KR20050022006A - Compounds useful for the treatment of cancer, compositions thereof and methods therewith - Google Patents

Abstract

The present invention relates to novel compounds and pharmaceutical compositions useful for the modulation of ligase activity. The present invention provides a compound of the invention for preventing or treating cancer, neoplastic disease, acute or chronic renal failure, inflammatory disorders, immune disorders, cardiovascular disease, aging or infectious diseases, comprising administering an effective amount of a compound of the invention, It relates to compositions and methods. The present invention relates to the use of the compounds of the present invention as agents for regulating stem cells or preservatives of cells, blood, tissues or organs.

Description

COMPOSITIONS USEFUL FOR THE TREATMENT OF CANCER, COMPOSITIONS THEREOF AND METHODS THEREWITH

The present invention relates to novel compounds and pharmaceutical compositions useful for the modulation of ligase activity. The present invention relates to a method for preventing or treating cancer, neoplastic disease, acute or chronic renal failure, inflammatory disorders, immune abnormalities, cardiovascular disease, aging or infectious disease comprising administering to a patient in need thereof an effective amount of a compound of the present invention. will be. The present invention relates to the use of the compounds of the present invention as agents for regulating stem cells or preservatives of cells, blood, tissues or organs.

Ubiquitin-mediated proteolysis includes p27, p53, p300, cyclins, E2F, STAT-1, c-Myc, c-Jun, EGF receptors, IkBa, NfkB and b-catenin Is an important pathway of non-lysosomal protein degradation that regulates the destruction of many cell regulatory proteins (Pagano (1997) FASEB J.11: 1067). Ubiquitin is a well-conserved 76-amino acid polypeptide present in eukaryotic cells. The ubiquitin pathway forms a polyubiquitin chain covalently attached to target substrates and is degraded by the multi-catalyst proteosome complex (Goldberg et al. (1995) Science 269: 682-685). Many steps are known to control protein ubiquitation. Initially, ubiquitin activating enzyme (E1) catalyzes the formation of high energy thioester bonds with ubiquitin and is delivered to the reactive cysteine residue of one of many ubiquitin attached enzymes. Final delivery of ubiquitin to e-amino groups or reactive lysine residues to the target protein occurs in reactions that do not require the ubiquitin ligase (E3) protein.

p27 / Kipl is a cyclin-dependent kinase (CDK) inhibitor that is primarily regulated through ubiquitin mediated proteolytic pathways. Degradation of the regulatory protein p27 / Kip1 is known to be required for S phase transition in Gl (Sheaff et al. (1997) Gene Dev. 11: 1464-1478). In both S-phase kinase related protein 2 (SKP2) and cyclin-dependent kinase subunit 1 (CKS1) knockout mice, p27 / Kipl accumulated at high levels and cell proliferation was stopped at S phase transition at Gl. In addition, overexpression of p27 / Kipl in Hela cells resulted in growth inhibition associated with cell cycle Gl arrest (Tang and Nordin (1997) Bioch. And Biophys. Res. Comm. 238: 534-538).

238: 534 (538)). Overexpression of p27 / Kipl also induced cell cycle arrest in the G1 phase in HCC-9204 cell lines (human liver cancer) and lung cancer and subsequently induced epoptosis (Yu et al. (1998) PNAS 95: 11324 ( 11329)). In addition, overexpression of p27 / Kipl has an anti-angiogenic effect (Goukassian et al. (2001) FASEB J. 15: 1877-1885).

The phosphorylation of Thrl87 of p27 / Kipl by CDK2 creates a binding site for SKP2 containing the E3 ubiquitin-protein ligase known as the skpl-cull-f-box ("SCF") protein.

Subsequent ubiquitination of p27 / Kipl by SCF causes degradation of p27 / Kipl by proteosome complexes (Alessandrini et al. (1997) Leukemia 11: 342-345). In addition, SKP2, which acts as a receptor component of the SCF1 ubiquitin ligase complex, binds to CKS1 and binds to p27 / Kipl only when Thrl87 of p27 / Kipl is phosphorylated. This important binding and interaction is known to be necessary for the ubiquitination and degradation of p27 / Kipl. Thus the regulation of ubiquitination of p27 / Kipl by E3 ubiquitin-protein ligase, which subsequently leads to the degradation of p27 / Kipl, provides an opportunity for the prevention and treatment of cancer, neoplasms and other proliferative diseases.

In addition, compounds having the general ability to extend cells at points in the cell cycle without affecting long-term survival of the cells are useful as preservatives for cells, blood, tissues or organs that require such conservation. Many 60% of stored human blood and blood-products may be lost due to limited "shelf-life". Degradation of biological products such as all cells is the result of the cellular level of catabolism and is inversely proportional to storage temperature. Compounds capable of stopping the cells in the G1 phase can increase the shelf life of biological products or allow biological products to be stored or delivered at elevated temperatures without increasing catabolism.

Thus there is a need for compounds that have the ability to preserve biological products.

2.1. Cancer and neoplastic disease

Cancer affects approximately 20 million adults and children worldwide and 9 million new cases have been diagnosed this year (International Cancer Research Institute; www.iarc.fr). Survey will be analyzed. According to the American Cancer Society, approximately 563,100 Americans are expected to die from more than 1500 cancers a day this year. Since 1990, nearly 5 million people have died of cancer in the United States alone, and about 12 million new cases have been diagnosed.

Recent cancer treatments include surgery, chemotherapy and / or radiation therapy to remove neoplastic cells from a patient (see, eg, Stockdale, 1998, "Principles of Cancer Patient Management", in Scientific American: Medicine , vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). All these approaches have drawbacks to the patient. For example, surgery may be contraindicated or difficult to accept due to the patient's health. In addition, surgery may not completely remove neoplastic tissue. Radiation therapy is effective only when the irradiated neoplastic tissue is more sensitive to radiation than normal tissue and radiation therapy can often cause severe side effects. (Id.) Chemotherapy in the treatment of neoplastic disease Several chemotherapy agents are possible. However, despite the use of various chemotherapy agents, chemotherapy has many drawbacks (see, for example, Stockdale, 1998, "Principles Of Cancer Patient Management" in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds. Almost all chemotherapy agents are toxic and chemotherapy causes serious and often dangerous side effects, including severe nausea, diarrhea, bone marrow suppression, and immunosuppression. In addition, many tumor cells are resistant to or develop resistance to chemotherapeutic agents through multi-drug resistance.

Therefore, there is an important need in the art for new compounds, compositions and methods useful for preventing or treating cancer or neoplastic disease with or without the mentioned side effects. In addition, there is a need for cancer therapeutics that provide cancer cell specific therapies with increased specificity and reduced toxicity.

Citation or identification of a reference in Chapter 2 of this specification is not an admission that such reference is available in the prior art.

3. Summary of the Invention

The present invention relates to compounds of the invention, such as compounds of formula (I).

The variables here are prodrugs, cladrates, hydrates, solvates, polymorphs and pharmaceutically acceptable salts thereof, as defined below.

The invention also relates to pharmaceutical compositions comprising an effective amount of a compound of the invention.

Compounds of the present invention and compositions thereof can be used to modulate ligase activity; Prevention or treatment of diseases responsive to modulating ligase activity; Prevention or treatment of a disease in response to inhibition of ligase activity; Prevention or treatment of a disease in response to activation of ligase activity; Modulating E3 ubiquitin-protein ligase activity; E3 ubiquitin-protein ligase mediated ubiquitation regulation of p27 / Kipl; Cellular p27 / Kipl regulation; Stop of growth of cells; Preventing or treating side effects associated with chemotherapy or radiation therapy; Increased survival time of cryopreserved cells, blood, tissues, organs or organisms; Regulating or controlling the differentiation and maturation of mammals, particularly human stem cells, following specific cell or tissue lineages for the differentiation of embryonic-like stem cells derived from placenta or post-partum placement, or of stem cells derived from such origin as cord blood; Preventing or treating cancer or neoplastic disease in a patient in need of such treatment or prevention; Or inhibiting the growth of cancer cells or neoplastic cells.

The present invention provides a method for preventing or treating cancer, neoplastic abnormalities, acute or chronic renal failure, inflammatory abnormalities, immune abnormalities, cardiovascular disease, the consequences of aging or infectious diseases comprising administering to a patient in need thereof an effective amount of a compound of the present invention. It is about. The present invention relates to the use of a compound of the invention as a preservative of cells, blood, tissues or organs or as a medicament for regulating stem cells.

4. Detailed description of the invention

4.1. Justice

As used herein, the term “patient” is an animal (eg, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig) and preferably non Mammals such as primates and primates (eg monkeys and humans), most preferably humans.

 "Alkyl" means a saturated straight or branched non-cyclic hydrocarbon having 1 to 10 carbon atoms. "Lower alkyl" means alkyl having 1 to 4 carbon atoms as defined above. Representative saturated linear alkyls are -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and -n- Decyl; While saturated branched alkyls are -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, isopentyl, 2-methylhexyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4- Methylpentyl, 2-methylhexyl, 3-methylhexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5- methylhexyl, 2, 3-dimethylbutyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylpentyl, 2,2 -Dimethylhexyl, 3,3-dimethylpentyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-decylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-methyl-4 Ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2-diethylhexyl, 3,3-diethylhexyl and analogs thereof.

"Alkenyl" means a saturated straight or branched non-cyclic hydrocarbon containing from 2 to 10 carbon atoms and at least one carbon-carbon double bond. Representative linear and branched (C ₂ -C ₁₀ ) alkenyls are -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl , -3-methyl-1-butenyl, -2-methyl-2-butenic, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3 -Hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3 octenyl, -1-nonenyl, -2 Nonnonyl, 3-nonenyl, -1-disenyl, -2-disenyl, -3-disenyl and analogs thereof. Alkenyl groups may or may not be substituted. A "cyclic alkylidene" is a ring having 3 to 8 carbon atoms and contains at least one carbon-carbon double bond, which ring may have 1 to 3 heteroatoms.

"Alkynyl" means a straight or branched, non-cyclic hydrocarbon having 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative straight chain and branched (C ₂ -C ₁₀ ) alkynyl are -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3 -Methyl-1-butynyl, -4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, -1-heptinyl, -2-heptinyl, -6-heptinyl, -1-octynyl, -2-octynyl, -7-octinyl, -1-noninyl, -2-noninyl, -8-noninyl, -1-decynyl, -2-decynyl, -9 -Decinyl and analogs thereof. Alkynyl groups may not be substituted or may be substituted.

The terms "halogen" and "halo" mean fluorine, chlorine, bromine or iodine. "Haloalkyl" means an alkyl group substituted with one or more halogen atoms, wherein alkyl as defined above is -CH ₂ F, -CH ₂ Cl, -CH ₂ Br, -CH ₂ I,-( CH ₂ ) ₂ F,-(CH ₂ ) ₂ Cl,-(CH ₂ ) ₂ Br,-(CH ₂ ) ₂ I, -CF ₂ , -CH ₂ CF ₂ ,-(CH ₂ ) ₂ CF ₂ and Substituted with one or more halogen atoms, including the like.

"Acyloxy" means -OC (O) CH ₃ , -OC (O) CH ₂ CH ₃ , -OC (O) (CH ₂ ) ₂ CH ₃ , -OC (O) (CH ₂ ) ₃ CH -OC (O) alkyl group comprising ₃ , -OC (O) (CH ₂ ) ₄ CH ₃ , -OC (O) (CH ₂ ) ₅ CH ₃ , and the like, wherein alkyl is Same as defined.

"Alkoxy" means -OCH ₃ , -OCH ₂ CH ₃ , -O (CH ₂ ) ₂ CH ₃ , -O (CH ₂ ) ₃ CH ₃ , -O (CH ₂ ) ₄ CH ₃ , -O (CH ₂ ) -O- (alkyl) including ₅ CH ₃ , and the like, wherein alkyl is as defined above.

"Lower alkoxy" means -O- (lower alkyl), where lower alkyl is as defined above.

"Aryl" means a carbocyclic aromatic group containing 5 to 10 ring atoms. Representative examples include phenyl, tolyl, anthracenyl, fluorenyl (fluorenyl), indenyl, azulenyl, pyridinyl and 5,6,7,8-tetrahydronaphthyl Including but not limited to benzo-fused cyclic cyclic moieties including naphthyl. Carbocyclic aromatic groups may be unsubstituted or substituted. In one embodiment, the carbocyclic aromatic group is a phenyl group.

"Cycloalkyl" means a monocyclic or polycyclic saturated ring having carbon and hydrogen atoms and no carbon-carbon multiple bonds. Examples of cycloalkyl groups include (C ₃ -C ₇ ) cycloalkyl groups, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes Including but not limited to. Cycloalkyl groups may be unsubstituted or substituted. In one embodiment, the cycloalkyl group is a monocyclic or bicyclic ring (ring).

"Mono-alkylamino" means -NHCH ₃ , -NHCH ₂ CH ₃ , -NH (CH ₂ ) ₂ CH ₃ , -NH (CH ₂ ) ₃ CH ₃ , -NH (CH ₂ ) ₄ CH ₃ , -NH ( CH ₂ ) ₅ CH ₃ , and analogs thereof, such as —NH (alkyl), wherein alkyl is as defined above.

"Di-alkylamino" means -N (CH ₃ ) ₂ , -N (CH ₂ CH ₃ ) ₂ , -N ((CH ₂ ) ₂ CH ₃ ) ₂ , -N (CH ₃ ) (CH ₂ CH ₃ ) And -N (alkyl) (alkyl), including the like, wherein each alkyl is independently alkyl as defined above.

"Alkylamino" means NHCH ₃ , -NHCH ₂ CH ₃ , -NH (CH ₂ ) ₂ CH ₃ , -NH (CH ₂ ) ₃ CH _3, -NH (CH ₂ ) ₄ CH ₃ , -NH (CH ₂ ) ₅ CH _3, and -N (alkyl) (alkyl) wherein each alkyl is independently -NH (alkyl) as defined above and each alkyl in the margin is independently an alkyl group as defined above -N (CH ₃ ) ₂ , -N (CH ₂ CH ₃ ) ₂ , -N ((CH ₂ ) ₂ CH ₃ ) ₂ , -N (CH ₃ ) (CH ₂ CH ₃ ) and the like, Wherein each alkyl independently means mono-alkylamino or di-alkylamino as defined above.

 "Carboxyl" and "carboxy" mean -COOH.

"Aminoalkyl" means CH ₂ -NH ₂ ,-(CH ₂ ) ₂ -NH ₂ ,-(CH ₂ ) ₃ -NH ₂ ,-(CH ₂ ) ₄ -NH ₂ ,-(CH ₂ ) ₅ -NH ₂ And-(alkyl) -NH ₂ , including the like, wherein alkyl is as defined above.

"Mono-alkylaminoalkyl" means -CH ₂ -NH-CH ₃ , -CH ₂ -NHCH ₂ CH ₃ , -CH ₂ -NH (CH ₂ ) ₂ CH ₃ , -CH ₂ -NH (CH ₂ ) ₃ CH -(Alkyl) including ₃ , -CH ₂ -NH (CH ₂ ) ₄ CH ₃ , -CH ₂ -NH (CH ₂ ) ₅ CH ₃ ,-(CH ₂ ) ₂ -NH-CH ₃ , and the like -NH (alkyl), wherein each alkyl is independently alkyl as defined above.

"Heteroaryl" has 5 to 10 membered aromatic heterocycles having at least one heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and comprising at least one carbon atom including mono- and bicyclic ring systems Heterocycle ring. Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl, pyridyl, furyl, benzofuranyl, thiophenyl, benzothiophenyl, quinolinyl, pyrrolyl, indolyl, oxa Zolyl, benzoxazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl , Pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, quinazolinyl, pyrimidyl, oxetanyl, azepinyl, piperazinyl, morpholinyl, dioxanyl, thietanyl And oxazolyl.

"Heterocycle" is a saturated, unsaturated 5- to 7-membered monocyclic, or 7- to 10-, containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Bicyclic, heterocyclic ring (ring) of a member, wherein nitrogen and sulfur heteroatoms can be selectively oxidized, nitrogen heteroatoms can be optionally quaternized, and the heterocycle Some of these include bicyclic rings fused to benzene rings. Heterocycles may be attached by heteroatoms or carbon atoms. Heterocycles include heteroaryls as defined above. Representative heterocycles are morpholinyl, pyrrolidinyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl, oxanyl, oxetanyl, Tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyra Nil, and the like.

"Heterocycle fused to phenyl" means a heterocycle attached to two adjacent carbon atoms of a phenyl ring, wherein the heterocycle is as defined above.

"Haloalkyl" means -CF ₃ , -CHF ₂ , -CH ₂ F, -CBr ₃ , -CHBr ₂ , -CH ₂ Br, -CC1 ₃ , -CHC1 ₂ , -CH ₂ CI, -CI ₃ , -CHI ₂ , -CH ₂ I, -CH ₂ -CF ₃ , -CH ₂ -CHF ₂ , -CH ₂ -CH ₂ F, -CH ₂ -CBr ₃ , -CH ₂ -CHBr ₂ , -CH ₂ -CH ₂ Br , -CH ₂ -CC1 ₃ , -CH ₂ -CHC1 ₂ , -CH ₂ -CH ₂ CI, -CH ₂ -CI ₃ , -CH ₂ -CHI ₂ , -CH ₂ -CH ₂ I, and the like Which means alkyl in which one or more hydrogen atoms have been replaced by halogen, wherein alkyl and halogen are as defined above.

"Hydroxyalkyl" means -CH ₂ OH, -CH ₂ CH ₂ OH,-(CH ₂ ) ₂ CH ₂ OH,-(CH ₂ ) ₃ CH ₂ 0H,-(CH ₂ ) ₄ CH ₂ 0H,-( CH ₂ ) ₅ CH ₂ OH, —CH (OH) —CH ₃ , —CH ₂ CH (OH) CH ₃ , and the like, alkyl having one or more hydrogen atoms replaced with hydroxy, wherein Alkyl is as defined above.

"Hydoxy" means -OH.

"Sulfonyl" means -S0 ₃ H.

"Sulfonylalkyl" means -SO ₂ -CH ₃ , -SO ₂ -CH ₂ CH ₃ , -SO _2- (CH ₂ ) ₂ CH ₃ , -SO _2- (CH ₂ ) ₃ CH ₃ , -SO _{2- (CH 2) 4 CH 3,} - S0 2 - (CH 2) 5 CH 3, -S0 _2, and the like, comprising - means (alkyl), and where alkyl is as defined above.

"Sulfinylalkyl" refers to -SO-CH ₃ , -SO-CH ₂ CH ₃ , -SO- (CH ₂ ) ₂ CH ₃ , -SO- (CH ₂ ) ₃ CH ₃ , -SO- (CH ₂ ) -SO- (alkyl), including ₄ CH ₃ , -SO- (CH ₂ ) ₅ CH ₃ , and the like, wherein alkyl is as defined above.

"Thioalkyl" means -S-CH ₃ , -S-CH ₂ CH ₃ , -S- (CH ₂ ) ₂ CH ₃ , -S- (CH ₂ ) ₃ CH ₃ , -S- (CH ₂ ) ₄ CH ₃ , -S- (CH ₂ ) ₅ CH ₃ , and the like, wherein alkyl is as defined above.

As used herein, the term "substituted" means any of the groups (eg, aryl, heteroaryl, heterocycle or cycloalkyl) in which at least one hydrogen atom of the portion to be substituted is replaced with a substituent. . In one embodiment, each carbon atom of the group being substituted is not substituted more than two substituents. In other embodiments, each carbon atom of the group being substituted is not substituted more than one substituent. In the case of ketosubstituents, the two hydrogen atoms are replaced by oxygen attached to the carbon by a double bond. Substituents include halogen, hydroxyl, alkyl, haloalkyl, mono- or di-substituted alkylamino, aryl, heterocycle, -NR _a R _b , -NR _a C (= 0) R _b , -NR _a C (= O) NR _a R _b , -NR _a C (= 0) OR _b , -NR _a SO ₂ R _b , -OR _a , -C (= 0) R _a , C (= 0) OR _a , -C ( = O) NR _a R _b , -OC (= O) R _a , -OC (= O) OR _a , -OC (= O) NR _a R _b , -NR _a SO ₂ R _b , wherein R _a and R _b are the same or different and are independently hydrogen, amino, alkyl, haloalkyl, aryl or heterocycle, or R _a and R _b , like the attached nitrogen atom, are in heterocycle form.

As used herein, an "effective amount" refers to the destruction, modification, control or removal of primary, local or metastatic cancer cells or cancer tissues; Slowing or minimizing cancer expansion; Or an amount of a compound of the present invention sufficient to provide a therapeutic benefit in the treatment or management of cancer, neoplastic disease, acute or chronic renal failure, inflammatory disease, immune disease, cardiovascular disease, outcome of aging or infectious disease. An "effective amount" also includes an amount of a compound of the present invention sufficient to cause cancer or neoplastic cell death. An "effective amount" also includes a compound of the present invention sufficient to modulate (eg, an activator or inhibitor, preferably an inhibitor), ligation activity, either in vitro or in vivo.

As used herein, "modulation of ligation activity" refers to an increase in the rate of activity of one or more proteins with inhibition, activity or delay, preferably inhibition or ligation activity, of the activity rate. In one embodiment, "modulation of ligase activity" means inhibiting the rate of activity of one or more proteins with ligase activity. In another embodiment “modulation of lagerase activity” refers to the regulation of a ligase mixture (eg, a P27 / Kip1 mixture). In another embodiment, "modulation of ligase activity" refers to the regulation of one or more proteins in the ligase complex. In another embodiment, "modulation of ligation activity" means one or more active proteins with ligation activity. In another embodiment, "modulation of ligation activity" means a delay in the rate of activity of one or more proteins with ligation activity. In another embodiment, "modulation of ligation activity" means an increase in the rate of activity of one or more proteins with ligation activity. Modulation of ligase activity can be achieved at the mRNA level, protein expression level and kinase activity.

As used herein, "responsive to modulation" of ligation activity indicates that the activity of one or more proteins with ligation activity is inhibited or activated, preferably inhibited by the compounds of the present invention. it means.

As used herein, "modulation of E3 ubiquitin-protein ligase activity" means that the activity of E3 ubiquitin-protein ligase is inhibited or activated, preferably inhibited by the compounds of the present invention. Means that.

As used herein, "modulation of p27 / Kip1 levels" refers to an increase or decrease in the amount of p27 / Kip1 in a cell in contact with a compound of the invention, preferably to a cell that has not been contacted with a compound of the invention. It means to increase.

As used herein, a “prophylactically effective amount” of the present invention is sufficient to inhibit the development of cancer in a patient, including but not limited to cancer recurrence or cancer expansion or cancer susceptible to or previously exposed to carcinogens. Mean amount of compound. An effective amount for disease prevention also means an amount of a compound of the invention that provides a prophylactic benefit in the prevention of cancer. Furthermore, an effective amount of disease prevention for another disease prevention agent means an amount of disease prevention agent combined with a compound of the present invention that provides the benefit of disease prevention in the prevention of cancer. An “effective amount for disease prevention” used in connection with the amount of a compound of the present invention may include an amount that improves overall prevention or increases the effectiveness of the prevention of another disease prevention agent or provides a synergistic effect with another disease prevention agent. .

As used herein, the term “neoplastic” refers to abnormal growth of cells or tissues (eg, boils) that may be benign or cancerous.

The term "management" as used herein includes the supply of one or more beneficial effects derived from a compound of the present invention in which the patient does not treat the disease in one embodiment. In some embodiments, administering to a patient a compound of the invention that manages the disease to prevent the progression or exacerbation of the disease.

As used herein, "prevention" includes preventing, expanding or preventing the recurrence of cancer in a patient.

As used herein, “treatment” includes eradication, removal, modification, or control of primary, local or metastatic cancer tissue; Minimize or delay cancer expansion.

As used herein, the phrase “compound of the invention” is a compound of formula (I), formula (II), formula (III) (including specific examples of each compound of formula (I)-(III)) As well as clathrates, hydrates, solvates, polymorphs or pharmaceutically acceptable salts. In one embodiment the compound of the invention is substantially free of stereoisomerically pure compounds such as other stereoisomers (eg, at least 85% ee, at least 90% ee, at least 95% ee, at least 97% ee). Above, or above 99% ee).

As used herein, the term “pharmaceutically acceptable salts” means salts prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts for the compounds of the present invention are metallic salts prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or lysine, N, N'-dibenzylenediamine, Organic salts prepared from chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include, but are not limited to, acetic, alginic, anthranilic, benzensulfonic, benzoic, camphorsulfonic, citric , Etenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic (glutamic), glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandeli mandelic, methanesulfonic, mucic, nitric, pamoic, pentothenic, phenylacetic, phosphoric, propionic ), Salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulphonic acid (p- inorganic and organic acids such as toluenesulfonic acid). Specific non-toxic acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and methanesulphonic acid. Examples of specific salts include hydrochloride and mesylate salts. Others are well known in the art, for example Remington's Pharmaceutical Sciences , l8 ^th eds., Mack Publishing, Easton PA (1990) or Remingtora : The Science and Practice of Pharmacy , 19 ^th eds., Mack Publishing, Easton PA ( 1995).

As used herein and unless otherwise indicated, the term "polymorph" means a solid crystalline form of a compound of the invention or a complex thereof. Different polymorphs of the same compound exhibit different physical, chemical and / or spectral characteristics. Differences in physical properties include, but are not limited to, stability (eg, to heat or light), compressibility and density (important for formulation and product manufacture), and dissolution rate (which may affect bioavailability). . Differences in stability can be attributed to chemical reactivity changes (eg, differential oxidation, such as discoloration faster when composed of one polymorph than when composed of another polymorphism) or mechanical features (eg kinematically Tablet fragments stored as preferred polymorphs convert to thermodynamically more stable polymorphs) or both (purification of one polymorph is more susceptible to degradation at higher humidity). Other physical properties of polymorphs can affect their processing. For example, one polymorph may be more likely to form a solvate, for example due to its shape or particle size distribution, compared to another polymorph, or filtration or washing without impurities may be more difficult.

As used herein, the term “solvent compound” further means a compound or salt of the present invention comprising a stoichiometric or non-stoichiometric amount of solvent bound by force between non-covalent molecules. Preferred solvents may be volatile, non-toxic and / or administered in very small amounts to humans.

As used herein, the term "hydrate" further refers to a compound or salt of the present invention comprising a stoichiometric or non-stoichiometric amount of water bound by force between non-covalent molecules.

The term "clathrate" as used herein refers to a compound of the present invention in the form of a crystal lattice comprising a space (e.g., a channel) that traps a guest molecule (e.g., a solvent or water). Or salts thereof.

Unless otherwise used and indicated herein, the term “prodrug” is hydrolyzed, oxidized, and other reactions under biological conditions (in vitro or in vivo) to supply the active compound, in particular the compound of the present invention. It means the compound of the present invention that can be. Examples of prodrugs include biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, and biohydrolyzable ureides. ureides) and metabolites of the compounds of the invention, including but not limited to biohydrolyzable moieties, such as biohydrolyzable phosphate analogs. Preferably the prodrug of the compound having a carboxyl group is a lower alkyl ester of carboxylic acid. Carboxylic esters are typically formed by esterifying a portion of the carboxylic acid present in the molecule. Prodrugs are Burger's Medicinal Chemistry and Drug Discovery 6 ^th ed. (Donald J. Abrahamed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed ., 1985, Harwood Academic Publishers Gmfh) can generally be prepared using well known methods.

In one embodiment, the compounds of the present invention are administered in isolated form when administered to a patient, such as, for example, a mammal for veterinary use or a human for clinical use. As used herein, the term “isolated” means that a compound of the invention is (a) a natural resource, such as a plant, cell, or bacterial culture, or (b) separated into components of one of the synthetic organic chemical reaction mixtures. Means that. Preferably, through the technique of tracing, the compound of the present invention is purified. As used herein, when "purified" is isolated, the separator is at least 90% pure, in one embodiment at least 95% pure, in other embodiments at least 99% pure and in other embodiments It means more than 99.9% pure.

4.2. Compounds of the Invention

As detailed above, the present invention relates to compounds of the present invention such as compounds of formula (I) and their prodrugs, clathrates, hydrates, solvates, polymorphs and pharmaceutically acceptable salts thereof:

Wherein X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl , Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted hetero Cycle, substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) ( CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ;

R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl;

Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5.

In one embodiment, the compound of formula (I) is (4-{[3- (2,2-dimethyl-tetrahydro-4-pyranyl) -4-phenyl-butylamino] -methyl} -phenyl) -dimethyl -Amine).

In another embodiment, the compound of formula (I) is a compound wherein Z is O.

In another embodiment, the compound of formula (I) is a compound wherein Q is O.

In other embodiments, p is 1 or 2. In the above embodiment, p is 2.

In another embodiment, the compound of formula (I) is a compound in which both carbons α for Z are not substituted with Z. In another embodiment, the compound of formula (I) is a compound in which two carbons are substituted with Z. In another embodiment, the compound of formula (I) is a compound in which both carbons are di-substituted with Z. In another embodiment, the compound of formula (I) is a compound in which both carbons α are mono-substituted with Z. In another embodiment, the compound of formula (I) is a compound in which one carbon α is di-substituted with Z and the other carbon α is a compound mono-substituted with Z.

In another embodiment W is H, halogen, hydroxy, carboxy, alkoxy, alkylamino, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, haloalkyl, acyloxy, thioalkyl, sulfonyl, Sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, -C ( = O) OR ₁ , -OC (= O) R ₁ ,-C (= O) NR ₁ R ₂ , -C (= O) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , -NR ₁ C (= 0) (CH ₂ ) qR ₂ , NR ₁ C (= 0) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ .

In another embodiment, U and X are hydrogen, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, substituted or substituted Unsubstituted cycloalkyl, -C (= O) OR _l , -OC (= O) R _l , -C (= O) NR _l R ₂ , -C (= O) NR _l OR ₂ , -SO ₂ NR _l R ₂ , -NR _l SO ₂ R ₂ , -CN, -NO ₂ , -NR _l R ₂ , -NR ₁ C (= 0) R ₂ , -NR _l C (= 0) (CH ₂ ) _q OR ₂ , -NR ₁ C (= 0) (CH ₂ ) _q R ₂ , NR ₁ C (= 0) (CH ₂ ) _q NR ₁ R ₂ , or -O (CH ₂ ) _q NR ₁ R ₂ .

In another embodiment, the invention relates to compounds of formula (II) and compounds of the invention, such as prodrugs, clathrates, hydrates, solvates, polymorphs, and pharmaceutically acceptable salts thereof:

From here:

X, W and U are independently at each occurrence hydrogen, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= O) OR _l , -OC (= O) R _l , -C (= O) NR _l R ₂ , -C (= O) NR _l OR ₂ ,- SO ₂ NR _l R ₂ , -NR _l SO ₂ R ₂ , -CN, -NO ₂ , -NR _l R ₂ , -NR ₁ C (= 0) R ₂ , -NR _l C (= 0) (CH ₂ ) _q OR ₂ , -NR _l C (= O) (CH ₂ ) _q R ₂ , NR _l C (= O) (CH ₂ ) _q NR _l R ₂ ,-O (CH ₂ ) _q NR _l R ₂ ;

X 'is hydrogen, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, haloalkyl, acyloxy , Thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cycloalkyl, -C (= O) OR _l , -OC (= O) R _l , -C (= O) NR _l R ₂ , -C (= O) NR _l OR ₂ , -SO ₂ NR _l R ₂ , -NR _{l SO 2 R 2, -CN,} -NO 2, -NR l R 2, -NR 1 C (= O) R 2, -NR l C (= O) (CH 2) q OR 2, -NR l C (═O) (CH ₂ ) _q R ₂ , NR ₁ C (= 0) (CH ₂ ) _q NR ₁ R ₂ , or —O (CH ₂ ) _q NR ₁ R ₂ ;

R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl;

Each occurrence of Y is independently hydrogen, branched or unbranched C ₁ -C ₁₀ alkyl, or

When O is 1,

Y can be (= O);

Z is C or O;

Q is hydrogen, pulverized or unground C ₁ -C ₁₀ alkyl;

n is 0-8;

o is 0-2;

p is 0-2;

q is 0-5; And

r is 0-5;

In one embodiment, the compound of formula (II) is (4-{[3- (2,2-dimethyl-tetrahydro-4-pyranyl) -4-phenyl-butylamino] -methyl} -phenyl) -dimethyl -Amine).

In another embodiment, the compound of formula (II) is a compound wherein Z is O.

In another embodiment, the compound of formula (II) is a compound wherein Q is H.

In another embodiment, the compound of formula II is a compound wherein one of X, X ', U or W is not hydrogen.

In another embodiment, the compound of formula (II) is a compound in which both carbons α for Z are not substituted with Z. In another embodiment, the compound of formula (II) is a compound in which two carbons are substituted with Z. In another embodiment, the compound of formula (II) is a compound in which both carbons a are di-substituted with Z. In another embodiment, the compound of formula (II) is a compound in which both carbons are mono-substituted with Z. In another embodiment, the compound of formula (I) is a compound in which one carbon α is di-substituted with Z and the other carbon α is a compound mono-substituted with Z.

In another embodiment, the present invention provides in another embodiment, the present invention provides a compound of formula (III) and its prodrugs, clathrates, hydrates, solvates, polymorphs and pharmaceutically acceptable salts thereof Regarding the compound:

X, W and U are independently at each occurrence hydrogen, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= O) OR _l , -OC (= O) R _l , -C (= O) NR _l R ₂ , -C (= O) NR _l OR ₂ ,- SO ₂ NR _l R ₂ , -NR _l SO ₂ R ₂ , -CN, -NO ₂ , -NR _l R ₂ , -NR ₁ C (= 0) R ₂ , -NR _l C (= 0) (CH ₂ ) _q OR ₂ , -NR _l C (= O) (CH ₂ ) _q R ₂ , NR _l C (= O) (CH ₂ ) _q NR _l R ₂ ,-O (CH ₂ ) _q NR _l R ₂ ;

R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl;

Each occurrence of Y is independently hydrogen, branched or unbranched C ₁ -C ₁₀ alkyl, or

When 0 is 1

Y can be (= O);

Z is C or O;

Q is hydrogen, pulverized or unground C ₁ -C ₁₀ alkyl;

o is 0-2;

p is 0-2; And

q is 0-5;

In one embodiment, the compound of formula III is (4-{[3- (2,2-dimethyl-tetrahydro-4-pyranyl) -4-phenyl-butylamino] -methyl} -phenyl) -dimethyl -Amine).

In another embodiment, the compound of formula (III) is a compound wherein Z is O.

In another embodiment, the compound of formula (III) is a compound wherein Q is H.

In another embodiment, the compound of formula (III) is a compound in which both carbons α for Z are not substituted with Z. In another embodiment, the compound of formula (III) is a compound in which two carbons are substituted with Z. In another embodiment, the compound of formula (III) is a compound in which both carbons are di-substituted with Z. In another embodiment, the compound of formula (III) is a compound in which both carbons are mono-substituted with Z. In another embodiment, the compound of formula (III) is a compound in which one carbon α is di-substituted with Z and the other carbon α is mono-substituted with Z.

The invention also relates to pharmaceutical compositions comprising an effective amount of a compound of the invention and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is suitable for treating a disease associated with ligase control. In another embodiment, the pharmaceutical composition is suitable for treating a disease associated with inhibition of ligase.

Compounds of the present invention and pharmaceutical compositions thereof may modulate ligase activity; Treating or preventing a disease responsive to the regulation of ligase activity; Treating or preventing a disease in response to inhibition of ligase activity; Treating or preventing a disease in response to the activity of ligase activity; Modulating E3 ubiquitin-protein ligase activity; regulating p3 / Kipl's E3 ubiquitin-protein ligase mediated ubiquitination; Regulation of cell p27 / Kipl; Arrest of growth of cells; Treating or preventing side effects associated with chemotherapy or radiation therapy; Increased lifespan of cryopreserved cells, blood, tissues, organs or organisms; Regulating or controlling the differentiation and maturation of mammals, particularly human stem cells, following specific cell or tissue lineages for the differentiation of embryonic-like stem cells derived from placenta or post-partum placement, or of stem cells derived from such origin as cord blood; Preventing or treating cancer or neoplastic disease in a patient in need of such treatment or prevention; Or inhibiting the growth of cancer cells or neoplastic cells.

In another embodiment, the present invention is directed to a method of treating or preventing a disease responsive to the modulation of ligase activity in a patient comprising administering an effective amount of a compound of the present invention to a patient in need thereof. Diseases that respond to the regulation of ligase in patients include cancer, neoplastic diseases, inflammatory diseases, infectious diseases, cardiovascular and immune diseases.

In another embodiment, the invention relates to a method for treating or preventing a disease responsive to modulation of the cellular level of p27 / Kipl in a patient comprising administering an effective amount of a compound of the invention to a patient in need of treatment or prevention will be. Diseases that respond to the regulation of the cellular level of p27 / Kipl include cancer, neoplastic diseases, inflammatory diseases, infectious diseases, cardiovascular diseases and immune diseases.

In another embodiment, the present invention relates to a method of inhibiting cell growth, including contacting a cell in need of growth inhibition with a compound of the present invention.

In another embodiment, the present invention is directed to a method of causing cancer or neoplastic cell death, including contacting cancer or neoplastic cells with a compound of the present invention.

In another embodiment, the present invention relates to a method of treating or preventing side effects associated with chemotherapy or radiation therapy comprising administering to a patient in need thereof an effective amount of a compound of the present invention. Side effects associated with chemotherapy or radiation treatment include low blood cell counts, nausea, diarrhea, oral disorders, and alopecia (reduced hair).

In another embodiment, the present invention relates to a method for maintaining a cell, blood, tissue, organ or organism comprising contacting an effective amount of a compound of the present invention with a cell, blood, tissue, organ or organism.

In another embodiment, the present invention relates to a method for controlling and controlling differentiation or maturation of mammalian stem cells comprising contacting the cells with an effective amount of a compound of the invention.

In another embodiment, the invention relates to methods for treating or preventing cancer or neoplastic disease in a patient comprising administering an effective amount of a compound of the invention to a patient in need thereof.

In another embodiment, the present invention relates to a method for preventing or treating cancer or neoplastic disease in a patient comprising contacting cancer or neoplastic cells with an effective amount of a compound of the present invention.

In another embodiment, the present invention relates to a method of inhibiting the growth of cancer cells or neoplastic cells comprising contacting cancer cells or neoplastic cells with an effective amount of a compound of the present invention.

In another embodiment, the present invention is directed to a method of treating or preventing acute or chronic renal failure patients comprising administering an effective amount of a compound of the present invention to a patient in need thereof.

In another embodiment, the present invention relates to a method of treating or preventing a patient with an inflammatory disease comprising administering an effective amount of a compound of the present invention to a patient in need of treatment or prevention.

In another embodiment, the invention is directed to a method of treating or preventing aging effects, including administering an effective amount of a compound of the invention to a patient in need thereof. Aging action includes sarcopenia (loss of muscle mass) and memory loss.

In another embodiment, the present invention relates to a method of treating or preventing a patient with an infectious disease comprising administering an effective amount of a compound of the present invention to a patient in need thereof.

In another embodiment, the present invention is directed to a method of treating or preventing a patient with an immune disease, comprising administering an effective amount of a compound of the present invention to a patient in need thereof.

In another embodiment, the present invention relates to a method of treating or preventing a patient with cardiovascular disease, comprising administering an effective amount of a compound of the present invention to a patient in need of treatment or prevention.

4.3 Preparation of Compounds of the Invention

The compounds of the present invention can be prepared using commercially available starting materials and conventional organic reactions and reagents.

Compounds of the present invention can be readily prepared by those skilled in the art to which the present invention pertains, as illustrated in Schemes I-III below.

                             Table Ⅰ

Wherein variable Z represents carbon or oxygen, and variable R ₁ represents alkyl, in certain instances lower alkyl (eg methyl), and in each case of variable X and R independently one or more arbitrary Substituents (e.g., halogen, alkyl, haloalkyl, alkoxy, amino, alkylamino, or other suitable substituents known by one of ordinary skill in the art) and variable n range from 0-2 Represents an integer.

Those skilled in the art will appreciate that small changes in Table II are inevitable depending on the particular starting material and reagents used.

Table II

Where variable Z represents carbon or oxygen and represents variable R'substituted or unsubstituted phenyl.

Example 1 Compound of ((4- [3- (2,2-Dimethyl-tetrahydro-pyran-4-yl) -4-phenyl-butylamino] -methyl-phenyl) -dimethyl-amine), an example of a compound of the present invention Can be prepared by Scheme III shown below.

Scheme Ⅲ

The compound of Example 1 can be obtained commercially from ChemBridge Corporation (16981 Via Tazon, suite G, San Diego, CA 92127; catalog no, 5936317). The compound of Example 1 obtained commercially is shown as one peak by HPLC (20-100% gradient: acetonitrile / water / 1% trifluoroacetic acid).

4.4. An illustrative compound

Examples of compounds of the invention relate to compounds of the invention, such as prodrugs, clathrates, hydrates, solvates, polymorphs, and pharmaceutically acceptable salts thereof:

4.5 Therapeutic / prophylactic administration and compositions

The compounds of the present invention are useful for the treatment of livestock diseases and for the treatment of humans. For example, the compounds of the present invention are effective in cancer, neoplastic disease, acute or chronic renal failure, inflammatory disease, immune disease, cardiovascular disease, side effects of chemotherapy or radiation therapy, aging effects or infectious diseases. The compounds of the present invention are also effective in inhibiting the growth of cancer cells or myocytes.

The pharmaceutical compositions comprising an effective amount of a compound of the present invention may be conveniently administered, for example, by infusion of a pill or pill, by epithelial or mucosal skin (eg, oral mucosa, rectal and intestinal mucosa). It can be administered and together with other therapeutic agents. The action can be systemic or local. Various means of transport, such as, for example, lysosomal encapsulation, microparticles or microcapsules or capsules, can be used to administer the compounds of the invention. In certain embodiments, one or more compounds of the invention are administered to a patient. Methods of administration may include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, cerebral, intravascular, transdermal, rectal, inhaled, or topical ears, nose Including but not limited to eyes or skin. Preferred methods of administration are at the discretion of the expert and depend, in part, on the site of the medical condition (such as the cancer site).

In certain embodiments, it may be desirable to administer one or more compounds of the invention in part to the area in need of treatment. This can be achieved by, for example, but not limited to, topical application during surgery, topical application such as post-operative wound dressing, injection, catheter, suppository means, implantation means, The implant is a pore, non-pore, or gelatinous material comprising a membrane, such as a siaastic membrane, or a fiber.

In one embodiment, the administration may be by injection directly to a site (or form site) of cancer, neoplastic or neoplastic or pre-neoplastic tissue.

 In certain embodiments, it is preferred to administer one or more compounds of the invention using suitable methods, including intraventricular and intradural infusion. Intraventricular injection will be enabled by an intraventricular catheter attached to a reservoir such as, for example, an Ommaya reservor.

Pulmonary infusions can be employed through the use of inhalers and nebulizers and formulations consisting of aerosolizing agents or fluorocarbons or synthetic pulmonary surfactants through perfusion. In certain embodiments, the compounds of the present invention may be prescribed as suppositories with carriers and traditional adhesives such as triglycerides.

In another embodiment, the compounds of the present invention may be prepared in small blisters, in particular in liposomes (Langer, Science 249: 1527-1533 (1990); Trea et al. eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid ., pp. 317-327; see generally ibid .

In another embodiment, the compounds of the present invention are delivered by a release control system. In one embodiment, a pump is used (Langer, supra ; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al. , N. Engl. J. Med. 321: 574 (1989). In other embodiments, polymeric materials can be used. (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton , Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23: 61 (1983); see also Levy et al., Sceienc 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); Howard et al., J. Neurosurg. 71: 105 (1989 ) Reference). In another embodiment, the release control system is in proximity of the target of the compound of the invention and thus only some systemic dose (e.g. Goodson, in Medical Applications of Controlled Release). supra , vol. 2, pp. 115-138 (1984)) Other emission control systems are discussed by Langer (Science 249: 1527-1533 (1990)) used.

The pharmaceutical composition of the invention comprises an effective amount of a compound of the invention, preferably in a purified state, with an appropriate amount of pharmaceutically acceptable carrier to provide the patient with a condition for proper administration.

In one embodiment, the term “pharmaceutically acceptable” is either approved by a federal agency or the US government, or contained in a US pharmacopeia or generally accepted in a pharmacopeia for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient, or excipient with which the compound of the present invention is administered. Such pharmaceutical carriers may be liquids such as oil, oil, animal, plant or synthetic origin, such as water, peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical carriers may be physiological saline, acacia gum, gelatin, starch, talc, keratin, colloidal silica, urea. In addition, auxiliary, stable, thick, slippery, pigmented drugs can be used. When administered to a patient. The compound of the present invention is preferably sterilized. Water is the preferred carrier when the compounds of the invention are administered intravenously. Saline, aqueous glucose and glycerol solutions can be employed as liquid carriers, in particular as injectable solutions. Suitable pharmaceutical carriers also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried Excipients such as skim milk, glycerol, propylene, glycol, water, ethanol. The present invention also preferably includes small amounts of wet or emulsion or pH buffered drugs.

The composition is a solution, suspension, emulsion, pill, pill, capsule, liquid capsule, powder, sustained-release formulation, suppository, emulsion, aerosol, spray, suspension, or use It may take other forms suitable for. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see, eg, US Pat. No. 5,698,155). Other examples of pharmaceutically suitable carriers are given in "Remington's Pharmaceutical Sciences" by E. W. Martin.

In another embodiment, the compounds of the present invention are formulated according to a defined procedure as a pharmaceutical composition adapted for intravenous administration of humans. In general, the compounds of the invention for intravenous administration are dissolved in isotonic aqueous buffer.

Here, necessarily, the composition comprises a soluble drug. Compositions for intravenous administration include local anesthetics, such as lignocaine, used for mild pain at the injection site. Generally, the components are separated or mixed together in unit dosage form, such as a moisture-free concentrate such as an ampoule or sachet, which requires a large amount of active agent in a dry stored powder or welded hermetically sealed container, for example. It is supplied. When the compounds of the invention are administered in infusions, they are dispersed, for example, into infusion bottles containing sterile pharmaceutical grade water or physiological saline. When the compound of the present invention is administered in infusion, an ampoule of sterile water or physiological saline for infusion may be provided for mixing before the composition is administered.

Compositions for oral delivery are, for example, in the form of pills, lozenges, water soluble or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Compositions that are administered in the past may be, for example, sweeteners such as fructose, aspartame, or saccharin; Flavoring compositions such as wintergreen or cherry oil; coloring agent; And one or more preservative drugs to provide pharmaceutically suitable pharmaceuticals. In addition, in the case of pills or pills, the composition is water-coated to slow down degradation and absorption in the gastrointestinal system, thereby leaving the action for long periods of time. Permeable membranes, optionally surrounding the osmotic propellant, are also suitable for oral administration of a compound of the present invention.

In the latter platform, the encapsulated fluid is absorbed by the propellant compound into the propellant compound that expands to withdraw the drug or pharmaceutical composition through the aperture. Such a delivery platform can provide a substantially zero order rate delivery profile as opposed to the curved profile of the immediate release formulation.

Time delay substances such as glycerol monostearate or gglycerol stearate are also used. Oral compositions may include standard carriers such as mannitol, lactose, sodium saccharin, fibrin, magnesium, carbonate and the like. Such carriers are preferably pharmaceutical grade.

A large amount of a compound of the invention that will be effective in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, ex vivo or in vivo assays help define the optimal dosage range. The exact dosage also depends on the constant dosage, the severity of the disease and disorder, and should be determined by the judgment of the expert and the situation of each patient. However, a suitable dosage range, especially intravenous administration, is generally about 20-500 micrograms of a compound of this invention per kilogram of body weight. In a preferred embodiment of the invention, the i.v. dosage is about 10-40, 30-60, 60-100 or 100-200 micrograms per kilogram of body weight. In another embodiment, i.v. The dosage is about 75-150, 150-250, 250-375 or 375-500 micrograms per kilogram of body weight. Appropriate dosages are intranasal administration, generally from about 0.01 pg to 1 mg / kg body weight. Suppositories generally contain a compound of the invention in the range of 0.5% to 10% by weight. Oral compositions preferably comprise from about 10% to about 95% of the compounds of the present invention. In a preferred embodiment of the invention, a suitable dosage range for oral administration is generally on the order of 1-500 micrograms of a compound of the invention per kilogram of body weight. In a preferred embodiment, the oral dosage is on the order of 1-10, 10-30, 30-90 or 90-150 micrograms per kilogram of body weight. In another embodiment, the oral dosage is on the order of 150-250, 250-325, 325-450 or 450-1000 micrograms per kilogram of body weight. Effective doses are extrapolated from dose-response curves derived from ex vivo or animal model test systems. Such animal model systems are known in the art.

The present invention also provides a pharmaceutical kit comprising a container containing a compound of the present invention. Such containers, optionally associated, may include the use or sale of a governmental authority that controls manufacture, the use or sale of a pharmaceutical or biological agent that reflects the manufacturer's approval, the use or sale of human administration; Or in the form prescribed by the instructions for use. The key may also contain a container containing a useful chemotherapeutic composition for treating cancer or neoplastic disease. The compounds of the present invention are preferably assayed in vitro and in vivo for the desired therapeutic or activity of disease prevention prior to use in humans. For example, ex vivo assays are used to determine which compounds of the invention are preferred in one or more administrations.

In another embodiment, a patient tissue sample is grown in culture, contacted or administered with a compound of the invention, and the effect of the compound of the invention in the tissue sample is observed and compared with the non-contacted tissue. In another embodiment, a cell-culture model is used for contacting or administering cells of a cell culture with a compound of the present invention, wherein the effect of the compound of the present invention on cell-culture is observed and compared with non-contact cell culture. In general, low levels of proliferation or survival of contacted cells as compared to non-contacted cells indicate that the compounds of the present invention are effective in treating patients. Such compounds of the invention have also proven effective and safe for use in animal model systems.

Other methods will be known by the skilled practitioner and are within the scope of the present invention.

4.6. Cancer and neoplastic cells and disease suppression

The compounds of the present invention can be shown to inhibit tumor cell proliferation, cell transformation and tumorigenesis in vitro or in vivo using various assays known or referred to in the art. Such an assay may use cells of a cancer cell line or cells of a patient. Many assays well known in the art can be used to assess survival and / or growth; For example, cell proliferation can be determined by ( ³ H) -thymidine insertion measurements, direct cell counting, proto-genes (eg fos, myc) or cell cycle markers (Rb, cdc2, cyclin A, D 1). , D2, D3 or E) can be analyzed by examining changes in transcription, translation or activity of known genes. The level and activity of such proteins and mRNA can be determined by methods well known in the art. For example, proteins may be used for Western blotting or immunoprecipitation using commercially available antibodies (eg, many cell cycle marker antibodies manufactured by Santa Cruz, Inc.). Can be quantified by known immunodiagnostic methods. mRNA is well known in the art and can be quantified by routine methods such as Northern blot, RNase protection, polymerase chain reaction involving reverse transcription, and the like. Cell viability can be assessed by using trypan-blue staining or other cell death or vitality markers known in the art. Differentiation can be assessed visually based on changes in morphology and the like.

The present invention provides, but is not limited to, cell cycle and cell proliferation assays by various techniques known in the art, including:

According to one example, bromodeoxyuridine ("BRDU") incorporation is used as an assay to identify proliferating cells. BRDU analysis identifies cell populations affected by DNA synthesis by BRDU insertion into newly synthesized DNA. Recently synthesized DNA is detected by using anti-BRDU antibodies (see Hoshino et al., 1986, Int. J. Cancer 38,369; Campana et al., 1988, J. hmunol. Meth. 107,79).

Cell proliferation is described by (3h) -thymidine insertion (see, eg, Chen, J., 1996, Oncogene 13: 1395-403; Jeoung, J., 1995, J. Biol. Chem. 270: 18367-73). It is observed by using

This assay allows for quantitative characterization of S-phase DNA synthesis. Cells that synthesize DNA in this assay will insert (3H) -thymidine in the newly synthesized DNA. Insertion is measured by standard techniques in the field of the present invention, such as radioisotope number counting at scintillation counters (eg, Beckman LS 3800 Liquid Scintillation Counter).

Detection of proliferating cell nuclear antigen (PCNA) is also used to measure cell proliferation. PCNA is a 36 kilodalton protein and its expression is particularly enhanced in proliferating cells, which are early G1 and S phases of the cell cycle. Positive cells were immunostained using an anti-PCNA antibody (Li et al., 1996, Curr. Biol. 6: 189-199; Vassilev et al., 1995, J. Cell Sci. 108: 1205-15). It is confirmed.

Cell proliferation can be measured by counting a sample of a group of cells over time of cell proliferation (eg, counting cells daily). Cells are counted using a hemacytometer and a light microscope (eg, HyLite hemacytometer, Hausser Scientific). The cell number is located by time coordinates to obtain the growth curve of the group of interest.

In one preferred embodiment, the cells counted by this method are first mixed with the staining agent Trypan-blue (Sigma), and the living cells exclude the staining agent and are counted as surviving members of the group.

The DNA content and / or mitotic index of the cells is determined based on, for example, the DNA ploidy value of the cell. For example, cells on G1 of the cell cycle generally contain 2N DNA ploidy values. Cells in the replicating DNA that do not progress through mitosis (eg, cells on S) will exhibit ploidy values higher than 2N and 4N DNA amount. Drainage values and cell-cycle kinetic are measured using propidium iodide analysis (eg, Turner, T., et al., 1998, Prostate 34: 175-81). Or DNA ploidy is DNA Foilgen staining in a computerized microdensitometry staining system (e.g., Bacus, S., 1989, Am. J. Pathol. 135: 783-92). Quantitatively binding to DNA). In another embodiment, the DNA content is analyzed by preparation of chromosomal diffusion (Zabalou, S., 1994, Hereditas. 120: 127-40; Pardue, 1994, Meth. Cell Biol. 44: 333-351).

Expression of cell-cycle proteins (eg, CycA. CycB, CycE, CycD, cdc2, Cdk4 / 6, Rb, p21 or p27) provides deterministic information related to the state of cell proliferation or group of cells. For example, confirmation in anti-proliferative signaling pathways may be indicated by induction of p21 cip 1. Increased levels of p21 expression in cells result in delayed entry into G1 of the cell cycle (Harper et al., 1993, Cell 75: 805-816; Li et al., 1996, Curr. Biol. 6: 189 -199). p21 induction is confirmed by immunostaining by using certain commercially available anti-p21 antibodies (eg, Santa Cruz, Inc.). Likewise, cell-cycle proteins can be observed by Western blot analysis using commercially available antibodies. In another embodiment, the cell population is synchronized prior to the detection of cell cycle proteins. Cell-cycle proteins are also detected by FACS (Fluorescence Activated Cell Separator) assays using antibodies against proteins of interest.

The discovery of changes in the length of the cell cycle or the rate of the cell cycle is used to measure cell proliferation inhibition by the compounds of the present invention. In one embodiment, the cell cycle length is determined by the passage time of the cell population (eg, using cells that are in contact with or without contact with one or more compounds of the invention). In another embodiment, FACS analysis is used to analyze cell cycle processes or to purify Gl, S, and G2 / M fractions (Delia, D. et al., 1997, Oncogene 14: 2137-47).

The passage of the cell cycle checkpoint and / or the induction of the cell cycle checkpoint is observed by the methods described herein or by methods known in the art. Without limitation, cell cycle checkpoints are a mechanism that ensures that certain cells occur in a particular order. Checkpoint genes are defined as mutations that allow later events to occur without complete completion of the initial events (Weinert, T., and Hartwell, L., 1993, Genetics, 134: 63-80). Induction or inhibition of cell cycle checkpoint genes is assessed, for example, by western blot analysis or by immunostaining. The progression of the cell cycle checkpoint is assessed by the progression of cells that pass through the checkpoint without the occurrence of certain events (eg, processes in mitosis without complete replication of genomic DNA).

In addition to the effects of expression of specific cell cycle proteins, the activity and post-translational modifications of the proteins involved in the cell cycle can play an important role in the normal and proliferative state of the cell. The present invention demonstrates an assay comprising post-translational modifications (eg phosphorylation) detected by methods known in the art. For example, antibodies in which phosphorylated tyrosine residues are detected are commercially available and used in western blot analysis to detect proteins with such mutations. In other embodiments, variations such as myristylation can be performed using thin monolayer chromatography or reversed phase hplc (Glover, C., 1988, Biochem. J. 250: 485-91; Paige, L., 1988, Biochem J .; 250: 485-91).

The activity of signal and cell cycle proteins and / or protein complexes is often mediated by kinase activity. The present invention provides an analysis of kinase activity by assays such as histone H1 assays (eg, Delia, D. et al., 1997, Oncogene 14: 2137-47).

Compounds of the present invention can be shown to improve cell proliferation in cells cultured ex vivo using methods well known in the art. Specific examples of cell culture models include, but are not limited to, primary rat lung tumor cells (Swafford et al., 1997, Mol. Cell. Biol., 17: 1366-1374) and large cell undifferentiated cancer cell lines for lung cancer. (Mabry et al., 1991, Cancer Cells, 3: 53-58); Colorectal cell lines for colon cancer (Park and Gazdar, 1996, J. Cell Biochem. Suppl. 24: 131-141); Multiple settled cell lines for breast cancer (Hambly et al., 1997, Breast Cancer Res. Treat. 43: 247-258; Gierthy et al., 1997, Chemosphere 34: 1495-1505; Prasad and Church, 1997, Biochem. Biophys. Res. Commun. 232: 14-19); Well characterized cell models of many prostate cancers (Webber et al., 1996, Prostate, Part 1,29: 386-394; Part 2,30: 58-64; and Part 3,30: 136-142; Boulikas, 1997, Anticancer Res. 17: 1471-1505); Urogenital, persistent human bladder cancer cell lines (Ribeiro et al., 1997, Int. J. Radiat. Biol, 72: 11-20); Organ cultures of transient cellular malignancies (Booth et al., 1997, Lab Invest. 76: 843-857) and mouse process models (Vet et al., 1997, Biochim. Biophys Acta 1360: 39-44); Settling cell lines of leukemia and lymphoma (Drexler, 1994, Leuk. Res. 18: 919-927, Tohyama, 1997, Int. J. Hematol. 65: 309-317).

Compounds of the invention can be provided for inhibiting cellular alteration (or progress to a malignant phenotype) in vitro. In one embodiment, cells with a transformed cell phenotype are contacted with one or more compounds of the invention and transformation (a characteristic ex vivo set associated with in vivo carcinogenic capacity) is observed, for example Colony formation in soft agar, more rounded cell morphology, more substratum adsorption, loss of contact inhibition, loss of fixation dependence, secretion of protease such as plasminogen activator, increased sugar transport, reduction Serum requirements, or expression of fetal antigens (Luria et al., 1978, General Virology, 3d Ed., John Wiley & Sons, New York, pp. 436-446).

Loss or reduced adhesion of invasiveness is used to demonstrate the anticancer effect of the compounds of the present invention. For example, an absolute aspect in the formation of metastatic cancer is the ability of pre-cancer or cancer cells to grow new colonies away from the first stage of the disease at the second stage of the disease. The ability of cells to invade peripheral sites is a condition of cancer. Reflects the potential for Loss of invasiveness is measured by various techniques known in the art, including, for example, induction of E-cadherin-mediated cell-cell adhesion. Such E-cadherin-mediated cell-cell adhesion can be obtained from phenotypic alteration and loss of invasiveness (Hordijk et al., 1997, Science 278: 1464-66). Loss of invasiveness is observed by inhibition of cell migration. Various secondary and tertiary cell matrices are commercially available (Calbiochem-Novabiochem Corp. San Diego, CA). Cell migration between or into matrices is examined by methods in the art to which the present invention pertains to microscopic, time-lapse photography or videographic or measurement of cell migration. In one related embodiment, the loss of invasiveness is examined according to hepatocyte growth factor (HGF). HGF- induced in cellular scattering is associated with invasion of cells such as Madin-Darby canine kidney (MDCK) cells. This assay identifies cell populations that have lost cell dispersion activity in response to HGF (Hordijk et al., 1997, Science 278: 1464-66).

Or loss of invasiveness is measured by cell migration through the chemotactic chamber (Neuroprobe / Precision Biochemicals Inc. Vancouver, BC). In this assay, the chemical-affinity composition is lurked in one part of the room (eg, the bottom of the room) and the cells are plated in the opposite room (eg, the room) where the filters are separated. In order for the cells to pass from the top of the room to the bottom of the room, the cells must actively move through small holes in the strainer. Tile analysis of many cells that have migrated is associated with invasiveness (eg, Ohnishi, T., 1993, Biochem. Biophys. Res. Commun. 193: 518-25).

Compounds of the invention appear to inhibit tumor formation in vivo. Models of large animal hyperproliferative disorders, including tumor development and metastatic distribution, are well known in the art (Table 317-1, Chapter 317, "Principals of Neoplasia," in Harrison's Principals of Internal Medicine, 13th Edition, Isselbacher et al. , eds., McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J. Pathol. 181: 130-135). Specific examples include transplantation of small tumor nodes in mice (Wang et al., 1997, Ann. Thorac. Surg. 64: 216-219) or lung cancer metastases in SCID mice bleeding from NK cells (Yono and Sone). , 1997, Gan To Kagaku Ryoho 24: 489-494); In the case of colon cancer, colon cancer cell transplantation of human colon cancer cells in hairless rats (Gutman and Fidler, 1995, World J. Surg. 19: 226-234), a cotton top-tamari model of human ulcerative colitis (Warren, 1996 , Aliment. Pharmacol. Ther. 10 Supp 12: 45-47) and a mouse model with mutations in adeno polyp tumor suppressor genes (Polakis, 1997, Biochim. Biophys. Acta 1332: F127-F147); For breast cancer, in transgenic models of breast cancer (Dankort and Muller, 1996, Cancer Treat. Res. 83: 71-88; Amundadittir et al., 1996, Breast Cancer Res. Treat. 39: 119-135) and in rats Chemical induction of tumors (Russo and Russo, 1996, Breast Cancer Res. Treat. 39: 7-20); For prostate cancer, chemically induced and hemotransformed rodent models and human xenograft models (Royai et al., 1996, Semin. Oncol. Biophys. 23: 35-40); For urogenital cancer, xenografts of induced bladder tumors in mice and rats (Oyasu, 1995, Food Chem. Toxicol 33: 747-755) and human transitional cell malignant tumors in hairless mice (Jarrett et al., 1995, J Endourol. 9: 1-7); And hematopoietic cancers include genetically different allogeneic bone marrow transplanted from animals (Appelbaum, 1997, Leukemia 11 (Suppl. 4): S15-S17). In addition, common animal models to which many forms of cancer can be applied include p53-deficient mouse models (Donehower, 1996, Semin. Cancer Biol. 7: 269-278), Min rats (Shoemaker et al., 1997, Biochem. Biophys. Acta, 1332: F25-F48), and immunization with tumors in mice (Frey, 1997, Methods, 12: 173-188) have been described.

For example, a compound of the present invention can be administered to a test animal in which one type of tumor develops in one embodiment of the test animal, wherein the test animal has reduced tumorigenicity compared to an animal not administered the compound of the present invention. Or a compound of the invention may be administered to a test animal with a tumor (e.g., an animal with a tumor induced by the induction of malignant, tumor or modified cells or administration of carcinogenesis) and Subsequent investigations of the test animal for tumor degeneration as compared to animals not administered the compound.

4.7. of ubiquitination of p27 in vitro  control

Compounds of the present invention may appear to inhibit ubiquitination in vitro using assays known in the art, or are described herein. Exemplary in vitro assays are described by Alessandrini et al. (1997) Leukemia 11: 342-345, wherein purified recombinant hexahistidine-labeled p27 (p27-his6) is used as a substrate for ubiquitination. Rabbit reticulum lysate (RRL) is used as a source of ubiquitination enzymes and proteosome complexes. With and without inhibitors, the degree of ubiquitination can be compared to determine the titer of the inhibitor.

Another exemplary in vitro ubiquitination assay was described by Chiaur et al. In PCT International Patent WO 00/12679, which is incorporated herein by reference in its entirety. Algebraically grown KeLa-S3 cells were collected at a density of 6 × 10 ⁵ cells / ml. Cells were inhibited on G1 by treatment with 70 mM lovastatin for 48 hours. In vitro translated [ ³⁵ S] p27 1 ml contains 40 mM Tris pH7.6, 5 mM MgCl ₂ , 1 mM DTT, 10% glycerol, 1 mM ubiquitin aldehyde, 1 mg / ml methyl ubiquitin, 10 mM creatine phosphate, 0.1 mg / ml creatine phosphokinase Incubated at 30 ° C. for another hour (0-75 min) in 10 ml ubiquitination mixture containing 0.5 mM ATP, 1 mM okadaic acid, 20-30 mg HeLa cell extract. Ubiquitin aldehydes may be added to the ubiquitination reaction to inhibit isopeptidase, which removes the chain of ubiquitin from p27. The added methyl ubiquitin competes with ubiquitin present in the cell extract and terminates the p27 ubiquitin chain. Such chains appear as bands of separability instead of high molecular weight smears. These short polyubiquitin chains have low affinity for proteosomes and are therefore more stable. The reaction is terminated with Laemmli sampler buffer containing b-mercaptoethanol and the product can be analyzed on protein gels under denaturing conditions. Polyubiquitinated p27 forms are identified by autoradiography.

4.8. Treatment or prevention of cancer or tumor disease in combination with chemotherapy or radiation therapy

Cancer or tumor diseases, including but not limited to any disease or disorder characterized by neoplasms, tumors, metastasis, or unregulated cell growth, are effective amounts of the compounds of the present invention. It can be treated or prevented by administering. In one embodiment, a compound comprising an effective amount of one or more compounds of a compound of the invention, or a pharmaceutically acceptable salt thereof, is administered.

In certain embodiments, the invention includes a method of treating or preventing a cancer or a tumor disease comprising administering to a patient in need thereof an effective amount of a compound of the invention and other therapeutic ingredients. In one embodiment, the therapeutic ingredient is methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin , Carboplatin, mitomycin, dacarbazine, procarbazine, etososide, camppatin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase Chemotherapeutic components, including but not limited to, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In one embodiment, the compounds of the present invention exhibit their activity while other therapeutic ingredients exhibit their activity. Other therapeutic ingredients are listed in Table 1 showing chemotherapeutic agents and other anti-cancer components.

In another embodiment, the present method for treating or preventing cancer comprises administering radiation therapy. The cancer may be refractory or non-refractory. The compounds of the present invention can be administered to patients who have undergone surgery for cancer treatment.

In certain embodiments, the compounds of the present invention may be administered to a patient who has undergone surgery as cancer treatment concurrent with chemotherapy or radiation therapy. In another specific embodiment, the chemotherapy component or radiotherapy is administered before or after administration of the compound of the invention, preferably at least 1 hour, 5 hours, 12 hours, 1 day, 1 week, 1 month, more preferably Preferably several months (for example up to three months).

The chemotherapy component or radiotherapy administered concurrently with, or before or after administration of a compound of the invention may be carried out by any method known in the art. The chemotherapeutic component is preferably administered in a series of sessions, and any one or mixture of chemotherapeutic components mentioned above may be administered. With regard to radiation therapy, any radiation treatment protocol can be used depending on the type of cancer to be treated. For example, but not limited to, x-ray radiation may be administered; In particular, high-energy ultrahigh pressure radiation (radiation greater than 1 MeV energy) can be used for deep tumors, and electron beams and tolerated x-ray radiation can be used for skin tumors. Gamma-ray emitting radioisotopes, such as radioactive isotopes of radium, cobalt and other elements, may also be administered to expose the tissue to radiation.

Additionally, the present invention provides chemotherapy or radiation to a patient being treated when chemotherapy or radiotherapy can prove to be too toxic or prove to be severe, for example when it results in unacceptable or intolerable side effects. As an alternative to treatment, a method of treatment of a cancer or a tumor disease is provided. Patients being treated can be selectively treated with other cancer therapies such as surgery, radiation therapy or chemotherapy, and it has been found that relying on such therapies can be acceptable or tolerated.

4.9. Cancer and Tumor Diseases That Can Be Treated or Preventable

Cancer or tumor diseases and related disorders that can be treated or prevented by the administration of a compound of the invention include those mentioned in Table 2 below as well as brain, neck, eye, skin, oral cavity, neck, esophagus, chest, bone, Lung, large intestine, sigmoid, rectal, stomach, prostate, breast, ovary, testes, kidneys, liver, pancreas, brain, small intestine, heart or adrenal cancers, including, but not limited to, such disorders For a review, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia): Cancer and tumor disorders are shown in Table 2.

In certain embodiments, cancer, malignant or dysproliferative changes (such as metastases and dysplasia), or hyperproliferative disorders are treated in the ovary, breast, colon, lung, skin, pancreas, prostate, tract, or uterus Possible or preventable. In another specific embodiment, the cancer treatable or preventable by administration of an effective amount of a compound of the invention is sarcoma, melanoma, or leukemia. In another specific embodiment, the cancer treatable or preventable by administration of an effective amount of a compound of the invention is multiple myeloma, leukemia, myelodysplastic syndrome or myeloproliferative disorder. In another specific embodiment, the cancer treatable or preventable by administration of an effective amount of a compound of the invention is glioma.

In a preferred embodiment, the compounds of the present invention comprise prostate (more preferably hormone-insensitive), neuroblastoma, lymphoma (preferably follicular or diffuse large B-cells), breast ( Cancers, including estrogen-receptor positive), rectal colon, endometrium, ovary, lymphoma (preferably non-Hodgkin), lung (preferably small cells), or testicles (preferably germ cells) It is useful for treating or preventing the disease.

In another embodiment, the compounds of the present invention may be used for the prostate (more preferably hormone-insensitive), neuroblastoma, lymphoma (preferably follicular or diffuse large B-cells), breast ( Preferably cancer such as estrogen-receptor positive), rectal colon, endometrium, ovary, lymphoma (preferably non-Hodgkin's), lung (preferably small cells), or testicles (preferably germ cells) or Useful for inhibiting growth of cells derived from tumors.

In certain embodiments of the invention, the compounds of the invention are useful for inhibiting the growth of cells, which cells are from Table 2 or from cancer or tumors herein.

Example 1 Compound ((4- [3- (2,2-Dimethyl-tetrahydro-pyran-4-yl) -4-phenyl-butylamino] -methyl-, which is an illustrative example of a compound of the present invention) Phenyl) -dimethyl-amine) has been shown to induce cell (programmed) mission in the following cell lines: Hela (neck adenocarcinoma), MDA-MB-435 (breast cancer), MDA-MB-231 (breast cancer), MCF-7 (breast cancer), HL-60 (leukemia), A172 (malignant glioma), NCIH1703 (non-small cell lung adenocarcinoma), A357 (lung cancer), DU145 (prostate cancer), MMls (multiple myeloma), DF15 (multiple myeloma), H929 (multiple myeloma), U266 (multiple myeloma), ANB6 (multiple myeloma).

4.10. Treatable or Preventable Infectious Diseases

Infectious diseases and related disorders that can be prevented or treated by administration of a compound of the invention include, but are not limited to, those listed in Table 3.

Useful therapeutic ingredients for the treatment of infectious diseases that may be used in combination with the compounds of the invention include penicillin, cephalosporin, vancomycin, aminoglycosides, quinolones, polymyxins, erythromycin, tetracycline, chloramphenicol, clindamycin, lincomycin , But not limited to, clarithromycin, azithromycin, sulfonamide, idoxuridine, vidarabine, trifluorothymidine, acyclovir, phencyclovir, and baracyclovir.

4.11. Treatable or Preventable Inflammatory Disease

Inflammatory diseases and related disorders that can be prevented or treated by administration of a compound of the present invention include but are not limited to rheumatoid arthritis, connective tissue disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis and ileitis It is not limited.

Useful therapeutic ingredients for the treatment of inflammatory diseases that can be used in combination with the compounds of the present invention include anticholinergic, diphenoxylate, loperamide, deodorized, opiate tincture, codeine and water soluble. Including but not limited to mucilloids.

4.12. Treatable or Preventable Cardiovascular Disease

Cardiovascular diseases and related disorders that can be prevented or treated by the administration of a compound of the invention include hypercholesterolemia, arterial hypertension, arteriosclerosis, coronary artery disease, arrhythmia, valve heart disease, endocarditis and pericardial disease. Including but not limited to.

Useful therapeutic ingredients for the treatment of cardiovascular diseases that can be used in combination with the compounds of the present invention include, but are not limited to, antibiotics, folic acid and antihypertensive agents.

4.13. Treatable or Preventable Immune Disorder

Immune disorders that can be prevented or treated by administration of a compound of the present invention are allergic, asthma, chronic granulomatous, autoimmune disorders, Wegener granulomatosis, systemic lupus erythematosus, multiple sclerosis, type 1 diabetes , Rheumatoid arthritis, graft-versus-host disease, rheumatic heart disease, and DiGeorge malformations.

Useful therapeutic ingredients for the treatment of immune disorders that can be used in combination with the compounds of the present invention include, but are not limited to, gamma interferon, glucocorticoids and cyclophosphamide.

4.14. Compounds of the Invention Useful as Preservatives for Cells, Blood, Tissues, Organs or Organisms

The use of the compounds of the present invention as cell growth inhibitors makes them useful for preserving blood, tissue or organs in a state suitable for use with a patient. In particular, the compounds of the present invention are cryopreserved, eg cryopreserved cells, blood, tissue, such as freezing in liquid nitrogen, freezing in dry ice, freezing in ice water or freezing in cold-packs. It is useful for extending the longevity of organs or living bodies.

The following examples are set forth to assist in understanding the present invention and, of course, should not be construed as specifically limiting the invention described and claimed herein. Various modifications of the invention, including substitutions of all equivalents presently known or later developed, are within the perception of one of ordinary skill in the art to which the invention pertains, and minor changes in formulation or experimental design are incorporated herein. It is recognized that it falls within the scope of the invention.

The following conditions were used to obtain the HPLC retention time in connection with Synthesis Examples 1-24:

Column :

YMC Pack-Pro C18 250mm × 4.6mm ID

Total run time 20 minutes, 0-10 minutes following gradient, 10-20 minutes isocratic

Detection wavelengths 214 nm, 254 nm and 290 nm.

Gradients :

25-75% acetonitrile-water / 0.1% TFA

25-95% acetonitrile-water / 0.1% TFA

Example 1

5.1 Example 1 (4-{[3- (2 ', 2'-Dimethyltetrahydropyran-4'-yl) -4-phenylbutylamino] -methyl} -phenyl) -dimethylamine dihydrochloride

1 (A). 2,2-dimethyl-tetrahydropyran-4-one (one)

Mesityl oxide (Mesityl oxide, 11.6 mmol) and water-soluble formaldehyde (11.6 mmol) were mixed and heated at 165 ° C. for 2 hours. The reaction mixture was cooled, partitioned between ethyl acetate and brine, dried using sodium sulphate and evaporated to oil. The crude reaction product was subjected to flash column chromatography (silica gel) eluting with ethyl acetate-hexane (1: 9) to obtain an oil. The oil was dissolved in chloroform (250 ml), Amberlyst 15 resin (11 g) was added and the mixture was stirred for about 16 hours. Filtration and evaporation gave the crude product, which was subjected to flash column chromatography (silica gel) eluting with diethyl ether-hexane (1: 9) to obtain the desired product.

1 (B). (E) and (Z) methyl (2,2-dimethyl-tetrahydropyran-4-ylidene) acetate

Trimethyl phosphonoacetate (22.86 mmol) was added to sodium hydride (23.8 mmol) in 0 ° C THF (80 ml). The reaction mixture was stirred for 15 minutes and a THF (20 ml) solution (20.6 mmol) of Example 1 (A) was added. After standing at room temperature for 20 hours, the reaction was terminated by adding a water-soluble ammonium chloride solution. The crude product was obtained after extraction with ethyl acetate, drying (sodium sulfate) and evaporation. Purification by flash column chromatography (silica gel) eluting with ethyl acetate-hexane (1: 5) afforded the desired product.

1 (C). Methyl (2 ', 2'-dimethyltetrahydropyran-4'-yl) acetate

Example 1 (B) (17.9 mmol) 10% palladium-on-carbon (10 mol%) was added to the ethyl acetate (35 ml) liquor and then hydrogenated. After standing at room temperature for 3 hours, the catalyst was filtered off and the reaction mixture was then evaporated under vacuum to afford the desired material.

1 (D). Methyl 2- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -3-phenylpropionate

Butyllithium (22.4 mmol) was added to the di-isopropylamine (22.5 mmol) THF (10 ml) liquor at -78 ° C, and the reaction was warmed to 0 ° C for 5 minutes and recooled to -78 ° C. THF (25 ml) solution (16.1 mmol) of Example 1 (C) was then slowly added to the preformed LDA solution. After 1 hour benzyl bromide (17.6 mmol) was added at −78 ° C. and the reaction mixture was allowed to warm to room temperature for 30 minutes. The crude product was obtained by evaporation after addition of aqueous ammonium chloride solution, extraction with ethyl acetate, brine washing and drying (sodium sulfate). Purification by flash column chromatography (silica gel) eluting with ethyl acetate-hexane (1: 3) afforded the desired product.

1 (E). 2- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -3-phenylpropan-1-ol

1.0 M DIBAL (27.0 mmol) was added to a solution of Example 1 (D) (10.9 mmol) in dichloromethane (35 ml) at -78 ° C. The reaction was immediately warmed to room temperature, whereupon the reaction was terminated with 1N HCl. Extraction with ethyl acetate, brine washing and drying (sodium sulfate) were carried out followed by evaporation to afford crude product. Purification by flash column chromatography (silica gel) eluting with diethyl ether-hexane (1: 1) gave the desired product.

1 (F). 2- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -3-phenylpropane-l-ol methanesulfonate

To dichloromethane (40 ml) solution of Example 1 (E) (10.4 mmol) was added di-isopropylethylamine (15.5 mmol) followed by methanesulfonyl chloride (11.6 mmol). After 2 hours the reaction was partitioned between water and ethyl acetate, washed with 1N HCl, brine, dried (sodium sulfate) and evaporated under vacuum to afford the crude product. Purification by flash column chromatography (silica gel) eluting with diethyl ether-hexane (1: 1) gave the desired product.

1 (G). 3- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -4-phenylbutyronnit

Sodium cyanide (10.6 mmol) was added to a solution of Example 1 (F) (9.90 mmol) in DMF (20 ml). The reaction was stirred at 100 ° C. for 6 hours, then partitioned between water and ethyl acetate, washed with brine, dried (sodium sulfate) and evaporated to afford the crude product. Purification by flash column chromatography (silica gel) eluting with diethyl ether-hexane (1: 1) gave the desired product.

1 (H). 3- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -4-phenylbutylamine

Example 1 (G) (8.37 mmol) To a solution of THF (40 mL) was added lithium aluminum hydride (11.85 mmol) at 0 ° C. The reaction was stirred for 16 hours, and after warming to room temperature, the reaction was terminated by successively adding water (0.45 ml), 3N NaOH (0.45 ml), and water (1.3 ml). The reaction was filtered through Celite® and evaporated to give an oil. Purification by flash column chromatography (silica gel) eluting with ethyl acetate: methanol: ammonium hydroxide (90: 10: 1) afforded the desired product.

1 (I). (4-{[3- (2 ', 2'-dimethyltetrahydropyran-4'-yl) -4-phenylbutylamino] -methyl} -phenyl) -dimethylamine dihydrochloride

Example 1 (H) (1.09 mmol) Magnesium sulfate and 4-dimethylamino-benzaldehyde (1.09 mmol) were added to a solution of dichloromethane (5 ml) and the reaction stirred at room temperature for 16 hours. The reaction mixture was filtered to remove magnesium sulphate and evaporated to obtain crude imine product. The resulting product was immediately dissolved in methanol (5 ml) and sodium borohydride (4.20 mmol) was added. The reaction was terminated by the addition of water, evaporated to low volume and extracted with ethyl acetate. After brine washing and drying (sodium sulfate), the crude product was obtained via evaporation. Purification by flash column chromatography (silica gel) eluting with ethyl acetate: methanol: ammonium hydroxide (90: 10: 1) afforded the desired free base. It was dissolved in diethyl ether and HCl (gas) solution was added to diethyl ether to form a precipitate. This was filtered, triturated with acetone and successive re-crystallization of the solid using boiling propan-2-ol to give the desired hydrochloride salt in white solid, the data as follows: Melting point 191-193 ° C .; The calculated value of C ₂₆ H ₃₈ N ₂ 0.2HCl is C 66.80 H 8.62 N 5.99 Cl 15.17%, found C 66.94 H 8.45 N 5.92 Cl 14.95%; HPLC: 9.00 mins (25-75%); MS (e / z): 395 (M + l).

Following the schematic procedure of Example 1, using one of the 1 (A) to 1 (H) intermediates, commercially available substituted acetic acid, substituted acetic esters, or cyclic ketones, examples of compounds of the present invention The following example, which is an exemplary embodiment, can be prepared using a protocol described above by those skilled in the art.

Example 2

5.2 Example 2 : (4-{[3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl]-(4-pyrrolidin-1-yl-benzyl) -amine: HPLC : 12.45 mins (25-75%); MS (e / z): 421 (M + 1).

Example 3

5.3 Example 3 : (4-Chloro-benzyl)-[3- (2,2-dimethyltetrahydropyran-4-yl) -4-phenyl-butyl] -amine: HPLC: 10.37 mins (25-95%) ; MS (e / z): 385 (M + l).

Example 4

5.4 Example 4 : [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl]-(4-methoxy-benzyl) amine: HPLC: 9.49 mins (25-95%) ; MS (e / z): 382 (M + l).

Example 5

5.5 Example 5 : (3,4-Dichloro-benzyl- [3- (2,2-dimethyltetrahydropyran-4-yl) -4-phenyl-butyl] -amine: HPLC: 11.21 mins (25-95% MS (e / z): 420 (M + I).

Example 6

5.6 Example 6 : [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl]-(4-methyl-benzyl) amine: HPLC: 11.79 mins (25-75%); MS (e / z): 366 (M + 1).

Example 7

5.7 Example 7 : [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl]-(4-fluoro-benzyl) amine: HPLC: 11.34 mins (25-75%) ; MS (e / z): 370 (M + l).

Example 8

5.8 Example 8 : (4-{[4- (3,4-Dichloro-phenyl) -3- (2,2-dimethyltetrahydropyran-4-yl) -4-phenyl-butylamino] -methyl}- Phenyl) -dimethyl-amine: HPLC: 10.75 mins (25-75%); MS (e / z): 463 (M + l).

Example 9

5.9 Example 9 : 4-dimethylamino-N- [3- (2,2-dimethyltetrahydropyran-4-yl) -4-phenyl-butyl] -benzamide: HPLC: 14.27 mins (25-75%) ; MS (e / z): 409 (M + 1).

Example 10

5.10 Example 10 N- [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl] -4-fluoro-benzamide: HPLC: 16.80 mins (25-75%) ; MS (e / z): 384 (M + l).

Example 11

5.11 Example 11 N- [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl] -4-methyl-benzamide: HPLC: 17.42 mins (25-75%); MS (e / z): 380 (M + l).

Example 12

5.12 Example 12 : Dimethyl- (4-{[4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butylamino] -methyl} -phenyl) -amine: HPLC: 8.13 mins ( 25-75%); MS (e / z): 367 (M + l).

Example 13

5.13 Example 13 [3- (2,2-Dimethyltetrahydropyran-4-yl) -4-phenyl-butyl]-(4-isopropyl-benzyl-amine: HPLC: 14.32 mins (25-75%) MS (e / z): 394 (M + l).

Example 14

5.14 Example 14 (4-Chloro-benzyl)-[4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butyl] -amine: HPLC: 11.14 mins (25-75%) ; MS (e / z): 358 (M + l).

Example 15

5.15 Example 15 (4-Methoxy-benzyl)-[4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butyl] -amine: HPLC: 10.54 mins (25-75% ); MS (e / z): 354 (M + 1).

Example 16

5.16 Example 16 (4-Methyl-benzyl)-[4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butyl] -amine: HPLC: 11.03 mins (25-75% ); MS (e / z): 338 (M + 1).

Example 17

5.17 Example 17 Benzyl- [4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butyl] -amine: HPLC: 10.45 mins (25-75%); MS (e / z): 324 (M + l).

Example 18

5.18 Example 18 (4-Fluoro-benzyl)-[4-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -butyl] -amine: HPLC: 10.84 mins (25-75% ); MS (e / z): 342 (M + l).

Example 19

5.19 Example 19 {4-[(3-cyclohexyl-4-phenyl-butylamino) -methyl] -phenyl} -dimethyl-amine: HPLC: 11.60 mins (25-75%); MS (e / z): 365 (M + 1).

Example 20

5.20 Example 20 : (3-Cyclohexyl-4-phenyl-butyl)-(4-methoxy-butyl) -amine: HPLC: 13.63 mins (25-75%); MS (e / z): 352 (M + 1).

Example 21

5.21 Example 21 : (3-Cyclohexyl-4-phenyl-butyl)-(4-fluoro-benzyl) -amine: HPLC: 13.65 mins (25-75%); MS (e / z): 340 (M + l).

Example 22

5.22 Example 22 : (4-{[3-cyclohexyl-4- (3,4-dichlorophenyl) -butylamino] -methyl-phenyl) -dimethyl-amine: HPLC: 12.94 mins (25-75%); MS (e / z): 435 (M + l).

Example 23

5.23 Example 23 [3-Cyclohexyl-4- (3,4-dichlorophenyl) -butyl]-(4-fluoro-benzyl) -amine: HPLC: 14.73 mins (25-75%); MS (e / z): 408 (M + l).

Example 24

5.24 Example 24 : Dimethyl- (4-{[3-phenyl-3- (2,2-dimethyltetrahydropyran-4-yl) -propylamino] -methyl} phenyl) -amine: HPLC: 7.90 mins (25 -75%); MS (e / z): 353 (M + l).

5.25 Example 25

In vitro analysis showing that the compound of Example 1 induces G1 arrest in MDA-435 cells

Way

In vitro cell cycle assay was used to show that the compounds of the present invention induce cell-cycle arrest in MDA-435 human breast cancer cells. Tumor cells were synchronized with serum deprivation for 24 hours, followed by compound of Example 1 ((4-{[3- (2,2-dimethyl-tetrahydro-pyran-4-yl)- 4-phenyl-butylamino] -methyl} -phenyl) -dimethyl-amine) (obtained from ChemBridge Corporation; single peak in HPLC: 20-100% gradient: acetonitrile / water / 1% trifluoroacetic acid) or It was released by adding 10% serum to cell culture in the presence of vehicle (dimethylsofloxide). Cells were cultured and stained with 0.04% digitonin working solution with 50 mg / ml propidium iodide. DNA content was analyzed by Flow Cytometry (Coulter). The cell population of each phase is Coulter EPICS? It was measured using Expo 32-ADC software, version 1.1B (2001) for the XL ^TM Cytometer.

result

MDA-435 human breast cancer cells treated with the compound of Example 1 blocked the transition from the G ₀ / G _l phase to the S phase more efficiently than the cells treated with the vehicle (28% of the cells treated with vehicle had metastasis) phase transition blocked while cells treated with the compound of Example 1 blocked 38%. Thus, the compound of Example 1, which is an illustrative example of the compound of the present invention, is significantly useful in stopping the cell cycle and thus treating or preventing human breast cancer.

5.26 Example 26

In vitro assay showing whether the compound of Example 1 induces apoptosis in MDA-435 tumor cells

Way

MDA-435 human breast cancer cells were treated with the compound of Example 1 at vehicle or at various doses. Cells were cultured, stained with Annexin V-FITC and PI, and flow cytometry was performed using ApoAlert Annexin V-FITC apoptosis kit (Clonetech, BD). What percentage of apoptotic cells did EPICS? Measured using XL ^™ and XL-MCL, System II ^™ software, version 1.0 (1996).

result

The compound of Example 1 increases the intensity of Annexin V-FITC staining by a percentage depending on the dose corresponding to 30% of the cell population. Annexin V is a 36kD Ca ²⁺ protein with a strong affinity for phosphatidylserine (PS). In non-apoptic cells, most PS molecules are located in the inner layer of the plasma membrane. During apoptosis, PS relocates to the outer layer of the membrane. Externalization of PS can be detected by fluorochromes bound to Annexin V. This indicates that the compound of Example 1, which is an exemplary example of the compound of the present invention, induces apoptosis and is thus useful for treating or preventing cancer, in particular human breast cancer.

PI is a fluorescent dye that stains DNA. PI cannot cross the plasma membrane in viable cells. In contrast, cells at the late stage of apoptosis lose their integrity and therefore PI is passable. The increase in PI staining in cells treated with the compound of Example 1 is evidence of its ability to induce apoptosis and its usefulness in treating or preventing cancer.

The invention is not to be limited in scope by the specific embodiments disclosed in the embodiments which are intended to illustrate some aspects of the invention, and any embodiments that are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein are intended to be within the scope of the appended claims and be apparent to those skilled in the art.

Many citations have been mentioned, and the entire disclosure of such documents is incorporated herein by reference in its entirety.

Claims (24)

X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5. A method of preventing or treating cancer or neoplastic disease comprising administering to a patient in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: The method of claim 1, wherein the cancer is breast, ovary, testicle, prostate, brain, neck, eye, skin, mouth, throat, esophagus, chest, bone, lung, colon, sigmoid colon, rectum, stomach, kidney, liver. , Cancer of the pancreas, head, intestine, heart or adrenal gland. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5, wherein the method inhibits the growth of cancer cells or neoplastic cells, comprising contacting cancer cells or neoplastic cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5, resulting in an acute or chronic renal failure, inflammatory disease, aging, comprising administering to a patient in need thereof such an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: , Methods of preventing or treating infectious diseases, immune disorders or cardiovascular diseases. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5 to treat a disease responsive to the modulation of ligase activity comprising administering to a patient in need thereof such an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: How to prevent or treat. The method of claim 5, wherein the ligase activity is inhibited. The method of claim 5, wherein the ligase activity is activated. 6. The method of claim 5, wherein said ligase is an E3 ubiquitin-protein ligase. The method of claim 8, wherein the ligase activity is inhibited. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5 in response to the regulation of intracellular p27 / Kip1 levels comprising administering to a patient in need thereof such an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: How to prevent or treat a disease. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5, wherein the method comprises regulating cell growth comprising administering to a patient in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. The method of claim 11, wherein said cell growth is inhibited. The method of claim 11, wherein said cell is a non-tumor cell. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5 an effective amount of a method of preserving a cell, blood, tissue, organ or organism comprising contacting blood, tissue or organ with a compound of formula (I) or a pharmaceutically acceptable salt thereof . The method of claim 14, wherein said cell, blood, tissue or organ is cryopreserved. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5, a method for preventing or treating side effects of chemotherapy or radiation therapy comprising administering to a patient in need thereof such an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: . The method of claim 16, wherein the side effect is alopecia. The method of claim 16, wherein the side effect is low blood count. The method of claim 16, wherein the side effect is nausea. The method of claim 16, wherein the side effect is diarrhea. The method of claim 16, wherein the side effect is an oral lesion. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5 to control or control the differentiation or growth of mammalian stem cells comprising administering to a patient in need thereof such an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: How to. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; And r is 0-5, wherein the pharmaceutical composition comprises an effective amount of a compound of formula (I): or a pharmaceutically acceptable salt thereof. X, W and U are each independently H, halogen, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, Haloalkyl, acyloxy, thioalkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle , Substituted or unsubstituted cycloalkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , —NR ₁ C (═O) (CH ₂ ) qR ₂ , NR ₁ C (═O) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; X 'is H, hydroxy, carboxy, alkoxy, alkylamino, branched or unbranched C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, haloalkyl, acyloxy, thio Alkyl, sulfonyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, substituted or unsubstituted cyclo Alkyl, -C (= 0) OR ₁ , -OC (= 0) R ₁ , -C (= 0) NR ₁ R ₂ , -C (= 0) NR ₁ OR ₂ , -SO ₂ NR ₁ R ₂ , -NR ₁ S0 ₂ R ₂ , -CN, -NO ₂ , -NR ₁ R ₂ , -NR ₁ C (= 0) R ₂ , -NR ₁ C (= 0) (CH ₂ ) qOR ₂ , -NR ₁ C (= 0) (CH ₂ ) qR ₂ , NR ₁ C (= 0) (CH ₂ ) qNR ₁ R ₂ , —O (CH ₂ ) qNR ₁ R ₂ ; R ₁ and R ₂ are independently hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl; Each Y independently is hydrogen or branched or unbranched C ₁ -C ₁₀ alkyl or when o is 1 Y can be (= O); Z is C or O; Q is H, branched or unbranched C ₁ -C ₁₀ alkyl; m is 0-5; n is 0-8; o is 0-2; p is 0-2; q is 0-5; r is 0-5; a disease associated with the regulation of ligation comprising an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein one of X, X ', U or W is not H: Pharmaceutical compositions suitable for the treatment of.