Novel class of cytodifferentiating agents and histone deacetylase inhibitors, and methods of use the

专利摘要:
The present invention provides a compound of formula (I) The present invention also provides a method of inhibiting the proliferation of such cells by selectively inducing growth arrest, terminal differentiation and / or apoptosis of tumor cells. The invention also provides a method of treating a patient with a tumor characterized by the proliferation of tumor cells. Finally, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable amount of the compound described above: Where R 1 and R 2 are each substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy or pyridine group; A is an amido residue, -O-, -S-, -NH- or -CH 2- ; n is an integer of 3-8.
公开号:KR20020059393A
申请号:KR1020027003114
申请日:2000-08-24
公开日:2002-07-12
发明作者:빅토리아 엠. 리치온；폴 에이. 마크스；리차드 에이. 리프킨드；로날드 브레슬로；산드로 벨버디어；레란드 거쉘；토마스 에이. 밀러
申请人:제임스 에스. 쿼크；슬로안-케테링인스티튜트퍼캔서리서치；추후제출；더 트러스티스 오브 컬럼비아 유니버시티 인 더 시티 오브 뉴욕；
IPC主号:

专利说明:

A new class of cell differentiators and histone deacetylases, and methods of using the same {NOVEL CLASS OF CYTODIFFERENTIATING AGENTS AND HISTONE DEACETYLASE INHIBITORS, AND METHODS OF USE THEREOF}
[1] Throughout the specification of the present invention, various references are referred to in Arabic numerals in parentheses. Full citations for these documents are given at the end of the specification immediately preceding the claims. The entire contents of these documents are incorporated herein by reference in order to more fully describe the technology to which the present invention belongs.
[2] Cancer is a disorder in which a cell's individual becomes unresponsive at varying degrees to regulatory mechanisms that normally regulate proliferation and differentiation. A recent approach to cancer treatment has been to study the induction of terminal differentiation of tumor cells (1). In cell culture models, differentiation has been reported by exposure to various stimuli including cyclic AMP and retinoic acid (2,3), aclarubicin and other anthracyclines (4).
[3] There is much evidence that tumor transformation does not necessarily destroy the potential for the differentiation of cancer cells (1, 5, 6). There are many examples of tumor cells that do not respond to normal proliferative regulators and are believed to block upon expression of their differentiation progression and are further induced to differentiate and stop replication. Some relatively simple polar compounds (5, 7-9), derivatives of vitamin D and retinoic acid (10-12), steroid hormones (13), growth factors (6, 14), proteases (15, 16), tumor promoters ( 17, 18), and various agents, including inhibitors of DNA or RNA synthesis (4, 19-24), can induce a variety of transformed cell lines and primary human tumor explants that exhibit more differentiated characteristics.
[4] Initial studies conducted by some of the inventors have identified a series of polar compounds that are effective inducers of multiple transformed cell lines (8, 9). One such effective inducer is a mixed polar / nonpolar compound N, N'-hexamethylene bisacetamide (HMBA) (9), and another inducer is subveroylanilide hydroxyamic acid (SAHA) (39, 50). Was. The use of these compounds to induce murine red leukemia (MEL) cells in which erythrocytes are differentiated by carcinogenic inhibition has proven to be a useful model for studying inducer mediated differentiation of transformed cells (5, 7-9). ).
[5] HMBA-induced terminal erythrocyte differentiation in MEL cells occurs in a multistep process. When HMBA is added to MEL cells (745A-DS19) in culture, it undergoes a 10-12 hour incubation period before a commitment to terminal differentiation is observed. Commitments are defined as the ability of cells to exhibit terminal differentiation despite removal of inducers (25). Upon continuous exposure to HMBA, the differentiated cells gradually recover. We have reported that MEL cell lines that are resistant to relatively low levels of vincristine may be significantly sensitive to the inducing action of HMBA and may be induced differentiation with little or no latency (26).
[6] HMBA can induce phenotypic changes consistent with differentiation in a wide range of cell lines (5). Characterization of drug-induced effects has been studied most extensively in murine erythroleukemic cell systems (5, 25, 27, 28). MEL cell induction of differentiation depends on both time and concentration. The minimum concentration required to demonstrate in vitro effects in most strains is 2 to 3 mM and is a sequential normally required to induce differentiation in the substantial part (> 20%) of the subject without sustained exposure to the drug. The minimum duration of exposure is about 36 hours.
[7] Protein kinase C has been shown to be involved in the pathway of inducer mediated differentiation (29). In vitro studies have provided a basis for evaluating the efficacy of HMBA as a cell differentiator in treating human cancers (30). Several Phase I clinical studies by HMBA have been completed (31-36). Clinical studies have shown that these compounds can induce therapeutic responses in patients with cancer (35, 36). However, these Phase I clinical studies are limited in part by the potential efficacy of HMBA by dose related toxicity that prevents optimal blood levels from being achieved and by intravenous administration of large amounts of the agent over a long period of time. As such, some inventors have turned to synthesizing compounds that are more potent and possibly less toxic than HMBA (37).
[8] Recently, a class of compounds that induce differentiation has been found to inhibit histone deacetylases. Several antitumor compounds obtained experimentally, such as trichostatin A (TSA), trapoxine, subveroylanilide hydroxyamic acid (SAHA) and phenylbutyrate, may at least partially inhibit histone deacetylases. It has been shown to work (38, 39, 42). In addition, diallyl sulfide and related molecules (43), oxamplatin (44), MS-27-275, synthetic benzamide derivatives (45) butyrate derivatives (46), FR901228 (47), depudecine (48) and m-carboxycinnamic acid bishydroxyxamide (39) has been found to inhibit hydrotone deacetylase. In vitro, these compounds can inhibit the growth of fibroblasts by inducing cell cycle arrest in the G1 and G2 phases, leading to the loss of the possibility of transformation and the terminal differentiation of various transformed cell lines. In vitro, phenylbutyrate is effective for the treatment of acute promyelocytic leukemia associated with retinoic acid (53). SAHA is effective in inhibiting the formation of breast cancer in rats and lung cancer in mice (54, 55).
[9] Some of the inventors of the inventors of U.S. Patent 5,369,108 (41) discloses compounds useful for selectively inducing terminal differentiation of tumor cells, having two polar end groups separated by methylene groups, one or both of which are large hydrophobic groups. Phosphorus compounds are disclosed. This patent states that such compounds are more active than HMBA and HMBA related compounds.
[10] However, U.S. Patent 5,369,108 does not describe that another large hydrophobic group at the same terminus of the molecule as the first hydrophobic group will further reduce the differentiation activity by about 100-fold in the enzyme assay and about 50-fold in the cell differentiation assay.
[11] This new class of compounds of the present invention may be useful for helping to treat tumors in patients by selectively inducing terminal differentiation of tumor cells.
[12] Summary of the Invention
[13] The present invention provides a compound having the formula: or a pharmaceutically acceptable salt thereof:
[14]
[15] Wherein R ₁ and R ₂ are the same or different and each is a hydrophobic moiety; Wherein R ₃ is a hydroxylsamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group; n is an integer of 3-4.
[16] The present invention also provides a compound having the formula: or a pharmaceutically acceptable salt thereof:
[17]
[18] Wherein R ₁ and R ₂ are substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branch Alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, or pyrimidine groups; R ₃ is a hydroxylsamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group; R ₄ is hydrogen, halogen, phenyl or cycloalkyl moiety; A can be the same or different and represents an amide residue, -O-, -S-, -NR ₅ -or -CH _2- ; R ₅ is substituted or unsubstituted C ₁ -C ₅ alkyl; n is an integer of 3 to 10.
[19] The present invention also provides a method of inhibiting proliferation of these cells by selectively inducing terminal differentiation of tumor cells, including contacting tumor cells with an effective amount of the compound under appropriate conditions.
[20] Brief description of the drawings
[21] 1 is a diagram showing the effect of compound 1 according to the present invention on MEL cell differentiation.
[22] 2 is a diagram showing the effect of compound 1 according to the present invention on histone deacetylase 1 activity.
[23] 3 is a diagram showing the effect of compound 2 according to the present invention on MEL cell differentiation.
[24] 4 is a diagram showing the effect of compound 3 according to the present invention on MEL cell differentiation.
[25] 5 is a diagram showing the effect of compound 3 according to the present invention on histone deacetylase 1 activity.
[26] 6 is a diagram showing the effect of compound 4 according to the present invention on MEL cell differentiation.
[27] 7 is a diagram showing the effect of compound 4 according to the present invention on histone deacetylase 1 activity.
[28] 8 shows a photoaffinity label (3H-498) directly bound to HDAC 1.
[29] Figure 9 shows that SAHA induces the accumulation of acetylated histones H3 and H4 in CWR22 tumor xenografts of mice.
[30] Figure 10 shows that SAHA causes the accumulation of acetylated histones H3 and H4 in peripheral blood mononuclear cells of a patient. SAHA was administered three times daily by IV infusion. It is a figure which shows the sample isolate | separated before injection, after injection, and 2 hours after injection.
[31] 11A-11F show the effect of selected compounds on human epitope-tagged (Flag) HDACI purified by affinity.
[32] Detailed description of the invention
[33] The present invention provides a compound having the formula: or a pharmaceutically acceptable salt thereof:
[34]
[35] Wherein R ₁ and R ₂ are the same or different and each is a hydrophobic moiety, wherein R ₃ is hydroxylsamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group and n is 3 to 10 Is an integer.
[36] In the aforementioned compounds, R ₁ and R ₂ are each directly or by a linker and are substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purine -6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[37] If a linker is used, the linker is an amide residue, -O-, -S-, -NH- or -CH-.
[38] According to the invention, n is 3 to 10, preferably 3 to 8, more preferably 3 to 7, even more preferably 4, 5, 6, most preferably 5.
[39] In another embodiment of the invention, the compound has the formula:
[40]
[41] In the above formula, R ₄ is each substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branch Alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups. R ₂ is -amide-R ₅ , wherein R ₅ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group , Hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[42] In another embodiment of the invention, the compound has the formula or a pharmaceutically acceptable salt thereof:
[43]
[44] Wherein R ₁ and R ₂ are each substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, Branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups; R ₃ is a hydroxylsamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group; R ₄ is hydrogen, halogen, phenyl or cycloalkyl moiety; A is the same or different and represents an amide moiety, —O—, —S—, —NR ₅ — or —CH ₂ —, wherein R ₅ is substituted or unsubstituted C ₁ -C ₅ alkyl; n is an integer of 3 to 10.
[45] In another embodiment, the compound has the formula
[46]
[47] In another embodiment, the compound has the formula
[48]
[49] In yet another embodiment, the compound has the formula
[50]
[51] Wherein R ₁ and R ₂ are substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, t-butyl, aryloxy, arylalkyloxy or pyrimidine groups ; n is an integer of 3-8.
[52] Aryl or cycloalkyl groups are methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro, 2,4- Difluoro, 2,5-difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro, 2,3, 6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5,6-pentafluoro Furnace, azido, hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenyloxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methoxyoxycarbonyl, methylaminocarbonyl, dimethylamino, It may be substituted with a dimethylaminocarbonyl or hydroxylaminocarbonyl group.
[53] In further embodiments, the compounds have the formula below or enantiomers thereof:
[54]
[55] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[56]
[57] In another embodiment, the compounds have the formula below or enantiomers thereof:
[58]
[59] In yet further embodiments, the compounds have the formula: or enantiomers thereof:
[60]
[61] In another embodiment, the compounds have the formula below or enantiomers thereof:
[62]
[63] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[64]
[65] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[66]
[67] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[68]
[69] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[70]
[71] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[72]
[73] In yet another embodiment, the compounds have the formula: or enantiomers thereof:
[74]
[75] In addition, the present invention will include enantiomers and salts of the compounds listed above.
[76] In another embodiment, the compound has the formula below or a pharmaceutically acceptable salt thereof:
[77]
[78] In the above formulae, R ₁ and R ₂ are the same or different and each is a hydrophobic moiety, R ₅ is —C (O) —NHOH (hydroxysamic acid), —C (O) —CF ₃ (trifluoroacetyl), -NH-P (O) OH-CH ₃ , -SO ₂ NH ₂ (sulfonamide), -SH (thiol), -C (O) -R ₆ , wherein R ₆ is hydroxyl, amino, alkylamino Or an alkyloxy group; n is an integer of 3 to 10.
[79] In the foregoing compound, R_OneAnd R₂Is Aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, each directly or attached by a linker, substituted or unsubstituted , Branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[80] The linker is an amide residue, -O-, -S-, -NH- or -CH _2- .
[81] In another embodiment, the compound has the formula
[82]
[83] Wherein each of R ₇ is substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or Unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[84] In the foregoing compounds, R ₂ is -sulfonamide-R ₆ or -amide-R ₈ , wherein R ₈ is substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[85] R ₂ is —NH—C (O) —Y, —NH—SO ₂ —Y, wherein Y is selected from the group of compounds:
[86]
[87] R ₇ may be selected from the group consisting of:
[88]
[89] In another embodiment, the compound has the formula below or a pharmaceutically acceptable salt thereof:
[90]
[91] Wherein R ₁ and R ₂ are the same or different and each is a hydrophobic moiety, and R ₅ is —C (O) —NHOH (hydroxysamic acid), —C (O) —CF ₃ (trifluoroacetyl), —NH -P (O) OH-CH ₃ , -SO ₂ NH ₂ (sulfonamide), -SH (thiol), -C (O) -R ₆ , wherein R ₆ is hydroxyl, amino, alkylamino or alkyloxy Group; L is a linker consisting of-(CH ₂ )-, -C (O)-, -S-, -O-,-(CH = CH)-, -phenyl- or -cycloalkyl- or any combination thereof .
[92] And L is-(CH ₂ ) _n- , -C (O)-, -S-, -O-,-(CH = CH) _m- , -phenyl- or -cycloalkyl- or any combination thereof Wherein n is an integer of 3 to 10 and m is an integer of 0 to 10.
[93] In the foregoing compounds, n is 4-7 and m is 0-7. Preferably n is 5 or 6, most preferably n is 6. Preferably m is 1 to 6, even more preferably m is 2 to 5, and most preferably m is 3 or 4.
[94] In the formula, R_OneAnd R₂ Each attached directly or by a linker, substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxide Hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[95] The linker is an amide residue, -O-, -S-, -NH- or -CH _2- .
[96] The invention will also include enantiomers, salts and prodrugs of the compounds disclosed herein.
[97] In another embodiment, the compound has the formula
[98]
[99] In the above formula, L is a linker selected from the group consisting of-(CH ₂ )-,-(CH = CH)-, -phenyl-, -cycloalkyl- or any combination thereof; Each of R ₇ and R ₈ is independently substituted or unsubstituted, aryl, cycloalkyl, cycloalkylamino, naphtha, pyrimidineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl group , Branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyrimidine groups.
[100] In a preferred embodiment, the linker L comprises the following residues:
[101]
[102] In another preferred embodiment, the compound has the formula:
[103]
[104] Any disclosed compound may form a pharmaceutical composition with a pharmaceutically acceptable carrier.
[105] In addition, any compound may be formed into a pharmaceutically acceptable salt of the compound using well known pharmacological techniques.
[106] In addition, prodrugs of any compounds can be prepared using known pharmaceutical techniques.
[107] Any compound can be used in a method of causing differentiation of tumor cells in a tumor, including contacting the cells with an effective amount of the compound thereby thereby differentiating the tumor cells.
[108] In addition, any compound may be used in a method of inhibiting the activity of histone deacetylase, including contacting histone deacetylase to an effective amount of the compound thereby inhibiting the activity of the histone deacetylase.
[109] In addition to the compounds listed above, the present invention will also include the use of homologs and analogs of these compounds. In this regard, homologues are molecules having substantial structural similarity to the compounds described above and analogues are molecules having substantial biological similarity regardless of structural similarity.
[110] In a further embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of any of the foregoing compounds and a pharmaceutically acceptable carrier.
[111] In a still further embodiment, the present invention provides for selective growth arrest, terminal differentiation and / or apoposis of tumorous cells, including contacting the cells with an effective amount of any one of the foregoing compounds at appropriate conditions. And thereby inhibit the proliferation of these cells.
[112] The contact should be continued for an extended period of time, for example, for at least 48 hours, preferably about 4-5 days or longer.
[113] The method may be carried out in vivo or ex vivo. If the method is carried out ex vivo, contact is effective for culturing the cells with the compound. The concentration of the compound in contact with the cell should be from about 1 nM to about 25 mM, preferably from about 20 nM to about 25 mM, even more preferably from about 40 nM to about 100 μΜ, even more preferably from about 40 nM to about 200 nM. The concentration depends on the individual compound and the condition of the tumorous cell.
[114] In addition, the method can firstly treat the cells with an anti-tumor agent to give them a resistance to the anti-tumor agent and then to effect any of the effective amounts of any effective amount to selectively cause terminal differentiation of these cells under appropriate conditions. Contacting the compound.
[115] The invention also includes administering to a patient an effective amount of any of the above compounds effective to selectively induce growth arrest, terminal differentiation and / or epoptosis of tumorous cells, thereby inhibiting their proliferation, A method of treating a patient having a tumor characterized by the proliferation of tumorous cells is provided.
[116] The method of the invention is for the treatment of a human having a tumor. However, this would also be an effective way to treat tumors of other mammals. The term tumor will include any cancer caused by the proliferation of tumorous cells such as prostate cancer, lung cancer, acute leukemia, multiple myeloma, bladder carcinoma, kidney cancer, breast cancer, rectal cancer, neuroblastoma or melanoma.
[117] Routes of administration of the compounds of the invention include, for example, oral administration, pulmonary administration, parenteral administration (intramuscular injection, intraperitoneal injection, intravenous injection (IV), subcutaneous injection), (fine powder preparation, micromixture). Prepared in a form suitable for each route of administration, including conventionally known and physiologically acceptable routes, such as administration by inhalation, percutaneous administration, nasal administration, hard administration, rectal administration or sublingual medication Can be.
[118] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier such as sterile pyrogen-free distilled water and a therapeutically acceptable amount of any of the above compounds. Preferably, the effective amount is an amount that is less than or equal to that which causes toxicity to the patient and which is effective to selectively induce terminal differentiation of the appropriate neoplastic cells.
[119] The present invention provides the pharmaceutical composition in admixture with an antitumor agent, hormone, steroid or retinoid.
[120] Antitumor agents include alkylating agents, anti-metabolites, hormonal agents, antibiotics, colchicin, vinca alkaloids, L-asparagine, procarbazine, hydroxyurea, mitotan, nitrosourea, already One of various chemotherapeutic agents such as dozol carboxamide. Suitable agents are those that promote depolarization of tubulin. Preferably, the antitumor agent is colchicine or vinca alkaloids, particularly preferably vinblastine and vincristine. In embodiments wherein the anti-tumor agent is vincristine, the cells are administered at a concentration of about 5 mg / ml in order to make the cells resistant to vincristine. The formulation is basically administered as described above for the administration of any compound. Preferably, the formulation is administered for at least 3 to 5 days. Any of the above compounds are administered as described above.
[121] The pharmaceutical composition may be administered as an infusion of 2 to 6 hours daily for 3 to 21 days, such as 4 hours of infusion for 5 days.
[122] The invention will be better understood through the following examples. However, one of ordinary skill in the art will readily recognize that the particular methods and results discussed are merely examples of the invention as described more fully in the claims below.
[123] Examples 1-5 show the synthesis of substituted L-α-aminosuberic hydroxamic acid according to the present invention and Examples 6 and 7 show the effects of compounds 1-5 on MEL cell differentiation and histone deacetylase activity. Indicates.
[124] Example 1-Synthesis of Compound 1
[125] N-Boc-ω-methyl- (L) -α-aminosuberate, Boc-Asu (OMe), was prepared according to the published method (40). ("Boc" = t-butoxycarbonyl; "Asu" = α-aminosuberate (or α-aminosuberic acid)
[126] N-Cbz-ω-t-butyl- (L) -α-aminosuberate, dicyclohexylamine salt was obtained from Research Plus, Bayonne, NJ.
[127] N-Boc-ω-methyl- (L) -α-aminosuberateanilide, Boc-Asu (OMe) -NHPh
[128]
[129] N-Boc-ω-methyl- (L) -α-aminosuberate (493 mg, 1.63 mmol) was dissolved in 7 mL of anhydrous CH ₂ Cl ₂ under argon. EDC (470 mg, 2.45 mmol) was added and aniline (230 μl, 2.52 mmol) was added. The solution was stirred at rt for 2 h 30 min and washed with dilute HCl (pH 2.4, 2 × 5 mL), saturated NaHCO ₃ (10 mL) and H ₂ O (2 × 10 mL). The product was purified by column chromatography (silica gel, hexanes: AcOEt 3.5: 1). The yield obtained was 366 mg (60%).
[130] ¹ H-NMR and mass spectrometry were consistent with the product.
[131] N-benzoyl-ω-methyl- (L) -α-aminosuberateanilide, PhCOHN-Asu (OMe) -NHPh.
[132]
[133] 90 mg of N-Bloc-ω-methyl- (L) -α-aminosuberateanilide (0.238 mmol) was treated with 3.2 ml of 25% trifluoroacetic acid (TFA) CH ₂ Cl ₂ for 30 minutes. The solvent was removed and the remaining residue was left under high vacuum for 12 hours. The residue was purified with 3 mL of anhydrous CH ₂ Cl ₂ and benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorourophosphate (PyBOP) (149 mg, 0.286 mmol), benzic acid (22 mg , 0.357 mmol) and diisopropylethylamine (114 μl, 0.655 mmol) were dissolved under argon. The solution was stirred for 1 hour at room temperature. The product was purified by column chromatography (silica gel, hexanes: AcOEt 3: 1 to 2: 1) to give a white solid (75 mg, 82%).
[134] ¹ H-NMR and mass spectrometry were consistent with the product.
[135] The above coupling reaction was successfully carried out even when 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was used as a reagent.
[136] N-benzoyl- (L) -α-aminosuberoylanilide, PhCONH-Asu (OH) -NHPh
[137]
[138] 75 mg (0.196 mmol) of N-benzoyl-aminosuberateanilide was stirred for 6 h at 0 ° C. in 1 M NaOH: THF: MeOH (1: 1: 1). After complete disappearance of the starting material the solution was neutralized (1M HCl) and extracted with AcOEt. The organic phase was collected and dried. Removal of solvent gave the product (67 mg, 93%) as a white solid.
[139] ¹ H-NMR and mass spectrometry were consistent with the product.
[140] N-benzoyl- (L) -α-aminosuberoylanilide-ω-hydroxysamic acid, PhCONH-Asu (NHOH) -NHPh:
[141]
[142] In a suspension of 26 mg of N-benzoyl-ω-methyl- (L) -α-aminosuberateanilide (I2) in 1 mL of anhydrous CH ₂ Cl ₂ H ₂ NOTBDPS (H ₂ NO-t-butyldiphenylsilyl) 58 Mg was added and EDC 22 mg was added. The reaction is stirred at room temperature for 4 hours. The intermediate protected by hydroxamic acid was purified by column chromatography (silica gel, CH ₂ Cl ₂ : MeOH 100: 0 to 98: 2). Deprotection was done with 5% TFA in CH ₂ Cl ₂ for 1 h 30 min. The product was precipitated in acetone-pentane.
[143]
[144] ESI-MS: 384 (M + 1), 406 (M + Na), 422 (M + K)
[145] Example 2 Synthesis of Compound 2
[146] N-Nicotinoyl- (L) -α-aminosuberoylanilide-ω-hydroxysamic acid, C ₅ H ₄ NCO-Asu (NHOH) -NHPh:
[147]
[148] Prepared from N-Boc-ω-methyl-L-α-aminosuberate according to the method used for the benzoyl analog. Yields and chromatographic patterns were similar.
[149]
[150] Example 3- Synthesis of Compound (3)
[151] N-benzyloxycarbonyl-ω-t-butyl- (L) -aminosuberic acid,
[152] N-Cbz- (L) -Asu (OtBu) -OH.
[153]
[154] Partitioned between 1M HCl (5 mL) and EtOAc (10 mL) using N-Cbz- (L) -Asu (OtBu) -OH, dicyclohexylamine salt (100 mg, 0.178 mmol). The organic layer was removed, the aqueous portion was washed with EtOAc (3 × 3 mL) and the organic portions combined, washed with brine (1 × 2 mL) and dried (MgSO ₄ ). The mixture was filtered and concentrated to give a colorless film (67 mg, 0.176 mmol, 99%). This compound was used directly in the next step.
[155] N-benzyloxycarbonyl-ω-t-butyl- (L) -α-aminosulfateanilide,
[156] N-Cbz- (L) -Asu (OtBu) -NHPh.
[157]
[158] N-Cbz- (L) -Asu (OtBu) -OH (67 mg, 0.176 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (2.5 mL). Aniline (17 μl, 0.187 mmol), PyBOP (97 mg, 0.187 mmol) and iPr ₂ NEt (46 μl, 0.266 mmol) were added and the mixture was stirred for 2 hours. The reaction was terminated as indicated by TLC. The mixture was diluted with EtOAc (5 mL) and water (5 mL) and the layers separated. The aqueous portion was washed with EtOAc (3 × 3 mL) and the organic portions were combined. The solution was washed with 1M HCl (1 × 2 mL) and brine (1 × 2 mL), dried (MgSO ₄ ), and then concentrated by filtration to give a crude oil. This was passed through a plug of silica gel (30% EtOAc / hexanes) to remove base impurities, yielding compound (76 mg, 0.167 mmol, 94%).
[159]
[160] N-benzyloxycarbonyl- (L) -α-aminosulfateanilide,
[161] N-Cbz- (L) -Asu (OH) -NHPh.
[162]
[163] N-Cbz- (L) -Asu (OtBu) -anilide (76 mg, 0.167 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (5 mL) and TFA (0.5 mL) was added dropwise. After 3 hours the reaction was terminated using TLC. The mixture was concentrated in vacuo to afford the title compound (80 mg, crude). This compound was used in the next step without purification.
[164]
[165] N-benzyloxycarbonyl- (L) -α-aminosulfateanilide ω-hydroxyamic acid, N-Cbz- (L) -Asu (NH-OH) -NHPh.
[166]
[167] N-Cbz- (L) -Asu (OH) -anilide (80 mg, crude) and Ot-butyldiphenylsilyl-hydroxyamine (60 mg, 0.221 mmol) were dissolved in CH ₂ Cl ₂ (4 mL). To this, PyBOP (125 mg, 0.241 mmol) and iPr ₂ NEt (52 µl, 0.302 mmol) were added and stirred overnight. The reaction was terminated using TLC and the mixture was concentrated in vacuo and then passed through a plug of silica gel (50% EtOAc / hexanes) to remove baseline impurities. After evaporating the volatiles to yield 107 mg of material, it was dissolved in anhydrous CH ₂ Cl ₂ (5 mL) and TFA (0.25 mL) was added. Monitored using TLC after 1.5 hours to terminate the reaction. Concentration drying removed all volatiles. The residue was dissolved in EtOAc (3 mL) and then hexane was added slowly to precipitate a white gelled material. The supernatant was removed and the precipitate was washed with hexane (3 × 2 mL). The material was dried under reduced pressure to give the title compound (40 mg, 0.097 mmol, 59%).
[168]
[169] Example 4- Synthesis of Compound (4)
[170] N-benzyloxycarbonyl- (L) -α-aminocuberoyl-8-quinolinamide-ω-hydroxyamic acid.
[171]
[172] Compound (3) was prepared using a method similar to the above.
[173]
[174] Example 5- Synthesis of Compound (5)
[175] N-benzoyl- (L) -α-aminosulberoyl-β-quinolinamide-ω-hydroxyamic acid:
[176]
[177] A sample of N-Cbz-ω-t-butyl L-α-aminosulberoyl-β-quinolineamide (90 mg, 0.178 mmol) was obtained from this synthesis. This Cbz group was removed by hydrogenation in MeOH with 5% Pd on carbon. The resulting free amine was coupled to benzoic acid using EDC (69% via two steps) in anhydrous CH ₂ Cl ₂ . The t-butyl ester was deprotected using TFA and then conventionally coupled using H ₂ NOTBDPS to afford the desired hydroxyamic acid.
[178]
[179] Example 6 Synthesis of Compounds Using Converted Amide Groups
[180] Chemical formula Or a compound of formula wherein R is removed by reaction with an amine and a carbodiimide reagent In order to form a compound of the formula wherein R 'is removed in the same manner as in the above examples and converted to hydroxylamine acid (NHOH), Compound represented by the formula The malonic acid ester of is treated with a base and then By the addition of a compound of which X is halogen.
[181] In the above scheme, R may be t-butyl, which is removed using trifluoroacetic acid; R 'may be methyl, which is removed using a base or LiI; Each R ″ may be the same or different depending on the reagent used.
[182] Example 7 Compound 1 for MEL Cell Differentiation and Histone Deacetylase Activity Effect on NHPh)
[183] Murine Erythroleuemia (MEL) Cell Differentiation
[184] To assess the ability of compound (1) to induce terminal differentiation, MEL cell differentiation assays were used. MEL cells (divided logarithmic) were incubated at the indicated concentrations of compound (1). After incubation for 5 days, cell growth was measured using a Coulter counter, and differentiation was measured under a microscope using a benzidine assay to determine the accumulation of hemoglobin protein per cell basis.
[185] As shown in Figure 1, it was confirmed that Compound (1) (200nM) can induce MEL cell differentiation.
[186] Histone polyacetylase (HDAC) enzyme activity
[187] Effect of compound (1) on the affinity of purified human epitope labeled (flag) HDAC1 by incubating the prepared enzyme on ice for 20 minutes in the absence of substrate, using indicated amounts of compound (1) Was analyzed. Substrate ([ ³ H] acetyl-labeled murine erythroleuemia cell-derived histone) was added and these samples were incubated at 37 ° C. for 20 minutes with a total volume of 30 μl. Then, the reaction was stopped, the separated acetate was extracted, and the amount of separated radioactivity was measured by scintillation counting.
[188] As shown in Figure 2, it was confirmed that Compound (1) is a potential inhibitor of HDAC1 enzyme activity (ID ₅₀ = 1 nM).
[189] Example 8 MEL Cells of Compound (2) (N-Nicotinoyl- (L) -α-aminosulferoylanilide-ω-hydroxyamic acid, C ₅ H ₄ NCO-Asu (NHOH) -NHPh) Effect on Differentiation
[190] Murine Erythroleuemia (MEL) Cell Differentiation:
[191] To assess the ability of compound (2) to induce terminal differentiation, MEL cell differentiation assays were used. MEL cells (divided logarithmic) were incubated at the indicated concentrations of compound (2). After incubation for 5 days, cell growth was measured using a Coulter counter, and differentiation was measured under a microscope using a benzidine assay to determine the accumulation of hemoglobin protein per cell basis.
[192] As shown in Figure 3, it was confirmed that Compound (2) (800nM) can induce MEL cell differentiation.
[193] Example 9-MEL Cells of Compound (3) Effect on Differentiation and Histone Deacetylase Activity
[194] Murine Erythroleuemia (MEL) Cell Differentiation:
[195] To assess the ability of compound (3) to induce terminal differentiation, MEL cell differentiation assays were used. MEL cells (divided logarithmic) were incubated at the indicated concentrations of compound (3). After incubation for 5 days, cell growth was measured using a coulter counter, and differentiation was measured under a microscope using a benzidine assay to determine the accumulation of hemoglobin protein per cell basis.
[196] As shown in FIG. 4, it was observed that Compound 3 (400 nM) can induce MEL cell differentiation.
[197] Histone deacetylase (HDAC) enzyme activity:
[198] The effect of Compound 3 on affinity purified human epitope labeled HDAC1 was assayed by incubating the enzyme preparation in the absence of substrate on ice for 20 minutes with the indicated amount of HPC. Substrate ([ ³ H] acetyl-labeled murine erythroleukemic cell induced histones) was added and samples were incubated at 37 ° C. for 20 minutes with a total volume of 30 μl. The reaction was then stopped, the released acetate was extracted and the amount of radiation emitted was measured by scintillation counting.
[199] As shown in FIG. 5, compound 3 was observed to be an potent inhibitor of HDACE1 enzyme activity (ID _50-100 nM).
[200] Example 10 Effect of Compound 4 (N-benzyloxycarbonyl- (L) -α-aminoxumberoyl-80quinolinamide-ω-hydroxysamic acid) on MEL Cell Differentiation and Histone Deacetylase Activity
[201] Murine Red Leukemia (MEL) Cell Differentiation:
[202] MEL cell differentiation assays were used to assess the ability of Compound 4 to induce terminal differentiation. MEL cells (log split) were incubated at the indicated concentrations of compound 4. After 5 days, the period differentiation of the cultures was determined by microscopy using a benzidine assay to measure hemoglobin protein accumulation on each cell substrate.
[203] As shown in Figure 6, it was observed that Compound 4 (40 nM) can induce MEL cell differentiation.
[204] Histone deacetylase (HDAC) enzyme activity:
[205] The effect of Compound 4 on affinity purified human epitope labeled HDAC1 was assayed by incubating the enzyme preparation in the absence of substrate on ice for 20 minutes with the indicated amount of HPC. Substrate ([ ³ H] acetyl-labeled murine erythroleukemic cell induced histones) was added and samples were incubated at 37 ° C. for 20 minutes with a total volume of 30 μl. The reaction was then stopped, the released acetate was extracted and the amount of radiation emitted was measured by scintillation counting.
[206] As shown in FIG. 7, it was observed that Compound 4 was an potent inhibitor of HDAC1 enzyme activity (ID ₅₀ <10 nM).
[207] SAHA inhibits the activity of affinity purified HDAC1 and HDAC3 39. Crystallographic studies with SAHA and HDAC related proteins have shown that SAHA inhibits HDAC by direct interaction with catalyst site 66. Further studies have demonstrated that tritium-labeled photoaffinity SAHA analogs ( ³ H-498) (67) containing azide moieties directly bind to HDAC1 (FIG. 8). These results suggest that the above-mentioned hydroxamic acid based compounds inhibit HDAC activity through direct interaction with HDAC proteins.
[208] SAHA results in the accumulation of acetylated histones H3 and H4 in vivo. The in vivo effects of these SAHAs were studied using CWR22 human prostate xenografts 68 in mice. SAHA (50 mg / kg / day) resulted in an average 97% reduction in final tumor volume compared to the control which was clearly not toxic. SAHA administration at this dose resulted in an increase in acetylated histones H3 and H4 in tumor xenografts (FIG. 9).
[209] SAHA is in phase I clinical trials in patients with parenchyma tumors at the same time. SAHA results in the accumulation of acetylated cystones H3 and H4 in peripheral blood mononuclear cells isolated from patients undergoing treatment (FIG. 10).
[210] Table 1 summarizes the results of Examples 7-10 for testing Compounds 1-4 and also compares these results with the results obtained using SAHA.
[211] Table 1. Summary of Test Results for Compound 1-4, and Comparison with SAHA Results
[212]
[213] Example 12 Modified Inhibitors of HDAC
[214] In a further study, we found that compounds 6 and 7 described below are very effective inhibitors of the enzyme HDAC. Compound 6 had an ID ₅₀ of 2.5 nM and Compound 7 had an ID ₅₀ of 50 nM. This contrasts with 1 μM, ID ₅₀ for much higher SAHD. Note that 1 μM ID ₅₀ for SAHA as an inhibitor of HDAC is the same general size as the 2.5 μM optimal dose for cell differentiation of MEL cells, but this close similarity is not the case for all compounds investigated. In some cases a highly effective HDAC inhibitor is less effective as a cell differentiator, since the drug is metabolized during cell assay. In addition, not all cell types are the same, and some compounds are much better for human tumor cells such as HT-29 than they are for MEL cells. Thus, inhibition of HDAC cells is a preliminary indicator.
[215]
[216] Example 13 Progress on Compounds Without the Hydroxamic Acid Part
[217] In the above compounds which are hydroxamic acids, the inventors have found that these compounds undergo enzymatic hydrolysis more rapidly to carboxylic acids, so that their biological life is short. We are interested in related compounds that may be more stable in vivo. Therefore, we have developed inhibitors of HDAC that are not hydroxamic acid but can be used as cell differentiating agents with longer biological life. In addition, the inventors have found that novel related compounds have better selectivity for HDAC than, for example, SAHA.
[218] The inventors have developed compounds with double bonds similar to Trichostatin A (TSA) to see if the compounds formed have much greater efficacy. Also, the chain in TSA is only five carbon atoms, not the six carbons of SAHA. In oxamplatin, there are four carbon chains containing double bonds and ethynyl bonds between the hydroxamic acid and the first phenyl ring, and oxamplatin is claimed to be an effective inhibitor of HDAC. We have incorporated some of these features into the compounds of the present invention, including those that are not hydroxamic acid.
[219] Also described are simple combination methods for screening for many of these compounds for efficacy and selectivity with respect to HDAC inhibition.
[220] In addition, because there are many important enzymes containing Zn (II), some of the coordination groups of hydroxamic acid, and other metals, can also bind to Zn (II) and other metals.
[221]
[222] Because the target for HDAC is the acetyl lysine side chain of histones, we prepare compounds in which the transition state analogs of the substrate are present. For example, the present inventors synthesize compounds such as SAHA in which a hydroxamic acid group, -CO-NHOH is substituted with a trifluoroacetyl group, and -CO-CF ₃ . Compound 8 formed will readily form a hydrate, thus binding to Zn (II) of HDAC of mimic compound 9 in transition state 10 for deacetylation. This relates to a document 56 published by Lippscomb concerning binding to carboxypeptidase a of substrate analog 11 which contains CF ₃ —CO—CH ₂ groups instead of regular amides. Hydrates of ketones are coordinated to Zn (II) as mimic compounds in transition state for catalyzed hydrolysis of amide substrates. The synthesis of specific Example 12 in the series of fluoroketones of the present invention is described in the following scheme.
[223]
[224]
[225] After malonic ester alkylation, an aldehyde was prepared and then converted to trifluoromethyl carbinol using Ruperts reagent [57,58]. Malonic acid bis-anilide was prepared and carbinol was oxidized to ketone (12) using Dess-Martin reagent. I tried another method, but it didn't succeed. In particular, no tests have been done for the direct conversion of carboxylic acid derivatives to trifluoromethyl ketones.
[226] Compound 12 was tested with HDAC and found to be an enzyme inhibitor. Thus, the inventors also applied this synthesis to prepare analogs of compound (12) having unsaturated groups or the like in the chain and other groups at the left end of the molecule.
[227] Example 14 Generation of Compounds When Hydroxamic Acid Groups Are Replaced with NH-P (O) OH-CH ₃
[228] Analogs of SAHA in which the CH ₂ -CO-NHOH group is substituted with NH-P (O) OH-CH ₃ can be synthesized by the general scheme shown below. The resulting compound, 13 , binds to Zn (II) of HDAC in such a way that the relevant group is bound to Zn (II) of the carboxypeptidase in the analog, as prepared by Bartlett [60].
[229]
[230]
[231] Zn (II) enzyme The classic inhibitor of carbonic anhydrase is sulfonamide, the anion of which binds to Zn (II) [61]. Thus, analogs of compound 14, SAHA with sulfonamide groups, were synthesized as set out below. In the last step, carboxy sulfone bis-chloride was reacted with aniline and ammonia. Since the chlorinated carboxylic acid reacts faster, it was used in the order of aniline, then ammonia, but the order could be reversed or the mixture could be separated if the two had similar reactivity.
[232] In the course of the synthesis of 14, thiol 15 which was readily prepared from the corresponding haloic acid was used. Thiols are also inhibitors of Z (II) enzymes, such as related peptidases such as Carboxypeptidase A and Angiotensin Converting Enzyme (ACE), thus converting 15 to 16 as inhibitors of HDAC. Similar synthetic methods can be used to attach the NH-P (O) OH-CH ₃ group to other compounds, especially compounds 6 and 7.
[233]
[234] Example 15 - Changing the Linker Between a Zn (II) Bonder and a Hydrophobic Bonder
[235] Based on the results with oxamplatin, the phenyl ring can be part of the chain between the Zn (II) linker and the left part of the molecule shown, especially when substituted with meta. Thus, a synthetic method for incorporating such meta substituted chains into other compounds has been provided. Although not described in detail, a simple synthetic method only requires the preparation of aryl amides of 17 and 18 in place of the hydroxamic acid attached to the phenyl ring.
[236]
[237] Additional compounds such as 19 and 20 can be synthesized to incorporate trifluoromethyl ketone group 12, which is known to be effective as Zn (II) binder in HDAC. Synthesis involves preparing compounds 21 and 22 and adding CF ₃ to form carbinol and then oxidizing as in the synthesis of 12. A simple synthetic method involves the Heck bond of compounds 23 and 24 with ethyl acrylate, and reduction with carbinol to convert the ester to aldehyde and then reoxidize.
[238] All the chains presented so far contain only carbon atoms, but thioether bonds can be tolerated and even useful, which facilitates synthesis. Thus, sulfonamides such as 25 and 26 are associated with 19 and 20 from the corresponding thiophenols and bromomethylsulfonamides. Related synthetic methods can be used to prepare the corresponding phosphonamidates 27 and 28, if they prove to be useful HDAC inhibitors and cytodifferentiators. In this case, (N-protected) m-aminobenzoic acid is used to acrylate the arylamine and then to phosphorylate the anilino group.
[239]
[240]
[241]
[242] Example 16 - Changing the left part of a molecule with a hydrophobic group
[243] Compound 29 was synthesized as an intermediate product that can be treated with various amines to produce compound 30 to change the hydrophobic group. The hydroxamic acid group will then be deprotected to produce 31. The synthesis method is shown in the following scheme.
[244]
[245]
[246] In the synthesis, after O-protected hydroxylamine is acylated with bromohexanoic acid, this compound alkylates the bispentafluoro ester of malonic acid. The resulting compound 29 then reacts with various amines and the protecting group is removed by acid.
[247] Using these compounds as starting materials, we synthesized related libraries containing other Zn (II) linking groups. For example, alkylation of malonic acid with Compound 32 allows the inventors to prepare phosphonamidate libraries, and alkylation with Compound 33 enables the preparation of CF ₃ -CO libraries. In a similar manner, sulfonamide libraries can be prepared if the above described work indicates that this is a promising Zn (II) linker to HDAC. Of course, after malonate alkylation and aminolysis, the compound from compound 32 is demethylated and the compound from compound 33 is oxidized.
[248]
[249] This allows for expansion into the structure of compound 6 , which is a derivative of aminosuberic acid. As explained, this was one of the most effective HDAC inhibitors we investigated. We prepared this compound using enzymatic hydrolysis to achieve selectivity and optical cleavage between the two carbomethoxy groups of compound 34 , thereby converting one of them to the aminoquinoline amide of compound 6 Nitrogen was protected as a carbobene group. At the end of the synthesis, we converted the remote carbomethoxy group to hydroxysamate. However, compound 6 is an intermediate that can be used to prepare other derivatives. Carbobenzone groups from compound 6 can be removed and amine 35 can be acetylated with various carboxylic acids to produce library 36, or acetylated with sulfonic acid chloride to produce the corresponding sulfonamides.
[250]
[251]
[252] We also synthesized different libraries of amide 37 associated with compound 6 and then expanded them using other libraries of amide 38 by acylating amino groups after deprotection. In addition, we synthesized one group of compound 39 which allowed us to prepare a library of sulfonamides using various sulfonyl chlorides after the carbobenzone group of compound 37 was removed. In all of these, the hydroxamic acid groups can be protected.
[253]
[254] The synthetic schemes can be used to produce compounds with many variations. Some substituents that are believed to produce compounds with potentially good affinity for HDAC or having distinctive activities are:
[255] Some amines which may be incorporated in place of aniline in SAHA or as X groups of compounds 37 and 38 :
[256]
[257] Some carboxylic acids and sulfonic acids that may be incorporated as the Y-CO group of compound 38 or 39 :
[258]
[259] Example 17 Synthesis Using the Scheme
[260] Reagents and starting materials were obtained from commercial suppliers and used without further purification unless otherwise indicated. For the water sensitive reaction, the solvent was freshly distilled before use: tetrahydrofuran was distilled under argon from sodium metal using benzophenone as indicator; Dichloromethane and acetonitrile were distilled from powdered calcium hydride. Anhydrous benzene, anhydrous DIEA and anhydrous pyridine were withdrawn from a sealed bottle purchased from Aldrich by syringe. Tert-butanol was dried with 4 mm molecular sieves before use. Sodium hydride was purchased as a 60% dispersion in mineral oil. Aniline, diisopropylamine, N -methylaniline and benzyl alcohol were freshly distilled before use. Deuterated solvents were obtained from Cambridge Isotope Laboratories. Air sensitive and / or moisture sensitive reactions were carried out in a dry argon atmosphere in an oven dried or flame dried glass apparatus fitted with fitted rubber diaphragms. The reaction at 0 ° C. was carried out in an ice bath / water bath. The reaction at −78 ° C. was carried out in a dry ice bath / acetone bath.
[261] Chromatography
[262] Analytical thin layer chromatography (TLC) was performed on glass plates precoated with a silica gel 60 F-254 with a thickness of 0.25 mm prepared by EM Science (EM Science, Germany). Eluted compounds were visualized using one or more of shortwave ultraviolet light, I ₂ vapor, KMnO ₄ staining, or FeCl ₃ staining. Preparative TLC was performed on Whatman precoated plates with silica gel thickness of 500 μm or 1000 μm. Flash column chromatography was performed on Merck Kieselgel 60 of 230-400 mesh.
[263] Instrumentation
[264] NMR spectra were measured with Bruker DPX300 and DRX400 spectrometers; ¹ H was observed at 300 and 400 MHz and ¹⁹ F was observed at 376 MHz. Chemical shifts are reported as δ values (ppm) for solvent residual peaks. Mass spectra were obtained with a Nermag R-10-1 instrument for chemical ionization (CI) or electron impact ionization (EI) spectra and with a Jeol JMSLCmate for electrospray ionization (ESI +) spectra. . CI spectra were obtained using ammonia (NH ₃ ) or methane (CH ₄ ) as the ionizing gas.
[265] ( E, E ) -7- t -butoxycarbonyl-octa-2,4-dienodioic acid 8- t -butylester 1-methyl ester (40)
[266]
[267] To a stirred solution of NaH (60% dispersion, 234 mg, 5.85 mmol) in THF (35 mL) was added dropwise di-t-butyl malonate (1.20 mL, 5.37 mmol) at 0 ° C. Gas evolution was observed and the solution warmed to room temperature and stirred for 6 hours. A solution of methyl 6-bromo-2,4-hexadienoate 62 (1.00 g, 4.88 mmol) in THF (20 mL) was prepared in a separate flask and stirred in a water bath. To this the malonate mixture was added dropwise using a cannula and the reaction proceeded overnight. The reaction was quenched with saturated NH ₄ Cl (5 mL), then H ₂ O (10 mL) was added and the mixture was extracted with Et ₂ O (3 × 15 mL). The organic fractions were combined, washed with H ₂ O (10 mL) and then brine, dried over MgSO ₄ and filtered. Flash chromatography (0-20% EtOAc / hexanes) after evaporation under reduced pressure gave compound 40 as a clear colorless oil (850 mg, 2.49 mmol, 51%). TLC R _f 0.66 (20% EtOAc / hexanes); ¹ H-NMR (CDCl ₃ , 400 MHz) δ 7.26 (dd, 1H), 6.26 (dd, 1H), 6.10 (m, 1H), 5.82 (d, 1H), 3.78 (s, 3H), 3.12 (t , 1H), 2.64 (t, 2H), 1.41 (s, 18H).
[268] (E, E) -7-carboxy-octa-2,4-dienedioic acid 1-methyl ester (41)
[269]
[270] To a stirred solution of 40 (200 mg, 0.59 mmol) in CH ₂ Cl ₂ (10 mL) was added TFA (1 mL). The reaction proceeded overnight. The volatiles were removed under reduced pressure to give 41 as a white solid (112 mg, 0.49 mmol, 83%). ¹ H-NMR (CD ₃ OD, 400 MHz) δ 7.11 (dd, 1H), 6.33 (dd, 1H), 6.16 (m, 1H), 5.81 (d, 1H), 3.76 (s, 3H), 3.15 ( t, 1H), 2.70 (t, 2H).
[271] 4-pentenoic acid phenylamide (42)
[272]
[273] 4-pentenoic acid (2.25 mL, 22.0) in a stirred solution of oxalyl chloride (2.0 M in CH ₂ Cl ₂ , 11.5 mL, 23.1 mmol) in CH ₂ Cl ₂ (100 mL) and DMF (1 drop) at 0 ° C. mmol) was added. It was warmed up to ambient temperature. As soon as gas evolution ceased, the mixture was returned to 0 ° C. and a solution of aniline (2.00 mL, 22.0 mmol) and TEA (6.72 mL, 26.3 mmol) in CH ₂ Cl ₂ (5 mL) was added dropwise. After warming up to ambient temperature, the reaction was allowed to proceed for 3 hours. The mixture was concentrated under reduced pressure, then partitioned between HCl (IN, 10 mL) and EtOAc (30 mL) and the layers separated. The aqueous portion was extracted with EtOAc (3 × 15 mL) and the organic layers combined, washed with brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave a yellowish solid which was recrystallized from toluene to give 42 as white crystals (1.97 g, 11.24 mmol, 51%). TLC R _f 0.68 (50% EtOAc / hexanes); ¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.49 (d, 2H), 7.29 (t, 2H), 7.08 (t, 1H), 5.88 (m, 1H), 5.10 (dd, 2H), 4.42 (br s, 4H).
[274] (E, E) -octa-2,4-dienedioic acid 8-t-butyl ester 1-methyl ester (43)
[275]
[276] To a stirred solution of diisopropylamine (2.06 mL, 14.7 mmol) in THF (25 mL) at −78 ° C. add n-BuLi (2.0 M in hexanes, 6.2 mL, 12.4 mmol) and at this temperature for 20 minutes Stirred. Then a solution of phosphonate 43a (63) (2.66 g, 11.3 mmol) in THF (4 mL) was added dropwise and a dark yellow color appeared upon addition. After 20 minutes at −78 ° C., the mixture was warmed to 0 ° C. and a solution of aldehyde 43b (64) (1.78 g, 11.3 mmol) in THF (4 mL) was added dropwise. After addition the solution was warmed to ambient temperature and stirred overnight. It was diluted with Et ₂ O (30 mL) and washed with H ₂ O (3 × 10 mL). The aqueous washes were combined and extracted with Et ₂ O (2 × 10 mL), the organic portions were combined, washed with brine, dried over MgSO ₄ and filtered. Evaporation under reduced pressure followed by flash chromatography (10-20% EtOAc / hexanes) gave 43 (1.54 g, 57%) as a clear oil. TLC R _f 0.56 (20% EtOAc / hexanes); ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.22 (dd, 1H), 6.19 (dd, 1H), 6.08 (m, 1H), 5.77 (d, 1H), 2.42 (m, 2H), 2.32 (t , 2H), 1.42 (s, 9H).
[277] (E, E) -7-phenylcarbamoyl-hepta-2,4-dienoic acid methyl ester (44)
[278]
[279] To a stirred solution of diester 43 (1.00 g, 4.61 mmol) in CH ₂ Cl ₂ (40 mL) was added TFA (4.0 mL) and reacted for 6 h. The mixture was concentrated under reduced pressure to remove volatiles. A white solid consisting of crude acid (710 mg, 3.85 mmol) remained. Dissolve this acid (400 mg, 2.17 mmol) in CH ₂ Cl ₂ (20 mL) and add DMAP (13 mg), aniline (218 μL, 2.39 mmol), and EDC (500 mg, 2.61 mmol) to this stirred solution. It was. After 1.5 h, the mixture was diluted with EtOAc and washed with H ₂ O. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 15 mL). The organic portions were combined and washed with HCL (IN, 1 x 5 mL) and brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave a brown solid. It was dissolved in a minimal amount of CH ₂ Cl ₂ and then passed through a plug of silica gel (20-30% EtOAc / hexanes, 200 mL) to remove the base impurities. The eluate was concentrated to give a light brown oil which was dissolved in a small amount of CH ₂ Cl ₂ from which precipitated crystals upon addition of hexane / diethyl ether. The mother liquor was drained off, the crystals were washed with ether, the liquid fractions were concentrated and the process repeated several times to give 44 as ultimately greyish white crystals (324 mg, 1.25 mmol, 58%). TLC R _f 0.44 (50% EtOAc / hexanes); ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.47 (d, 1H), 7.30 (t, 2H), 7.24 (m, 1H), 7.09 (t, 1H), 6.24 (dd, 1H), 6.14 (m , 1H), 5.81 (d, 1H), 3.72 (s, 3H), 2.60 (m, 2H), 2.47 (t, 2H).
[280] (E, E) -7- (methyl-phenyl-carbamoyl) -hepta-2,4-dienoic acid methyl ester (45)
[281]
[282] The crude acid intermediate and N-methylaniline (130 μL, 1.19 mmol) from the first step of the preparation ( 44 mg, 1.09 mmol) were dissolved in CH ₂ Cl ₂ (10 mL) and stirred. Then EDC (271 mg, 1.41 mmol) and DMAP (5 mg) were added and the reaction was performed overnight. The mixture was partitioned between H ₂ O and EtOAc and the layers separated. The aqueous layer was extracted with EtOAc (3 × 10 mL), the organic portions combined and washed with HCl (1N, 1 × 5 mL) followed by brine, dried over MgSO ₄ and filtered. Evaporation under reduced pressure gave 45 as pure as a brown oil (286 mg, 1.05 mmol, 96%). TLC R _f 0.81 (5% MeOH / CH ₂ Cl ₂ ); ¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.40 (t, 2H), 7.35 (t, 1H), 7.20 (d, 2H), 7.15 (dd, 1H), 6.20 (m, 2H), 5.76 (d , 1H), 3.70 (s, 3H), 3.24 (s, 3H), 2.42 (m, 2H), 2.18 (t, 2H).
[283] (E, E) -7-phenylcarbamoyl-hepta-2,4-dienoic acid (46)
[284]
[285] Ester 45 (260 mg, 0.95 mmol) was dissolved in MeOH (7.5 mL). Then a solution of LiOH.H ₂ O (200 mg, 4.76 mmol) in H ₂ O (2.5 mL) was added and the mixture was stirred for 6 h. The reaction was acidified with HCl (1N) to pH 2 and then extracted with EtOAc (3 x 10 mL). The organic fractions were combined and washed with H ₂ O and brine, dried over MgSO ₄ and filtered. Evaporation under reduced pressure gave the product pure 46 as a brown solid (200 mg, 0.77 mmol, 81%). TLC R _f 0.13 (40% EtOAc / hexanes); ¹ H-NMR (300 MHz, CD ₃ OD) δ 7.47 (t, 2H), 7.41 (d, 1H), 7.28 (d, 2H), 7.19 (dd, 1H), 6.18 (dd, 1H), 6.05 ( m, 1H), 3.27 (s, 3H), 3.40 (m, 2H), 2.22 (t, 2H).
[286] (E, E) -octa-2,4-dienedioic acid 1-hydroxyamide 8-phenylamide (47)
[287]
[288] Acid 46 (200 mg, 0.77 mmol) and TBDPSO-NH ₂ (220 mg, 0.81 mmol) were dissolved in CH ₂ Cl ₂ (8 mL). To this stirred solution was added EDC (178 mg, 0.93 mmol) and DMAP (5 mg) and the reaction proceeded overnight. The mixture was concentrated and then passed through a plug of silica gel (EtOAc). Evaporation under reduced pressure gave a light brown oil (383 mg, 0.75 mmol, 97%). Protected hydroxamate (270 mg, 0.53 mmol0) was dissolved in CH ₂ Cl ₂ (10 mL) and TFA added (0.5 mL) The solution was stirred for 2 h and new spots observed on TLC stained with FeCl ₃ . The solution was concentrated under reduced pressure and diethyl ether was added to give a residue attached to the flask The liquid phase was drained, the residue was triturated with EtOAc, the liquid was removed and all volatiles were removed from the residue. Evaporation gave 47 as brown gum (23 mg, 0.084 mmol, 16%) TLC R _f 0.22 (5% MeOH / CH ₂ Cl ₂ ); ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.50 ( t, 2H), 7.40 (t, 1H), 2.27 (d, 2H), 7.08 (m, 1H), 6.11 (m, 1H), 5.97 (m, 1H), 5.80 (m, 1H), 3.23 (s , 3H), 3.39 (m, 2H), 2.21 (t, 2H).
[289] Octanedioic Acid Hydroxyamide Phenylamide (48)
[290]
[291] The title compound 48 was obtained as a brown gum (9 mg) by a series of steps similar to the preparation of 47 . TLC R _f 0.20 (5% MeOH / CH ₂ Cl ₂ ); ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.51 (t, 2H), 7.419t, 1H, 7.30 (d, 2H), 3.29 (s, 3H), 2.11 (m, 4H), 1.58 (m , 4H), 1.22 (m, 4H).
[292] Octanedioic acid benzylamide (49)
[293]
[294] To a stirred solution of subveroyl chloride (1.00 mL, 5.55 mmol) in THF (40 mL) at 0 ° C. add benzylamine (0.61 mL, 5.55 mmol) and DIEA (1.45 mL, 8.33 mmol) in THF (10 mL). Added dropwise. The mixture was warmed to ambient temperature and stirred for 1 hour. Then HCl (10 mL, 1N) was added and the mixture was stirred for 0.5 h. The contents were diluted with EtOAc (30 mL) and the layers separated. The aqueous portion was extracted with EtOAc (3 × 10 mL), the organic portions combined, washed with brine (5 mL) and dried over MgSO ₄ . Filtration and concentration under reduced pressure gave 49 as a grayish white solid. ¹ H-NMR (300 MHz, DMSO-d ₆ ) δ 11.98 (br s, 1H), 9.80 (t, 1H0, 7.32 (m, 2H), 7.23 (m, 3H), 4.259d, 2H), 2.19 ( t, 2H), 2.12 (t, 2H), 1.50 (m, 4H), 1.25 (m, 4H).
[295] Octanedioic Acid Benzylamide Hydroxyamide (50)
[296]
[297] The compound was prepared from 49 via its protected hydroxamate as described for the previous compounds. 50 obtained was a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 10.30 (s, 1H0, 8.27 (t, 1H), 7.28 (m, 2H), 7,23 (m, 3H), 5.65 (d, 2H), 2.11 (t, 2H), 1.91 (t, 2H), 1.46 (m, 4H), 1.23 (m, 4H).
[298] (7S) -7-benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid t-butyl ester (51)
[299]
[300] N-Cbz- _L -2-aminosuberic acid 8-t-butyl ester, dicyclohexylamine salt (100 mg, 0.18 mmol) was dissolved in HCl (5 mL, 1N) and added with EtOAc (3 x 10 mL). Extracted. The extracts were combined, washed with brine and dried over MgSO ₄ . Evaporation gave the free acid as a white solid (68 mg, 0.179 mmol). It was dissolved in CH ₂ Cl ₂ (2.5 mL), to which aniline (17 μL, 0.19 mmol), DIEA (46 μL, 0.27 mmol), and finally PyBOP (97 mg, 0.19 mmol) were added. The solution was stirred for 1 h, then concentrated and the residue was partitioned between H ₂ O (5 mL) and EtOAc (10 mL). The layers were separated and the aqueous portion extracted with EtOAc (3 x 10 mL). The extract was pooled and washed with HCl (1N) followed by brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave a solid residue which was passed through a plug of silica gel (30% EtOAc / hexanes). The collected eluate was evaporated to give 51 as a white solid (76 mg, 0.167 mmol, 94%). TLC R _f 0.38 (30% EtOAc / hexanes); ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.21 (s, 1H), 7.48 (d, 2H), 7.32 (m, 5H), 7.28 (t, 2H), 7.08 (t, 1H), 5.39 (br d, 1H), 5.10 (m, 2H), 4.26 (br dd, 1H), 2.07 (t, 2H), 1.92 (m, 1H), 1.66 (m, 1H), 1.55 (m, 2H), 1.42 ( s, 9H), 1.38 (m, 4H).
[301] (7S) -7-Benzyloxycarbonylamino-7-phenylcarbamoyl-heptanoic acid (52)
[302]
[303] To a solution of ester 51 (76 mg, 0.167 mmol) in CH ₂ Cl ₂ (5 mL) was added TFA (0.5 mL) and the resulting reaction solution was stirred for 5 h. The solution was concentrated under reduced pressure to afford crude Compound (52) (80 mg) as a white solid, which was used in the next step without purification.
[304]
[305] (1S)-(6-hydroxycarbamoyl-1-phenylcarbamoyl-hexyl) -carbamic acid benzyl ester (53)
[306]
[307] To the crude acid (52) (80 mg) and TBDPSO-NH ₂ (60 mg, 0.221 mmol) solution in CH ₂ Cl ₂ was added DIEA (52 μL, 0.302 mmol), followed by PyBOP (125 mg, 0.241 mmol) was added. The solution was stirred for 3 hours and then concentrated under reduced pressure. The residue was passed through a silica gel plug (EtOAc / hexanes) and the collected eluate was evaporated. White foam (107 mg, 0.164 mmol, 82%) was obtained, which was dissolved in CH ₂ Cl ₂ (5 ml), TFA (0.25 ml) was added and the solution was stirred for 2 hours. New sites stained with FeCl ₃ were shown by TLC analysis. The mixture was concentrated under reduced pressure, the residue was solvated in a minimum amount of EtOAc and the product precipitated with hexanes. The resulting white gel was washed with hexanes and dried under vacuum to afford compound 53 (40 mg, 0.097 mmol, 58% excess in 3 steps) as a white solid.
[308]
[309] (7S) -7-benzyloxycarbonylamino-7- (quinolin-8-ylcarbamoyl) -heptanoic acid t-butyl ester (54)
[310]
[311] N-Cbz-L-2-aminosuberine acid 8-t-butyl ester, dicyclohexylamine salt was prepared by a method similar to compound (51). Flash chromatography (0-1% MeOH / CH ₂ Cl ₂ ) afforded compound (54) as a pale brown solid (70 mg, 0.138 mmol, 82%).
[312]
[313] (7S) -7-benzyloxycarbonylamino-7- (quinolin-8-ylcarbamoyl) -heptanoic acid (55)
[314]
[315] Prepared from compound (54) in a similar manner to that for compound (52). Compound (55) was obtained as a brown solid (72 mg, 0.129 mmol).
[316]
[317] (1S)-[6-hydroxycarbamoyl-1- (quinoline-8-ylcarbamoyl) -hexyl] -carbamic acid benzyl ester (56)
[318]
[319] Prepared from compound (55) in a similar manner to that for compound (53). Compound (56) was obtained as a white solid (15 mg, 0.032 mmol, 44%).
[320]
[321] (7S)-(Cyclohexanecarbonyl-amino) -7-phenylcarbamoyl-heptanoic acid methyl ester (57)
[322]
[323] To a solution of compound 5 (81 mg, 0.214 mmol) in CH ₂ Cl ₂ (10 mL) was added TFA (0.5 ml) and the solution was stirred for 2 hours. The mixture was concentrated under reduced pressure. To a solution of said amine (62 mg, 0.223 mmol) and cyclohexane carboxylic acid (31 μl, 0.245 mmol) in CH ₂ Cl ₂ (4 ml) PyBOP (140 mg, 0.268 mmol) and DIEA (58 μl) , 0.335 mmol) was added. The solution was stirred for 2 hours, concentrated under reduced pressure, and then the product was purified by flash chromatography (40% EtOAc / hexanes). Evaporation left crude compound (57) as a white solid containing a small amount of unreacted cyclohexane impurity. The material was used for the next step without further purification.
[324]
[325] (7S)-(Cyclohexanecarbonyl-amino) -7-phenylcarbamoyl-heptanoic acid (58)
[326]
[327] To a solution of ester (57) in MeOH (2.5 ml) was added a solution of NaOH (1 M, 2.5 ml) at 0 ° C. THF (2.5 mL) was added to the white precipitate produced at the time of redissolution. Further NaOH (1 M, 0.1 mL) was added after 3 hours and the temperature was kept at 0 ° C. Immediately after the starting material was completely removed in TLC analysis, the reaction components were oxidized with HCl (IN) to give a white precipitate. The supernatant was drained off and the solid was filtered off with suction. The combined liquid was extracted with EtOAc (3 × 5 ml) and the combined extracts were washed with brine and then dried over MgSO 4 and filtered. Concentration under reduced pressure yielded a white solid combined with the filter cake which was dried under vacuum to give carboxylic acid 58 (75 mg, 0.200 mmol, 90%).
[328]
[329] (2S) -2- (Cyclohexanecarbonyl-amino) -7-octanedioic acid 8-hydroxyamide 1-phenylamide (59)
[330]
[331] Acid 58 (70 mg, 0.187 mmol), TBDPSO-NH ₂ (61 mg, 0.224 mmol), and DMAP (5 mg) are dissolved in CH ₂ Cl ₂ (4 mL) and EDC (47 mg, 0.243 mmol ). The solution was dried overnight. After concentration under reduced pressure, the material was purified by flash chromatography (50% EtOAc / hexanes). The combined product fractions were evaporated to give a white foam (80 mg, 0.131 mmol, 70%). To this solution of protected hydroxyxamate in CH ₂ Cl ₂ and THF (3 mL) was added TFA (0.25 mL) and stirred for 1.5 h. A new spot stained immediately with FeCl ₃ was observed on TLC. The solution was concentrated and all volatiles were removed in vacuo. The residue was triturated with EtOAc 롸 to give a white gel which was then transferred to a plastic tube with EtOAc (5 ml). The tube was centrifuged to form pellets, the supernatant was dried and EtOAc (10 mL) was added. The pellet was resuspended with sonication, recentrifuged, the supernatant was removed and the residue was dried under vacuum. White solid (59) (18 mg, 0.046 mmol, 35%) was obtained.
[332]
[333] Octanedioic Acid Hydroxyamide Quinolin-8-ylamide (60)
[334]
[335] The compound was prepared in a similar manner to compound (48) using a 8-aminoquinoline as a suberic acid monomethyl ester. The crude residue obtained after TFA deprotection of the protected hydroxyxamate was taken up in a small volume of EtOAc and precipitated with hexanes to give compound 60 as a white solid (18 mg, 0.057 mmol, 21 from carboxylic acid). %).
[336]
[337] 2-t-butoxycarbonyl-octanediionic acid 1-t-butyl ester 8-ethyl ester (61)
[338]
[339] To a stirred suspension of NaH (60%, 197 mg, 4.913 mmol) in THF (25 mL) was added di-t-butyl malonate (1.00 mL, 4.466 mmol) at 0 ° C. and the mixture was heated to ambient temperature. After 1 hour, gas rotation was stopped and 6-bromohexanoate (0.88 mL, 4.913 mmol) was added gradually. The reaction was refluxed overnight. The reaction was quenched with H ₂ O (10 mL) and diluted with EtOAc. After separating the layers, the aqueous portion was extracted with EtOAc (3 × 10 mL). The extract was pooled, washed with H ₂ O, washed with brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure yielded a yellow oil which was passed through a plug of silica gel (10% EtOAc / hexanes). Evaporation gave a light yellow syrup (compound 61) (1.52 g, 4.24 mmol, 95%). TLC R _f 0.44 (10% EtOAc / hexanes);
[340]
[341] 2-carboxy-octanedioic acid 8-ethyl ester (62)
[342]
[343] To a solution of triester (Compound 61) (500 mg, 1.395 mmol) in CH ₂ Cl ₂ (2025 mL) was added TFA (2.0 mL) and the reaction mixture was stirred overnight. The volatile components were evaporated in vacuo and the residue was repeatedly dissolved in CH ₂ Cl ₂ and evaporated to remove all residual amounts of TFA. Solid (Compound 62) (327 mg, 1.33 mmol) was obtained and used directly in the next step without further purification.
[344]
[345] 7,7-bis- (quinoline-8-ylcarbamoyl) -heptanoic acid ethyl ester (65)
[346]
[347] Dieside (Compound 62) (150 mg, 0.609 mmol), 8-aminoquinoline (211 mg, 1.462 mmol), and DMAP (5 mg) were dissolved in THF (6 mL). EDC (350 mg, 1.827 mmol) was added to this solution and the reaction proceeded overnight. The mixture was concentrated under reduced pressure and the product was purified by flash chromatography (40% EtOAc / hexanes). The recovered product fractions were evaporated to yield compound 63 as a light brown solid (100 mg, 0.201 mmol, 14%).
[348]
[349] 7,7-bis- (quinoline-8-ylcarbamoyl) -heptanoic acid (64)
[350]
[351] To a solution of ester (Compound 63) (94 mg, 0.212 mmol) in MeOH (3 mL) and THF (1 mL) was added LiOH.H ₂ O (44 mg, 1.062 mmol) in H ₂ O (1 mL) and The mixture was stirred for 5 hours. Acidified with HCl (IN) to pH 7, then EtOAc (10 mL) was added and the layers separated. The aqueous portion was extracted with EtOAc (3 × 5 mL) and the extracts were recovered, washed successively with saturated NH ₄ Cl (3 mL), H ₂ O (3 mL) then brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave 64 as a white solid (94 mg, 0.200 mmol, 94%). TLC R _f 0.21 (50% EtOAc / hexanes);
[352]
[353] 2- (Quinoline-8-ylcarbamoyl) -octanoic acid 8-hydroxyamide 1-quinolin-8-ylamide (65)
[354]
[355] Acid (Compound 64) (94 mg, 0.200 mmol), TBDPSO-NH ₂ (74 mg, 0.272 mmol), and DMAP (5 mg) were dissolved in CH ₂ Cl ₂ (4 mL) and EDC (57 mg, 0.295 mmol) Was added. The solution was stirred overnight and then concentrated under reduced pressure. Purification by flash chromatography (30-50% EtOAc / hexanes) and the recovered product fractions were evaporated to give a white foam. To this solution protected with hydroxysamate in CH ₂ Cl ₂ (4 mL) was added TFA (0.2 mL) and the solution was stirred for 4 h. TLC showed that the starting material was consumed completely and showed new dots colored with FeCl ₃ . The solution was concentrated under reduced pressure and the residue was dissolved in a minimum amount of EtOAc. Hexane was added to give a white precipitate, from which the mother solution was removed. After rinsing with hexane, the residue was dried under vacuum to give compound 65 as a white solid (30 mg, 0.061 mmol, 22% from carboxylic acid).
[356]
[357] 2- (Quinoline-3-ylcarbamoyl) -octanoic acid 8-hydroxyamide 1-quinolin-3-ylamide (68)
[358]
[359] The title compound was obtained by treatment similar to compound 65 from dieside (compound 62).
[360]
[361] 6-Bromohexanoic acid phenylamide (76)
[362]
[363] To a solution of 6-bromohexanoyl chloride (1.00 mL, 6.53 mmol) in THF (35 mL) at 0 ° C. of aniline (0.60 mL, 6.53 mmol) and TEA (1.09 mL, 7.84 mmol) in THF (5 mL). The solution was added dropwise. The reaction mixture was warmed to room temperature and stirred for 2 hours. The mixture was filtered, the solid was rinsed with EtOAc and the filtrate was reduced in vacuo. The residue was partitioned between H ₂ O (15 mL) and EtOAc (20 mL) and the layers separated. The aqueous portion was extracted with EtOAc (3 × 10 mL) and the organic layer was recovered, washed with HCl (1 N), brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave a brown oil which was passed through a plug of silica gel (30% EtOAc / hexanes) under intake. Concentration under reduced pressure gave compound 67 as a solid (1.55 g, 5.74 mmol, 88%). TLC R _f 0.36 (25% EtOAc / hexanes);
[364]
[365] Thioacetic acid s- (5-phenylcarbamoyl-pentyl) ester (68)
[366]
[367] Bromid (Compound 67) (200 mg, 0.74 mmol), potassium thioacetate (110 mg, 0.96 mmol), and sodium iodide (10 mg) were recovered in THF (6 mL) and the vigorously stirred mixture was refluxed overnight. I was. The reaction mixture was concentrated and passed through a plug of silica gel (20% EtOAc / hexanes, 200 mL) under intake. Concentration under reduced pressure gave 68 as an orange crystalline solid (190 mg, 0.72 mmol, 97%). TLC R _f 0.22 (25% EtOAc / hexanes);
[368]
[369] 6-Methanesulfonylamino-hexanoic acid (69)
[370]
[371] 6-aminohexanoic acid (904 mg, 6.89 mmol) and NaOH (415 mg, 10.34 mmol) were dissolved in H ₂ O (30 mL) and cooled to 0-5 ° C. Methanesulfonyl chloride (0.586 mL, 7.58 mmol) was added dropwise and the reaction mixture was stirred for 2 hours, then warmed to ambient temperature and stirred for a further 2 hours. The mixture was acidified with HCl (IN) and extracted with EtOAc (3 × 15 mL). The extract was recovered, washed with H ₂ O, washed with brine, dried over MgSO ₄ and filtered. Evaporation under reduced pressure yielded compound 69 as a white crystalline solid (207 mg, 0.99 mmol, 14%).
[372]
[373] 6-Methanesulfonylamino-hexanoic acid phenylamide (70)
[374]
[375] To a solution of acid (Compound 69) (100 mg, 0.48 mmol), aniline (60 μl, 0.66 mmol), and DMAP (5 mL) in THF (5 mL) was added EDC (119 mg, 0.57 mmol). The reaction mixture was stirred overnight and then partitioned between H ₂ O (10 mL) and EtOAc (15 mL). The layers were separated and the aqueous portion extracted with EtOAc (3 × 10 mL). The organic fractions were recovered, washed with saturated NH ₄ Cl (5 mL), then brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave 70 as a white crystalline solid (130 mg, 0.46 mmol, 95%).
[376]
[377] 9,9,9-Trefluoro-8-oxononanoic acid methyl ester (71)
[378]
[379] To a solution of suberic acid monomethyl ester (1.00 g, 5.31 mmol) in THF (15 mL) was added oxalyl chloride (2 mL) followed by DMF (1 drop). The solution was stirred for 2 hours and then concentrated under reduced pressure. The volatiles were removed overnight under high vacuum to yield a yellow oil (1.08 g, 5.22 mmol, 98%). This crude acid chloride was then transformed to trifluoromethyl ketone by the following literature method. (Compound 65) To a solution of acid chloride (1.08 g, 5.22 mmol) in CH ₂ Cl ₂ (45 mL) was added trifluoro acetic anhydride (4.64 mL, 32.81 mmol) and pyridine (3.54 mL, 43.74 mmol) at 0 ° C. Added. The mixture was warmed to ambient temperature and stirred for 2 hours. Return to 0 ° C. and ice cold H ₂ O (20 mL) was added carefully. Additional H ₂ O (100 mL) was added to separate the layers. The aqueous phase was extracted with CH ₂ Cl ₂ (2 × 30 mL) and the organic phase was recovered, washed with brine, dried over MgSO ₄ and filtered. Evaporation under reduced pressure gave a brown oil, which was purified by flash chromatography (2-4% MeOH / CH ₂ Cl ₂ ) to give compound 71 as a clean oil (641 mg, 2.67 mmol, 49%).
[380]
[381] 9,9,9-trifluoro-8-oxo-nonanoic acid phenylamide (72)
[382]
[383] To a solution of ester 71 (300 mg, 1.25 mmol) in THF (18 mL) was added a solution of LiOH.H ₂ O (262 mg, 6.24 mmol) in water (6 mL) and the suspension was stirred overnight. The mixture was then acidified with HCl (1N) to pH 2 and then extracted with EtOAc (3x15 mL). The extracts were collected, washed with brine, dried over MgSO ₄ and filtered. Concentration under reduced pressure gave a white solid (211 mg, 0.93 mmol, 75%). To a solution of the acid (109 mg, 0.48 mmol), EDC (111 mg, 0.58 mmol), and DMAP (5 mg) in CH ₂ Cl ₂ (5 mL) was added aniline (49 μl, 0.53 mmol) and the reaction proceeded overnight. The solution was partitioned between H ₂ O (5 mL) and EtOAc (10 mL). The layers were separated and the aqueous phase extracted with EtOAc (3x5 mL). The organic portion was collected, washed with brine, dried over MgSO ₄ and filtered. Filtration under reduced pressure gave a solid which was purified by preparative TLC (30% EtOAc / hexanes) and the lowest polar band separated by EtOAc extraction. The extract was concentrated to give compound 72 as a yellow solid (92 mg, 0.31 mmol, 65%).
[384]
[385] (5-phenylcarbamoyl-pentyl) -carbamic acid t-butyl ester (73)
[386]
[387] Aniline (1.04 mL, 11.35 mmol) in a solution of N-bok-6-aminohexanoic acid (2.50 g, 10.81 mmol), EDC (2.69 g, 14.05 mmol), and DMAP (20 mg) in CH ₂ Cl ₂ (100 mL). Was added and the mixture was stirred overnight. The solution was evaporated under reduced pressure to a small volume, then partitioned between H ₂ O (20 mL) and EtOAc (30 mL). The layers were separated and the aqueous phase extracted with EtOAc (3x5 mL). The organic portion was collected, washed with saturated NH ₄ Cl solution (5 mL), then brine, dried over MgSO ₄ and filtered. Filtration under reduced pressure gave pure compound (73) as a white solid (3.14 g, 10.25 mmol, 95%).
[388]
[389] 6-Aminohexanoic acid phenylamide, TFA salt (74)
[390]
[391] To a solution of carbamate (73) (300 mg, 0.98 mmol) in CH ₂ Cl ₂ (15 mL) was added TFA (0.75 mL) and the solution was stirred overnight. TLC confirmed that the starting material was consumed completely. The mixture was evaporated under reduced pressure to remove all volatiles, yielding a pale white solid (295 mg, 0.92 mmol, 94%). Crude compound (74) was used without further purification.
[392] N- (N-phenylcarbamoyl-5-pentyl) phosphoramic acid dimethyl ester (75)
[393]
[394] Dimethyl chlorophosphate (77 μl, 0.72 mmol) was added dropwise to a stirred suspension of ammonium salt (74) (197 mg, 0.62 mmol) and DIEA (148 μl, 0.85 mmol) in CH ₂ Cl ₂ (7 mL) at 0 ° C. The mixture was allowed to warm to rt and stirred overnight. The solution was diluted with H ₂ O (10 mL) and the layers separated. The aqueous phase was extracted with CH ₂ Cl ₂ (3 × 10 mL) and the organic portion collected and washed with saturated NH ₄ Cl solution (5 mL), then brine, dried over MgSO ₄ and filtered. After concentration, the residue was purified by flash chromatography (2-5% MeOH / CH ₂ Cl ₂ ), and the fractions containing the more polar of the two UV-active bands on TLC were collected and concentrated to give a compound (75 ) Was obtained as a clear oil (40 mg, 0.13 mmol, 20%).
[395]
[396] Methyl N- (5-N-phenylcarbamoylpentyl) methylphosphonamidate (76)
[397]
[398] To a suspension of ammonium salt (74) (155 mg, 0.48 mmol) in CH ₃ CN (8 mL) was added DIEA (0.21 mL) and methyl methylphosphonochlorate (77 mg, 0.600 mmol). The reaction mixture was stirred overnight during which time the mixture became clear. The solution was partitioned between H ₂ O (10 mL) and EtOAc (15 mL) and the layers separated. The aqueous portion was extracted with EtOAc (3 × 10 mL) and the organic portion collected and washed with saturated NH ₄ Cl solution (1 × ₅ mL), then brine, dried over MgSO ₄ and filtered. The product was purified by flash chromatography (3-10% MeOH / CH ₂ Cl ₂ ), and the fractions containing more polar spots were collected and concentrated to give compound 76 as a clear oil (102 mg, 0.34 mmol, 71%). Obtained).
[399]
[400] Example 18 Synthesis of Compound (77)
[401] Diethyl 3-bromophenylmalonate
[402]
[403] Diethyl 3-bromophenyl malonate is described in Cehnevert, R. and Desjardins, M. Can. J. Chem. 1994, 72, 3212-2317.
[404]
[405] 3-bromophenyl malonyl di (phenylamide)
[406]
[407] Diethyl 3-bromophenyl malonate (1 g, 3.2 mmol) was added to aniline (5 mL). The reaction mixture was purged with Ar (g) and refluxed for 2 hours. After cooling, the reaction mixture was diluted with 10% HCl (20 mL) and ethyl acetate (50 mL). The organic layer was separated and concentrated to give 3-bromophenyl malonyl di (phenylamide) as a white powder (540 mg, 1.3 mmol, 42%).
[408]
[409] 3- (malonyl di (phenylamide)) cinnamic acid
[410]
[411] 3-bromophenyl malonyl di (phenylamide) (500 mg, 1.22 mmol), acrylic acid (115 mg, 1.6 mmol, 1.3 equiv), Pd (OAc) ₂ (2 mg), tri- O -tolyl phosphone (20 mg), Tributyl amine (0.6 mL) and xylene (5 mL) were heated to 120 ° C. for 6 hours in a closed vessel. After cooling, the reaction was diluted with 5% HCl (10 mL) and ethyl acetate (50 mL). The organic layer was separated, filtered and, upon standing, 3- (malonyl di (phenylamide)) cinnamic acid precipitated as a white powder (450 mg, 1.12 mmol, 92%).
[412]
[413] 3- (malonyl di (phenylamide)) cinnamil hydroxamic acid (77)
[414]
[415] 3- (malonyl di (phenylamide)) cinnamic acid (200 mg, 0.5 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (10 mL). Isobutylchloroformate (0.10 mL, 0.77 mmol) and triethyl amine (0.20 mL) were added with stirring at 0 ° C. After 2 hours at 25 ° C., O- (t-butyldiphenylsilyl) hydroxylamine was added and the mixture was stirred for an additional 4 hours. The crude reaction mixture was applied directly to pad silica gel (15 g) and eluted with 20% ethyl acetate / hexanes to afford the corresponding silyl protected hydroxamic acid (Rf = 0.58, 50% ethyl acetate / hexanes) as a foam. It was treated directly with 10% trifluoroacetic acid in dichloromethane (10 mL) for 4 hours. The solvent was concentrated by rotavap at 50 ° C. and the residue was suspended in ethyl ether (10 mL). The resulting precipitate was filtered to give compound (77) as a white powder (150 mg, 0.365 mmol, 73%).
[416]
[417] The effect of compound (77) on MEL cell differentiation and histone deacetylase activity is shown in Table 2. Compound (77) corresponds to structure 683 in Table 2. As is apparent from Table 2, compound 77 is expected to be a very effective cell differentiating agent.
[418] result
[419] All compounds prepared were tested. Table 2 below shows only the results of testing one compound subgroup. Table 2 was prepared from experiments similar to the experiments described in Examples 7-10 above. The compounds tested were designated by the structure numbers shown in Table 2. The structure numbers are assigned randomly and are independent of the compound numbers used herein.
[420] The results presented in Table 2 demonstrate that the principles of prediction for designing compounds with cell differentiation and HDAC inhibitory activity discussed above herein are generally accurate. Based on the disclosed principles and synthetic schemes, many additional compounds can be readily designed and prepared for testing for cell differentiation and HDAC inhibitory activity.
[421] 11A-F show the effect of selected compounds on affinity purified human epitope-tag HDAC1. In the absence of a substrate, the enzyme preparation was incubated for 20 minutes on ice with the indicated amount of compound to analyze the effect. Substrates (histones from [ ³ H] acetyllabeled murine leukemia cells) were added and samples incubated at 37 ° C. in a total volume of 30 μl for 20 minutes. The reaction was then stopped and the released acetate was extracted to determine the amount of radioactivity released by scintillation counting. This is a variation of the HDAC analysis described in Richon et al., 1988 (39).
[422] Table 2-Inhibition Data of Selected Compounds
[423]
[424]
[425]
[426]
[427]
[428]
[429]
[430]
[431]
[432]
[433]
[434]
[435]

权利要求:
Claims (54)
[1" claim-type="Currently amended] Compounds of the formula: or pharmaceutically acceptable salts thereof

Where
R ₁ and R ₂ are the same or different and each is a hydrophobic moiety;
R ₃ is a hydroxylamine acid, hydroxylamino, hydroxyl, amino, alkylamino, or alkyloxy group;
n is an integer of 3 to 10.
[2" claim-type="Currently amended] The compound of claim 1, wherein each of R ₁ and R ₂ is attached directly or through a linker and is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-. A purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine group.
[3" claim-type="Currently amended] The compound of claim 2, wherein the linker is an amide moiety, —O—, —S—, —NH—, or —CH ₂ —.
[4" claim-type="Currently amended] A compound according to claim 1 having the formula:

Where
Each of R ₄ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, Alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine groups.
[5" claim-type="Currently amended] 5. The compound of claim 4, wherein R ₂ is -amide-R ₅ , and R ₅ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, Thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine group.
[6" claim-type="Currently amended] Compounds of the formula: or pharmaceutically acceptable salts thereof

Where
Each of R ₁ and R ₂ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or branched Alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine groups;
R ₃ is a hydroxylamine acid, hydroxyamino, hydroxyl, amino, alkylamino or alkyloxy group;
R ₄ is hydrogen, halogen, phenyl or cycloalkyl moiety;
A may be the same or different and is an amide moiety, —O—, —S—, —NR ₅ — or —CH ₂ —, wherein R ₅ is substituted or unsubstituted C ₁ -C ₅ alkyl;
n is an integer of 3 to 10.
[7" claim-type="Currently amended] A compound according to claim 6 having the formula:

[8" claim-type="Currently amended] A compound according to claim 6 having the formula:

[9" claim-type="Currently amended] A compound according to claim 6 having the formula:

Where
Each of R ₁ and R ₂ is a substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, t-butyl, aryloxy, arylalkyloxy or pyridine group;
n is an integer of 3-8.
[10" claim-type="Currently amended] 10. The compound of claim 9, wherein the aryl or cycloalkyl group is methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-di Fluoro, 2,4-difluoro, 2,5-difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-tree Fluoro, 2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4 , 5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenyloxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methyloxycarbonyl, methyl A compound characterized in that the aminocarbonyl, dimethylamino, dimethylaminocarbonyl or hydroxylaminocarbonyl group.
[11" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[12" claim-type="Currently amended] 12. The compound of claim 11, wherein n is 5.
[13" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[14" claim-type="Currently amended] 14. Compounds according to claim 13, wherein n is 5.
[15" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[16" claim-type="Currently amended] 16. The compound of claim 15, wherein n is 5.
[17" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[18" claim-type="Currently amended] 18. The compound of claim 17, wherein n is 5.
[19" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[20" claim-type="Currently amended] 20. The compound of claim 19, wherein n is 5.
[21" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[22" claim-type="Currently amended] 22. The compound of claim 21, wherein n is 5.
[23" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[24" claim-type="Currently amended] 24. The compound of claim 23, wherein n is 5.
[25" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[26" claim-type="Currently amended] 26. The compound of claim 25, wherein n is 5.
[27" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[28" claim-type="Currently amended] 28. The compound of claim 27, wherein n is 5.
[29" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[30" claim-type="Currently amended] 30. The compound of claim 29, wherein n is 5.
[31" claim-type="Currently amended] The compound or enantiomer thereof according to claim 6, having the formula:

[32" claim-type="Currently amended] 32. The compound of claim 31, wherein n is 5.
[33" claim-type="Currently amended] A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of any one of claims 1 to 9 and a pharmaceutically acceptable carrier.
[34" claim-type="Currently amended] A method of inhibiting proliferation of tumor cells by selectively inducing terminal differentiation of tumor cells, including contacting the tumor cells with an effective amount of a compound of any one of claims 1 to 9 under appropriate conditions.
[35" claim-type="Currently amended] A method of treating a patient with a tumor characterized by the proliferation of neoplastics, comprising administering to the patient an effective amount of a compound of any one of claims 1 to 9.
[36" claim-type="Currently amended] Compounds of the formula: or pharmaceutically acceptable salts thereof

Where
R ₁ and R ₂ are the same or different and each is a hydrophobic moiety;
R ₅ is —C (O) —NHOH (hydroxyamic acid), —C (O) —CF ₃ (trifluoroacetyl), —NH-P (O) OH—CH ₃ , —SO ₂ NH ₂ ( Sulfonamide), -SH (thiol), -C (O) -R ₆ , wherein R ₆ is a hydroxyl, amino, alkylamino or alkyloxy group;
n is an integer of 3 to 10.
[37" claim-type="Currently amended] 37. The compound of claim 36, wherein each of R ₁ and R ₂ are bonded directly or through a linker, substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6 -Amines, thiazoleamino groups, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine groups.
[38" claim-type="Currently amended] 38. The compound of claim 37, wherein the linker is an amide moiety, -O-, -S-, -NH- or -CH _2- .
[39" claim-type="Currently amended] The compound of claim 36 having the formula:

Where
Each R ₇ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl , Alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine groups.
[40" claim-type="Currently amended] 40. The compound of claim 39, wherein R ₂ is -sulfonamide-R ₈ or -amide-R ₈ wherein R ₈ is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino , 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine group.
[41" claim-type="Currently amended] The compound of claim 39, wherein R ₂ is —NH—C (O) —Y, —NH—SO ₂ —Y, wherein Y is selected from the group consisting of:

[42" claim-type="Currently amended] 40. The compound of claim 39, wherein R ₇ is selected from the group consisting of:

[43" claim-type="Currently amended] Compounds of the formula: or pharmaceutically acceptable salts thereof

R ₁ and R ₂ are the same or different and each is a hydrophobic moiety;
R ₅ is —C (O) —NHOH (hydroxyamic acid), —C (O) —CF ₃ (trifluoroacetyl), —NH-P (O) OH—CH ₃ , —SO ₂ NH ₂ ( Sulfonamide), -SH (thiol), -C (O) -R ₆ , wherein R ₆ is a hydroxyl, amino, alkylamino or alkyloxy group;
L is selected from the group consisting of-(CH ₂ )-, -C (O)-, -S-, -O-,-(CH = CH)-, -phenyl- or -cycloalkyl- or combinations thereof It is a linker.
[44" claim-type="Currently amended] 44. The compound of claim 43, wherein n is 4-7 and m is 1-3.
[45" claim-type="Currently amended] 44. The compound of claim 43, wherein each of R ₁ and R ₂ is attached directly or through a linker and is substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-. A purine-6-amine, thiazoleamino group, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine group.
[46" claim-type="Currently amended] The compound of claim 43, wherein the linker is an amide moiety, —O—, —S—, —NH—, or —CH ₂ —.
[47" claim-type="Currently amended] 44. A compound of claim 43 having the formula:

Where
L is a linker selected from the group consisting of-(CH ₂ )-,-(CH = CH)-, -phenyl- or -cycloalkyl- or combinations thereof;
Each of R ₇ and R ₈ is independently substituted or unsubstituted aryl, cycloalkyl, cycloalkylamino, naphtha, pyridineamino, piperidino, 9-purin-6-amine, thiazoleamino group, hydroxyl, branched or Unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy or pyridine groups.
[48" claim-type="Currently amended] 48. The compound of claim 47, wherein the linker L comprises the following residues:

[49" claim-type="Currently amended] 44. A compound of claim 43 having the formula:

[50" claim-type="Currently amended] A pharmaceutical composition comprising the compound of claim 1, 36 or 43 and a pharmaceutically acceptable carrier.
[51" claim-type="Currently amended] 44. A pharmaceutically acceptable salt of a compound of claim 1, 36 or 43.
[52" claim-type="Currently amended] 44. A prodrug of a compound of claim 1, 36 or 43.
[53" claim-type="Currently amended] A method of inducing differentiation of tumor cells in a tumor, comprising differentiating the tumor cells by contacting the tumor cells with an effective amount of the compound of claim 1, 36, or 43.
[54" claim-type="Currently amended] A method of inhibiting the activity of histone deacetylase, comprising inhibiting the activity of histone deacetylase by contacting histone deacetylase with an effective amount of the compound of claim 1, 36, or 43.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-09-08|Priority to US15275599P

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1999-09-08|Priority to US60/152,755

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2000-06-01|Priority to US20868800P

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2000-06-01|Priority to US60/208,688

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2000-08-24|Application filed by 제임스 에스. 쿼크, 슬로안-케테링인스티튜트퍼캔서리서치, 추후제출, 더 트러스티스 오브 컬럼비아 유니버시티 인 더 시티 오브 뉴욕

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2000-08-24|Priority to PCT/US2000/023232

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2002-07-12|Publication of KR20020059393A

优先权:

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申请号 | 申请日 | 专利标题

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US15275599P| true| 1999-09-08|1999-09-08|

---

US60/152,755|1999-09-08|

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US20868800P| true| 2000-06-01|2000-06-01|

---

US60/208,688|2000-06-01|

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PCT/US2000/023232|WO2001018171A2|1999-09-08|2000-08-24|Novel class of cytodifferentiating agents and histone deacetylase inhibitors, and methods of use thereof|