Histone deacetylases (HDACs) inhibitors are disclosed according to the following structural formula. The moiety A is a benzene ring, optionally substituted. The moiety B is a benzene ring attached at the 1,4 or 1,3 position, or a cyclohexane ring attached at the 1,4 position, optionally substituted. R and Z are further substituents. The HDACs inhibitors possess cytotoxicities to various cancer cell lines. They are useful for treating a tumor associated with deregulation of the activity of histone deacetylases in a subject in need thereof, in one embodiment, the HDACs inhibitors of the invention are useful for treating glioma, breast cancer, colon cancer, target cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promyiocytic leukemia, chronic myelocytic leukemia, or acute lymphocytic leukemia in a subject in need thereof.

FIELD OF THE INVENTION

The present invention relates generally to histone deacetylases inhibitors.

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein

The heterocyclic moiety

is optionally substituted with one or more Raor Rb, or Raand Rb, and is selected from the group consisting of

The moiety

is selected from the group consisting of

in which Rcis optionally present and is hydrogen, halogen, (C1-C6)alkyl, or (C1-C6)alkoxyl;

Y is absent or is selected from the group consisting of —CH2—, —CF2—, —CFH—, —CH═CH—, and —CH2CH2—; and

In another embodiment, the moiety

In another embodiment, a compound or a pharmaceutically acceptable salt thereof according to the invention is as listed in Table 5.

In another aspect, the invention relates to use of a compound or a pharmaceutically acceptable salt thereof according to the invention in the manufacture of a medicament for the treatment of a tumor associated with deregulation of the activity of histone deacetylases in a subject in need thereof. In one embodiment, the tumor is selected from the group consisting of glioma, pancreatic carcinoma, hepatocellular carcinoma, colon tumor, breast tumor, prostate tumor, lymphoma and cutaneous tumor. The cutaneous tumor may be melanomas or basal carcinomas.

In another aspect, the invention relates to use of a compound or a pharmaceutically acceptable salt thereof according to the invention in the manufacture of a medicament for the treatment of glioma, breast cancer, colon cancer, large cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, stomach cancer, liver cancer, ovary adenocarcinoma, pancreas carcinoma, prostate carcinoma, promylocytic leukemia, chronic myelocytic leukemia, or acute lymphocytic leukemia in a subject in need thereof.

In another aspect, the invention relates to use of a compound or a pharmaceutically acceptable salt thereof according to the invention in the manufacture of a medicament for treatment of a disease or a condition wherein inhibition of HDAC provides a benefit.

These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the singular forms “a,” “an” and “the” include plural reference useless the context dearly dictates otherwise.

A dash (“-”) that is not between two letters or symbols is used to indicate a point of attachment for a moiety or substituent. For example, the moiety —CONH2is attached through the carbon atom.

The term “amino” refers to —NH2. The amino group can be optionally substituted as defined herein for the term “substituted.”

The alkyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., alkylene).

The term “alkenyl” refers to a C2-C18hydrocarbon containing normal, secondary, tertiary or cyclic carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp2double bond. Examples include, hut are not limited to: ethylene or vinyl (—CH═CH2), allyl, (—CH2CH═CH2), cyclopentenyl (—C5H7), and 5-hexenyl (—CH2CH2CH2CH2CH═CH2). The alkenyl can be a monovalent hydrocarbon radical, as described and exemplified above, or it can be a divalent hydrocarbon radical (i.e., alkenylene).

The term “alkylene” refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (—CH2—) 1,2-ethylene (—CH2CH2—), 1,3-propylene (—CH2CH2CH2—), 1,4-butylene (—CH2CH2CH2CH2—), and the like.

The term “aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at least one ring is aromatic (e.g., naphtyl, dihydrophenanthrenyl, fluorenyl, or anthryl). Preferred aryls include phenyl, naphthyl and the like. The aryl can optionally be a divalent radical, thereby providing an aryl ene.

The terms “aryloxy” and “arylalkoxy” refer to, respectively, an aryl group bonded to an oxygen atom and an aralkyl group bonded to the oxygen atom at the alkyl moeity. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy.

The term “carboxyl” refers to —COOH.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

As used herein, the term “halogen” or “halo” refer to fluoro, chloro, bromo, and iodo, the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

As used herein, the term “heteroaryl” is defined herein as a monocycle, bicycle, or tricyclic ring system containing one, two, or three aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted. The heteroaryl can optionally be a divalent radical, thereby providing a heteroarylene.

The term “oxo” refers to ═O.

By “(C1-C6)”, it means that all integer unit amounts within the range 1 to 6 are specifically disclosed as part of the invention. Thus, C1, C2, C3, C4, C5, C6, (C1-C2), (C1-C3), (C1-C4), (C1-C5), (C1-C6); (C2-C3), (C2-C4), (C2-C5), (C2-C6); (C3-C4), (C3-C5), (C3-C6); (C4-C5), (C4-C6); and (C5-C6) units amounts are included as embodiments of this invention.

By “(C3-C6)”, means that all integer unit amounts within the range 3 to 6 are specifically disclosed as part of the invention. Thus, C3, C4, C5, C6; (C3-C4), (C3-C5), (C3-C6); (C4-C5), (C4-C6); and (C5-C6) units amounts are included as embodiments of this invention.

Methods of Making Compounds of Formula I

Synthesis

Compounds of formula I were prepared using the following schemes:

EXAMPLES

Steps 1 to 3: Preparation of 7-fluoro-3-phenethylquinazoline-2,4(1H,3H)-dione

4-fluoro anthranilic acid (10.00 g, 63.17 mmol) mixed with phenyl chloroformate (9.6 mL, 75.51 mmol) in dioxane (120 mL) was added dropwise with 1 N NaOH (126 mL) under ice bath for 1 h. The resulting solution was poured to ice water (200 mL) and filtered to get crude solid. Then the solid mixed with EDCI (13.30 g, 69.45 mmol) and HOBt (9.58 g, 69.48 mmol) in dichloromethane (150 mL) was stirred at it for 0.5 h.

Then phenethylamine (8.44 mL, 66.33 mmol) was added to reaction mixture and kept stirring for 4 h. The mixture was evaporated to dry and added with triethylamine (8.67 mL, 62.55 mmol) and DMF (50 mL), and the mixture was irradiated with microwave to reflux for 20 min. The resulting solution was poured to ice water (250 mL) to get solid formed. The solution was filtered and washed with excess of water to give tide compound as beige solid (6.80 g, three steps 37.9%).

Step 4: Preparation of ethyl 4-((7-fluoro-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)benzoate

The beige solid (2.40 g, 8.44 mmol) from previous step suspended in DMF (35 mL) under ice bath was added with NaH (0.46 g, 11.5 mmol) and kept stirring for 1 h. After 1 h, ethyl 4-(bromomethyl)benzoate (4.45 g, 17.57 mmol) was added to the reaction mixture and kept stirring for 9 h from ice bath to room temperature. Alter 9 h. the resulting mixture was poured to ice water (150 mL) and then filtered to get beige solid. The solid was purified by column chromatography eluting with EtOAc/Hexanes=¼ to get title compound as white solid (2.35 g, 62.4%).

Steps 5 and 6: Preparation of N-(benzyloxy)-4-((7-fluoro-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)benzamide

The white solid (1.40 g, 3.14 mmol) from step 4 dissolved in mixture of THF (20 mL) and MeOH (4 mL) was added with aqueous 2.5 M LiOH (5 mL) and stirred at rt for 17 h. After 17 h, the solution was neutralized with 1 N (10 mL) and evaporated to remove most organic solvent. Then the rest solution was extracted with dichloromethane (3×30 mL). The dichloromethane solution dried over MgSO4was evaporated to get white solid. The dried solid was mixed with EDCI (0.66 g, 3.45 mmol) and HOBt (0.47 g, 3.41 mmol) in dichloromethane (30 mL) and stirred at rt for 0.5 h. After 0.5 h, NH2OBn.HCl (0.50 g, 3.13 mmol) and tritely famine (0.48 mL 3.46 mmol) were added to reaction mixture and kept stirring tsar 12 h. After 12 h, the solution was washed with water (3×30 mL) and dried over MgSO4. Then the solution was evaporated and purified by column chromatography eluting with EtOAc/Hexanes=⅔ to get white solid (0.9 g, two steps 54.7%)

Steps 1 to 3: Preparation of 7-chloro-3-phenethylquinazoline-2,4(1H,3H)-dione

Step 4: Preparation of Ethyl 4-((7-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)benzoate

The title compound was prepared from the solid (6.00 g, 19.95 mmol) of previous step by using the similar procedure described above of the step 4 of example 1 to give white solid (6.87 g, 74.4%).

Steps 5 and 6: Preparation of N-(benzyloxy)-4-((7-chloro-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)ethyl)benzamide

The title compound was prepared from the solid (8.00 g, 17.28 mmol) of previous step by using the similar procedure described above of the step 5 and step 6 of example 1 to give white solid (9.01 g, 96.6%).

Step 1: Preparation of 1-(4-iodobenzyl)-3-phenethylquinazoline-2,4(1H,3H)-dione

NaH (0.63 g, 15.75 mmol) was added portionwise to 3-phenethylquinazoline-2,4(1H,3H)-dione (3.5 g, 13.14 mmol) in DMF (0.45 mL), and the solution was stirred under ice bath for 0.5 h. Then 4-iodobenzyl bromide (4.22 z, 13.79 mmol) was added to the above solution and stirred from ice bath to rt for 4 h. The resulting solution was poured to water and filtered to get title compound as white solid. (5.40 g, 85%).

Step 2: Preparation of Ethyl 2-(4-((2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)-2,2-difluoroacetate

Cu (0.1 g, 1.57 mmol) was added to a solution of 1-(4-iodobenzyl)-3-phenethylquinazoline-2,4(1H,3H)-dione (0.3 g, 0.62 mmol) and BrCF2CO2Et (0.08 mL, 0.61 mmol) DMSO (3.00 mL) and stirring at 60° C. for 15 h. The resulting solution was poured to ice water and filtered to get blue solid. The blue solid was purified by column doted with EtOAc/Hexanes (1:4) to get the title compound as white solid (60 mg, 20%).

NH2OH.HCl (3.0 g, 41.9 mmol) suspended in MeOH (14 mL) was added by solution of KOH (2.3 g, 41.0 mmol) dissolved in MeOH (30 mL), and the mixed solution was filtered and added dropwise for 20 mill to solution of ethyl 2,2-difluoro-2-(4-((3,4-dihydro-2,4-dioxo-3-phenethylquinazolin-1(2H)-yl)methyl)phenyl)acetate (1.0 g, 2.1 mmol) under ice bath. The reaction mixture was stirred from ice bath to rt for 11 h. The resulting solution was poured to ice water (150 mL) and filtered to get white solid. The solid was purified by column eluted by MeOH/DCM= 4/96 to get title compound as white solid (0.5 g, 51%).

Step 1: Preparation of (E)-3-(4-((7-fluoro-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)acrylic Acid

1-(4-bromobenzyl)-7-fluoro-3-phenethylquinazoline-2,4(1H,3H)-dione (3.00 g, 6.62 mmol) was mixed With Herrmann's Palladacycle (0.12 g, 0.02 eq), [(t-Bu)3PH]BF4(0.08 g, 0.04 eq), Cy2NMe 97% (1.61 mL, 1.1 eq), acrylic acid (0.45 mL, 1 eq) in DMF (20 mL) under Argon and irradiated with μW 100 W to reflux for 10 min. The resulting solution was filtered by celite and then poured into excess water (100 mL). The mixture solution was neutralized with NaHCO3(aq)to pH 3-4. The precipitation was filtered to get carboxylic acid solid. The crude solid was put to next step without further purification.

Step 2: Preparation of (E)-N-(benzyloxy)-3-(4-((7-fluoro-2,4-dioxo-3-phenethyl-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)acrylamide

The crude carboxylic acid (2.00 g, 4.50 mmol) was mixed with EDCI (1.29 g, 1.5 eq) and HOBt (0.62 g, 1 eq) in DMF (15 mL) and stirred at rt for 30 min. Then NH2OTHP (1 eq) was added and continued stirring for 5-8 hr at rt. The resulting solution was evaporated and extracted with DCM/H2O. The mixture of DCM layer was purified by flash column chromatography (silica gel: ϕ3.5×9.5 cm; eluted by EtOAc/Hexanes=1/1) to get white solid, 1.24 g.

Step 1: Preparation of 3-(2-fluorophenyl)quinazoline-2,4(1H,3H)-dione

Step 2: Preparation of Ethyl 4-((3-(2-fluorophenyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)benzoate

To a solution of ethyl 4-(bromomethyl)benzoate (165.1 g, 1.1 eq) in acetone (1240 mL, 0.5 M), 3-(2-fluorophenyl)quinazoline-2,4(1H,3H)-dione (158.9 g, 620 mmol, 1.0 eq), and K2CO3(258.4 g, 3.0 eq) was added and stirred under 60° C., 1.5 h. The crude mixture was concentrated in vacuo and extracted with DCM/H2O=1.5 L/1.5 L. The organic layer was dried over MgSO4 and evaporated under vacuum till little precipitate was observed, Et3O (500 mL) and pentane (250 mL) was added and filtrated. The solid was washed with pentane and dried under vacuum to provide white fine solid product 232.4 g, 93%

The following compounds were prepared according to the procedure given in abo e Examples.

Step 1: Preparation of Ethyl 4-((3,4-dihydro-7-hydroxy-2,4-dioxo-3-phenethylquinazolin-1(2H)-yl)methyl)benzoate

Example 148. Enzymatic Assay

The IC50values for aforementioned compounds against HDACs were determined. HDAC 1 to 11 can be assayed by using acetylated AMC-labeled peptide substrate. The Substrate 1, a fluorogenic peptide from p53 residues 379-382 (RHKKAc) is used for all MAC 1 to 11 but HDAC8, which has a substrate II (RHKAcKAc), a fluorogenic diacyl peptide based on residues 179-382 of p53. Compounds were tested in 10-dose 1050 mode in duplicate with 3-fold serial dilution starting at 10 μM.

Human HDAC1, Human HDAC2, Human HDAC3/NcoR2, Human HDAC6, Human HDAC10, and Human HDAC11 were all expressed by baculovirus expression system in Sf9 cells.

Materials

Tables 1 and 2 illustrate HDAC inhibition activities of the compounds according to the invention.

Cells were seeded in 96-well plates at a density of 5×103cells per well and allowed to attach for 24 h before compound treatment. A series dilutions of the testing compounds, SAHA, and Tubastatin A were added to the culture medium so that the final concentration of DMSO) was 0.1% in all reactions. At end of the treatment period 72 h, 20 μl (5 mg/mL) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reagent was added each well. After 4 h incubation at 37° C., the supernatant was aspirated and the formazan crystals were dissolved in 100 μl of DMSO at 37° C. for 10 min with gentle agitation. The absorbance was measured at 570 nm using the Molecular Devices Microplate Reader. Results were presented as mean±standard error mean (SEM) from at least three independent experiments. IC50values were calculated from the relative viability values and concentrations by regression analysis. Tables 3 and 4 show the cytotoxicity of HDAC inhibitors against cancer cell lines and normal human cell line.

Xenografted nude mice model of prostate cancer cell line. Forty-five male nude mice (BALB/cAnN.Cg-Foxnlnu/CrINarl, 4-6 weeks old) were used. Mice were subcutaneously injected in the left flank with 1×106LNCaP human prostatic carcinoma in PBS through a 1 cm long 25 G needle. When LNCaP xenografts had reached an average volume of 150 mm3, animals were randomized into groups of five mice. Compounds were prepared in PBS daily fresh and injected i.p. (10 mL/kg of body weight). The tumor sizes were measured by two perpendicular diameters (Length and Width) and tumor volumes (me) were determined by the formula length×width2×1/2. The mouse body weight was determined as an indicator of tolerability on the same days. TGI was calculated according to the formula [1−(T−T0)/(C−C0)]×100, where T and T0 are the mean tumor volumes on Day 30 and Day 1, respectively, for the experimental group, and C and C0 are those for the vehicle control group. After 14 or 30 days of treatment, the animals were sacrificed on Day 30 by cervical dislocation. Tumor samples were harvested from animals and post-fixed in 4% paraformaldehyde and weighted. Animal studies were carried out in accordance with the guidelines.

LL/2 syngeneic xenograft model. The 7 weeks old B6 mice were injected s.c. with 100 μL of LL/2 cell suspensions, equivalent to 5×106cells. Paclitaxel was treated via i.p. injection at 10 mg/kg once daily for five days. AJ20064 was dosed orally, at 20 mg/kg, 40 mg/kg and 80 mg/kg once daily for 21 consecutive days. Tumor volumes were measured twice weekly throughout the duration of the experiment and tumor growth inhibition (TGI) was assessed at the end of the third cycle of therapy.