Title of Invention

ASCOCHLOPvIN DERIVATIVE AND USE THEREOF AS AMPK ACTIVATOR

Technical Field

[0001 ] The present invention is directed to a novel ascochlorin derivative which activates adenosine monophosphate-activated protein kinase (AMPK), and is useful in the treatment or prevention of diseases or conditions that involve AMPK dysregulation, such as cancer, type 2 diabetes, metabolic syndrome-associated diseases, and Alzheimer disease. The invention is also directed to a pharmaceutical composition comprising the compound for the treatment or prevention of diseases or conditions that involve AMPK dysregulation.

Background Art

[0002] The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered as a master switch of glucose and lipid metabolisms in various organs, especially in skeletal muscle, liver and fat tissue.

[0003] AMPK is also known to have critical roles in regulating cell growth and autophagy through inhibition of m-TOR, as well as mitochondrial biogenesis [Physiological Rev. 2009, vol. 89, page 1025, Nature Reviews/Molecular Cell Biology, 2012, vol.13 page 251, Nature Cell Biology, 2011, vol.13 (9),1016, Molecular and Cellular Endocrinology, 2013 vol. 366, page 152, J. Physiol., 2006, vol 574.1 page 33.].

[0004] When intracellular ATP levels becomes lower, AMP or ADP can directly bind to the γ regulatory subunit of AMPK, leading to a conformational change that promotes AMPK phosphorylation at Thrl72 by LKB1 (known as a tumor suppressor) or CAMKK2 (Ca ⁺⁺ dependent kinase). Further, the conformational change also protects AMPK from dephosphorylation by protein phosphatases 2A and C so that AMPK remains activated.

[0005] The dysregulation of AMPK is considered as a pathogenic factor for the diseases such as cancer, type 2 diabetes, metabolic syndrome-associated diseases (dyslipidemia, adiposity, atherosclerotic cardiovascular disease, hypertension, and nonalcoholic fatty liver disease (NAFLD)), inflammation and Alzheimer disease, and thus AMPK is considered as a target for the prevention and therapy of these diseases [metabolic syndrome: Science in Medicine, 2013, Vol. 123 (7) page 2764, Molecular and Cellular Endocrinology, 2013, vol. 366 page 135; cancer and inflammation: Oncotarget, 2015, vol. 6 (10), page 7365;

cardiovascular disease: Circ Res., 2009 vol. 104 (3), page 282; inflammation, Diabetologia, 2010 vol. 53, page 2406, Am. J. Respir. Cell Mol. Biol, 2010, vol 42, page 676; Alzheimer disease: J Alzheimer s Dis. 2009, vol. 16(4) page 731].

[0006] Metformin is known as an antihyperglycemic agent to indirectly activate AMPK through inhibition of respiratory chain complex 1 [Clinical Science, 2012, vol. 122, page 253]. Metformin, however, is reported to activate AMPK in HepG2 cell only at surprisingly high concentration of mM level for in vitro cellular experiment.

[0007] 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) is known as an AMP mimic. AICAR directly activates AMPK through binding to a regulatory γ-subunit to promote phosphorylation of AMPK and to protect AMPK from dephosphorylation. A- 769662 is reported to be a direct AMPK-activator with a different binding mode [Cell Metabolism, 2006, vol. 3, page 403]. There are other AMPK-activators reported in literatures, but none of them is clinically used up to now, partly because of the insufficient in vivo efficacy and/or safety profile.

Therefore, novel AMPK activators with good efficacy and safety profile would provide therapeutic benefits to the patients with diseases that involve AMPK dysregulations.

[0008] 4-O-Methy-ascochlorin (MAC) is a derivative of a fungal metabolite of ascochrolin, which is reported to have ability for activating AMPK in HepG2 cells [Biochemical and Biophysical Research Communications, 2011, vol. 406, page 353]. 4-O-Substituted ascochlorin derivatives including MAC are described as an antidiabetic agent in JP H06- 305959 A (published in 1994), and prodrugs of MAC that are Schiff base derivatives formed with natural amino acids are described in WO2004/074236. However, the development of any ascochrolin derivatives has not become successful.

Citation List Patent Documents

[0009] Patent Document 1 : JP H06-305959 A;

Patent Document 2: WO 2004/074236

Non-Patent Documents

[0010] Non-Patent Document 1 : Physiological Rev. 2009, vol. 89, page 1025,

Non-Patent Document 2 : Nature Reviews/Molecular Cell Biology, 2012, vol.13 page 251,

Non-Patent Document 3: Nature Cell Biology, 201 1, vol.13 (9), 1016,

Non-Patent Document 4: Molecular and Cellular Endocrinology, 2013 vol. 366, page 152,

Non-Patent Document 5: J. Physiol., 2006, vol 574.1 page 33

Non-Patent Document 6: Science in Medicine, 2013, 123 (7), 2764;

Non-Patent Document 7: Mol. Cell. Endocrinol., 2013, 366; 135;

Non-Patent Document 8 : Oncotarget, 2015 , 6 ( 10), 7365 ;

Non-Patent Document 9: Circ Res., 2009, 104 (3), page 282;

Non-Patent Document 10: Diabetologia, 2010, 53, 2406;

Non-Patent Document 11 : Am. J. Respir. Cell Mol. Biol, 2010, 42, 676;

Non-Patent Document 12: J Alzheimers Dis. 2009, 16(4), 731 ;

Non-Patent Document 13: Clinical Science, 2012, 122, 253;

Non-Patent Document 14: Cell Metabolism, 2006, 3, 403.

Non-Patent Document 15 : Biochemical and Biophysical Research Communications, 2011, vol. 406, page 353

Summary of Invention

Technical Problem

[0011] The present inventors have found that MAC has critical drawbacks as a therapeutic agent in the physicochemical and pharmacokinetic profiles. Namely, MAC has very low water-solubility (0.07μΜ in pH7.4 phosphate buffer) and extremely low metabolic stability that was confirmed in an assay using the liver microsomes of human and mouse in the presence of NADPH in vitro (only 6% of MAC remained after lhr incubation).

[0012] Regarding the Schiff base derivatives of MAC described in WO2004/074236, the inventors found that these compounds are extremely labile in acidic pH solution. A representative compound (compound #13 in WO2004/074236) which is a Schiff base of MAC with glycine amide was confirmed to be almost instantly hydrolyzed to MAC at pH 4.0 phosphate buffer solution (the pH similar to that in human stomach after meal) at room temperature. Therefore, the Schiff base derivatives of MAC have the same problem in the metabolic stability as the parent compound, MAC.

Solution to Problem

[0013] The present inventors have conducted diligent studies in light of the problems described above and consequently completed the present invention by finding a number of ascochlorin derivatives, which are confirmed to have AMPK activation profile, and significantly improved metabolic stability as compared with MAC. Particularly, the compounds of the present invention also show good in vivo efficacy in several disease models especially for oncology and metabolic diseases.

[0014] The present invention is directed to an ascochlorin derivative which activates adenosine monophosphate-activated protein kinase (AMPK), and that is useful in the treatment or prevention of diseases or conditions that involve AMPK dysregulation, such as cancer, type 2 diabetes, metabolic syndrome-associated diseases, and Alzheimer disease. The invention is also directed to a pharmaceutical composition comprising the compound, and the use of the compound for the treatment or prevention of diseases or conditions that involve AMPK dysregulation.

[0015] The present invention relates to a new ascochlorin derivative that is useful as an activator of AMP-activated protein kinase.

[0016] According to one aspect of the present invention, there is provided the following inventions:

[0017] ( 1 ) A compound represented by formula I :

wherein

R ¹ is formyl, or -CH=N-0-Y, in which Y is a hydrogen atom or Ci _-6 alkyl;

R ² is C\4 alkyl, in which the alkyl may be substituted with one to five fluorine atoms;

R ³ is a hydrogen atom or Ci _-6 alkyl;

R ⁴ is hydroxy, Ci _-6 alkylamino, C _3-7 cycloalkylamino, or 3- to 7-membered heterocycloalkylamino which contains -0-, -S-, -NR ⁶ -, -SO- or -S0 ₂ - as a ring atom, or R ³ and R ⁴ together with the carbon atom to which they are attached form >C=N-0-Z;

Z is a hydrogen atom, Ci _-6 alkyl, -CO(CH ₂ ) _n -R ⁵ or -(CH ₂ ) _n -R ⁵ ;

n is an integer selected from 1 to 4;

R ⁵ is -C0 ₂ R ⁶ , -CONR ⁷ R ⁸ , -OCONR ⁷ R ⁸ , -S0 ₂ NR ⁷ R ⁸ , -S0 ₂ R ⁹ , hydroxy, -NHS0 ₂ R ⁹ , or -NR ⁷ R ⁸ ;

R ⁶ , R ⁷ and R ⁸ are each independently selected from a group consisting of a hydrogen

7 &

atom, C _1-6 alkyl, and C _3-7 cycloalkyl; or R and R together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclic ring which may further contain - 0-, -S-, -NR ⁶ -, -SO- or -S0 ₂ - as a ring atom, in which the heterocyclic ring may be substituted with one or more substituents selected from hydroxy and Ci _-6 alkyl, and

R ⁹ is C _1-6 alkyl, or C _3-7 cycloalkyl;

a pharmaceutically acceptable salt or a solvate thereof.

[0018] (2) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (1), wherein R ⁴ is selected from the group consisting of hydroxy, methylamino, ethylamino, cyclopropylamino, and oxetan-3-ylamino.

[0019] (3) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (1), wherein R ³ and R ⁴ together with the carbon atom to which they are attached form >C=N-0-Z in which Z is selected from the group consisting of a hydrogen atom, Ci. ₆ alkyl, -CH ₂ C0 ₂ H, -CH ₂ C0 ₂ R ⁵ , morpholinoethyl, piperazinylethyl, N-methyl- piperazinylethyl, and N,N-dimethylamino-ethyl.

[0020] (4) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (2) or (3), wherein R ¹ is formyl.

[0021] (5) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (2) or (3), wherein R ¹ is -CH=N-OH.

[0022] (6) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (2) or (3), wherein R ¹ is -CH=N-OMe.

[0023] (7) The compound, a pharmaceutically acceptable salt or a solvate thereof according to (1 ), which is selected from the group consisting of

3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R)-3-hydroxy-l,2,3, 6- tetramethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methox y-2-methylbenzaldehyde,

3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxyi mino)-l,2,6- trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde,

(E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxy imino)-l,2,6- trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde oxime,

3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2R ,6R,E)-3- (hydroxyimino)- 1 ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien- 1 -yl)-2- methylbenzaldehyde oxime,

3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R ,E)-3- (hydroxyimino)- 1 ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien- 1 -yl)-2- methylbenzaldehyde,

[(lE,2R,3R,4R)-3-[(lE,3E)-5-(3-chloro-5-formyl-6-hydroxy-2-m ethoxy-4- methylphenyl)-3 -methylpenta- 1 ,3 -dien- 1 -yl]-2 ,3 ,4-trimethylcyclohexylidene] amino 2- (dimethylamino)acetate,

(E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3S,6R)-3-hydroxy -l,2,3,6- tetramethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methox y-2-methylbenzaldehyde oxime,

(E)-3-chloro-6-hydroxy-4-methoxy-5-((2E,4E)-5-((lR,2R,6R,E)- 3-(methoxyimino)- 1 ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien-l -yl)-2-methylbenzaldehyde O-methyl oxime,

(E)-3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2 R,6R,E)-3- (methoxyimino)- 1 ,2,6-trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-2- methylbenzaldehyde O-methyl oxime,

(E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxy imino)-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methoxy- 2-methylbenzaldehyde O- methyl oxime,

(E)-3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2 R,6R,E)-3- (hydroxyimino)-l,2,6-trimethylcyclohexyl)-3-methylpenta-2,4- dien-l-yl)-2- methylbenzaldehyde O-methyl oxime,

3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-((2E,4E)-3-m ethyl-5- (( 1 R,2R,6R,E)- 1 ,2,6-trimethyl-3 -(2-mo holinoethoxyimino)cyclohexyl)penta-2,4-dien- 1 - yl)benzaldehyde,

3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(cyclopropylamino)-l,2,6 - trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-(fluoromethoxy)-6-hydroxy-2- methylbenzaldehyde,

(E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3S,6R)-3-hydroxy -l,2,3,6- tetramethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde O- methyl oxime,

(E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R,E)-3-(2-(dimethylamino) ethoxyimino)-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde O-methyl oxime,

(E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5-((lR,2R,6R,E)- l,2,6-trimethyl-3-(2-morpholinoethoxyimino)cyclohexyl)penta- 2,4-dien-l-yl)benzaldehyde O-methyl oxime, (E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5-((lR,2R,6R,E)- 1 ,2,6-trimethyl-3-(2-(4-methylpiperazin-l -yl)ethoxyimino)-cyclohexyl)penta-2,4-dien-l - yl)benzaldehyde O-methyl oxime,

ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methox y-5-[(lE)- (methoxyimino)methyl]-4-methylphenyl]-3-methylpenta-l ,3-dien-l -yl]-2,3,4- trimethylcyclohexylidene]amino]oxy)acetate,

2- ([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy- 5-[(lE)- (methoxyimino)methyl]-4-methylphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]-2,3 ,4- trimethylcyclohexylidene]amino]oxy)acetic acid,

3- chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(ethylamino)-l,2,6-trimeth ylcyclohexyl)-3- methylpenta-2,4-dien- 1 -yl)-6-hydroxy-4-methoxy-2-methylbenzaldehyde,

(E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(ethylamino)-l,2,6-t rimethylcyclohexyl)- 3 -methylpenta-2,4-dien- 1 -yl)-6-hydroxy-4-methoxy-2-methylbenzaldehyde O-methyl oxime,

3-chIoro-5-((2E,4E)-5-((lR,2R,6R)-3-(cyclopropylamino)-l, 2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde,

(E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(cyclopropylamino)-l ,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien- 1 -yl)-6-hydroxy-4-methoxy-2- methylbenzaldehyde O-methyl oxime,

2-[(2E,4E)-5-[(lR,2R,6R)-3-amino-l,2,6-trimethylcyclohexyl]- 3-methylpenta-2,4- dien- 1 -yl]-4-ehloro-3 -methoxy-6-[( 1 E)-(methoxyimino)methyl]-5-methylphenol,

(E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5-((lR,2R,6R)- l,2,6-trimethyl-3-(oxetan-3-ylamino)cyclohexyl)penta-2,4-die n-l-yl)benzaldehyde O-methyl oxime,

2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-fluoro methoxy-5-formyl-4- methylphenyl] -3 -methylpenta- 1 ,3-dien- 1 -yl] -2,3 ,4-trimethylcyclohexylidene] - amino]oxy)acetic acid,

2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methox y-5-[(lE)- (hydroxyimino)methyl]-4-methylphenyl]-3-methylpenta-l ,3-dien-l -yl]-2,3,4- trimethylcyclohexylidene]amino]oxy)acetic acid,

2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-methoxy-6-hydrox y-5-[(lE)- (hydroxyimino)methy 1] -4-methy lphenyl] -3 -methy lpenta- 1 ,3 -dien- 1 -yl] -2 ,3 ,4- trimethylcyclohexylidene] amino] oxy)acetic acid,

and pharmaceutically acceptable salts thereof.

[0024] (8) A method for preparing a compound represented by formula I,

a pharmaceutically acceptable salt or a solvate thereof, wherein R is formyl, and R , R , and R ⁴ are as defined in (1), which comprises:

i) converting a compound represented by formula VIII:

(VIII)

wherein R ¹⁰ is independently selected from Ci _-6 alkyl, and R ² is as defined above, to a compound represented by formula XXII:

(xxii);

10 2

wherein R is independently selected from Ci _-6 alkyl, and R is as defined above; and

ii) converting a compound represented by formula VIII to a compound represented by formula I.

[0025] (9) A method for preparing a compound represented by formula I,

a pharmaceutically acceptable salt or a solvate thereof, wherein R is -CH=N-0-Y, and R , R ³ , R ⁴ and Y are as defined in (1), which comprises:

i) converting a compound represented by formula IV: (IV)

10 2

wherein R is independently selected from C _1-6 alkyl, and R and Y are as defined above, to a compound represented by formula I.

[0026] (10) A compound represented by formula VIII:

wherein R 10 is independently selected from Ci _-6 alkyl, and R 2 is C _1-6 alkyl that is substituted with one to five fluorine atoms;.

[0027] (1 1) A compound represented by the formula IV: (IV)

wherein R is a hydrogen atom or Ci _-6 alkyl that may be substituted with one to five fluorine atoms; and Y is a hydrogen atom or Ci _-6 alkyl.

[0028] (12) A pharmaceutical composition comprising a compound according to any one of (1) to (7) and a pharmaceutically acceptable carrier.

[0029] (13) The pharmaceutical composition according to (12), for use in treatment of cancer, diabetes, dyslipidemia, hypercholesterolemia, obesity and a fatty liver condition, a cardiovascular disease, atherosclerosis or Alzheimer's disease.

[0030] (14) The pharmaceutical composition according to ( 12) or ( 13 ), further comprising a therapeutic agent for treatment of cancer, diabetes, dyslipidemia,

hypercholesterolemia, obesity and a fatty liver condition, a cardiovascular disease, atherosclerosis or Alzheimer's disease.

[0031 ] (15) A method for treating a disease that involves AMPK dysregulation in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound according to any one of (1) to (7).

[0032] (16) The method according to (15), wherein the disease is selected from cancer, type 2 diabetes, and dyslipidemia.

Advantageous Effects of Invention

[0033] The compound of the present invention is useful as a preventive or therapeutic agent for the disease in which AMPK is dysregulated; such as metabolic syndrome, type 2 diabetes, dyslipidemia, adiposity, atherosclerotic cardiovascular disease, hypertension, and

nonalcoholic fatty liver disease (NAFLD), inflammation and Alzheimer disease.

Furthermore, the compound of the present invention is useful as a preventive or therapeutic agent for cancers such as hepatoma, glioma, breast, prostate and non-small cell lung carcinoma and cancer-associated inflammation. Examples of the cancers include cancers that involve activation of PI3K/AKT/mTOR pathway and/or loss of tumor suppressor LKBl, such as breast cancer (e.g. triple negative breast cancer), hepatoma (especially sorafenib resistant HCC), colon cancer, prostate cancer, glioma and non-small cell lung carcinoma [Oncotarget, vol. 6 (10), page 7365, 2015].

Brief Description of The Drawings

[0034] Figure 1 shows the results of western blot analysis to indicate AMPK activation and downstream signal, ACC in HepG2 cell.

Figure 2 shows pharmacokinetic profile of Compound 19 in SD rats.

Figure 3 shows the results of assay indicating the effect of Compound 5 on the level of triglyceride in blood of HFD-STZ model mice. Figure 4 shows the results of assay indicating the effect of the compounds of the present invention on the level of total cholesterol in blood of mice.

Figure 5 shows the result of assay indicating the effect of Compound 5 on the blood glucose level of mice (HFD-STZ model) in a fasting state.

Figure 6 shows the result of in vivo assay using human glioma, U87MG xenograft model for assessing anti-cancer activity of Compound 5.

Figure 7 shows the result of in vivo assay using human hepatocellular carcinoma, Hep3B xenograft model for assessing anti-cancer activity of Compound 5 in combination with sorafenib.

Description of Embodiments

[0035] In the subject specification, the term "C _1-6 alkyl" refers to a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Examples thereof include methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl , pentan-3-yl and the like.

[0036] The term "C _1- alkylamino" means a group: -NH-(C _1-6 alkyl), in which C _1-6 alkyl is as defined above.

[0037] The term "C _3-7 cycloalkyl" refers to a saturated carbocyclic group having 3 to 7 carbon atoms. Examples thereof include cyclopropyl (cPr), cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.

[0038] The term "C _3-7 cycloalkylamino" means a group: -NH-(C ₃ . ₇ cycloalkyl), in which "C _3- 7 cycloalkyl" is as defined above. Examples thereof include cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino and the like.

[0039] The term "3- to 7-membered heterocycloalkylamino" refers to an amino group substituted with a saturated heterocyclic group having 3 to 7 ring atoms, which includes one or more hetero atoms selected from -0-, -S-, -NR ⁶ -, -SO- and -S0 ₂ -. Examples thereof include pyrrolidinylamino, piperidinylamino, piperazinylamino, N-methylpiperazinylamino, morpholinylamino, thiomo holinylamino, imidazolidinylamino, homopiperizynylamino, homopiperazinylamino, and the like.

[0040] The term "a nitrogen-containing 3- to 7-membered heterocyclic ring" refers to a heterocyclic ring having 3 to 7 ring atoms, which includes at least one nitrogen atom and one or more hetero atoms selected from -0-, -S-, -NR. ⁶ -, -SO- and -S0 ₂ -. Examples thereof include pyrrolidine, piperidine, piperazine, N-methylpiperazine, morphorine, thiomorpholine, imidazolidine, imidazoline, homopiperidine, homopiperazine and the like.

[0041] In one embodiment of the present invention, R ¹ is selected from aldehyde, -CH=N- OH, -CH=N-OMe, and -CH=N-OEt.

[0042] In another embodiment of the present invention, R is Ci _-6 alkyl which may be substituted with 1 to 3 fluorine atoms, for example, CH ₃ , CH ₂ CH ₃ , CHF ₂ , CH ₂ F, CF ₃ , and CH ₂ CF ₃ , preferably CH ₃ or CH ₂ F.

[0043] In another embodiment of the present invention, R is selected from hydrogen, methyl and ethyl.

[0044] In another embodiment of the present invention, R ⁴ is selected from a group consisting of hydroxy, methylamino, ethylamino, cyclopropylamino, oxetan-3-ylamino, (3- morpholinopropyl)amino, pirrolidin-3-ylamino or (3-(2-methIypipelidin-l-yl)propyl)amino. Preferably, R ⁴ is selected from hydroxyl, ethylamino, cyclopropylamino, and oxetan-3- ylamino.

[0045] In another embodiment of the present invention, R ³ and R ⁴ form a group =N-0-Z, in which Z is a hydrogen atom, C _1-6 alkyl or,-(CH ₂ ) _n -R ⁵ , and R ⁵ is as defined above. Examples of the group =N-0-Z include hydroxyimino, methoxyimino, (2-(N,N- dimethylamino)ethoxy)imino, (2-morpholinoethoxy)imino, (2-(4- methylpiperazinyl)ethoxy)imino, (ethoxycarbonylmethoxy)imino, (carboxymethoxy)imino, ((N,N-dimethylamino)acetoxy)imino.

[0046] Specifically, examples of the present invention include the compounds

(Compound 1 to 28) indicated in Table 1.

[Table 1]

-CH=N-OMe (E-

23 Me H

form) H

-CH=N-OMe (E-

24 Me H NH ₂

form)

-CH=N-OMe (E-

25 Me H

form) H

26 -CHO CH ₂ F =N-OCH ₂ -C0 ₂ H (E-form)

27 -CH=N-OH (E-form) CH ₂ F =N-OCH ₂ -C0 ₂ H (E-form)

28 -CH=N-OH (E-form) Me =N-OCH ₂ -C0 ₂ H (E-form)

[0047] More specifically, examples of the present invention include the following compounds:

1) 3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R)-3-hydroxy-l,2,3, 6- tetramethyIcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde

2) 3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxyimin o)- 1,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methoxy- 2-methylbenzaldehyde;

3) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxy imino)-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methoxy- 2-methylbenzaldehyde

4) (E)-3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2 R,6R,E)-3- (hydroxyimino)-l ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien-l -yl)-2- methylbenzaldehyde oxime;

5) 3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R ,E)-3- (hydroxyimino)-l,2,6-trimethylcyclohexyl)-3-methylpenta-2,4- dien-l-yl)-2- methylbenzaldehyde;

6) [(lE,2R,3R,4R)-3-[(lE,3E)-5-(3-chloro-5-formyl-6-hydroxy-2-m ethoxy-4- methylphenyl)-3-methylpenta-l ,3-dien-l -yl] -2,3, 4-trimethylcyclohexylidene] amino 2- (dimethylamino)acetate;

7) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R)-3-hydroxy- 1,2,3,6- tetramethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehydi oxime; 8) (E)-3-chIoro-6-hydroxy-4-methoxy-5-((2E,4E)-5-((lR,2R,6R,E)- 3-(methoxyimino)- 1 ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien-l -yl)-2-methylbenzaldehyde O-methyl oxime;

9) (E)-3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2 R,6R,E)-3- (methoxyimino)-l ,2,6-trimethylcyclohexyl)-3-methylpenta-2,4-dien-l -yl)-2- methylbenzaldehyde O-methyl oxime;

10) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R,E)-3-(hydroxy imino)-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methoxy- 2-methylbenzaldehyde O- methyl oxime;

11) (E)-3-chloro-4-(fluoromethoxy)-6-hydroxy-5-((2E,4E)-5-((lR,2 R,6R,E)-3- (hydroxyimino)- 1 ,2,6-trimethylcycIohexyl)-3-methylpenta-2,4-dien- 1 -yl)-2- methylbenzaldehyde O-methyl oxime;

12) 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-((2E,4E)-3-m ethyl-5-

(( 1 R,2R,6R,E)- 1 ,2,6-trimethyl-3 -(2-morpholinoethoxyimino)cyelohexyl)penta-2,4-dien- 1 - yl)benzaldehyde;

13) 3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(cyclopropylamino)-l,2,6 - trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-(fluorom ethoxy)-6-hydroxy-2- methylbenzaldehyde ;

14) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,6R)-3-hydroxy-l, 2,3,6- tetramethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde O- methyl oxime;

15) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R,E)-3-(2-(dimethylamino) ethoxyimino)-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde O-methyl oxime;

16) (E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5- ((lR,2R,6R,E)-l,2,6-trimethyl-3-(2-morpholinoethoxyimino)cyc lohexyl)penta-2,4-dien-l - yl)benzaldehyde O-methyl oxime;

17) (E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5- (( 1 R,2R,6R,E)- 1 ,2,6-trimethyl-3 -(2-(4-methylpiperazin- 1 -yl)ethoxyimino)cyclohexyl)penta- 2,4-dien-l-yl)benzaldehyde O-methyl oxime;

18) ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methox y-5-[(lE)- (methoxyimino)methyl] -4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl] -2 ,3 ,4- trimethylcyclohexylidene] amino] oxy)acetate ;

19) 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methox y-5-[(lE)- (methoxyimino)methyl]-4-methylphenyl]-3-methylpenta-l,3-dien -l-yl]-2,3,4- trimethylcyclohexylidene] amino] oxy)acetic acid;

20) 3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(ethylamino)-l,2,6-trime thylcyclohexyl)-3- methylpenta-2,4-dien-l-yl)-6-hydroxy-4-methoxy-2-methylbenza ldehyde;

21) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(ethylamino)-l,2,6-t rimethylcyclohexyl)-

3- methylpenta-2,4-dien-l-yl)-6-hydroxy-4-methoxy-2-methylbenza ldehyde O-methyl oxime;

22) 3-chloro-5-((2E,4E)-5-(( 1 R,2R,6R)-3-(cyclopropylamino)-l ,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methy lbenzaldehyde ;

23) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,6R)-3-(cyclopropylamino)-l ,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methy lbenzaldehyde O-methyl oxime;

24) 2-[(2E,4E)-5-[(lR,2R,6R)-3-amino-l,2,6-trimethylcyclohexyl]- 3-methylpenta-2,4- dien- 1 -yl] -4-chloro-3 -methoxy-6-[( 1 E)-(methoxyimino)methyl] -5-methylphenol;

25) (E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5-((lR,2R,6R)-

1 ,2,6-trimethyl-3-(oxetan-3-ylamino)cyclohexyl)penta-2,4-dien - 1 -yl)benzaldehyde O-methyl oxime;

26) 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-fluoro methoxy-5-formyl-

4- methylphenyl] -3 -methylpenta- 1 , 3 -dien- 1 -y 1] -2,3,4- trimethylcyclohexylidene]amino]oxy)acetic acid;

27) 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-fluoro methoxy-5-[(lE)- (hydroxyimino)methyl] -4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl] -2,3 ,4- trimethylcyclohexylidene]arnino]oxy)acetic acid;

28) 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-methoxy-6-hydrox y-5-[(lE)- (hydroxyimino)methyl] -4-methylpheny 1] -3 -methy lpenta- 1 ,3 -dien- 1 -yl] -2 ,3 ,4- trimethylcyclohexylidene] amino] oxy)acetic acid;

and pharmaceutically acceptable salts thereof.

[0048] Examples of the present invention also include the following compounds:

7-1) (E)-3-chIoro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3S,6R)-3-hydroxy -l,2,3,6- tetramethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methox y-2-methylbenzaldehyde oxime;

7-2) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3R,6R)-3-hydroxy -l,2,3,6- tetramethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde oxime;

13-1) 3-chloro-5-((2E,4E)-5-(( 1 R,2R,3S,6R)-3-(cyclopropylamino)- 1 ,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-(fluorom ethoxy)-6-hydroxy-2- methylbenzaldehyde ;

13- 2) 3-chloro-5-((2E,4E)-5-((lR,2R,3R,6R)-3-(cyclopropylamino)-l, 2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-(fluorom ethoxy)-6-hydroxy-2- methylbenzaldehyde;

14- 1) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3S,6R)-3-hydroxy -l, 2,3,6- tetramethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-4-methox y-2-methylbenzaldehyde O- methyl oxime;

14-2) (E)-3-chloro-6-hydroxy-5-((2E,4E)-5-((lR,2R,3R,6R)-3-hydroxy -l,2,3,6- tetramethylcyclohexyl)-3-methylpenta-2,4-dien- 1 -yl)-4-methoxy-2-methylbenzaldehyde O- methyl oxime;

20- 1 ) 3 -chloro-5 -((2E,4E)-5 -(( 1 R,2R,3 S,6R)-3 -(ethylamino)- 1 ,2,6-trimethylcyclohexyl)-

3 -methylpenta-2 ,4-dien- 1 -y l)-6-hydroxy-4-methoxy-2 -methy lbenzaldehyde ;

20-2) 3-chloro-5-((2E,4E)-5-((lR,2R,3R,6R)-3-(ethylamino)-l,2,6-tr imethylcyclohexyl)-

3-methylpenta-2,4-dien-l-yl)-6-hydroxy-4-methoxy-2-methyl benzaldehyde; 21-1) (E)-3 -chloro-5-((2E,4E)-5 -(( 1 R,2R,3 S,6R)-3 -(ethylamino)- 1,2,6- trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-6-hydroxy-4-methoxy-2- methylbenzaldehyde O-methyl oxime;

21- 2) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,3R,6R)-3-(ethylamino)-l,2, 6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde O-methyl oxime;

22- 1 ) 3-chloro-5-((2E,4E)-5-(( 1 R,2R,3 S,6R)-3-(cyclopropylamino)- 1 ,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde;

22- 2) 3-chloro-5-((2E,4E)-5-(( 1 R,2R,3R,6R)-3-(cyclopropylamino)- 1 ,2,6- trimethylcyclohexyl)-3 -methylpenta-2,4-dien- 1 -yl)-6-hydroxy-4-methoxy-2- methylbenzaldehyde;

23- 1) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,3S,6R)-3-(cyclopropylamino )-l,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde O-methyl oxime;

23- 2) (E)-3-chloro-5-((2E,4E)-5-((lR,2R,3R,6R)-3-(cyclopropylamino )- 1,2,6- trimethylcyclohexyl)-3-methylpenta-2,4-dien-l-yl)-6-hydroxy- 4-methoxy-2- methylbenzaldehyde O-methyl oxime;

24- 1) 2-[(2E,4E)-5-[(lR,2R,3S,6R)-3-amino-l,2,6-trimethyIcyclohexy l]-3-methylpenta- 2,4-dien-l-yl]-4-chloro-3-methoxy-6-[(lE)-(methoxyimino)meth yl]-5-methylphenol;

24- 2) 2-[(2E,4E)-5-[(lR,2R,3R,6R)-3-amino-l,2,6-trimethylcyclohexy l]-3-methylpenta- 2,4-dien-l-yl]-4-chloro-3-methoxy-6-[(lE)-(methoxyimino)meth yl]-5-methylphenol;

25- 1 ) (E)-3 -chloro-6-hydroxy-4-methoxy-2-methyl-5 -((2E,4E)-3 -methyl-5 - ((lR,2R,3S,6R)-l,2,6-trimethyl-3-(oxetan-3-ylamino)cyclohexy l)penta-2,4-dien-l- yl)benzaldehyde O-methyl oxime;

25-2) (E)-3-chloro-6-hydroxy-4-methoxy-2-methyl-5-((2E,4E)-3-methy l-5- ((lR,2R,3R,6R)-l,2,6-trimethyl-3-(oxetan-3-ylamino)cyclohexy l)penta-2,4-dien-l- yl)benzaldehyde O-methyl oxime; [0049] According to one aspect of the present invention, there is provided a compound selected from the compounds of Nos. (5), (8), (9), (10), (11), (12), (14), (15), (16), (19), (21), (24), (25), (26), (27), (28) indicated above, and particularly preferably a compound selected from the compounds of Nos. (5), (10), (12), (15), (16), (19), (21), and (25) indicated above.

[0050] The compound of the present invention has activity to increase phosphorylation of AMPK and its downstream effector, acetyl-CoA carboxylase (ACC) [Test Example 1].

Further, the compound has significantly improved metabolic stability against degradation by liver microsome (human and mouse) [Test Example 3], improved PK profile that allows once a day oral treatment [Test Example 4], and improved solubility [test Example 2] as compared with 4-O-methyl-ascoclorin (MAC) and its prodrug, a Schiff base of MAC with glycine amide (compound #13 in WO2004/074236).

[0051] The compound of the present invention exhibited blood glucose lowering activity as well as anti-hyperlipidemic activity (lowering blood triglyceride and total cholesterol) [Test Example 6].

[0052] The compound of the present invention represented by formula (I) showed antiproliferative activity against various human cancer cell lines at low μΜ concentrations [Test Example 5], and also exhibited potent antitumor activity in human cancer xenograft models by the oral administration [Test Example7].

[0053] The compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a salt, which is non-toxic and is prepared with a pharmaceutically acceptable base or acid. When the compound of the present invention is basic, the pharmaceutically acceptable salt thereof is generally prepared by adding the compound with a suitable organic or inorganic acid. Examples of the acid-addition salt include acetate, benzenesulfonate, benzoate, citrate, fumarate, glutamate, hydrobromide, hydrochloride, lactate, maleate, mandelate, mesylate, oxalate, palmitate, phosphate/diphosphate, salicylate, stearate, sulfate, succinate, tartrate, and tosylate.

[0054] Furthermore, when the compound of the present invention is acidic, the pharmaceutically acceptable salt thereof is generally prepared by adding the compound with a suitable organic or inorganic base. Examples of the base-addition salt include salts derived from inorganic bases including aluminum, ammonium, calcium, potassium, sodium and the like. Particularly preferred are the ammonium, calcium, potassium, and sodium salts. Examples of the base-addition salt also include salts derived from primary, secondary, and tertiary amines such as arginine, betaine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, lysine, methylglucamine, morpholine, piperazine, piperidine, triethylamine, tripropylamine, and the like.

[0055] The compound of the present invention may be administered as a prodrug. The prodrug can be prepared in line with technical knowledge commonly available to a person skilled in the art. For example, in the case of a carboxy group (-COOH) or hydroxy group being present in the compounds of the present invention, a prodrug can be prepared by converting the carboxy group to a pharmaceutically acceptable ester group, such as methyl, ethyl, or pivaloyloxymethyl ester, or by converting the hydroxy group to a pharmaceutically acceptable ester group, such as acetate or maleate. Use of a prodrug may be beneficial for modifying the solubility or hydrolysis characteristics for use as sustained-release or prodrug formulations.

[0056] In one embodiment, the compound of the present invention may be used in an amorphous form or a crystalline form. The crystalline form may include solvent to form a solvate such as a hydrate. It should be understood that the present invention covers any use of such a solvate or any mixture such as a solution, suspension or solid mixture containing the compound of the present invention.

[0057] According to one aspect of the present invention, there is provided a method for treating or preventing a disease that involves AMPK dysregulation, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. The disease that involves AMPK dysregulation is exemplified as described above. An appropriate route of the administration may be determined by a person skilled in the art. Examples thereof include oral, endorectal, parenteral (intravenous, intramuscular, or subcutaneous),

intracisternal, vaginal, intraperitoneal, intravesical, topical (drip infusion, powder, ointment, gel, or cream) application, and inhalation (intraoral or nasal spray). The dosage form for the administration includes tablets, capsules, granules, powders, pills, aqueous and non-aqueous solutions or suspensions for oral use, and parenteral solutions loaded into containers.

[0058] According to one aspect of the present invention, there is provided a pharmaceutical composition comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or additive. The composition may be administered in an appropriate route, for example, oral, endorectal, parenteral (intravenous, intramuscular, or subcutaneous), intracisternal, vaginal, intraperitoneal, intravesical, topical (drip infusion, powder, ointment, gel, or cream) application, and inhalation (intraoral or nasal spray). The dosage form for the

administration includes tablets, capsules, granules, powders, pills, aqueous and non-aqueous solutions or suspensions for oral use, and parenteral solutions loaded into containers.

[0059] The compound or pharmaceutical composition may be administered as a

pharmaceutical formulation. The pharmaceutical formulation can be produced by well- known methods, using additives such as excipients, coating agents lubricants, binders, disintegrators, stabilizers, corrigents, diluents, solvent and surfactants or emulsifiers.

[0060] Examples of excipients include starches such as potato starch and cornstarch, lactose, crystalline cellulose, and calcium hydrogen phosphate.

[0061] Examples of coating agents include ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, shellac, talc, carnauba wax, and paraffin.

[0062] Examples of lubricants include magnesium stearate, and stearic acid.

[0063] Examples of binders include polyvinylpyrrolidone, macrogol, and the same substances exemplified as the excipients.

[0064] Examples of disintegrators include the same substances exemplified as the excipients and croscarmellose sodium, sodium carboxymethylstarch, and chemically modified starch or celluloses.

[0065] Examples of stabilizers include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzylalcohol, and phenylethylalcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and ascorbic acid.

[0066] Examples of corrigents include sweeteners which are conventionally used.

[0067] Examples of diluents include the substances exemplified as excipients.

[0068] Examples of solvents include ethanol, phenol, chlorocresol, purified water, and distilled water.

[0069] Examples of surfactants or emulsifiers include polysorbate 80, polyoxyl 40 stearate, lauromacrogol, soybean oil, Maisin 35-1 (Gattefosse, France) and Cremophor (BASF, Germany).

[0070] The dose of the compounds or pharmaceutically acceptable salts thereof differs depending on the symptoms, age, body weight, relative health condition, presence of other medication, method of administration, and the like. For example, a generally effective dose for an oral agent as the active ingredient for a patient (a warmblooded animal, especially a human) is a daily dose of preferably 0.1 to 1000 mg per kg body weight, more preferably 1 to 30 mg per kg body weight. The daily dose for an adult patient with a normal body weight is preferably in the range of 10 to 1000 mg. For a parenteral agent, the daily dose is preferably 0.1 to 1000 mg per kg body weight, more preferably 10 to 800 mg per kg body weight.

Preferably such a dose is administered at one time or in several portions per day, or intermittent schedule depending on the symptoms.

[0071 ] The compound of the present invention is further useful in a method for the prevention or treatment of aforementioned diseases, disorders and conditions in combination with administration of one or more other drugs. Namely, the compounds of the present invention may be administered in combination with one or more other drugs in the treatment, prevention, suppression or amelioration of diseases or conditions such as cancer, type 2 diabetes, metabolic syndrome-associated diseases, and Alzheimer disease. The other drug may be administered, by a route and in an amount that are commonly known therefor, simultaneously or sequentially with a compound of formula (I). When the compound is administered simultaneously with one or more other drugs, these ingredients may be contained in a single unit dosage form or two or more separate dosage forms. However, in the combination therapy , the compound of the present invention and one or more other drugs are administered on different overlapping schedules. It is also contemplated that in the combination therapy, the compounds of the present invention and the other active ingredients may be administered in lower doses than that administered each alone. Accordingly, the pharmaceutical compositions of the present invention may further comprise one or more other active ingredients, in addition to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0072] Examples of the other active ingredients that may be administered in the

combination therapy include, but are not limited to:

1) a drug for diabetes: a) dipeptidyl peptidase IV (DPP IV) inhibitors, b) PPAR γ agonist such as pioglitazone, c) biguanides such as metformin, d) insulin or insulin mimetics, sulfonylureas, d) a-glycosidase inhibitors such as acarbose, e) GLP-1, GLP-1 analogues or mimetics, and GLP-1 receptor agonists such as exenatide, and f) sodium-glucose

cotransporter-2 inhibitors (SGLT-2 inhibitors: e.g. dapagliflozin).

2) a drug for hyperlipidemia: a) choresterol lowering agents such as i) HMGA CoA reductase inhibitors (eg. atorvastatin calcium, simvastatin), b) PPARcc/γ dual agonists, c) inhibitors of choresterol absorption,

3) a drug for cancer: a) cytotoxic agents (e.g. taxanes, anthracyclines, CPT-11, 5-FU, capecitabine, platinum agents), ii) molecular targeting agents such as kinase inhibitors (e.g. sorafenib, lapatinib, rapamycin, erlotinib, sunitinib, vemurafenib), iii) antibodies

(trastuzumab, rituximab, cetuximab, bevacizumab, anti-PD-1 antibodies, anti-PD-Ll antibodies)

4) a drug for Alzheimer disease: agents such as galantamine, rivastigmine, donepezil, memantine. [0073] Synthetic processes for preparing the compounds represented by formula (I) are illustrated in the following schemes and examples.

[0074] A starting material, ascochhlorin can be obtained by fermentation of Ascochyts viciae Libert as described in JP S45-009832 B published in 1970. In the following schemes, , R ² , R ³ , R ⁴ , R ⁵ , R ⁷ , Y, and Z are as defined hereinbefore.

[0075] Scheme 1 shows a method for producing compound (IV), (Via) or (VIb). The compound (II) in which R is methyl (4-O-methyl -ascochlorin) can be prepared by the method described in JP H6-305959 A. The compound (II) in which R is fluorinated alkyl group can be obtained by O-alkylation with a fluoroalkylating agent in the presence of base in an appropriate solvent. Examples of the base include potassium carbonate, cesium carbonate and the like. Examples of fluoroalkylating agents include bromofluoromethane, bis(trifluoromethyl)-bis(trifluoromethyloxy)-suIfane, 5-tert-butyl-2'- (trifluoromethoxy)biphenylyl-2-diazonium hexafluoroantimonate, 2,2,2-tris(fluoranyl)ethyl methanesulfonate, 2-diazo-l,l,l-tris(fluoranyl)ethane, tris(fluoranyl)methane, 1 -chloranyl-4- [chloranyl-bis(fluoranyl)methyl]sulfonyl -benzene and the like.

[0076] The compound (IV) can be prepared by reacting a compound (II) with NH ₂ OY (III) in the presence of base such as pyridine, sodium carbonate, sodium hydrogencarbonate and the like. The compound (VIb) can be prepared by reacting compound (IV) with NH ₂ OZ (V) in the presence of base such as pyridine, sodium carbonate, sodium hydrogencarbonate and the like. The compound (Via) can be directly prepared from the compound (II) by reacting two or more equivalents of hydroxyamine (III) in the same reaction condition as described above.

[0077] The compound (VIb) wherein Z is -(CH ₂ ) _n C0 ₂ H can be prepared by basic hydrolysis of the corresponding ester derivative (VIb) wherein Z is -(CH ₂ ) _n C0 ₂ R ⁶ .

Scheme 2

[0078] Scheme 2 shows a method for producing compound (X). The compound (VIII) in which R ² is methyl or fluonnated alkyl can be prepared from ascochlorin via O-acetyl derivative (VII) wherein R is methyl or fluonnated alkyl by the method described in JP 2005- 225851 A. The intermediate (IX) can be prepared by reacting the intermediate (VIII) with NH ₂ 0-Z (V) in the presence of base such as pyridine, sodium carbonate, sodium hydrogencarbonate and the like.. The compound (X) can be prepared by acidic hydrolysis of the acetal group of compound (IX) in an appropriate solvent by the method known to a person skilled in the art.

Scheme 3

[0079] Scheme 3 shows another method for producing compound (X). The compound (X) can be prepared from the corresponding oxime derivative (VI) by treatment with

hydrochloric acid in THF/H ₂ 0 at 100 ^° C.

Scheme 4

[0080] Scheme 4 shows a method for producing compounds (XII) and (XIII). The intermediate (XI) can be prepared by reacting the intermediate (VIII) with an alkylating agent, such as alkyl lithium, alkyl Grignard agent or alkyl cupper lithium reagent, in an appropriate solvent. The compound (XII) can be prepare by acidic hydrolysis of the acetal group of the intermediate (XI) in an appropriate solvent.

[0081] The compound (XIII) can be prepared by reacting the intermediate (IV) with an alkylating agent stated above in an appropriate solvent. The compound (XIII) can be also prepared by reacting the compound (XII) with NH ₂ -OY (III) in the presence of base such as pyridine, sodium carbonate, sodium hydrogencarbonate and the like. The compound (XII) can be also prepared from the compound (XIII) under similar reaction condition as described in Scheme 3.

[0082] Scheme 5a shows a method for producing compound (XV) and (XVI) wherein R ¹¹ is Ci _-6 alkyl, C _3-7 cycloalkyl, or 3- to 7-membered heterocycloalkyl which contains -0-, -S-, - NR ⁶ -, or -S0 ₂ - as a ring atom. The intermediate (XIV) can be prepared by reductive amination of the intermediate (VIII) with a primary amine, R ^H -NH ₂ , in one step or stepwise via intermediate imine (XVII), using the reducing agent such as sodium cyanoborohydride, sodium triacetoxyborohydride and the like.

[0083] The compound (XV) can be prepared by acidic hydrolysis of the acetal group of compound (XIV) in an appropriate solvent by the method known to the skilled art. The compound (XVI) can be prepared by reductive amination of the intermediate (IV) with a primary amine, R ⁿ -NH ₂ by the same method as described above either directly or step wise fashion via intermediate (XVIII).

[0084] The compound (XV) can be also prepared from the oxime ether derivative represented by the formula (XVI) under similar reaction condition as described in Scheme 3. Scheme 5b

[0085] The compound (XVI) wherein R ¹¹ is Ci _-6 alkyl, C _3-7 cycloalkyl or 3- to 7-membered heterocycloalkylamino which contains -0-, -S-, -NR ⁶ -, -SO- or -SG ₂ - as a ring atom such as oxetane can be prepared from the intermediate (IV) in 2 steps: (i) by reductive amination with ammonium acetate/sodium cyanoborohydride in ethanol, (ii) followed by second reductive amination of the resulting intermediate (XIX) with a ketone corresponding to R ¹¹ such as oxetan-3-one and sodium triacetoxyborohydride in dichloromethane.

Scheme 6

[0086] Scheme 6 shows a method for producing compound (XXI), wherein R ^{1 1} is Ci. ₆ alkyl, C _3-7 cycloalkyl, or 3- to 7-membered heterocycloalkyl which contains -0-, -S-, -NR ⁶ -, or - S0 ₂ - as a ring atom. The intermediate (XX) can be prepared by Scheme 2 or 3. The compound (XXI) can be prepared by esterification of XX with a carboxylic acid of formula R ¹² C0 ₂ H by an active ester method or with condensation agent that are known to those skilled in the art.

[0087] When the compound of the present invention represented by formula (I) is obtained in a free form, it can be converted to a salt is a conventional method.

Examples

[0088] Herein below, the present invention will be more specifically described using

Examples, however, it is not to be construed as being limited thereto.

[0089] NMR analysis was performed using Brucker, AVANCE 400MHz or 300MHz, and NMR data were expressed as chemical shifts in ppm (parts per million) (δ), and the deuterium lock signal from the sample solvent was referenced. Mass spectrum data was obtained using a LC-MS, Shimazu LCMS-2020 or -2010 equipped with a photodiode array, SPD-M20A, ESI-SQD 2020 or 2010 (Shimadzu)

[0090] Commercially available reagents were used without further purification "Room temperature" means temperatures ranging from about 20°C. to 25°C. All non-aqueous reactions were carried out under a nitrogen atmosphere. Concentration or distillation of solvents under reduced pressure means that a rotary evaporator was used.

[0091] In the preparation of compounds, a functional group was protected with a protecting group as required to obtain a target compound, followed by removal of the protecting groups. Selection of the protecting group, as well as procedure for the introduction or the removal of the protecting group were carried out, for example, according to a method described in Greene and Wuts, "Protective groups in Organic Synthesis" (second edition, John Wiley & Sons, 1991).

Example 1

[0092] Synthesis of 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-[(2E,4E)-3-m ethyl- 3-oxocyclohexyl]penta-2,4-dien- 1 -yl]benzaldehyde.

[0093] A solution of 3-chloro-4,6-dihydroxy-2-methyl-5-[(2E,4E)-3-methyl-5-[(l R,2R,6R)- l,2,6-trimethyl-3-oxocyclohexyl]penta-2,4-dien-l-yl]benzalde hyde, (ascochlorine, 6 g, 14.82 mmol) in acetonitrile (120 mL), bromofluoromethane (excess), cesium carbonate (2.4 g, 7.38 mmol) was placed in a 250-mL sealed tube. The resulting mixture was stirred for 16 h at 25°C. The mixture was filtered, and the filtrate was concentrated under vacuum. The resulting residue was purified by silica gel column chromatography with Petroleum ether/ethyl acetate (5: 1). The product was recrystallized from dichloromethane/hexane in the ratio of 1 :5 to give the titled compound (4.5 g, 70%) as an off-white solid. LC-MS (ES, m z): 435 [M-H] ^"

H-NMR (400MHz, DMSO, ppm): δ 12.60 (s, 1H), 10.30 (s, 1H), 5.86 (m, 2H), 5.72 (s, 1H), 5.42 (m, 2H), 3.50 (d, J = 7.2 Hz, 2H), 2.64 (s, 3H), 2.54-2.49 (m, 2H), 2.17 (m, lH), 2.05 (m, 1H), 1.84 (s, 4H), 1.52 (m, 1H), 0.73-0.67 (m, 6H), 0.59 (s, 3H)

F-NMR (400MHz, DMSO, ppm): 5 -148.31.

Example 2

[0094] Synthesis of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl)-2- (fluoromethoxy)-6-hydroxy-4-methylphenyl] -3-methylpenta- 1 ,3 -dien- 1 -yl] -2,3 ,4- trimethylcyclohexan- 1 -one

[0095] Step l

A solution of 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-[(2E,4E)-3-m ethyl- 5-[(lR,2R,6R)-l,2,6-trimethyl-3-oxocyclohexyl]penta-2,4-dien -l-yl]benzaldehyde [Example 1] (600 mg, 1.37 mmol) in pyridine (12 mL) was placed in a 50-mL round-bottom flask purged and maintained with an atmosphere of nitrogen, and acetic anhydride (196 mg, 1.92 mmol) was added at 25°C. The resulting solution was stirred for 1.5 h at 25°C. Then the mixture was diluted with of dichloromethane (50 mL) and washed with aqueous hydrochloric acid (0.3mol/L, 20 mL x5). The organic layer was dried with anhydrous sodium sulfate and concentrated under vacuum to give 4-chloro-3-(fluoromethoxy)-6-formyl-5-methyl-2- [(2E,4E)-3-methyl-5-[(lR,2R,6R)-l,2,6-trimethyl-3-oxocyclohe xyl]penta-2,4-dien-l- yl]phenyl acetate (500 mg, 76%) as a white solid.

[0096] Step 2

Into a solution of 4-chloro-3-(fluoromethoxy)-6-formyl-5-methyl-2-[(2E,4E)-3- methyl-5-[(l R,2R,6R)- 1 ,2,6-trimethyl-3-oxocyclohexyl]penta-2,4-dien- 1 -yl]phenyl acetate (500 mg, 1.04 mmol) in ethanol (22.5 mL) was placed in a 250-mL round-bottom flask purged and maintained with an atmosphere of nitrogen, and triethylamine (0.02 mL) was added at 25°C. The solution was stirred for 5 h at 25°C, and then concentrated under vacuum to give (2R,3R,4R)-3-[(lE,3E)-5-[3-chIoro-5-(diethoxymethyl)-2- (fluoromethoxy)-6-hydroxy-4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl] -2,3 ,4- trimethylcyclohexan- 1 -one (410 mg, 77%) as a yellow oil.

Example 3

[0097] Synthesis of 3-chloro-6-hydroxy-5-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hydroxy-l,2 ,3,6- tetramethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-methox y-2-methylbenzaldehyde (Compound 1-1)

[0098] The intermediate (VIII) wherein R ² is methyl: (4R,5R,6S)-5-[(lE,3E)-5-[3-chloro-5- (diethoxymethyl)-6-hydroxy-2-methoxy-4-methylphenyl]-3-methy lpenta-l ,3-dien-l-yl]- 4,5,6-trimethyloxan-3-one was prepared from ascochlorin by the procedures described in JP 2005-225851 A.

[0099] Stepl

A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl)-6-hydro xy-2- methoxy-4-methylphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]-2,3 ,4-trimethylcyclohexan- 1 -one ( 1 g, 2.03 mmol) in tetrahydrofuran (30 mL) was placed in a 50-mL 3-necked round-bottom flask purged and maintained with an atmosphere of nitrogen, followed by addition of methylmagnesium bromide (2 mL, 3.00 equiv, 3 M) dropwise with stirring at -78°C. The resulting solution was stirred for 3 h at 0°C. The reaction was then quenched by addition of sat. aqueous ammonium chloride (30 mL). The resulting mixture was extracted with diethyl ether (30 mL x3) and the organic layers were combined and washed with saturated aqueous sodium chloride (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting residue was purified by Flash-Prep- HPLC with the following condition to give 4-chloro-2-(diethoxymethyl)-6-[(2E,4E)-5- [(lR,2R,3S,6R)-3-hydroxy-l,2,3,6-tetramethylcyclohexyl]-3-me thylpenta-2,4-dien-l-yl]-5- methoxy-3-methylphenol (0.6 g, 60%) as a light yellow solid (mp. 39-40°C).

Flash-Prep-HPLC condition (IntelFlash-1): Column, CI 8 silica gel; mobile phase, acetonitrile/water =70/30 increasing to CH ₃ CN /water = 90/10 within 10 min and then CH ₃ CN within 10 min; Detector, UV 210 nm. LC-MS (ESI, m/z): 507 [M-H] ^"

H-NMR (CD ₃ CN, 400 MHz, ppm): δ 5.91 (d, J = 16.4 Hz, 1H), 5.83 (s, IH), 5.38- 5.43 (m, 1H), 5.30 (d, J = 16 Hz, 1H), 3.72-3.79 (m, 5H), 3.62-3.68 (m, 2H), 3.46 (d, J = 5.6 Hz, 2H), 2.33 (s, 3H), 1.87 (s, 3H), 1.45-1.70 (m, 3H), 1.29-1.35 (m, 2H), 1.20-1.25 (m, 7H), 1.12 (s, 3H), 0.90 (s, 3H), 0.80 (d, J = 5.6 Hz, 3H), 0.70 (d, J = 5.4 Hz, 3H).

[0100] Step 2

A solution of 4-chloro-2-(diethoxymethyl)-6-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hy droxy- 1 ,2,3 ,6-tetramethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl]-5-methoxy-3 -methylphenol (355 mg, 0.699 mmol) in tetrahydrofuran/water (10 mL /20 mL) was placed in a 50-mL round-bottom flask purged and maintained with an atmosphere of nitrogen, followed by addition of acetic acid (5 mL). The resulting solution was stirred for 5 h at 25°C. The resulting solution was concentrated under vacuum. The resulting residue was purified by Flash-Prep-HPLC with the following condition to give 3-chloro-6-hydroxy-5-[(2E,4E)-5- [( 1 R,2R,3 S,6R)-3 -hydroxy- 1 ,2,3 ,6-tetramethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl]-4- methoxy-2-methylbenzaldehyde (0.26 g, 76%, Compound 1) as an off-white solid (mp. 42- 43°C).

Flash-Prep-HPLC condition (IntelFlash-1): Column, CI 8 silica gel; mobile phase, CH ₃ CN/water = 70/30 increasing to CH ₃ CN /water =90/10 within 10 min; Detector, UV 210 nm.

LC-MS (ESI, m/z): 433 [M-H] ^"

H-NMR (CD ₃ CN, 400 MHz, ppm): δ 10.25 (s, 1H), 5.89 (d, J = 16.0 Hz, 1H), 5.40 (t, J = 1.2 Hz, 1H), 5.29 (d, J = 16.0 Hz, 1H), 3.84 (s, 3H), 3.48 (d, J = 7.2 Hz, 2H), 2.63 (s, 3H), 1.87 (s, 3H), 1.57-1.78 (m, 3H), 1.33-1.47 (m, 3H), 1.09 (s, 3H), 0.79 (d, J = 3.2 Hz, 3H), 0.68 (d, J = 6.8 Hz, 3H).

Example 4 [0101] Synthesis of 3-chloro-6-hydroxy-5-[(2E,4E)-5-[(lR,2R,3E,6R)-3- (hydroxyimino)- l,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-me thoxy-2-methylbenzaldehyde (Compound 2)

[0102] Stepl

A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl)-6-hydro xy-2- methoxy-4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl] -2 ,3 ,4-trimethylcyclohexan- 1 -one ( 1 g, 2.03 mmol) in pyridine (30 mL) was placed in a 50-mL 3 -necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, followed by addition of hydroxyamine hydrochloride (200 mg, 2.88 mmol). The mixture was stirred for 16 h at 25°C. The reaction mixture was concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give 4-chloro-2-(diethoxymethyl)-6- [(2E,4E)-5-[(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6-trimethylcy clohexyl]-3-methylpenta- 2,4-dien-l-yl]-5-methoxy-3-methylphenol (0.40 g, 39%) as an off-white solid (mp. 68-70°C).

Flash-Prep-HPLC condition (IntelFlash-1): Column, CI 8 silica gel; mobile phase, CH ₃ CN /water =70/30 increasing to CH ₃ CN /water = 90/10 within 15 min and then acetonitrile within 10 min; Detector, UV 210 nm.

LC-MS: (ESI, m/z): 506 [M-H] ^"

H-NMR: (CD3CN, 400 MHz, ppm): δ 5.95 (d, J = 16.0 Hz, 1H), 5.81 (s, 1H), 5.42 (q, J = 12.8 Hz, 4.8 Hz, 2H), 3.69-3.77 (m, 5H), 3.59-3.66 (m, 2H), 3.45 (d, J = 7.2 Hz, 2H), 3.27-3.32 (m, 1H), 2.47 (s, 3H), 2.27 (d, J = 6.8 Hz, 1H), 1.88 (s, 3H), 1.60-1.72 (m, 3H), 1.19-1.29 (m, 7H), 0.82 (d, J = 6.8 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H), 0.67 (s, 3H).

[0103] Step 2

A solution of 4-chloro-2-(diethoxymethyl)-6-[(2E,4E)-5-[(lR,2R,3E,6R)-3- (hydroxyimino)-l,2,6-trimethylcyclohexyl]-3-methylpenta-2,4- dien-l-yl]-5-methoxy-3- methylphenol (370 mg, 0.726 mmol) in tetrahydrofuran/water (10 mL /20 mL), was placed in a 100-mL 2-necked round-bottom flask purged and maintained with an atmosphere of nitrogen, followed by addition of acetic acid (10 mL). The mixture was stirred for 5 h at 25°C. The reaction mixture was concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give 3-chloro-6-hydroxy-5-[(2E,4E)-5- [(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6-trimethylcyclohexyl]-3 -methylpenta-2,4-dien-l-yl]- 4-methoxy-2-methylbenzaldehyde (220 mg, 70%, Compound 2) as a light yellow solid (mp. 158-160°C).

Flash-Prep-HPLC condition (IntelFlash-1): Column, C18 silica gel; mobile phase, CH ₃ CN / water = 70/30 increasing to CH ₃ CN /water = 90/10 within 10 min; Detector, UV 210 nm.

LC-MS (ESI, m/z): 432 [M-H] ^~

H-NMR (CD ₃ CN, 400 MHz, ppm): δ 10.25 (s, 1H), 5.94 (d, J = 16.4 Hz, 1H), 5.44 (q, J = 15.6 Hz, 6.8 Hz, 2H), 3.85 (s, 3H), 3.50 (d, J = 7.2 Hz, 2H), 3.29 (t, J = 8.4 Hz, 1H), 2.63 (s, 3H), 2.21 (q, J = 13.6 Hz, 6.8 Hz, 1H), 1.90 (s, 3H), 1.60-1.69 (m, 3H), 1.20-1.33 (m, 1H), 0.81 (d, J = 6.8 Hz, 3H), 0.71 (d, J = 6.8 Hz, 3H), 0.66 (s, 3H).

Example 5

[0104] Synthesis of 4-chloro-2-[(2E,4E)-5-[( 1 R,2R,3E,6R)-3-(hydroxyimino)- 1 ,2,6- trimethy lcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -6- [( 1 E)-(hydroxyimino)methyl] -3 - methoxy-5-methylphenol (Compound 3)

[0105] A solution of 3-chloro-6-hydroxy-4-methoxy-2-methyl-5-[(2E,4E)-3-methyl-5- [(lR,2R,6R)-l ,2,6-trimethyl-3-oxocyclohexyl]penta-2,4-dien-l-yl]benzaldeh yde (700 mg, 1.67 mmol, ascochlorine) in pyridine (14 ml) was placed in a 25-mL round-bottom flask, followed by addition of hydroxyamine hydrochloride (290 mg, 4.17 mmol) at 25°C. The mixture was stirred for 16 h at 25 °C, and then concentrated under vacuum. After dilution with dichloromethane (100 ml), the resulting solution was washed with aqueous hydrochloric acid (IN, 50 mLx2) and brine (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting residue was purified by Flash-Prep-HPLC with the following condition to give the titled compound (221.8 mg, 29.6%) as a white solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, silica gel; mobile phase, CH ₃ CN /water(0.08% ammonium bicarbonate) = 50/50 increasing to a CH ₃ CN / water (0.08% ammonium bicarbonate) = 80/20 within 25 min; Detector, UV 254 nm.

LC-MS (ES, m/z): 449 [M+H] ⁺

H-NMR (CD ₃ OD, 400MHz, ppm): 8.56 (s, 1H), 5.92 (d, J=16 Hz, lH), 5.46 (m, 2H), 3.78 (s, 3H), 3.52 (d, J=7.2 Hz, 2H), 3.41(m, 1H), 3.31 (m, 1H), 2.43 (s, 3H), 2.20 (m, 1H), 1.91(s, 3H), 1.67 (m, 3H), 1.33 (m, 1H), 0.91-0.69 (m, 9H)

Example 6

[0106] Synthesis of 3-chloro-4-(fluoromethoxy)-6-hydroxy-5-[(2E,4E)-5-[( 1 R,2R,3E,6R)- 3-(hydroxyimino)-l,2,6-trimethylcyclohexyl]-3-methylpenta-2, 4-dien-l-yl]-2- methylbenzaldehyde (Compound 5)

[0107] Step l

A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl)-2- (fluoromethoxy)-6-hydroxy-4-methylphenyl]-3-methylpenta-l,3- dien-l-yl]-2,3,4- trimethylcyclohexan-l-one [Example 2] (410 mg, 0.80 mmol) in pyridine (12.3 mL) was placed in a 25-mL round-bottom flask, followed by addition of hydroxyamine hydrochloride (83.7 mg, 1.20 mmol, 1.50 equiv) at 25°C. The mixture was stirred overnight at 25°C, and then diluted with dichloromethane (300 mL). The organic layer was washed with aqueous hydrogen chloride (0.3mol/L, 200 mLx5). The organic layer was combined and concentrated under vacuum to give 4-chloro-2-(diethoxymethyl)-5-(fluoromethoxy)-6- [(2E,4E)-5-[(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6-trimethylcy clohexyl]-3-methylpenta- 2,4-dien-l-yl]-3-methylphenol (330 mg, 78%) as a yellow solid.

[0108] Step 2

A solution of 4-chloro-2-(diethoxymethyl)-5-(fluoromethoxy)-6-[(2E,4E)-5- [(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6-trimethylcyclohexyl]-3 -methylpenta-2,4-dien-l-yl]- 3-methylphenol (330 mg, 0.63 mmol) in tetrahydrofuran (6.6 mL) was placed in a 250-mL round-bottom flask, and then a solution of glacial acetic acid (6.6 mL, 5.56 equiv) in water (13.2 mL) was added at 25°C. The mixture was stirred for 4 h at 25°C, and then extracted with of dichloromethane (100 mLx5). The organic layer was combined and the pH value of the solution was adjusted to 7-8 with aqueous sodium bicarbonate. The resulting organic layer was separated, dried and concentrated under vacuum. The resulting residue was purified by Flash-Prep-HPLC with the following condition to give 3-chloro-4- (fluoromethoxy)-6-hydroxy-5-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(hyd roxyimino)-l,2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-2-methylbe nzaldehyde (200 mg, 71%, Compound 5) as a yellow solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, C18 silica gel; mobile phase, CH ₃ CN/H ₂ 0 = 50/50 increasing to CH ₃ CN/H ₂ 0 = 80/20 within 28 min; Detector, UV 254 nm.

LC-MS (ES, m/z): 452 [M+H] ⁺

H-NMR (DMSO, 300MHz, ppm): 12.59(s, IH) , 10.34 (s, IH), 10.30 (s, IH), 5.85-5.90 (t, 2H), 5.70 (s, IH), 5.43 (s, IH), 5.38 (d, J=4.5Hz, 2H) , 3.48-3.51 (d, J=7.5Hz, 2H), 3.31-3.20 (m, IH), 2.72-2.64 (d, J=24.9Hz, IH), 2.26-2.18 (m, IH), 1.84 (s, 3H), 1.63- 1.54 (m, 3H), 1.24-1.19(d, J=13.2Hz, IH).

Example 7

[0109] Synthesis of [(lE,2R,3R,4R)-3-[(lE,3E)-5-(3-chloro-5-formyl-6-hydroxy-2- methoxy-4-methylphenyl)-3 -methylpenta- 1 ,3 -dien- 1 -yl] -2,3 ,4- trimethylcyclohexylidene]amino 2-(dimethylamino)-acetate (compound 6)

[0110] Step l

A solution of 2-(dimethylamino)acetic acid (15 mg, 0.15 mmol) in dichloromethane (0.4 mL) was placed in a 25-mL round-bottom flask, followed by addition of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide methiodide (45 mg, 0.15 mmol), 1- hydroxybenzotrizole (21 mg, 0.16 mmol) and N-methylmorpholine (15 mg, 0.15 mmol) at 25°C. The mixture was stirred for 30 min at 25°C. Then 3-chloro-6-hydroxy-5-[(2E,4E)- 5-[(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6-trimethylcyclohexyl] -3-methylpenta-2,4-dien-l- yl]-4-methoxy-2-methylbenzaldehyde, [Compound 2, Example 3] (60 mg, 0.14 mmol) was added. The resulting mixture was stirred for 2 h at 25°C, then washed with water (10 mL) and brine (10 ml). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The resulting residue was purified by Prep-HPLC with the following condition to give the titled compound (30 mg, 42%) as a yellow solid.

Prep-HPLC condition: Column, X Bridge CI 8, 19* 150mm 5um; mobile phase, CH ₃ CN / water (0.05% ammonium bicarbonate)=50/50 increasing to CH ₃ CN /water (0.05% ammonium bicarbonate)=100/0 within 30 min. Detector, UV 254nm.

LC-MS: (ES, m/z): 519 [M+H] ⁺

H-NMR: (CD ₃ OD, 400MHz, ppm): 10.30 (s, 1H), 5.96 (d, J=16Hz, 1H), 5.46 (m, 2H), 3.83 (s, 3H), 3.52 (d, J=7.2Hz, 2H), 3.34(s, 2H), 3.31 (m, 1H), 2.64 (s, 3H), 2.47-2.32 (m, 7H), 1.95 (m, 4H), 1.76 (m, 2H), 1.39 (m, 1H), 0.97 (d, J=6.8Hz, 3H), 0.75-0.88 (m, 6H).

[01 11] Step 2

A solution of hydrogen chloride in tetrahydrofuran(0.0225M, 13 mL) was added to [( 1 E,2R,3 R,4R)-3 - [( 1 E,3E)-5-(3 -chloro-5 -formyl-6-hydroxy-2-methoxy-4-methylphenyl)-3 - methylpenta-1 ,3-dien-l -yl]-2,3,4-trimethylcyclohexylidene]amino 2-(dimethylamino)acetate (150 mg, 0.29 mmol) in a 50-mL round-bottom flask. The mixture was stirred for 5 min at 25°C, then concentrated under vacuum to give [(lE,2R,3R,4R)-3-[(lE,3E)-5-(3-chloro-5- formyl-6-hydroxy-2-methoxy-4-methylphenyl)-3 -methylpenta- 1 ,3-dien- 1 -yl]-2,3 ,4- trimethylcyclohexylidene] amino 2-(dimethylamino)acetate hydrochloride (101.6 mg, 63%) as a light yellow solid.

LC-MS: (ES, m/z):519 [M+H] ⁺

H-NMR: (CD ₃ OD, 300MHz, ppm): 10.30 (s, IH), 5.96 (d, J=17.7Hz, 1H),4.34 (m, 2H), 3.83 (s, 3H), 3.52 (m, 2H), 2.81-3.15 (m, 6H), 3.31 (m, IH), 2.64 (s, 3H), 2.45 (m, IH), 1.60-2.11 (m, 7H), 1.39 (m, IH), 0.97 (m, 3H), 0.78 (m, 6H).

Example 8

[01 12] Synthesis of 4-chloro-3-(fluoromethoxy)-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3- (hydroxyimino)-l,2,6-trimethylcyclohexyl]-3-methylpenta-2,4- dien-l-yl]-6-[(lE)- (hydroxyimino)methyl]-5-methylphenol (Compound 4)

[0113] A solution of 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-[(2E,4E)-3- methyl-5-[(lR,2R,6R)-l,2,6-trimethyl-3-oxocyclohexyl]penta-2 ,4-dien-l-yl]benzaldehyde [Example 1] (250 mg, 0.57 mmol) in pyridine (7.5 mL) was placed in a 25 mL round-bottom flask, followed by addition of hydroxyamine hydrochloride (80 mg, 1.15 mmol) at 25°C. The mixture was stirred for 16 h at 25°C, and concentrated under vacuum. The resulting residue was purified by Flash-Prep-HPLC with the following condition to give the titled compound (201.7 mg, 75%, Compound 4) as a white solid.

Flash-Prep-HPLC condition (IntelFlash-1) to give: Column, silica gel; mobile phase, CH ₃ CN /water (0.08% ammonium bicarbonate) = 70/30 increasing to CH ₃ CN /water (0.08% ammonium bicarbonate) = 90/10 within 15 min; Detector, UV 220 nm.

LC-MS: (ES, m z): 467 [M+H] ⁺

H-NMR: (CD ₃ OD, 400MHz,ppm): 8.55 (s, IH), 5.90 (d, J=16Hz, IH), 5.72 (s, 5.58 (s, IH), 5.39-5.47 (m, IH), 3.58 (d, J=7.2Hz, 2H), 3.36-3.42 (m, IH), 2.43 (s, 3H), 2.18 (t, IH), 1.89 (s, 3H), 1.61-1.70 (m, 3H), 1.29-1.35 (m, IH).

Example 9

[0114] Synthesis of 4-chloro-2-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hydroxy-l,2,3,6- tetramethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl]-6- [( 1 E)-(hydroxyimino)methyl] -3 - methoxy-5-methylphenol (Compound 7-1)

[01 15] A solution of 3-chloro^6-hydroxy-5-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hydroxy-l,2 ,3,6- tetramethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-methox y-2-methylbenzaldehyde [Compound 1, Example 2] (160 mg, 0.37 mmol) in pyridine (9.6 mL) was placed in a 25-mL round-bottom flask, followed by addition of hydroxyamine hydrochloride (25.6 mg, 0.37 mmol, 1.00 equiv) at 25°C. The mixture was stirred for 16 h at 25°C and then concentrated under vacuum. The resulting residue was purified by Flash-Prep-HPLC with the following condition to give the titled compound (114.0 mg, 69%, Compound 7-1) as a white solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, silica gel; mobile phase, CH ₃ CN /water (0.08% NH ₄ HC0 ₃ ) = 70/30 increasing to CH ₃ CN /water (0.08% NH ₄ HC0 ₃ ) = 90/10 within 15 min; Detector, UV 254 nm.

LC-MS: (ES, m/z): 450 [M+H] ⁺

H-NMR: (CD ₃ OD, 400MHz, ppm): 8.55 (s, IH), 5.87 (d, J=16Hz, IH), 5.42 (t, IH), 5.24 (d, J=16Hz, IH), 3.78 (s, 3H), 3.47-3.53 (m, 2H), 2.44 (s, 3H), 1.88 (s, 3H), 1.46-1.72 (m, 3H), 1.30-1.37 (m, 2H), 1.21 (m, 3H), 1.15 (s, IH), 0.92 (s, 3H), 0.84 (d, J=7.2Hz, 3H), 0.72 (d, J=6.8Hz, 3H).

Example 10

[0116] Synthesis of 4-chloro-3-methoxy-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(methoxyimi no)- 1 ,2,6-trimethylcyclohexyl]-3 -methylpenta-2,4-dien- 1 -yl] -6- [( 1 E)-(methoxyimino)methyl] -5 - methylphenol (Compound 8)

[0117] The titled compound was prepared from 4-O-methyl-ascochlorine by the similar manner as described in Example 4 except that O-methyl hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS: (ES, m/z): 477 [M+H] ⁺

H-NMR: (DMSO, 400MHz, ppm): δ 10.80 (s, IH), 8.64 (s, IH), 5.89(d, J = 16.1 Hz, IH), 5.46-5.37(m, 2H), 3.96 (s, 3H), 3.75 (s, 3H), 3.71 (s, 3H), 3.45 (d, J=7.2Hz, 2H), 3.15- 3.11 (m, 1Ή), 2.41 (s, 3H), 2.223 (q, J=6.8Hz, IH), 1.84 (s, 3H), 1.66 (m, 3H), 1.20 (td, J = 14.4, 13.1, 6.3 Hz, IH), 0.80 (d, J = 6.8 Hz, 3H), 0.65 (m, 6H).

Example 11

[0118] Synthesis of 4-chloro-3-(fluoromethoxy)-2-[(2E,4E)-5-[( lR,2R,3E,6R)-3- (methoxyimino)- 1 ,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dien- 1 -yl]-6-[( 1 E)- (methoxyimino)methyl]-5-methylphenol (Compound 9)

[0119] The titled compound was prepared from 4-O-fluoromethyl-ascochlorine by the similar manner as described in Example 8 except that O-methyl hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): [M+H] ⁺ : 495.

Ή NMR (300 MHz, Methanol-d ₄ ) δ 8.60 (s, IH), 5.89 (d, J = 16.2 Hz, IH), 5.64 (d, J = 54.3 Hz, 2H), 5.42 (t, J = 7.2 Hz, IH), 5.36 (d, J = 16.2 Hz, IH), 3.97 (s, 3H), 3.73 (s, 3H), 3.58 (d, J = 7.2 Hz, 2H), 3.23-3.26 (m, IH), 2.43 (s, 3H), 2.17 (q, J = 6.9 Hz, IH), 1.87 (s, 3H), 1.60-1.71 (m, 3H), 1.30-1.33 (m, IH), 0.86 (d, J = 6.9 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H), 0.67 (s, 3H). Example 12

[0120] Synthesis of 4-chloro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(hydroxyimino)-l,2,6- trimethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -3 -methoxy-6- [( 1 E)- (methoxyimino)methyl]-5-methylphenol (Compound 10)

[0121] Ste l

A mixture of 3-chloro-6-hydroxy-4-methoxy-2-methyl-5-[(2E,4E)-3-methyl-5- [( 1 R,2R,6R)- 1 ,2,6-trimethyl-3 -oxocyclohexyl]penta-2,4-dien- 1 -yl]benzaldehyde (4-0- methyl-ascochlorine, 350 mg, 0.84 mmol) and NH ₂ OMe.HCl (69.3 mg, 0.83 mmol) in pyridine (4 mL) was placed in a 50-mL round-bottom flask purged and maintained with an atmosphere of argon. The mixture was stirred overnight at room temperature and concentrated under vacuum. The residue (400 mg) was purified by Prep-HPLC with the following condition to give (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy-5- [( 1 E)-(methoxyimino)methyl]-4-methylphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]-2,3 ,4- trimethylcyclohexan-l-one (200.9 mg, 54%) as an off-white solid.

Prep-HPLC condition (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, XBridge Prep CI 8 OBD Column, 5um, 19* 150mm; mobile phase, Water with

10mmolNH ₄ HCO ₃ and CH ₃ CN (hold 80.0% CH ₃ CN in 12 min); Detector, UV 254nm.

LC-MS (ES, m/z): 448 [M+H] ⁺

H-NMR (DMSO, 300MHz, ppm): δ 10.80 (s, 1H), 8.64 (s, 1H), 5.88 (d, J= 16.0 Hz, 1H), 5.37-5.51 (m, 2H), 3.96 (s, 3H), 3.75 (s, 3H), 3.46 (d, J=6.9Hz, 2H) , 2.56 (m, 2H), 2.41 (s, 3H), 2.15 (m, 1H), 1.80-2.06 (m, 4H), 1.41-1.59 (m, 1H), 0.70 (dd, J= 15.0, 6.7 Hz, 6H), 0.60 (s, 3H).

[0122] Step 2

A mixture of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy-5-[(lE )- (methoxyimino)methyl] -4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl]-2,3 ,4- trimethylcyclohexan-l-one (500 mg, 1.12 mmol) and NH20H.HC1 (84.9 mg, 1.23 mmol, 1.10 equiv) in pyridine (5 mL) was placed in a 50-mL round-bottom flask purged and maintained with an atmosphere of argon. The mixture was stirred overnight at room temperature and then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give 4-chloro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3- (hydroxyimino)-l,2,6-trimethylcyclohexyl]-3-methylpenta-2,4- dien-l-yl]-3-methoxy-6- [(lE)-(methoxyimino)methyl]-5-methylphenol (246.5 mg, 48%, Compound 10) as a white solid.

Prep-HPLC condition: Column: X Bridge CI 8, 19*250 mm, 5 urn; Mobile Phase A: Water/10mm NH ₄ HC0 ₃ , Mobile Phase B: CH ₃ CN; Flow rate: 30 mL/min; Gradient:

88%B to 88%B in 8 min; 254nm; Detector.

LC-MS (ES, m/z): 463 [M+H] ⁺

H-NMR (DMSO, 300MHz, ppm):5 10.80 (s, 1H), 10.35 (s, 1H), 8.65 (s, 1H), 5.90 (d, J = 16.1 Hz, 1H), 5.42 (m, 2H), 3.96 (s, 3H), 3.75 (s, 3H), 3.46 (d, J = 7.1 Hz, 2H), 3.23 (m, 1H), 2.41 (s, 3H), 2.23 (t, J = 6.8 Hz, 1H), 1.86 (s, 3H), 1.62 (d, J = 11.9 Hz, 3H), 1.18 (d, J = 10.5 Hz, 1H), 0.80 (d, J = 6.8 Hz, 3H), 0.68 (d, J = 6.6 Hz, 3H), 0.61 (s, 3H).

Example 13

[0123] Synthesis of 4-chloro-3-(fluoromethoxy)-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3- (hydroxyimino)- 1 ,2,6-trimethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -6- [( 1 E)- (methoxyimino)methyl]-5-methylphenol (Compound 11).

[0124] The titled compound was prepared from 4-O-fluoromethyl-ascochlorine by the similar manner as described in Example 12.

LC-MS (ES, m/z): [M+H] ⁺ : 481. 1H NMR (300 MHz, DMSO-t ₆ ) δ 10.36 (s, 1H), 8.66 (s, lH), 5.88 (d, J= 16.2 Hz, lH), 5.72 (d, J= 54.3 Hz, 2H) 5.42 (t, J= 7.5 Hz, 1H), 5.37 (d, J= 16.2 Hz, 1H), 3.95 (s, 3H) 3.50 (m, 2H), 3.23 (m, 2H), 2.43 (s, 3H), 2.21 (q, J= 6.6 Hz, 1H), 1.84 (s, 3H), 1.59 (m, 3H), 1.25 (m, 1H), 0.80 (d, J= 6.6 Hz, 3H), 0.68 (d, J= 6.9 Hz, 3H), 0.61 (s, 3H).

Example 14

[0125] Synthesis of 4-chloro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-[[2- (dimethylamino)ethoxy]imino]-l,2,6-trimethylcyclohexyl]-3-me thylpenta-2,4-dien-l-yl]-3- l (Compound 15)

[0126] The titled compound was prepared by the method similar to that described in Example 12 except that 0-(2-(dimethylamino)ethyl)hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): 534 [M+H] ⁺

H-NMR (DMSO, 400MHz, ppm): 510.79 (s, 1H), 8.63 (s, lH), 5.89 (d, J = 16.1 Hz, 1H), 5.33 - 5.48 (m, 2H), 3.98 (m, 5H), 3.74 (s, 3H), 3.45 (d, J = 7.2 Hz, 2H), 3.13 (m, 1H), 2.46 (m, 2H), 2.39 (s,3H), 2.23 (q, J = 6.8 Hz, lH), 2.14 (s, 6H), 1.85 (d, J = 1.3 Hz, 3H), 1.57 - 1.74 (m, 3H), 1.19 (m, lH), 0.78 (d, J = 6.7 Hz, 3H), 0.66 (d, J = 6.5 Hz, 3H), 0.61 (s, 3H).

Example 15

[0127] Synthesis of 4-chloro-3-methoxy-6-[(lE)-(methoxyimino)methyl]-5-methyl-2- [(2E,4E)-3-methyl-5-[(lR,2R,3E,6R)-l,2,6-trimethyl-3-[[2-(mo holin-4- yl)ethoxy]imino]cyclohexyl]penta-2,4-dien-l-yl]phenol (Compound 16). [0128] The titled compound was prepared by the method similar to that described in

Example 12 except that 0-(2-(morpholin-4-yl)ethyl)hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): 576 [M+H] ⁺

H-NMR (CD ₃ CN, 300MHz, ppm): δ 8.55 (s, 1H), 5.93 (d, J= 16.1 Hz, 1H), 5.36- 5.50 (m, 2H), 4.07 (t, J= 5.7 Hz, 2H), 3.94 (s, 3H), 3.75 (s, 3H), 3.51 (m, 6H), 3.14-3.28 (m, 1H), 2.55 (m, 2H), 2.46 (s, 7H), 2.20 (t, J= 6.8 Hz, 1H), 1.89 (d, J= 1.1 Hz, 3H), 1.58-1.72 (m, 3H), 1.25 (m, 1H), 0.81 (d, J= 6.8 Hz, 3H), 0.67 (m, 6H).

Example 16

[0129] Synthesis of 4-chloro-3-methoxy-6-[(lE)-(methoxyimino)methyl]-5-methyl-2- [(2E,4E)-3-methyl-5-[(lR,2R,3E,6R)-l,2,6-trimethyl-3-[[2-(4- methylpiperazin-l- yl)ethoxy]imino]cyclohexyl]penta-2,4-dien-l-yl]phenol (Compound 17).

[0130] The titled compound was prepared by the method similar to that described in

Example 12 except that 0-(2-(N-methylpipearzin-4-yl)ethyl)hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): 589 [M+H] ⁺

H-NMR (CDC1 ₃ , 400MHz, ppm): δ 10.80 (s, 1H), 8.50 (s, 1H), 5.92 (t, J=16.0Hz, 1H), 5.51 (m, 1H), 5.33 (m, 1H), 4.19 (m, 2H), 3.99 (s, 3H), 3.83 (s, 3H), 3.56 (d, J=7.2Hz, 2H), 3.28 (m, 1H), 2.42-2.70 (m, 13H), 2.32 (s, 3H), 2.14 (m, 1H), 1.93 (s, 3H), 1.63 (m, 3H), 1.29 (m, 1H), 0.88 (d, J=6.8Hz, 3H), 0.71 (m, 6H).

Example 17

[0131] Synthesis of ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2- methoxy-5-[(l E)-(methoxyimino)methyl]-4-methylphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]- 2,3,4-trimethylcyclohexylidene]amino]oxy)acetate (Compound 18).

[0132] The titled compound was prepared by the method similar to that described in Example 12 except that ethyl 2-(aminooxy)acetate was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): [M+H] ⁺ : 549

1H NMR (300 MHz, DMSO-i/ ₆ ): 5 10.74 (s, 1H), 8.60 (s, 1H), 5.86 (d, J= 16.2 Hz, 1H), 5.41 (t, J= 7.0 Hz, 1H), 5.35 (d, J= 16.2 Hz, 1H), 4.51 (s, 2H), 4.06 (q, J= 7.1 Hz, 2H), 3.71 (s, 3H), 3.42 (d, J= 7.2 Hz, 2H), 3.28 (s, 1H), 3.18 (dd, J= 12.9, 4.1 Hz, lH), 2.37 (s, 3H), 2.24 (q, J = 6.8 Hz, 1H), 1.81 (d, J= 1.3 Hz, 3H), 1.79 - 1.56 (m, 3H), 1.31 - 1.17 (m, 1H), 1.14 (t, J= 7.1 Hz, 3H), 0.71 (d, J= 6.7 Hz, 3H), 0.64 (d, J= 6.5 Hz, 3H), 0.58 (s, 3H). Example 18

[0133] Synthesis of 4-chIoro-2-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hydroxy-l,2,3,6- tetramethylcyclohexyl]-3 -methylpenta-2,4-dien- 1 -yl]-3 -methoxy-6- [( 1 E)- (methoxyimino)methyl]-5-methylphenol (Compound 14-1).

[0134] A mixture of 3-chloro-6-hydroxy-5-[(2E,4E)-5-[(lR,2R,3S,6R)-3-hydroxy-l,2 ,3,6- tetramethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-methox y-2-methylbenzaldehyde [Compound 1, Example 2] (350 mg, 0.80 mmol) and methoxylamine hydrochloride (85 mg, 1.02 mmol) in pyridine (5 mL) was placed in a 50-mL round-bottom flask. The mixture was stirred for 12 h at 10 to 15 °C and concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give the titled compound (209.3 mg, 55%) as a light yellow solid.

Prep-HPLC condition (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, XBridge BEH CI 8 OBD Prep Column, 5 μπι, 19 mm 250 mm; mobile phase, Water with lOmmol NH ₄ HCO3 and CH ₃ CN (hold 90.0% CH ₃ CN in 9 min); Detector, uv 254&220nm.

LC-MS (ES, m/z): [M-H] 462

H-NMR (DMSO, 400MHz ppm): δ 10.79 (s,lH), 8.64 (s,lH),5.82 (m,lH), 5.39 (m.lH), 5.22 (m,lH), 3.96 (s.3H), 3.74 (m,4H), 3.32-3.45 (m,2H), 2.50 (s,3H), 1.81 (m,3H), 1.47-1.59 (m,2H), 1.21-1.38 (m,3H), 1.13 (m,lH), 1.04 (s,3H), 0.85 (m,3H), 0.74 (m,3H), 0.64 (m,3H).

Example 19

[0135] Synthesis of 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methox y-5- [(lE)-(methoxyimino)methyl]-4-methylphenyl]-3-methylpenta-l, 3-dien-l-yl -2,3,4- trimethylcyclohexylidene]amino]oxy)acetic acid (Compound 19).

A solution of ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2- methoxy-5-[(lE)-(methoxyimino)methyl]-4-methylphenyl]-3-meth ylpenta-l,3-dien-l-yl]- 2,3,4-trimethylcyclohexylidene]amino]oxy)acetate [Compound 18, Example 17] (350 mg, 0.64 mmol) was placed in a 25-mL round-bottom flask, followed by addition of a solution of lithium hydroxide (76 mg, 3.17 mmol) in water/THF (0.5/1 mL). The mixture was stirred overnight at room temperature. The pH value of the mixture was adjusted to 4 with aqueous hydrochloride acid (IN). The reaction mixture was extracted with dichloromethane (2 mL x2). The organic layer was combined, and then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give 2-([[(lE,2R,3R,4R)-3- [(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy-5-[(lE)-(methoxyimi no)methyl]-4- methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl]-2,3 ,4- trimethylcyclohexylidene] amino] oxy)acetic acid (300 mg, 90%, Compound 19) as a white solid. Prep-HPLC (2#-AnalyseHPLC-SHIMADZU(HPLC- 10)): Column, XBridge Shield RP18 OBD Column, 5um,19* 150mm; mobile phase, WATER WITH 0.05%TFA and CH ₃ CN (hold 72.0% CH ₃ CN in 1 1 min); Detector, UV 254nm.

LC-MS: (ES, m/z): [M+H] ⁺ : 521.

Ή NMR (300 MHz, DMSO-c/ ₆ ) δ 10.76 (s, lH), 8.60 (s, 1H), 5.86 (d, J= 16.2 Hz, 1H), 5.41 (t, J= 6.9 Hz, 1H), 5.35 (d, J= 16.2 Hz, 1H), 4.42 (s, 2H), 3.92 (s, 3H), 3.42 (d, J = 7.1 Hz, 2H), 3.13 - 3.25 (m, 1H), 2.37 (s, 3H), 2.23 (q, J= 6.8 Hz, 1H), 1.82 (d, J= 1.4 Hz, 3H), 1.56 - 1.76 (m, 3H), 1.22 (s, lH), 0.71 (d, J= 6.8 Hz, 3H), 0.64 (d, J= 6.5 Hz, 3H), 0.59 (s, 3H).

Example 20

[0136] Synthesis of 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-[(2E,4E)-3-m ethyl- 5-[(lR,2R,3E,6R)-l,2,6-trimethyl-3-[[2-(morpholin-4-yl)ethox y]imino]cyclohexyl]penta-2,4- dien-l-yl]benzaldehyde (Compound 12)

[0137] The titled compound was prepared by the similar method as described in Example 6 except that 0-2-(morpholin-4-yl)ethyl hydroxyamine hydrochloride was used instead of hydroxyamine hydrochloride.

LC-MS (ES, m/z): [M+H] ⁺ : 565

'H NMR (300 MHz, Chloroform-tf) δ 12.55 (s, 1H), 10.29 (s, 1H), 5.91 (d, J= 16.0 Hz, 1H), 5.70 (d, J= 54.3 Hz, 1H), 5.46 (t, J= 7.4 Hz, 1H), 5.34 (d, J= 16.0 Hz, 1H), 4.19 (t, J= 5.8 Hz, 2H), 3.72 (m, 4H), 3.61 (d, J= 7.3 Hz, 2H), 3.21 - 3.36 (m, 1H), 2.67 (d, J= 9.0 Hz, 5H), 2.55 (m, 4H), 2.15 (q, J = 6.7 Hz, 1H), 1.52 - 1.74 (m, 3H), 1.30 (m, 1H), 0.89 (d, J = 6.7 Hz, 3H), 0.73 (d, J = 6.4 Hz, 3H), 0.69 (s, 3H).

Example 21

[0138] Synthesis of 4-chloro-2-[(2E,4E)-5-[(lR,2R,6R)-3-(ethylamino)-l,2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-3-methoxy- 6-[(lE)- (methoxyimino)methyl]-5-methylphenol (Compound 21).

[0139] Step l

A mixture of placed (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy-5- [( 1 E)-(methoxyimino)methyl] -4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl]-2,3 ,4- trimethylcyclohexan-l-one [Example 12, step 1] (3 g, 6.70 mmol), Ti(i-PrO) ₄ (15.2 g, 53.52 mmol), ethylamine hydrochloride (2.2 g, 26.83 mmol) and MS 4A (lg) in THF (30 niL) was placed in a 250-mL round-bottom flask. The mixture was stirred overnight at 68°C in an oil bath. The resulting mixture was diluted with 200 mL of ethyl acetate followed by addition of 10 mL of saturated ammonium chloride. The mixture was filtered, and the filtrate was dried over anhydrous magnesium sulfate and concentrated under vacuum to give 4-chloro-2- [(2E,4E)-5-[(lR,2R,3E,6R)-3-(ethylimino)-l,2,6-trimethylcycl ohexyl]-3-methylpenta-2,4- dien-l-yl]-3-methoxy-6-[(lE)-(methoxyimino)methyl]-5-methylp henol (1.2 g, crude) as a brown solid.

[0140] Step 2

A solution of 4-chloro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(ethylimino)-l,2,6- trimethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -3 -methoxy-6- [( 1 E)- (methoxyimino)methyl]-5-methylphenol (1.2 g, 2.53 mmol) and sodium

triacetoxyborohydride (1.07 g, 5.05 mmol, 2.00 equiv) in dichloromethane (20 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 5 h at room

temperature. The mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give a crude product (350 mg), which was purified by Chiral-Prep-HPLC with the following condition to give 4-chloro-2-[(2E,4E)-5-[(lR,2R,6R)-3-(ethylamino)-l,2,6-trime thylcyclohexyl]-3- methylpenta-2,4-dien- 1 -yl]-3-methoxy-6-[^

(211.4 mg, 18%, Compound 21 ) as an orange solid.

Flash-Prep-HPLC condition (CombiFlash-1): Column, CI 8 silica gel; mobile phase, water/acetonitrile=l :0 increasing to water/acetonitrile = 0:1 within 40 min; Detector, UV 254 nm.

Chiral-Prep-HPLC condition: Column: XBridge Shield RP 18 OBD Column, 5um, 19* 150mm; Mobile Phase A: Water with lOmmol NH ₄ HC0 ₃ , Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 75% B to 95% B in 10 min; Detector 254 nm.

LC-MS (ES, m/z): 477 [M+H] ⁺

H-NMR (DMSO, 300MHz, ppm): δ 10.71-10.87 (br, IH), 8.644 (s, IH), 5.82 (t, J=16.2Hz, IH), 5.39 (m, IH), 5.25 (m, IH), 3.96 (s, 3H), 3.75 (s, 3H), 3.44 (d, J=6.9Hz, 2H), 2.57-2.66 (m, 2H), 2.51 (s, 3H), 2.28-2.36 (m, IH), 1.74-1.82 (m, 4H), 1.12-1.50 (m, 6H), 0.98 (m, 3H), 0.86 (m, 3H), 0.77 (m, 3H), 0.63 (m, 3H).

Example 22

[0141] Synthesis of 4-chloro-2-[(2E,4E)-5-[(l R,2R,6R)-3-(cyclopropylamino)- 1 ,2,6- trimethy lcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -3 -methoxy-6- [( 1 E)- (methoxyimino)methyl]-5-methylphenol (Compound 23)

[0142] The titled compound was prepared by the similar method described in Example 21 except that cyclopropyl amine was used instead of ethylamine.

LC-MS (ES, m/z): 489 [M+H] ⁺

H-NMR (DMSO, 300MHz, ppm): δ 10.79 (s, IH), 8.64 (s, IH), 5.80 (t,

J=16.2Hz,lH), 5.37 (m, IH), 5.24 (m, IH), 3.96 (s, 3H), 3.74 (s, 3H), 3.44 (d, J=6.3Hz, 2H), 2.74 (m, IH), 2.41 (s, 3H), 1.96 (m, 2H), 1.82 (s, 3H), 1.41-1.57 (m, 3H), 1.15-1.40 (m, 3H), 0.80 (s, 3H), 0.73 (m, 3H), 0.64 (m, 3H), 0.39 (m, IH), 0.25 (m, 2H), 0.15 (m, IH). Example 23

[0143] Synthesis of 2-[(2E,4E)-5-[(lR,2R,6R)-3-amino-l ,2,6-trimethylcyclohexyl]-3- methylpenta-2,4-dien-l -yl]-4-chloro-3-methoxy-6-[(lE)-(methoxyimino)methyl]-5-

[0144] A mixture of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-6-hydroxy-2-methoxy-5-[(lE )- (methoxyimino)methyl]-4-methylphenyl]-3-methylpenta-l ,3-dien-l -yl]-2,3,4- trimethylcyclohexan-l-one (3 g, 6.70 mmol) [Example 12, Step 1], NaBH ₃ CN (2.75 g, 43.76 mmol) and NH ₄ OAc (20 g, 40.00 equiv) in ethanol (100 mL) was placed in a 250-mL round- bottom flask purged and maintained with an atmosphere of argon. The resulting mixture was stirred overnight at 70°C in an oil bath. The resulting mixture was concentrated under vacuum. The residue was diluted with H ₂ 0 (80 mL). The mixture was extracted with ethyl acetate (200 mL x3) and the organic layer was combined, then concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give the titled compound (1.5 g, 50%, Compound 24) as a yellow solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, CI 8 silica gel; mobile phase, CH ₃ CN: H ₂ O=0:95 increasing to CH ₃ CN: H ₂ 0 = 95:5 within 55 min; Detector, UV 254 nm.

LC-MS: (ES, m/z): [M+H] ⁺ 449

H-NMR: (CDCl ₃ ,400MHz ppm): δ 10.77 (s, 1H), 8.50 (s, 1H), 5.93 (d,

J=16.4Hz ,1H), 5.52 (m,lH), 5.19 (m, lH), 3.99 (s, 3H), 3.82 (s, 3H), 3.54 (m, 2H), 2.42 (s, 3H), 2.04-2.23 (m, 1H), 1.90 (m, 3H), 1.25-1.85 (m, 6H), 0.91 -1.04 (m, 5H), 0.70-0.78 (m, 4H).

Example 24

[0145] Synthesis of 4-chloro-3-methoxy-6-[(l E)-(methoxyimino)methyl]-5-methyl-2- [(2E,4E)-3 -methyl-5 - [( 1 R,2R,6R)- 1 ,2,6-trimethyl-3 - [(oxetan-3 -yl)amino]cyclohexyl]penta- 2,4-dien-l -yl]phenol (Compound 25)

[0146] Into a 50-niL sealed tube, a solution of 2 (2E,4E)-5-[(lR,2R,6R)-3-amino-l,2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-chloro-3 -methoxy-6-[(lE)- (methoxyimino)methyl]-5-methylphenol (Compound 24; 1 g, 2.23 mmol), sodium triacetoxyborohydride (STAB, 709,8 mg, 3.35 mmol) and oxetan-3-one (353.6 mg, 4.91 mmol) in DCM (12mL) were placed. The mixture was stirred for 18 h at room temperature and then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give the titled compound (239.2 mg, 21%, Compound 25) as an off- white solid.

Prep-HPLC condition (2#-AnalyseHPLC-SHIMADZU(HPLC-l 0)): Column, XBridge Shield RP18 OBD Column, 5um, 19* 150mm; mobile phase, Water with lOmmol NH ₄ HC0 ₃ and CH ₃ CN (hold 82.0% ACN in 10 min); Detector, UV 254nm.

LC-MS: (ES, m/z): [M+H] ⁺ 505

H-NMR: (DMSO, 400MHz ppm): 610.80 (s, IH), 8.66 (s, IH), 5.84 (d,

J=16.4Hz,lH), 5.41 (m,lH), 5.25 (d, J=15.6Hz, IH), 4.60 (m,2H), 4.27-4.36(m, 2H), 4.38 (s, 3H), 3.83 (m, IH), 3.76 (s, 3H), 3.46 (d, J=7.2Hz, 2H), 2.42 (s, 3H), 1.84 (s, 3H), 1.45-1.58 (m, 3H), 1.35 (m, 2H), 1.21 (m, IH), 0.89 (s, 3H), 0.81 (d, J=7.2Hz, 3H), 0.67 (m, 3H). Example 25

[0147] Synthesis of 3-chloro-5-[(2E,4E)-5-[(lR,2R,6R)-3-(ethylamino)-l,2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-6-hydroxy- 4-methoxy-2- methylbenzaldehyde (Compound 20).

[0148] A solution of 4-chloro-2-[(2E,4E)-5-[(lR,2R,6R)-3-(ethylamino> trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-3-methoxy- 6-[(lE)- (methoxyimino)methyl]-5-methylphenol [Compound 21, Example 21] (500 mg, 1.05 mmol), formaldehyde (425 mg, 5.25 mmol, 5.00 equiv, 37%) and hydrochloride acid (500 mg, 5.3 mmol, 5.00 equiv, 38%) in THF/water (20 mL/2 mL) was placed in a 30-mL sealed tube. The mixture was stirred overnight at 100°C and then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give the titled compound (55.4 mg, 9.4%, Compound 20) as an off-white solid.

Prep-HPLC condition (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Gemini-NX 5u CI 8 110A, AXIA Packed, 150 x 21.2 mm; mobile phase, Column: Mobile Phase A: Water with lOmmol CF ₃ COOH, Mobile Phase B: CH ₃ CN; Flow rate: 30 mL/min; Gradient: 60% B to 95% B in 10 min; 254 nm; Detector, uv 254nm.

LC-MS (ES, m/z) 448 [M+H] ⁺

H-NMR (DMSO, 300 MHz, ppm) 12.62 (br, IH), 10.27 (br, IH), 7.90 (br, IH), 7.43 (br, IH), 5.94 (d, J=16.2Hz, IH), 5.43 (t, J=7.5Hz, IH), 5.34 (d, J=15.9Hz, IH), 3.82 (s, 3H), 3.47 (d, J=7.5Hz, 2H), 3.28 (m, IH), 2.96 (m, 2H), 2.62 (s, 3H), 1.85 (m, 5H), 1.70 (m, IH), 1.50 (s, 3H), 1.20 (m, 3H), 0.88 (m, 3H), 0.83 (m, 3H), 0.74 (m, 3H).

Example 26

[0149] Synthesis of 3-chloro-5-[(2E,4E)-5-[(lR,2R,6R)-3-(cyclopropylamino)-l ,2,6- trimethylcyclohexyl] -3 -methylpenta-2,4-dien- 1 -yl] -6-hydroxy-4-methoxy-2- methylbenzaldehyde (Compound 22)

[0150] The titled compound was prepared from Compound 23 (Example 22) by the similar method as described in Example 25.

LC-MS (ES, m/z): 460 [M+H] ⁺

H-NMR (DMSO, 300 MHz, ppm): 12.60 (br, IH), 10.27 (s, IH), 5.80 (d, J=16.2Hz, 1H), 5.36 (t, J=7.2Hz, 1H), 5.25 (d, J=15.9Hz, 1H), 3.81 (s, 3H), 3.42 (d, J=6.9Hz, 2H), 2.75 (m, 1H), 2.62 (s, 3H), 1.96 (m, 2H), 1.82 (s, 3H), 1.20-1.61 (m, 6H), 0.80 (m, 3H), 0.71 (m, 3H), 0.63 (m, 3H), 0.35-0.47 (m, 1H), 0.24-0.31 (m, 2H), 0.06-0.18 (m, 1H).

Example 27

[0151] Synthesis of 3-chloro-5-[(2E,4E)-5-[(lR,2R,6R)-3-(cyclopropylamino)-l,2,6 - trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-(fluorom ethoxy)-6-hydroxy-2- methylbenzaldehyde (Compound 13)

[0152] Step l

Into a 50-niL round-bottom flask, was placed A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3- chloro-5-(diethoxymethyl)-2-(fluoromethoxy)-6-hydroxy-4-meth ylphenyl]-3-methylpenta- l,3-dien-l-yl]-2,3,4-trimethylcyclohexan-l-one [Example 2] (900 mg, 1.76 mmol), MS 4A (200 mg) and cyclopropanamine (2 g, 35.03 mmol, 20.00 equiv) in ethanol (10 mL). The resulting solution was stirred overnight at 30°C. The resulting mixture was concentrated under vacuum to give 4-chIoro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(cyclopropylimino)-l, 2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-6-(diethox ymethyl)-3-(fluoromethoxy)-5- methylphenol (0.9 g, 93%) as a solid.

[0153] Step 2

A solution of 4-chloro-2-[(2E,4E)-5-[(lR,2R,3E,6R)-3-(cyclopropylimino)-l, 2,6- trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-6-(diethox ymethyl)-3-(fluoromethoxy)-5- methylphenol (1.1 g, 2.00 mmol) and sodium borohydride acetate (464 mg, 2.19 mmol) in ethanol (20 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 3 h at room temperature and concentrated under vacuum. The residue was purified by silica gel column chromatography with ethyl acetate/petroleum ether (1 :10) to give 4-chloro-2- [(2E,4E)-5-[(lR,2R,6R)-3-(cyclopropylamino)-l,2,6-trimethylc yclohexyl]-3-methylpenta- 2,4-dien-l-yl]-6-(diethoxymethyl)-3-(fluoromethoxy)-5-methyl phenol (460 mg, 42%) as a solid.

[0154] Step 3

A solution of 4-chloro-2-[(2E,4E)-5-[(lR,2R,6R)-3-(cyclopropylamino)-l,2,6 - trimethylcyclohexyl]-3 -methylpenta-2,4-dien- 1 -yl] -6-(diethoxymethyl)-3 -(fluoromethoxy)-5 - methylphenol (460 mg, 0.83 mmol) in THF/water/acetic acid (10/20/10 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 3 h at room temperature and concentrated under vacuum. The residue was dissolved in water (10 mL). The mixture was extracted with ethyl acetate (20 mL) and the organic layer was combined, then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition of 3-chloro-5-[(2E,4E)-5-[(lR,2R,6R)-3-(cyclopropylamino)-l,2,6 - trimethylcyclohexyl]-3-methylpenta-2,4-dien-l-yl]-4-(fluorom ethoxy)-6-hydroxy-2- methylbenzaldehyde (206.7 mg, 52%, Compound 13) as a light yellow solid.

Prep-HPLC condition (2#-AnalyseHPLC-SHIMADZU(HPLC-10)): Column, Gemini-NX CI 8 AXAI Packed, 21.2 x 150mm 5um 1 lnm; mobile phase, Water with lOmmol NH ₄ HC0 ₃ and CH ₃ CN (hold 95.0% CH ₃ CN in 8 min); Detector, uv 254nm.

LC-MS (ES, m/z): [M+H] ⁺ : 478.

Ή NMR (300 MHz, DMSO-c¾) δ 10.26 (s, 1H), 5.79 (d, J= 154.3 Hz, 2H), 5.78 (d, J= 16.2 Hz, 1H) 5.34 (t, J= 7.2 Hz, 1H), 5.25 (d, J= 16.2 Hz, 1H), 3.48 (d, J= 6.9 Hz, 2H), 2.76 (m, 1H), 2.64 (s, 3H), 1.95-2.08 (m, 2H), 1.80(s, 3H), 1.20-1.48 (m, 5H), 0.79 (s, 3H), 0.69 (d, J= 7.2 Hz, 3H), 0.59 (d, J= 6.3 Hz, 3H), 0.35 (m, 1H), 0.24 (m, 1H), 0.1 1 (m, 1H). Example 28

[0155] Synthesis of 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)- 5- formyl-6-hydroxy-4-methylphenyl]-3-methylpenta-l,3-dien-l-yl ]-2,3,4- trimethylcyclohexylidene]amino]oxy)acetic acid (Compound 26)

[0156] Step l

A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl)-2- (fluoromethoxy)-6-hydroxy-4-methylphenyl]-3-methylpenta-l,3- dien-l-yl]-2,3,4- trimethylcyclohexan-l-one [Example 2] (1.5 g, 2.94 mmol), Cs ₂ C0 ₃ (479 mg, 1.47 mmol) and ethyl 2-(aminooxy)acetate oxalic acid salt (605 mg, 2.89 mmol) in ethanol (10 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 2 h at room

temperature. The reaction mixture was diluted with water (10 mL), and extracted with ethyl acetate (10 mL x2). The organic layer was combined, and then concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl) -2- (fluoromethoxy)-6-hydroxy-4-methylphenyl]-3 -methylpenta- 1 ,3 -dien- 1 -yl]-2,3 ,4- trimethylcyclohexylidene]amino]oxy)acetate (1.0 g, 56%) as a light yellow solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, C18 silica gel; mobile phase, CH ₃ CN/H ₂ 0 = 60:40 increasing to CH ₃ CN/H ₂ 0 = 95:5 within 40 min; Detector, UV 254 nm.

LC-MS (ES, m/z): 612 [M+H] ⁺

[0157] Step 2

A mixture of ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-5-(diethoxymethyl) - 2-(fluoromethoxy)-6-hydroxy-4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl] -2,3 ,4- trimethylcyclohexylidene] amino] oxy)acetate (1 g, 1.63 mmol), lithium hydroxide (117 mg, 3.00 equiv) in THF/H ₂ 0 (1 :1, 30 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 2 h at room temperature. Then (15 mL) was added to the solution. The resulting solution was stirred for an additional 6 h at room temperature. The resulting mixture was concentrated under vacuum and diluted with 20 mL of water. The mixture was extracted with of ethyl acetate (20 mL x2). The organic layer was combined, and then concentrated under vacuum. The residue was purified by Flash-Prep-HPLC with the following condition to give 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)- 5- formyl-6-hydroxy-4-methylphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]-2,3,4- trimethylcyclohexylidene]amino]oxy)-acetic acid (533.3 mg, 64%, Compound 26) as a light yellow solid.

Flash-Prep-HPLC condition (IntelFlash-1): Column, C18 silica gel; mobile phase, CH ₃ CN/H ₂ 0=55:45 increasing to CH ₃ CN/H ₂ 0 = 95:5 within 45 min; Detector, UV 254 ran.

LC-MS (ES, m/z): [M+H] ⁺ : 510.

H-NMR (300 MHz, DMSO-< ) 612.58 (s, 2H), 10.30 (s, 1H), 5.88 (d, J= 16.2 Hz, 1H), 5.79 (d, J= 53.7 Hz, 2H), 5.40 (d, J= 16.2 Hz, 1H), 5.38 (m, 1H), 4.46 (s, 2H), 3.50 (d, J= 7.2 Hz, 2H), 3.23 (dd, J= 12.7, 4.0 Hz, 1H), 2.64 (s, 3H), 2.27 (q, J= 6.7 Hz, 1H), 1.84 (s, 3H), 1.82-1.59 (m, 3H), 1.35-1.19 (m, 1H), 0.75 (d, J= 6.6 Hz, 3H), 0.68 (d, J= 6.6 Hz, 3H), 0.62 (s, 3H).

Example 29

[0158] Synthesis of 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)- 6- hydroxy-5-[( 1 E)-(hydroxyimino)methyl]-4-methyIphenyl]-3-methylpenta- 1 ,3-dien- 1 -yl]- 2,3,4-trimethylcyclohexylidene]amino]oxy)acetic acid (Compound 27)

[0159] Step l

A solution of 3-chloro-4-(fluoromethoxy)-6-hydroxy-2-methyl-5-[(2E,4E)-3-m ethyl- 5-[(lR,2R,6R)-l,2,6-trimethyl-3-oxocyclohexyl]penta-2,4-dien -l-yl]benzaldehyde [Example 1] (600 mg, 1.37 mmol) and hydroxyamine hydrochloride (93.5 mg, 1.35 mmol, 0.98 equiv) in pyridine (6 mL) was placed in a 100-mL round-bottom flask. The mixture was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum to give (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)-6-hydrox y-5-[(lE)- (hydroxyimino)methyl]-4-methylphenyl]-3-methylpenta-l ,3-dien-l-yl]-2,3,4- trimethylcyclohexan-l-one (500 mg, crude) as yellow oil.

[0160] Step 2

A solution of (2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)-6-hydrox y-5- [( 1 E)-(hydroxyimino)methyl] -4-methylphenyl] -3 -methylpenta- 1 ,3 -dien- 1 -yl]-2,3 ,4- trimethylcyclohexan-l -one (500 mg, 1.1 1 mmol) and ethyl 2-(aminooxy)acetate oxalic acid salt (925 mg, 4.42 mmol, 4.00 equiv) in pyridine (6 mL) was placed in a 100-mL round- bottom flask. The mixture was stirred for 1 h at room temperature. The resulting mixture was concentrated under vacuum to give resulted in of ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)- 5 - [3 -chloro-2-(fluoromethoxy)-6-hydroxy-5 - [( 1 E)-(hydroxyimino)methyl] -4-methylphenyl] - 3-methylpenta-l ,3-dien-l-yl]-2,3,4-trimethylcyclohexylidene]amino]oxy)aceta te (550 mg, crude) as yellow solid.

[0161] Step 3

A mixture of ethyl 2-([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)- 6- hydroxy- 5 - [( 1 E)-(hydroxyimino)methyl] -4-methy Iphenyl] -3 -methylpenta- 1 , 3 -dien- 1 -y 1] - 2,3,4-trimethylcyclohexylidene]amino]oxy)acetate (550 mg, 0.99 mmol), lithium hydroxide (853 mg, 35.62 mmol) in tetrahydrofuran (6 mL) and water (2 mL) was placed in a 100-mL round-bottom flask. The resulting solution was stirred for 1 h at room temperature. The mixture was concentrated under vacuum. The resulting mixture was extracted with ethyl acetate (10 mL x3) and the organic layer was combined, then concentrated under vacuum. The residue was purified by Prep-HPLC with the following condition to give 2- ([[(lE,2R,3R,4R)-3-[(lE,3E)-5-[3-chloro-2-(fluoromethoxy)-6- hydroxy-5-[(l E)- (hydroxyimino)methyl]-4-methylphenyl]-3-methylpenta-l ,3-dien-l -yl]-2,3,4- trimethylcyclohexylidene]amino]oxy)acetic acid (242.2 mg, 46%, Compound 27) as a solid.

Prep-HPLC condition (Prep_HPLC_MCl): Column: XBridge Shield RP18 OBD Column, 5um, 19* 150mm; Mobile Phase A: Waters(0.1 %FA), Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 56% B to 80% B in 8 min; Detector: 254 nm,.

LC-MS (ES, m/z): [M+H] ⁺ : 525, [M+Na-H] ⁺ : 547 H-NMR (300MHz, CD ₃ OD, ppm): δ 8.56(S, IH), 5.89(d, J=16.2 Hz, IH), 5.72(s, IH), 5.54(s, IH), 5.44-5.33(m, 2H), 4.48(s, 2H), 3.56(d, J=7.2 Hz, 2H), 3.39-3.32(m, IH), 2.43(s, 3H), 2.19(q, J=6.6 Hz, IH), 1.86(s, 3H), 1.77-1.59(m, 3H), 1.25-1.48(m, IH), 0.84(d, 3=6.0 Hz, 3H), 0.74-0.69(m, 6H).

Test Example 1

Effect on phosphorylation level of AMPK and ACC in HepG2 cells.

[0162] p-AMPK and p-ACC levels were measured by Western blot analysis using the antibodies for p-AMPK and p-ACC. Following reagents were purchased from the vendors, and the instruments listed below were used.

[0163] Reagents: AMPKa (D5 A2) rabbit mAb, phospho-acetyl-CoA carboxylase (Ser79) (D7D1 1) rabbit mAb, anti-rabbit IgG, HRP-linked antibody, and beta-actin (13E5) rabbit mAb were purchased from Cell Signaling Technology. ECL Western Blotting Detection Reagents were purchased from Thermo.

[0164] Instruments: Western Blot System (Invitrogen / Bio-Rad), and Imaging System (Cell Biosciences)

Compound treatment

[0165] HepG2 cells were cultured in MEM medium supplemented with 10% FBS at 37°C and under 5% C0 ₂ condition. HepG2 cells (1 X 10 ⁶ cells/well) were seeded into 6-well cell culture plates for 24hr. The cells were starved in serum-free MEM medium (3ml/well) for 24hr.

Test Compounds preparation:

[0166] The compounds were dissolved in DMSO to make the stock solution of 5, 25, 50mM respectively. The compounds were diluted to final concentration in serum-free MEM medium to be 5, 25, and 50 μΜ.

Metformin preparation:

[0167] Metformin was dissolved in PBS to make the stock concentration as 1M. Metformin was diluted to final concentration in serum-free MEM medium to be 5, and 50 mM

The serum-free MEM medium was discarded from each well. 3ml of 1 ^χ compounds was added to each well. Cells were incubated in a 37°C, 5% CG ₂ incubator for 6 h (Compound 5, Compound 19 and Metformin).

Western Blot Analysis

[0168] Total proteins (10 μg) from each sample was loaded to sample well of SDS-PAGE. Electrophoresis was run at 120 V constant voltage until the blue marker reaches the end of the gel. The proteins were transferred to a PVDF membrane using Bio-Rad's Trans blot for 40 min at 300 mA. After transfer, the membrane was blocked with blocking buffer for 2hr at room temperature. The membrane was incubated with the corresponding primary antibody solution at 4°C overnight. The membrane was washed for 5min ^χ 3 with 1 xTBST and incubated with secondary antibody solution at room temperature for 45 min. The membrane was washed for 5min ^χ 3 with 1 xTBST and the detection was conducted with chemiluminescence detection reagents. The results are indicated in Figure 1.

Test Example 2

Measurement of solubility in pH4.0 and pH7.4 phosphate buffer solution

1) Preparation of Stock Solutions

[0169] The stock solutions of test compounds and positive control compound were prepared in DMSO at the concentration of 30 mM. Diclofenac was used as positive control in the assay.

2) Procedures for Solubility Determination

[0170] The stock solution of each compound (10 μί) was placed in order into their proper 96-well rack, followed by adding 990 of PBS at pH 4.0 and pH 7.4 into each vial of the cap-less Solubility Sample plate. This study was performed in duplicate. One stir stick was added to each vial and then vials were sealed using a molded PTDE/SIL 96-Well Plate Cover. The Solubility Sample plate was transferred to the Thermomixer Comfort plate shaker and incubated at 25 °C for 2 hours with shaking at 1,100 rpm. After 2 hours incubation, stir sticks were removed using a big magnet and all samples from the Solubility Sample plate were transferred into the filter plate. All the samples were filtered by using the vacuum manifold. The filtered samples were diluted with methanol. 3) Samples analyzed by LC - MS/MS:

[0171] The LC system comprised a Shimadzu liquid chromatograph separation system equipped with degasser DGU-20A3, solvent delivery unit LC-20AD, system controller CBM-20 A, column oven CTO- 1 OAS VP and CTC Analytics HTC PAL System. Mass spectrometric analysis was performed using an API 4000 instrument from AB Inc (Canada) with an ESI interface. The data acquisition and control system were created using Analyst 1.6 software from ABI Inc. The results of the measurements are indicated in Table 2. [Table 2]

Test Example 3

Determination of stability against mouse and human mirosomes

[0172] Pooled human liver microsomes (Cat. 452161 ; Lot. 23418) were purchased from Corning and pooled male mouse liver microsomes (Cat. M1000; Lot. 1210302) were purchased from Xenotech. Microsomes were stored at - 80 °C prior to use. 40 of ultra-pure H ₂ 0 was used instead of NADPH solution in the negative control. The negative control was used to exclude the misleading factor that was resulted from instability of chemical itself. Samples with NADPH were prepared in duplicate. Negative controls were prepared in singlet. [0173] The reaction was started with the addition of 4 μΐ, of 200 μΜ compound solution to each master solution to get the final concentration of 2 μΜ. Verapamil was used as positive control in the assay. Aliquots of 50 were taken from the reaction solution at 0, 15, 30, 45 and 60 min. The reaction was stopped by addition of 4 volumes of cold acetonitrile with IS (200 nM imipramine, 200 nM labetalol and 2 μΜ ketoprofen). Samples were centrifuged at 3,220 g for 40 minutes. Aliquot of 90 μΐ, of the supernatant was mixed with 90 μί of ultra-pure H ₂ 0 and then was used for LC-MS/MS analysis. Results are shown in Table 3 by indicating remaining % of the test compound after 60 minutes incubation with liver misrosomes from human and mouse in the presence of NADPH.

[Table 3]

Test Example 4

[0174] PK profiles of Compound 19 in the blood were analyzed by LC MS/MS after dose administration of the test compounds in SD rats.

Test animal: SD rat (n=3 /group) Vehicle: 10 v/v% DMSO and 90 v/v% PEG400

Dose (single): iv 1 1 and 5mg/kg, po 1 5 and 25 mg/kg

Analytical instrument and condition: LC/MS/MS instrument [LC: Shimazu, MS: AB API4000 (ion source: ESI)], LC column: XSELECT™ CSH™ C18 2.5μιη (2.1 ^χ 50 mm), mobile phase: 5% acetonitrile (0.1% formic acid) and 95% acetonitrile (0.1% formic acid).

The results are shown in Figure 2.

Test Example 5

[0175] The anti-proliferative activities of the compounds against 6 human cancer cell lines were determined by CellTiterGlo assay kit. All cells were obtained from ATCC and cultured as recommended by the vendor. Cells were assayed in exponential growth phase. Sample solution was prepared by 3 -fold serial dilution from 30 mM stock for 10 doses in DMSO. Antiproliferative activities were measured by CellTiterGlo assay kit according to the protocol after cells were incubated for 96 hrs in a 37 °C, 5% C0 ₂ incubator.

[0176] On the 4 ^th day of incubation, i) 15μΙ reagent (CelltiterGlo assay kit) was added per well and plate was shaken (avoiding light) for 2 mins on a plate shaker, ii) the plate was kept (avoiding light) in room temperature for 30min, and iii) the luminescence value was recorded by Multiplate reader. The results are shown in Table 4.

[Table 4]

Antiproliferative activities (IC50: μΜ)

compound MDA-MB-

A549 HT29 BxPC-3 LNCaP U87-MG

number 231

4 4.3 6.4 11.4 4.2 5.1 9.1

5 2.7 2.8 3.2 1.8 2.6 5.5

7 6.5 10 12.8 7.2 6.8 13.1

12 2.9 2.8 2.3 0.9 5.8 3.6

13 2 3.6 2.2 0.8 5.3 3

15 6.7 7 1.7 4.2 4.8 4.4

17 6.8 5.2 1.7 4.1 5 4.5

19 22.3 22.5 17 14.6 47.4 20.4

20 4.8 2.7 2.3 1.9 4.1 2.3

21 5.3 5 1.7 3.8 4.3 3.1

22 1.9 3.8 2.7 1.1 6 3.2 Test Example 6

In vivo efficacy in HFD-STZ C57BL/6J mice model

Diet induced obesity model establishment:

[0177] C57BL/6J mice were fed with high fat diet (Research Diets, D12492) for 64 days, and streptozotocin (10 mg/kg) was given orally on day 22 and day 29. Compound treatment was done from day 36 to day 63.

Groups and Treatments

[0178] Based on the animal's body weight and oral glucose tolerance test (OGTT) on week 5, mice were randomly assigned to respective groups using a computer-generated randomization procedure.

Vehicle group: HFD/STZ + vehicle

Treatment group: HFD/STZ + test compound

Treatment dose: Compound 5 (po. qd x 28: 0.2mg/kg, Img/kg and 5mg/kg) Pioglitazone, a positive control (po. qd x 28: 30mg/kg)

Normal diet group: normal diet + vehicle

Number of animals per group: 8

Vehicle for Compound 5: PEG-400 (Sigma) / Tween 80 (Sigma) buffer = 95:5

Vehicle for pioglitazone: saline

Administration of test compounds:

[0179] Compound 5 and pioglitazone were administered orally once a day for 4 weeks with the dose indicated above.

Glucose Determination:

Blood glucose level was measured with the blood from tail vein every 2nd week by using Accu-Chek Aviva System.

Blood Chemistry Analysis:

[0180] The lipid profile of every 2nd week and terminal blood bio-chemistry parameters were tested as listed in Table 2. Blood samples were obtained from collection on Day 49 and 65, the samples were immediately processed by centrifugation at 4°C, 4,000 g for 15 minutes, and then they were transferred into a new test tube. Lipid profile and full panel blood bio-chemistry parameters were measured by using TOSHIBA TBA-40FR automated biochemical analyzer.

[0181] Figures 3 and 4 show the effect of the compounds of the present invention on the levels of triglyceride and total cholesterol in blood of mice. Figure 5 shows the effect of the compounds of the present invention on the blood glucose level of mice in fasted state.

Test Example 7

Antitumor activity of Compound 5 (po, qd x 14) in U87MG human glioblastoma xenograft model in BALB/c nude mice

Method for Tumor Inoculation

[0182] Each mouse was inoculated subcutaneously on the right flank with U87MG tumor cells (5 x 10 ⁶ ) in 0.1 ml of MEM - Matrigel mixture (7:3 ratio) for tumor development. The treatment was started when the mean tumor size reached approximately 113 mm ³ . Mice were then randomized into groups. The treatments were administrated to the tumor-bearing mice.

Compound treatment

[0183] Compound 5 was dissolved in the vehicle (95% v/v PEG400 + 5% Tween 80) and orally administered to the tumor-bearing mice with three dose levels (50, 100 and 200 mg/kg) for 14 days.

[0184] The . measurement of tumor size was conducted twice weekly with a caliper and the tumor volume (mm ) was estimated using the formula: TV=a ^χ b2/2, where "a" and "b" are long and short diameters of a tumor, respectively. The TVs were used for calculation of the tumor growth inhibition (TGI, an indicator of antitumor effectiveness) value using the formula: TGI = (1-T/C) * 100%, where "T" and "C" was the mean relative volumes (% tumor growth) of the tumors in the treated and the control groups, respectively, on a given day after tumor inoculation. The results are shown in Figure 6.

Test Example 8

Antitumor activity of Compound 5 (po, qd x 14) in Hep3B human liver cancer xenograft model in BALB/c nude mice: single agent and combination with sorafenib.

[0185] Each mouse was inoculated subcutaneously on the right flank with Hep3B tumor cells (5 x 106) in 0.1 ml of MEM - Matrigel mixture (1 :1 ratio) for tumor development. The treatment started when the mean tumor size reached approximately 137 mm ³ . Mice were then randomized into groups.

Compound treatment

[0186] Compound 5 was dissolved in the vehicle (95% v/v PEG400 + 5% Tween 80) and orally administered to the tumor-bearing mice with 200 mg/kg, once daily for the first 5 days, followed by treatment with 100 mg kg for 10 days. A positive control, sorafenib, was dissolved in the vehicle (20% 2-hydroxypropyl-P-cyclodextrin (HPBCD)) and orally administered to the tumor-bearing mice with 80 mg/kg, once daily for 15 days. The measurement of tumor size was conducted twice weekly with a caliper as described above. The results are shown in Figure 7. The results clearly show a significant effect of the compound of the present invention to reduce the proliferation of the liver cancer cells by administration of the compound alone or in combination of sorafenib.

Test Example 9

[0187] In a similar manner to Test Example 6, in vivo efficacy study of Compound 19 was carried out with HFD-STZ C57BL/6J mice model. As a result, Compound 19 (once a day oral treatment for 28 days at 0.25 to 16 mg/kg) significantly decreased blood glucose level (- 68%), and HbAlC level (-31%). In OGTT assay, a group treated with Compound 19 showed significantly improved glucose absorption, specifically blood glucose level after 2 hours of glucose administration was 30% lower and glucose AUC was 25% lower than control group (a group treated with vehicle).