS; 2mM Glutamine; 1% HEPES; and Pen/Strep at lOOIU/ml and lOO ⁇ g/ml, respectively).
- Cell lines were cultured at 37 0 C, 5% CO 2 in a 95% humidified incubator.
- HT-29 or PC-3 cells were seeded at 5,000 cells/well in 90 ⁇ l complete media into a Corning 3904 black- walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth.
- 20 mM stock solutions of dehydrophenylahistin and tBu-dehydrophenylahistin were prepared in 100% DMSO and stored at -20 °C.
- 1OX concentrated serial dilutions of the two compounds were prepared in appropriate culture medium for final concentrations ranging from 2.0 x 10 "5 M to 2.0 x 10 "10 JvL
- Ten ⁇ l volumes of the 1OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours.
- the final concentration of DMSO was 0.25% in all samples.
- HT-29 cells (ATCC; HTB-38) were maintained in ATCC recommended culture media and cultured in an incubator at 37 0 C in 5% CO 2 and 95% humidified air.
- HT-29 cells were seeded at 5x10 3 . cells/well in 90 ⁇ l complete media into a Corning 3904 black- walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth. 20 mM stock solutions of compounds were prepared in 100% DMSO and stored at -8 ' 0 ' 0 C. 1OX concentrated serial dilutions of the compounds were prepared in culture medium for final concentrations ranging from 20 ⁇ M to 20OpM, Ten ⁇ l volumes of the 1 OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
- the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (generated by XLfit 3.0, ID Business Solutions Ltd). Where the maximum inhibition of cell growth was less than 50%, an EC 50 value was not determined.
- derivatives of the compound may include the following substitutions at the phenyl ring (A): -CF 3 , -SO 2 NH 2 (-SO 2 NRiR 2 ), -SO 3 H, -CONH 2 (-CONR 1 R 2 ), -COOH, etc.
- Other ring systems (C) may also include the following:
- Evaluation of derivatives described above is assessed according to the methods described in Example 3. Additional evaluation of the derivatives are extended to specific activities such as determining the inhibiting effect on cell proliferation, the effects on a specific cellular mechanism (i.e. microtuble function), effects on cell cycle progression, evaluating in vitro anti-tumor activity against cancer cell lines, etc. Some evaluation method protocols are given below.
- Methanol solution of the derivative obtained by the above-listed examples are added to the wells of the uppermost row, specimens are diluted by the half-log dilution method and added, and the plate is incubated in a carbon dioxide gas incubator at 37 0 C for 48 hours.
- the result is added in lots of lO ⁇ l with MTT reagent (3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2H-tetra bromide)(lmg/ml • PBS), followed by incubation in a carbon dioxide gas incubator at 37 0 C for 6 hours.
- the culture medium is discarded and the crystal of produced in the cells are dissolved in lOO ⁇ l/well of dimethylsulfoxide.
- Cell strain A431 is derived from human lung cancer.
- EMEM culture medium containing 10% bovine fetal serum and 1% MEM nonessential amino acid solution (SIGMA M2025) is used to incubate A431 cells at 37 0 C in an incubator saturated with 5% carbon dioxide gas and water vapor.
- SIGMA M2025 MEM nonessential amino acid solution
- the refined specimen of dehydrophenylahistin obtained by the methods above is added to the cells in the log-growth phase and progression of the cell cycle is analyzed by flow cytometer and microscopic observation.
- Colchicine recognizes the same binding site on ⁇ -tubulin as PLH. Colchicine has four characteristic methoxy groups on its A and B rings. A series of substitutions with the single or multiple methoxy groups was performed and the results of cytotoxic activity are shown in Table 6. Table 6. Effect of the methoxy group substitution on the proliferation of HT-29 cells
- the compounds having effective functional groups which showed higher activity than tBu- dehydroPLH, may also be further modified. And since the migration of the stereochemistry from Z to E under the visible light irradiation was observed* substituents that decrease the electron density in the conjugated double bonds may contribute to the reduction of Z to £ migration by the light, results in more physicochemically stable structures. Temperature can also effect this migration.
- Modification at two parts of the ring can be preferred for the development of potent but also biologically stable compounds.
- the phenyl ring of phenylahistin is oxidized by cytochrome P-450. Double modification that reduces the electron density of the phenyl ring may therefore be effective to avoid P-450 oxidation.
- the combination of the small electron withdrawing group such as the fluorine atom to the element that can increase the activity such as -OMe, -Me, -Cl 3 -F and Br, may result in more potent and biologically stable drug compounds.
- the phenyl ring may also be replaced by heteroaxyl groups.
- the result of such replacements in terms of the cytotoxic activity is shown in Table 11. Since the arylic nitrogen atoms can . form a hydrogen bonding with a NH group of the diketopiperazine ring and restrict the conformation of the molecule between pyridine and diketopiperazine rings to an uniplanar structure, the active conformation of dehydroPLH would be required a certain level of dihedral angle formed by the steric repulsion between an amide hydrogen atom of the diketopiperazine ring and an o-hydrogen atom of the phenyl ring ( Figure 6). Table 11. Effect of the replacement with the heteroaryl ring on proliferation of HT-29 cells
- the physicochemical stability is one of the unfavorable problems of dehydroPLH.
- phenylahistin since there is no additional olefin structure at the benzyl part, there is no such problem.
- the benzylidene moiety can be easily activated, probably with the visible light, and the Z to E migration frequently occurs due to the existence of longer conjugation of the double bond. This migration occurred even under normal room light.
- some of the compounds migrate to E-form during the incubation, although this migration probably equilibrates at the 1 : 1 ratio in the case of dehydroPLH. This migration can be controlled.
- the Z to E migration is also known in combretastatin A4, a same type of tubulin inhibitor, and a few studies for improving this problem were reported.
- the J5-form may also be used as a prodrug of dehydroPLH or of one or more of its analogs, including those analogs described herein.
- One of the undesired properties of anti-tubulin drugs involves its low selectivity between tumor and intact tissues, although these drugs belong to one of the molecular target therapies. This causes undesired side effects. However, if the compounds functions selectively only in tumor tissues, negative side effects of anti-microtubule drugs can be reduced.
- the dehydroPLH (Z-form) can be produced from its ⁇ -isomer by visible light irradiation, the E-forrn is administered and photo irradiation is performed only at the tumor site, then only the tumor is damaged by photo-produced Z-form and the adverse effect to the intact tissues is reduced.
- the E-form can be protected chemically by the addition of a bulky but biodegradable acyl group, which is introduced into the diketopiperazine ring as a prodrug.
- This acyl group can be cleaved by the protease in the body. Therefore, the acylated-/?- compound is maintained before administration, then after administration it is changed to the real .E-form, which can migrate to the bioactive Z-form by the local photo irradiation.
- a mixture obtained by thoroughly blending 1 g of a compound synthesized by the method, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method.
- the granules are thoroughly dried and sifted to obtain a granule preparation suitable for packaging in bottles or by heat sealing.
- the resultant granule preparations are orally administered at between approximately 100ml/day to approximately 1000 ml/day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans.
- Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the com ⁇ ound(s) directly to the affected area of the skin of the individual.
- the compound administered or applied is in the form of a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
- topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
- Other materials such as anti-oxidants, hurnectants, viscosity stabilizers, and similar agents may be added as necessary.
- Percutaneous penetration enhancers such as Azone may also be included.
- the compound may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
- 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 murine melanoma; ATCC; CRL-6475
- CCD-27sk normal human fibroblast; ATCC; CRL- 1475
- the disclosed compounds are effective agents against a variety of different and distinct tumor cell lines.
- KPU-2 and KPU-35 were most effective on the HT-29 tumor cell line, both in terms of potency (active in the low nanomolar range) and efficacy (most responsive in terms of the maximum cytotoxic effept);
- t-butyl- phenylahistin exhibited its greatest potency against the PC-3 tumor cell line, although the greatest efficacy was displayed against the HT-29 cell line;
- KPU-2 and KPU-35 were generally 10-40 fold more potent than t-butyl-phenylahistin whereas the efficacy was similar for all three compounds in the different tumor cell lines;
- the HT-29, PC-3, MDA-MB-231 and NCI-H292 tumor cell lines all responded similarly to the NPI compounds, whereas the B16-F10 appeared to be somewhat less sensitive, t-butyl-phenylahistin displayed a marked differential between normal fibroblasts and the tumor cell lines
- chemotherapeutic agents in clinical oncology
- resistance to the drug effect by the tumor cells.
- mechanisms for the development of resistance include increased expression of ATP-dependent efflux pumps such as the P-glycoprotein encoded by MDRl or the multidrug-resistance associated protein 1 encoded by MRPl.
- Reduced drug uptake, alteration of the drug's target, increasing repair of drug-induced DNA damage, alteration of the apoptotic pathway and the activation of cytochrome P45Q enzymes are other examples of mechanisms by which cancer cells become resistant to anticancer drugs.
- the selected compounds were studied in three different cell lines that exhibit two different mechanisms of resistance; the overexpression of the P-glycoprotein and altered topoisomerase II activity.
- MES-SA Texol Sensitive
- MES-S A/Dx5 Texol Resistant
- This cell line expresses elevated mdr-1 mRNA and P-glycoprotein (an extrusion pump mechanism). Pretreatment with cyclosporin-A (CsA) blocks P-glycoprotein and reinstates activity in the resistant cell line for those compounds for which the resistance is due to elevated P-glycoprotein.
- CsA cyclosporin-A
- KPU-2, and KPU-35 have the same potency in the resistant cell line as in the sensitive line and the potency of t-butyl- phenylahistin was only slightly reduced.
- Cyclosporin A (CsA) pretreatment did not alter the potency of the selected compounds.
- taxol was virtually inactive in the MES- SAfDXS resistant cell line, whereas this compound was very potent in the sensitive cell line.
- CsA treatment restored the sensitivity to taxol of the MES-SA/DX5 cell line.
- the MES- SA/DX5 cell line also showed reduced susceptibility to etoposide (60 fold), doxorubicin (34 fold) and mitoxantrone (20 fold).
- This cell line is considered to have atypical drug resistance properties with altered topoisomerase II catalytic activity without overexpression of P-glycoprotein.
- KPU-2, KPU-35 and t-butyl-phenylahistin are not susceptible to the same resistance mechanisms as Mitoxantrone in this cell line, and there is no cross-resistance from Mitoxantrone to these selected novel compounds in this model, Table 14.
- MCF-7 Human Breast Carcinoma Cell Line Pair: MCF-7 (Taxol Sensitive) and MCF-7/ADR (Taxol Resistant)
- KPU-2 was virtually inactive in the resistant cell line whereas there was low nanomolar potency in the sensitive cell line (Table 15).
- Human umbilical vein endothelial cells (HuVEC from Cambrex) were used in this study, for evaluating the effects of KPU-2 and t-butyl-phenylahistin in comparison to colchicine and taxol on tubulin by staining for ⁇ -tubulin.
- PARP poly (ADP ribose) polymerase
- KPU-2 induced the typical morphological changes associated with early stages of apoptosis in human prostate tumor cells. A similar finding was also clear in the treatment of HuVEC cells with KPU-2.
- a late stage characteristic of apoptosis is internucleosomal DNA cleavage that results in a distinctive ladder pattern that can be visualized by gel electrophoresis.
- This approach was used to study the effect of KPU-2 on DNA laddering in Jurkat cells (human T cell leukemia line) in comparison to halimide and dehydrophenylahistin (KPU-I). KPU-2 induced DNA laddering at the 1 nM concentration whereas halimide and KPU-I were much less potent.
- PARP poly(ADP-ribose) polymerase
- PARP is a 116 kDa nuclear protein that is one of the main intracellular targets of Caspase-3. The cleavage of PARP generates a stable 89 kDa product, and this process can be easily monitored by western blotting. Cleavage of PARP by caspases is one of the hallmarks of apoptosis, and as such serves as an excellent marker for this process.
- KPU-2 at 100 nM induced cleavage of PARP in Jurkat cells 10 hours after exposure of the cells to the compound. KPU-2 appeared to be more active than either halimide or KPU-I .
- KPU-2, KPU-35 and t-butyl-phenylahistin all rapidly (within 1 hour) induced significant HuVEC monolayer permeability, to an extent similar to colchicine.
- the microtubule stabilizing agent taxol was inactive in this assay ( Figure 12).
- KPU-2 was initially screened at a concentration of 10 ⁇ M in a panel of 60 different kinases; the ATP concentration was 10 ⁇ M.
- kinases were inhibited by greater than 50% in the primary screen and the IC50's determined in secondary screening are presented in Table 17. All of the IC50 values are in the low micromolar range, which indicates that inhibition of these kinases is not related to the low nanomolar activities observed for tumor cell cytotoxicity.
- NPI-2421 , NPI-2463, NPI-2503 and NPI- 2504 were determined against selected human (colorectal adenocarcinoma, HT-29; breast adenocarcinoma, MDA-MB-231 ; non-small cell lung carcinoma, NCI-H292 and prostate carcinomas, PC-3 and DU 145) and mouse (melanoma, B16-F10) tumor cell lines.
- HT-29 (HTB-38), MDA-MB-231 (HTB-26), NCI-H292 (CRL-1848), B16-F10 (CRL-6475), PC-3 (CRL- 1435) and DU 145 (HTB-81) cells were all purchased from the ATCC. The cell lines were maintained in their respective ATCC recommended culture media and cultured in an incubator at 37°C in 5% CO 2 and 95% humidified air.
- HT-29, MDA-MB-231 , NCI-H292, B 16- FlO, PC-3 and DU 145 cells were seeded at 5xlO 3 , IxIO 4 , 4xlO 3 , 1.25xlO 3 , 5xlO 3 and 5xlO 3 cells/well respectively in 90 ⁇ l complete media into a Corning 3904 black- walled, clear- bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth. 20 mM stock solutions of compounds were prepared in 100% DMSO and stored at -80 °C.
- 1OX concentrated serial dilutions of the compounds were prepared in B16-F10 cell culture medium for final concentrations ranging from 20 ⁇ M to 20OpM. Ten ⁇ l volumes of the 1OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
- EC 50 values the drug concentration at which 50% of the maximal observed growth inhibition is established
- EC 50 values were determined using a standard sigmoidal dose response curve fitting algorithm generated by XLfit 3.0 (ID Business Solutions Ltd) or Prism 3.0 (GraphPad Software Inc). Where the maximum inhibition of cell growth was less than 50%, an EC 50 value was not determined.
- MES-SA Human uterine sarcoma
- MES-SA/Dx5 human acute promyelocyte leukemia cells
- HL-60 human acute promyelocyte leukemia cells
- HL-60/MX2 human acute promyelocyte leukemia cells
- CRL-2257 human acute promyelocyte leukemia cells
- MES-SA and MES-SA/Dx5 cells were both seeded at 3x10 3 cells/ well in 90 ⁇ l complete media into 96 well (Corning; 3904) black- walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth.
- HL-60 and HL-60/MX2 cells were both seeded at 5x10 4 cells/ well in 90 ⁇ l complete media into 96 well plates on the day of compound addition.
- 2OmM stock solutions of the compounds were prepared in 100% DMSO and stored at -8O 0 C.
- 1OX concentrated serial dilutions of the compounds were prepared in appropriate culture medium for final concentrations ranging from 2 ⁇ M to 632pM. Ten ⁇ l volumes of the 1OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
- the data were normalized to the average fluorescence of the cells treated with media + 0.25% DMSO (100% cell growth) and EC 50 values (the drug concentration at which 50% of the maximal observed growth inhibition is established) were determined using a standard sigmoidal dose response curve fitting algorithm (XLfit 3.0, ID Business Solutions Ltd).
- the multidrug resistant MES-SA/Dx5 tumor cell line was derived from the human uterine sarcoma MES-SA tumor cell line and expresses elevated P-Glycoprotein (P-gp), an ATP dependent efflux pump.
- P-gp P-Glycoprotein
- Table 19 summarize the growth inhibitory effects of NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI-2506 against MES- SA and its multidrug resistant derivative MES-S A/Dx ' 5.
- Paclitaxel, a known substrate of the P-gp pump was included as a control.
- the EC 50 values indicate that NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI-2506 retained equivalent cytotoxic activity against both MES-SA and MES-SA/Dx5 tumor cell lines.
- the multidrug resistant phenotype was confirmed by the observation that Paclitaxel was -400 times less active against the resistant MES-SA/Dx5 cells,
- HL-60/MX2 is a multidrug resistant tumor cell line derived from the human promyelocytic leukemia cell line, HL-60 and expresses reduced topoisomerase II activity.
- Table 20 summarize the growth inhibitory effects of NPI- 2421, NPI-2463, NPI-2503, NPI-2504 and NPI-2506 against HL-60 and its multidrug resistant derivative HL-60/MX2. Mitoxantrone, the topoisomerase II targeting agent was included as a control.
- the EC 50 values indicate that NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI-2506 retained cytotoxic activity against both HL-60 and HL-60/MX2 tumor cell lines.
- the multidrug resistant phenotype was confirmed by the observation that Mitoxantrone was ⁇ 26 times less active against the resistant HL-60/MX2 cells.
- Tumor vasculature differs from established vascular endothelium in normal tissues in that it has a reduced number of supporting pericytes and is highly permeable (Tozer, G. M.,, C. Kanthou, et al. (2002), "The biology of the combretastatins as tumour vascular targeting agents.” Int J Exp Pathol 83(1): 21-38; each of which is incorporated herein by reference in its entirety).
- proliferating human umbilical vein endothelial cells typically lack a well defined actin filament structure, making them more reliant on the microtubule network for maintenance of cell shape (Gotenden, A. I. (1990). "The endothelial cytoskeleton: organization in normal and regenerating endothelium.” Toxicol Pathol 18(4 Pt 1): 603-17; Ingher, D. E., D. Prusty, et al. (1995). "Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis.” J Biomech 28(12): 1471-84; each of which is incorporated herein by reference in its entirety). Consequently, proliferating HuVECs are used as an in vitro model of tumor vascular endothelial cells (Davis, Dougherty et al. 2002).
- NPI-2421 and NPI-2463 are cytotoxic against HuVECs
- NPI-2421 and NPI-2463 were determined against human umbilical cord endothelial cells, HuVECs.
- HuVECs (Cambrex Biosciences; CC2519A) were maintained in EGM-2 medium at 37 0 C in 5% CO 2 and 95% humidified air.
- HuVECs were seeded at 1x10 3 cells/well in 90 ⁇ l complete media into a Corning 3904 black- walled, clear-bottom tissue culture plates and the plates were incubated overnight to allow cells to establish and enter log phase growth.
- 20 mM stock solutions of compounds were prepared in 100% DMSO and stored at - 80 °C.
- 1OX concentrated serial dilutions of the compounds were prepared in culture medium for final concentrations ranging from 632nM to 20OpM.
- Ten ⁇ l volumes of the 1OX serial dilutions were added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO was 0.25% in all samples.
- NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI-2506 rapidly induce microtubule depolymerization in HuVECs
- HuVECs (Cambrex Biosciences; CC2519A) were maintained in EGM-2 medium at 37°C in 5% CO 2 and 95% humidified air.
- HuVEC cells were seeded at a density of 3x10 4 cells/ml in EGM-2 on tissue culture compatible coverslips (Fisher). The plates were returned to the incubator for 2 days.
- the cells were rinsed in dPBS before fixation in 10%(v/v) neutral buffered formalin for 10 minutes at room temperature. Following fixation, ⁇ -tubulin was visualized by indirect immunofluorescence. Specifically, the cells were permeabilized in 0.2%(v/v) triton X-100/dPBS for 10 minutes. The cells were washed prior to transferring the coverslips to a humidified chamber, the coverslips were blocked for two hours in antibody buffer [2%(w/v) BSA/ 0.1%(v/v) Tween 20/ dPBS].
- the coverslips were incubated with 50 ⁇ l of 0.1 ⁇ g/ml mouse ⁇ -tubulin (Molecular Probes) in antibody buffer for 1 hour before washing and incubation with 50 ⁇ l of 1 ⁇ g/ml goat anti-mouse FITC (Jackson ImmunoResearch Laboratories) for one hour in the dark. Finally, the cells were washed and treated with 2 ⁇ g/ml DAPI (Molecular Probes) for 10 minutes before rinsing in H 2 O and mounting with Vectashield (Vector Labs) mounting media. The cells were imaged using a 6Ox oil immersion objective on an upright microscope (Olympus BX51). The images were digitally captured using a CCD camera and Magnafire 2.0 software (Olympus). Post image processing was performed in Photoshop Elements 2.0 (Adobe) and in Microsoft Powerpoint.
- Figure 55 shows that NPI-2421, NPI-2463, NPI-2503, NPI-2504 and NPI- 2506 induce tubulin depolymerization within 30 minutes in HuVECs.
- the doses of the selected novel compounds were determined from the acute tolerability testing (Maximally Tolerated Dose, MTD) and were adjusted if necessary during each study.
- the doses of the clinically-used chemotherapeutic agents were selected on the basis of historical studies.
- KPU-2 was the first compound to be studied in these five tumor models. Following the initial results from this study, all three compounds were compared in the HT- 29 human colon tumor, the DU- 145 human prostate and the MCF-7 human breast tumor xenograft models. '
- mice used were (exceptions are indicated for individual studies): female nude mice (nu/nu) between 5 and 6 weeks of age ( ⁇ 20g, Harlan); group size was 9-10 mice per group unless otherwise indicated.
- Cell lines used for tumor implantation were: HT-29 human colon tumor; MCF-7 human breast tumor; A549 human non small cell lung tumor; MiaPaCa-2 human pancreas tumor; DU- 145 human prostate tumor.
- Vehicles used in these studies were: 12.5% DMSO, 5% Cremaphor and 82.5% peanut oil for the selected novel compounds; (1 :3) Polysorbate 80:13% ethanol for taxotere; (1:1) Cremapho ⁇ ethanol for paclitaxel; for CPT-I l each mL of solution contained 20 mg of irinotecan hydrochloride, 45 mg of sorbitol NF powder, and 0.9 mg of lactic acid, the pH being adjusted to 7.4 with NaOH or HCl. Saline dilutions are used to achieve the injection concentrations used for the reference compounds, HT-29 Human Colon Tumor Model
- mice Animals were implanted subcutaneously (s.c.) by trocar with fragments of HT-29 tumors harvested from s.c. growing tumors in nude mice hosts. When the tumor size reached 5 mm x 5 mm (about 10-17 days) the animals were matched into treatment and control groups. Mice were weighed twice weekly and tumor measurements were obtained using calipers twice weekly, starting on Day 1. The tumor measurements were converted to estimated mg tumor weight using the formula (W 2 xL)/2. When the estimated tumor weight of the control group reached an average of 1000 mg the mice were weighed, sacrificed and the tumor removed. The tumors were weighed and the mean tumor weight per group was calculated and the tumor growth inhibition (TGI) was determined for each group (100% minus the change in the mean treated tumor weight/the change in the mean control tumor weight x 100.
- TGI tumor growth inhibition
- mice Female nude mice ( ⁇ 20 g) were implanted s.c. with 21 -day release estrogen ' (0.25 mg) pellets 24 hours prior to the s.c. implantation with MCF-7 tumor fragments (harvested from s.c. tumors in nude mice hosts). The study then proceeded as described for the HT-29 model, using taxotere as the standard chemotherapy agent. [0526] In this model unless otherwise noted--for the individual study, the novel compounds were injected via the intraperitoneal route daily on Days 1-5, inclusive (qdx5); taxotere was administered intravenously on Days 1, 3 and 5 (qodx3).
- qdx549 Human Lung Tumor Model A549 Human Lung Tumor Model
- test compounds were administered every third day via the intraperitoneal route on Days 1, 4, 7, 10 and 15 (q3dx5); gemcitabine was administered via the intraperitoneal route on Days 1, 4, 7 and lO (q3dx4).
- DU- 145 Human Prostate Tumor Model DU- 145 Human Prostate Tumor Model
- mice Male mice were implanted s.c. by trocar with fragments of DU-145 tumors harvested from s.c. growing tumors in nude male mice hosts. When the tumors reached ⁇ 5 mm x 5 mm ( at about 13 -17 days) the animals were matched into treatment and control groups. The remainder of the study proceeded as for the HT-29 model, using taxotere as the standard chemotherapy agent. [0532] In this model unless otherwise- noted for the individual study, test compounds were administered via the intraperitoneal route on Days 1, 3, 5, 8 and 11 (q3dx5); taxotere was administered intravenously on Days 1, 3 and 5 (q2dx3). 2).
- mice used were: female nude mice (nu/nu) (MDA-231 study) or B6D2F1 (B16-F10 studies) mice between 5 and 6 weeks of age ( ⁇ 20g, Harlan); group size was 10 mice per group unless otherwise indicated.
- the cell lines used were : MD A-MB-231 human breast tumor and B 16-F 10 murine melanoma cells.
- NPI compounds were administered as monotherapy via the intraperitoneal route at the doses indicated for the individual study; for the combination studies the selected reference chemotherapy agents were injected 15-30 min prior to the NPI compound.
- mice Female nude mice were injected in the mammary fat pad with 2x10 6 MDA-231 cells harvested from in vitro cell culture. When the tumor size reached 5 mm x 5 mm (about 14-28 days) the animals were matched into treatment and control groups. The study then proceeded as described for the HT-29 model, using paclitaxel as the standard chemotherapy agent.
- test compounds were administered via the intraperitoneal route on Days 1, 4, 8, 11 and 15 (q3dx5); paclitaxel was administered via the intraperitoneal route on Days 1-5 (qdx5).
- mice received Bl 6-F 10 cells (prepared from an in vitro cell culture of Bl 6-F 10 cells) by the iv route on Day 0. On Day 1 mice were randomized into treatment and control groups and treatment commenced. Mice were weighed twice weekly, starting on Day 1. All mice are sacrificed on Day 16, the lungs removed, weighed and the surface colonies counted. Results are expressed as mean colonies of treated mice/mean colonies of control mice (T/C) x 100%). The metastasis growth inhibition (MGI) is this number subtracted from 100%. Paclitaxel was the standard chemotherapy agent used in this study. [0539] In this model unless otherwise noted for the individual study, the test compounds were administered via the intraperitoneal route on Days 1-5 (qdx5); paclitaxel was administered intravenously on Days l-5(qdx5).
- KPU-2 was administered at doses of 7.5 mg/kg ip daily for five days (qdx5), 3.75 mg/kg ip bid for five days, 7.5 mg/kg ip eveiy second day for 10 days (qodx5) and 7.5 mg/kg ip every third day for 15 days (q3dx5).
- the combination of CTP-11 with NPI- 2358 at a dose of 7.5 mg/kg ip q3dx5 resulted in a significantly greater effect than for either compound alone, which lasted for the duration of the study (3).
- the aim of the HT-29 arm of the study was to investigate a slightly higher dose of KPU-2 (10 mg/kg ip q3dx5) in the HT-29 human colon tumor xenograft model as compared to those used in the study described above, in which a marked synergy was observed between KPU-2 (7.5 mg/kg ip q3dx5) and CPT-11 (100 mg/kg ip qwx3).
- the tumor size for the combination group was generally smaller than for the other treated or control groups.
- the TGI of the combination group was 65.8%, indicating a positive effect by the NCI criterion, while monotherapy did not reach the NCI criterion of TGI > 58%.
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Abstract
La présente invention concerne des analogues de déshydrophenylahistines, ainsi que des procédés permettant de réaliser de tels composés. Cette invention concerne également des compositions et des méthodes permettant de traiter divers états pathologiques parmi lesquels le cancer et les maladies non cancéreuses associées à une prolifération vasculaire.
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WO2011084962A1 (fr) * | 2010-01-05 | 2011-07-14 | Nereus Pharmaceuticals, Inc. | Analogues de déshydrophénylahistines |
BR112014022780A8 (pt) | 2012-03-16 | 2021-06-15 | Vitae Pharmaceuticals Inc | composto modulador de receptor de fígado x, composição farmacêutica que o compreende e seu uso |
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RU2736045C2 (ru) | 2015-03-06 | 2020-11-11 | Бейондспринг Фармасьютикалс, Инк. | Способ лечения рака, ассоциированного с мутацией ras |
WO2017011399A1 (fr) | 2015-07-13 | 2017-01-19 | Beyondspring Pharmaceuticals, Inc | Compositions de plinabuline |
AU2017217426B2 (en) | 2016-02-08 | 2022-12-01 | Beyondspring Pharmaceuticals, Inc. | Compositions containing tucaresol or its analogs |
BR112018074990A2 (pt) | 2016-06-06 | 2019-03-12 | Beyondspring Pharmaceuticals, Inc. | composição, usos e método para reduzir neutropenia |
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US11400086B2 (en) | 2017-02-01 | 2022-08-02 | Beyondspring Pharmaceuticals, Inc. | Method of reducing chemotherapy-induced neutropenia |
CN108658945B (zh) * | 2017-03-31 | 2020-11-20 | 深圳海王医药科技研究院有限公司 | 一种微管蛋白抑制剂(vda-1)的a晶型 |
CN109498627B (zh) * | 2017-09-15 | 2021-06-04 | 深圳华大海洋科技有限公司 | 一种***的药物组合物及其应用 |
EP3743074A4 (fr) | 2018-01-24 | 2021-12-15 | Beyondspring Pharmaceuticals Inc. | Composition et procédé pour réduire la thrombocytopénie par l'administration de plinabuline |
CN108299399A (zh) * | 2018-02-01 | 2018-07-20 | 深圳海王医药科技研究院有限公司 | 一种小分子免疫化合物的晶型、其制备方法和含有其的药物组合物 |
CN111840291B (zh) * | 2020-07-14 | 2021-12-21 | 深圳海王医药科技研究院有限公司 | 一种在肿瘤治疗中具有增效作用的化合物的应用 |
CN114349740A (zh) * | 2022-01-17 | 2022-04-15 | 中国海洋大学 | 一种微管蛋白抑制剂普那布林异构体杂质的制备方法及其应用 |
WO2023232113A1 (fr) * | 2022-06-02 | 2023-12-07 | 上海旭成医药科技有限公司 | Composé de pipérazinone, son procédé de préparation et son utilisation |
CN115252616B (zh) * | 2022-08-09 | 2024-05-17 | 中国科学院长春应用化学研究所 | 一种自激活血管阻断剂前药及其制备方法和应用 |
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