WO2008069500A1 - Triazolopyridazine derivatives having inhibitory activity against acetyl-coa carboxylase - Google Patents

Triazolopyridazine derivatives having inhibitory activity against acetyl-coa carboxylase Download PDF

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WO2008069500A1
WO2008069500A1 PCT/KR2007/006136 KR2007006136W WO2008069500A1 WO 2008069500 A1 WO2008069500 A1 WO 2008069500A1 KR 2007006136 W KR2007006136 W KR 2007006136W WO 2008069500 A1 WO2008069500 A1 WO 2008069500A1
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Prior art keywords
triazolo
pyridazin
hydroxy
cyclohexyl
pyridazine
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PCT/KR2007/006136
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French (fr)
Inventor
Yong Deog Hong
Seung-Hyun Kang
Yeonjoon Kim
Kyoung Hee Byoun
Byoung Young Woo
Miyoung Park
Yung Hyup Joo
Jung Ju Kim
Doo Ok Jang
Jinsung Tae
Dongkyu Shin
Yong Eun Kim
Younghoon Cheon
Jae Il Lee
Young-Lan Hyun
Seonggu Ro
Joong Myung Cho
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Amorepacific Corporation
Industry-Academic Cooperation Foundation, Yonsei University
Crystalgenomics, Inc.
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Application filed by Amorepacific Corporation, Industry-Academic Cooperation Foundation, Yonsei University, Crystalgenomics, Inc. filed Critical Amorepacific Corporation
Publication of WO2008069500A1 publication Critical patent/WO2008069500A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems

Definitions

  • the present invention relates to a novel triazolopyridazine derivative or a pharmaceutically acceptable salt thereof, and An Acetyl-CoA
  • Carboxylase2 (ACC2) activity inhibitor and a pharmaceutical composition for preventing and treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising same as an active ingredient.
  • ACC is an enzyme involved in the synthesis and oxidation of a fatty acid, and composed of three distinct proteins-the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyltransferase (CT). It converts ace ⁇ yl-CoA to malonyl-CoA, a precursor of the fatty acid synthesis. The formation of the malonyl-CoA is the committed rate- determining step of the fatty acid synthesis.
  • ACC exists as two tissue specific isozymes ACCl and ACC2.
  • ACCl (265 kDa) present in lipogenic tissues is subjected to long-term regulation at the transcription stage, and short-term regulation by the allosteric effect brought about by serine phosphoration/dephosphoration or citrate. Further, the concentration and activity of ACCl are affected by food intake or hormone changes. ACCl is present in cytosol, and involved in a long chain fatty acid biosynthesis.
  • ACC2 (280 kDa) present in oxidative tissues (heart and muscle) is also regulated by the allosteric effect brought about by phosphorylation/dephosphorylation or a citrate.
  • ACC2 is localized in the mitochondrial membrane, and the malonyl-CoA formed therefrom inhibits carboxylpalmitoyltransferase-I (CPT-I), one of mitochondria enzymes, by the allosteric effect.
  • CPT-I transfers a long chain acyl-CoA from cytosol to mitochondria to make it possible for the fatty acid to undergo oxidation.
  • ACC is an isozyme involved in the synthesis and oxidation of a fatty acid, and it is reported that the inhibition of ACCl lowers the fatty acid synthesis, while the inhibition of ACC2 enhances the fatty acid oxidation.
  • a cyclohexanedione herbicide derivative which functions as a competitive inhibitor of the rat heart ACC may be used as an anti-obesity agent (Thomas W. Seng et ah, Bioorg. Med. Chem. Lett. 13, 3237, 2003); and a bipiperidine compound, a non-selective ACC inhibitor, may be used to treat metabolic syndrome including obesity, diabetes and arteriosclerosis by inhibiting both the ACC2 activity in oxidative tissues such as the skeletal muscle and the ACCl activity in tissues for the fatty acid synthesis such as liver and adipose tissues (US20030187254, and H. James Harwood, JR et ah, J.Biol.Chem.
  • the thiazole derivative developed by Abbott Lab. shows a high ACC2 selectivity (more than 1,000 times than normal), and in an in vivo experiment, it reduced the malonyl-CoA level in rodent muscle tissues.
  • the reduction of the malonyl-CoA level by inhibiting ACC2 is reported to enhance the fatty acid oxidation, leading to increased total energy consumption through decreased inhibition of CPT-I on the mitochondrial membrane, and as a result, the insulin sensitivity of a type 2 diabetes or obesity patient can be enhanced (Yu Gui Gu et ah, J.MedChem, 49, 3770, 2006).
  • ACC2 Acetyl-CoA Carboxylase2
  • X is hydrogen, pyridyl, thiophenyl, furanyl, or phenyl optionally substituted with Ci_ 5 alkyl, C 1-5 alkoxy, hydroxy, or halogen;
  • Y is pyridine, thiophene, , NHR 2 , in which, Z is O, S, NH, methylene, ethylene, or -CH(CH 3 )-; Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl, I is 1, 2, or 3, and when I is 2 or 3, two Ri 's may be fused together to form a phenyl or cyclohexane ring;
  • R 2 is hydrogen, C ⁇ 7 alkyl, hydroxy, C 3 . 8 cycloalkyl optionally substituted with Ci. 7 alkyl, or phenyl optionally substituted with C ⁇ 5 alkyl;
  • R 3 is selected from the group consisting of hydroxy, C 1-5 alkyl, Ci -5 alkoxy and halogen, and m is 1, 2, or 3;
  • R 4 is selected from the group consisting of hydroxy, C ⁇ . 5 alkyl, Ci -5 alkoxy, trifluoromethyl, Ci_ 5 alkoxycarbonyl and halogen, n is 1, 2, or 3, and when n is 2 or 3, two R 4 ⁇ s may be fused together to form a dioxolane ring; and
  • W is Ci_ 2 alkylene, alkenylene, alkynylene or a bond that directly
  • an ACC2 activity inhibitor comprising the triazolopyridazine derivative of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient.
  • a pharmaceutical composition for preventing or treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising the triazolopyridazine derivative of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient.
  • the preferred triazolopyridazine derivatives of formula (I) of the present invention are those wherein: X is hydrogen, thiophenyl, furanyl, or phenyl optionally substituted with methyl, hydroxy, bromo or chloro; and
  • Y is pyridine, thiophene, , NHR 2 ,
  • Z is O, S, NH, methylene, ethylene or -CH(CH 3 )-;
  • Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl, and I is 1, 2, or 3, and when I is 2 or 3, two R] 's may be fused together to form a phenyl ring;
  • R 2 is hydrogen, C ⁇ 1 alkyl, C 6 . ? cycloalkyl optionally substituted with hydroxy or methyl, or phenyl optionally substituted with methyl;
  • R 4 is selected from the group consisting of hydrogen, hydroxy, methyl, Ci -4 alkoxy, trifluoromethyl, methoxycarbonyl, fluoro, chloro and bromo, n is 1, 2, or 3 and when n is 2 or 3, two R 4 5 S may be fused together to form a dioxolane ring; and
  • W is Ci. 2 alkylene, alkenylene, alkynylene or a bond that directly
  • the more preferred compounds of formula (I) of the present invention are those wherein: X is hydrogen, thiophen-2-yl, furan-2-yl, phenyl optionally substituted with methyl, bromo or chloro; and
  • Ri is selected from the group consisting of methyl, hydroxy or hydroxymethyl, and I is 1, 2, or 3, and when I is 2 or 3, two Ri 's may be fused together to form a phenyl ring;
  • R 2 is hexyl, heptyl, cyclohexyl, cycloheptyl, methylcyclohexyl or hydroxy cyclohexyl;
  • R 4 is selected from the group consisting of hydrogen, methoxy, butoxy, trifluoromethyl. methoxycarbonyl and fluoro, n is 1, 2, or 3, and n is 2 or 3, two R 4 5 S may be fused together to form a dioxolane ring; and
  • the more preferred compounds of formula (I) according to the present invention are the following examples or the pharmaceutically acceptable salts thereof: Cyclohexyl-(3-phenyl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)-amine;
  • the compound of formula (I) of the present invention may be used in the form of a pharmaceutically acceptable salt derived using an inorganic or organic acid, and the preferred are an inorganic acid salt such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and acetic acid, and an organic acid salt such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.
  • an inorganic acid salt such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid
  • the compound of the present invention may be chemically synthesized by the procedure shown in Reaction Schemes (A) to (E), but these are not intended to limit the scope of the invention in any way.
  • R 5 and R 6 are each independently hydrogen, C 1 J 7 alkyl, or C 3 . 8 cycloalkyl optionally substituted with hydroxy or C 1 . 7 alkyl; or fused to each other together with the nitrogen atom they are attached to, to form Q 11 (J ⁇ wherein, Z and Rj have the same meanings as described above.
  • Reaction Scheme (A) i) dichloropyridazine is brought to react with aroylhydrazine in an organic solvent in the presence of a base to obtain 6-chloro-3-aryl-[l,2,4]triazolo[4,3-b]pyridazine (I.Collins et al.
  • the organic solvent may be para-xylene, toluene or benzene
  • the base may be triethylamine hydrochloride.
  • R 7 is hydrogen or C ⁇ 5 alkyl.
  • the solvent in the first step of the above reaction may be ethanol or acetonitrile, and the other solvent used in the second step, para-xylene, toluene or benzene together with a base such as triethylamine hydrochloride.
  • R 8 is hydrogen or Q. 5 alkyl.
  • R 9 is phenyl optionally substituted with hydroxy, C 1 . 5 alkyl, Ci_ 5 alkoxy, trifluoromethyl, C 1 . 5 alkoxycarbonyl or halogen; hydroxy cyclohexyl; or benzdioxolane.
  • the inventive triazolopyridazine derivative of formula (I) inhibiting the activity of ACC2 may prevent or treat obesity, diabetes, dyslipidemia
  • hypercholesterolemia e.g., hypercholesterolemia, hyperlipemia
  • diseases related to metabolic syndrome e.g., arteriosclerosis, hypertension, hyperlipemia
  • the inventive triazolopyridazine derivative of formula (I) can be used for an ACC2 activity inhibitor.
  • the present invention provides a pharmaceutical composition for inhibiting the activity of ACC2 comprising the compound of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient.
  • the compound of formula (I) as an active ingredient may be employed in an amount of 0.01 to 10 weight%, preferably 0.1 to 5 weight% based on the total weight of the inventive pharmaceutical composition.
  • the pharmaceutical composition of the present invention may be formulated for administration orally or parenterally.
  • the formulation for oral administration may include tablets, powder, soft and hard gelatin capsules, aqueous solutions, suspensions, emulsions, syrups and granules, and additionally include conventional additives such as a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose glycine), lubricant (e.g., silica, talc, stearic acid or magnesium or calcium salt thereof, and polyethyleneglycol) and the like.
  • a diluent e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose glycine
  • lubricant e.g., silica, talc, stearic acid or magnesium or calcium salt thereof, and polyethyleneglycol
  • the composition may further comprise a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally include a disintegrant such as starch, agar, alginic acid or a sodium salt thereof, boiling mixture, absorbent, colorant, flavoring agent, and sweetener.
  • a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine
  • a disintegrant such as starch, agar, alginic acid or a sodium salt thereof, boiling mixture, absorbent, colorant, flavoring agent, and sweetener.
  • the preferred formulation for parenteral administration may include injection formulations such as isotonic aqueous solutions and suspensions.
  • composition may be sterilized and/or contain an adjuvant such as a preservative, stabilizer, wetting agent, emulsifier, a salt for controlling an osmotic pressure and/or a buffer solution, and other pharmaceutically effective materials, and formulated in accordance with conventional mixing, granulating or coating methods.
  • an adjuvant such as a preservative, stabilizer, wetting agent, emulsifier, a salt for controlling an osmotic pressure and/or a buffer solution, and other pharmaceutically effective materials, and formulated in accordance with conventional mixing, granulating or coating methods.
  • inventive compound of formula (I) may be administered orally or parenterally as an active ingredient in an effective amount ranging from about 0.01 to 500 mg/kg, preferably from about 0.5 to 100 mg/kg body weight per day in case of mammals including human in a single dose or in divided doses.
  • Example 1 The procedure of Example 1 was repeated except for reacting the following compounds as shown in Table 1 as a starting material to obtain the compounds of Examples 2 to 10. Table 1
  • Example 11 The procedure of Example 11 was repeated except for reacting the following compounds as shown in Table 2 as a starting material to obtain the compounds of Examples 12 to 36.
  • Example 37 The procedure of Example 37 was repeated except for reacting the following compound as shown in Table 3 as a starting material to obtain the compound of Example 38.
  • Example 39 Preparation of cyclohexyl-[l,2,4]triazo ⁇ o[4,3-b]pyridazin-6- yl-amimne (39-1) Preparation of 6-chloro-[l,2,4]1xiazolo[4,3-b]pyridazine (Ic)
  • Example 39 The procedure of Example 39 was repeated except for reacting the following compound as shown in Table 4 as a starting material to obtain the compound of Example 40.
  • Table 4 The procedure of Example 39 was repeated except for reacting the following compound as shown in Table 4 as a starting material to obtain the compound of Example 40.
  • Example 41 The procedure of Example 41 was repeated except for reacting the following compounds as shown in Table 5 as a starting material to obtain the compounds of Examples 42 to 46.
  • Table 5 The procedure of Example 41 was repeated except for reacting the following compounds as shown in Table 5 as a starting material to obtain the compounds of Examples 42 to 46.
  • Example 48 to 51 The procedure of Example 47 was repeated except for reacting the following compounds as shown in Table 6 as a starting material to obtain the compounds of Examples 48 to 51.
  • Test Example 1 Assay for inhibiting the activity of human ACC2
  • Step 1) Cloning and expression of ACC2 gene cDNA cloning of human ACC2 (hACC2) without N-terminus, and the expression thereof in HEK293 cell (ATCC, #CRL- 1573) were carried out as follows.
  • Human ACC2 gene was cloned by PCR using cDNA library of human skeletal muscle (Clontech) as a template, and primers of SEQ ID NO.: 1 (hACC2F) and SEQ ID NO.: 2 (hACC2B).
  • the primers were prepared from a sequence of human ACC2 (hACC2; GenBank accession No.: BC028417) by adding Nhel/Xhol restriction site, and the primer sequence are as shown in Table 7.
  • PCR was carried out using BD Advantage2 PCR Enzyme System (Clontech, #S1798), and the expression and activity was confirmed by inserting the amplified DNA fragment into Nhel/Xhol restriction site of pcDNA3.1-Flag vector (Invitrogen, #V790-20) and transforming thereof into
  • the Flp-In-293 cell lines stably expressing hACC2 was cultured in a
  • DMEM Delbecco's modified eagle medium
  • FBS fetal bovine serum
  • the culture solution was centrifuged at l,000xg for 5 min to obtain the hACC2 expressing cell.
  • the cell was washed with PBS (CGXINC) 5 centrifuged under a same condition as described above, and cryopreserved at -70 TC .
  • the cell was melted at 4 °C , and Complete Protease Inhibitor (Roche, #1873580) was suspended in 50 niM HEPES (2-[4-(2-hydroxyethyl)-l- piperazinyl] ethanesulfonic acid) buffer (pH 7.5) containing 250 mM sucrose, 2 mM EDTA, 5% glycerol and 2 mM dithiothreitol (DTT) per 50 ml cell. The suspension was subjected to a sonicator (Fisher Scientific), centrifuged at 30,000 ⁇ g for 60 min, and filtered with a 0.45 ⁇ m filer.
  • 50 niM HEPES (2-[4-(2-hydroxyethyl)-l- piperazinyl] ethanesulfonic acid) buffer (pH 7.5) containing 250 mM sucrose, 2 mM EDTA, 5% glycerol and 2 mM
  • the supernatant was fractioned 3%, 5% and 10% concentration (w/v) using PEG8000 (Polyethylene glycol 8000), and centrifuged at 30,000 ⁇ g, 4 ° C for 60 min to obtain a supernatant and precipitate.
  • the precipitate was dissolved in salt free buffer (5OmM HEPES, pH7.5, 2mM DTT, 2mM EDTA, 5% glycerin, and protease inhibitor), and the samples expressing the enzyme activity were separated by the protein size using Superdex 200 (Pharmarcia, #17-1069-01) column.
  • a buffer containing 5OmM HEPES, pH 7.5, 2mM DTT, 5% glycerol, protease inhibitor and 125 mM NaCl was used, and the separated hACC2 protein was cryopreserved at -70 ° C .
  • the obtained hACC2 protein was melted, and preincubated in a buffer containing 50 mM Tris (pH7.5), 10 mM potassium citrate, 8 mM MgSO 4 , 1 mM DTT and fatty acid-free BSA at ) at 37 °C for 20 min.
  • the compounds prepared in Examples were dissolved in DMSO to the final concentration of 3 mM, 1 ⁇ i of each compounds was added to the polypropylene tube with 79 f ⁇ of the preincubated hACC2 solution.
  • the control group contained only 1 ⁇ i of DMSO (the final concentration of DMSO was 1%).
  • a substrate mixture containing 0.25 mM ATP, 0.2 mM acetyl-CoA and 0.5 mM NaHCO 3 (2.4 ⁇ Ci) was put into a test tube of the test group and control group to the final volume of 100 ⁇ l, and reacted at 37 ° C for 15 min.
  • % Inhibition ⁇ l-(cpm of the compound treated sample - cpm of Blank)/(cpm of Control - cpm of Blank) ⁇ xlOO wherein, Blank is treated with an equal amount of buffer instead of hACC2 protein, and Control is only treated with an equal amount of DMSO instead of the compound.
  • Test Example 2 Assay for inhibiting the activity of rodent ACC2
  • C3H mouse myoblast C2C12 (ATCC #CRL-1772) was cultured in a culture dish with DMEM containing 105 FBS until the cultured cells are filled up to 70% of the dish.
  • the medium was replace with DMEM containing 1% FBS, and the cell was cultured for 6 days again. The medium was removed, and the cell was washed with PBS.
  • cell lysis buffer a mixture of 50 mM Tris (pH7.5), 1 mM EDTA, 1 mM PMSF 5 0.25% sucrose, 0.4 mg/ml Digitonin, 0.5 mM Na 3 VU 4 and 50 mM NaF) and protease inhibitor, and centrifuged at l,000xg for 5 min. The obtained supernatant was used for the following test.
  • Step 2) Determination of rACC2 inhibitory activity
  • the C2C12 cell extract obtain in step 1) is diluted with 50 mM Tris (pH7.5), 10 mM potassium citrate, 8 mM MgSO 4 , 1 mM DTT and fatty acid- free BSA) to the final concentration of 0.38 mg/ml, and preincubated at 37 ° C for 20 min.
  • step 3 of Test Example 1 The procedure of step 3) of Test Example 1 was repeated except for using the preincubated C2C12 cell extract to determine the rACC2 inhibitory activity. (%) Inhibition of rACC2 was calculated according to the above formula 1, and IC 50 was calculated therefrom. The results are shown in
  • the triazolopyridazine derivatives of formula (I) showed the inhibitory activity ranging from 1 to 20 ⁇ M of IC 50 against rACC2.

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Abstract

The present invention relates to a novel triazolopyridazine derivative or a pharmaceutically acceptable salt thereof, and an Acetyl-CoA Carboxylase2 (ACC2) comprising same as an active ingredient. The triazolopyridazine derivative of the present invention effectively inhibits the activity of ACC2 and it may be used for preventing or treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome.

Description

TRIAZOLOPYRTOAZINE DERIVATIVES HAVING INHIBITORY ACTIVITY AGAINST ACETYL-COA CARBOXYLASE
Field of the Invention
The present invention relates to a novel triazolopyridazine derivative or a pharmaceutically acceptable salt thereof, and An Acetyl-CoA
Carboxylase2 (ACC2) activity inhibitor and a pharmaceutical composition for preventing and treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising same as an active ingredient.
Background of the Invention
ACC is an enzyme involved in the synthesis and oxidation of a fatty acid, and composed of three distinct proteins-the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyltransferase (CT). It converts aceτyl-CoA to malonyl-CoA, a precursor of the fatty acid synthesis. The formation of the malonyl-CoA is the committed rate- determining step of the fatty acid synthesis. ACC exists as two tissue specific isozymes ACCl and ACC2.
ACCl (265 kDa) present in lipogenic tissues (liver and adipose tissue) is subjected to long-term regulation at the transcription stage, and short-term regulation by the allosteric effect brought about by serine phosphoration/dephosphoration or citrate. Further, the concentration and activity of ACCl are affected by food intake or hormone changes. ACCl is present in cytosol, and involved in a long chain fatty acid biosynthesis.
Further, ACC2 (280 kDa) present in oxidative tissues (heart and muscle) is also regulated by the allosteric effect brought about by phosphorylation/dephosphorylation or a citrate. Unlike ACCl, ACC2 is localized in the mitochondrial membrane, and the malonyl-CoA formed therefrom inhibits carboxylpalmitoyltransferase-I (CPT-I), one of mitochondria enzymes, by the allosteric effect. CPT-I transfers a long chain acyl-CoA from cytosol to mitochondria to make it possible for the fatty acid to undergo oxidation. As described above, ACC is an isozyme involved in the synthesis and oxidation of a fatty acid, and it is reported that the inhibition of ACCl lowers the fatty acid synthesis, while the inhibition of ACC2 enhances the fatty acid oxidation.
Wakil et al. (Science, 291, 2613-2616, 2001) reported that ACC2- knockout mice showed normal longevity and fertility, and inspite of intake of a large amount of food, the mice showed a reduced body fat mass and increased fatty acid oxidation. It implies that the inhibition of ACC2 increase the fatty acid oxidation to consume the body fat, and enhances the biochemical index such as weight loss. Therefore, there have been continued studies to develop an effective ACC2 inhibitor.
It is reported that a cyclohexanedione herbicide derivative which functions as a competitive inhibitor of the rat heart ACC may be used as an anti-obesity agent (Thomas W. Seng et ah, Bioorg. Med. Chem. Lett. 13, 3237, 2003); and a bipiperidine compound, a non-selective ACC inhibitor, may be used to treat metabolic syndrome including obesity, diabetes and arteriosclerosis by inhibiting both the ACC2 activity in oxidative tissues such as the skeletal muscle and the ACCl activity in tissues for the fatty acid synthesis such as liver and adipose tissues (US20030187254, and H. James Harwood, JR et ah, J.Biol.Chem. 278(39), 30799, 2003). Further, the thiazole derivative developed by Abbott Lab. shows a high ACC2 selectivity (more than 1,000 times than normal), and in an in vivo experiment, it reduced the malonyl-CoA level in rodent muscle tissues. Thus, the reduction of the malonyl-CoA level by inhibiting ACC2 is reported to enhance the fatty acid oxidation, leading to increased total energy consumption through decreased inhibition of CPT-I on the mitochondrial membrane, and as a result, the insulin sensitivity of a type 2 diabetes or obesity patient can be enhanced (Yu Gui Gu et ah, J.MedChem, 49, 3770, 2006).
Summary of the Invention
Accordingly, it is an object of the present invention to provide a novel triazolopyridazine derivative or a pharmaceutically acceptable salt thereof, which effectively inhibits Acetyl-CoA Carboxylase2 (ACC2) activity. It is another object of the present invention to provide an ACC2 activity inhibitor comprising the compound as an active ingredient.
It is further another object of the present invention to provide a pharmaceutical composition for preventing or treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising the compound as an active ingredient.
In accordance with one aspect of the present invention, there is provided a triazolopyridazine derivative of formula (I) or a pharmaceutically acceptable salt thereof:
Figure imgf000004_0001
wherein:
X is hydrogen, pyridyl, thiophenyl, furanyl, or phenyl optionally substituted with Ci_5 alkyl, C1-5 alkoxy, hydroxy, or halogen; and
Y is pyridine, thiophene,
Figure imgf000004_0003
,
Figure imgf000004_0004
NHR2,
Figure imgf000004_0002
in which, Z is O, S, NH, methylene, ethylene, or -CH(CH3)-; Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl, I is 1, 2, or 3, and when I is 2 or 3, two Ri 's may be fused together to form a phenyl or cyclohexane ring;
R2 is hydrogen, Cμ7 alkyl, hydroxy, C3.8 cycloalkyl optionally substituted with Ci.7 alkyl, or phenyl optionally substituted with Cμ5 alkyl;
R3 is selected from the group consisting of hydroxy, C1-5 alkyl, Ci-5 alkoxy and halogen, and m is 1, 2, or 3;
R4 is selected from the group consisting of hydroxy, Cχ.5 alkyl, Ci-5 alkoxy, trifluoromethyl, Ci_5 alkoxycarbonyl and halogen, n is 1, 2, or 3, and when n is 2 or 3, two R4^s may be fused together to form a dioxolane ring; and
W is Ci_2 alkylene, alkenylene, alkynylene or a bond that directly
links to tne triazolopyridazine ring.
Figure imgf000005_0001
In accordance with another aspect of the present invention, there is provided an ACC2 activity inhibitor comprising the triazolopyridazine derivative of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient. In accordance with further another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising the triazolopyridazine derivative of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient.
Detailed Description of the Invention
The preferred triazolopyridazine derivatives of formula (I) of the present invention are those wherein: X is hydrogen, thiophenyl, furanyl, or phenyl optionally substituted with methyl, hydroxy, bromo or chloro; and
Y is pyridine, thiophene,
Figure imgf000006_0004
, NHR2,
Figure imgf000006_0001
in which;
Z is O, S, NH, methylene, ethylene or -CH(CH3)-;
Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl, and I is 1, 2, or 3, and when I is 2 or 3, two R] 's may be fused together to form a phenyl ring;
R2 is hydrogen, C^1 alkyl, C6.? cycloalkyl optionally substituted with hydroxy or methyl, or phenyl optionally substituted with methyl;
R4 is selected from the group consisting of hydrogen, hydroxy, methyl, Ci-4 alkoxy, trifluoromethyl, methoxycarbonyl, fluoro, chloro and bromo, n is 1, 2, or 3 and when n is 2 or 3, two R4 5S may be fused together to form a dioxolane ring; and
W is Ci.2 alkylene, alkenylene, alkynylene or a bond that directly
links to the triazolopyridazine ring.
Figure imgf000006_0002
The more preferred compounds of formula (I) of the present invention are those wherein: X is hydrogen, thiophen-2-yl, furan-2-yl, phenyl optionally substituted with methyl, bromo or chloro; and
Y
Figure imgf000006_0005
Figure imgf000006_0003
Figure imgf000007_0002
in which;
Z is O, methylene or ethylene; Ri is selected from the group consisting of methyl, hydroxy or hydroxymethyl, and I is 1, 2, or 3, and when I is 2 or 3, two Ri 's may be fused together to form a phenyl ring;
R2 is hexyl, heptyl, cyclohexyl, cycloheptyl, methylcyclohexyl or hydroxy cyclohexyl; R4 is selected from the group consisting of hydrogen, methoxy, butoxy, trifluoromethyl. methoxycarbonyl and fluoro, n is 1, 2, or 3, and n is 2 or 3, two R4 5S may be fused together to form a dioxolane ring; and
W is -CH=CH-, -C=C- or a bond that directly links
Figure imgf000007_0001
to the triazolopyridazine ring.
The more preferred compounds of formula (I) according to the present invention are the following examples or the pharmaceutically acceptable salts thereof: Cyclohexyl-(3-phenyl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)-amine;
Cyclohexyl-(3-furan-2-yl-[ 1 ,2,4]triazolo[4,3-b]ρyridazin-6-yl)- amine;
Cyclohexyl-(3-thioρhen-2-yl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)- amine; (2-Methylcyclohexyl)-(3-thioρhen-2-yl-[l,2,4]triazolo[453- b]pyridazin-6-yl)-amine
2-(3-Thiophen-2-yl-[l,2,4]triazolo[4,3-b]ρyridazin-6-ylamino)- cyclohexanol;
{ 1 -[3-(4-Chloroρhenyl)-[l ,2,4]triazolo[4,3-b]pyridazin-6-yl]- pipeiϊdin-2-yl}methanol; 3-(4-Chlorophenyl)-6-morpholin-4-yl-[l,2,4] triazolo [4,3- bjpyridazine;
3 -(3 -Bromophenyl)-6-piperidin- 1 -yl- [ 1 ,2,4]triazolo [4,3-b]ρyridazine;
[2-(3-Phenyl-[l ,2,4]1ήazolo[4,3-b]pyπdazin-6-yl)- 1 ,2,3,4- tetrahydroisoquinolin-3-yl]methanol;
6-Piperidin-l-yl-3-para-tolyl-[l,2,4]triazolo[4,3-b]pyridazine;
[l-(3-Para-tolyl-[l,2,4]triazolo[4,3-b]ρyridazin-6-yl)-ρiρeridin-3-yl]- methanol;
3 -(4-Chlorophenyl)-6-(3 ,5 -dimethylpiperidin- 1 -yl)- [l,2,4]triazolo[4,3-b] pyridazine;
Cyclohexyl-[1 ,2,4]triazolo[4,3-b]pyridazin-6-ylamine; and
(2-Methylcyclohexyl)-[l,2,4]triazolo[4,3-b]pyridazin-6-ylamine.
The most preferred compounds of formula (I) of the present invention are the following examples or pharmaceutically acceptable salts thereof:
Cyclohexyl-(3-furan-2-yl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)- amine;
Cyclohexyl-(3-thiophen-2-yl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)- amine;
(2-Methylcyclohexyl)-(3-thioρhen-2-yl-[l,2,4]triazolo[4,3- b]pyridazin-6-yl)-amine;
2-(3-Thioρhen-2-yl-[l,2,4]triazolo[4,3-b]pyridazin-6-ylamino)- cyclohexanol; 3-(4-Chlorophenyl)-6-morpholin-4-yl-[l,2,4] triazolo [4,3- b]pyridazine;
3 -(3 -Bromophenyl)-6-ρiperidin- 1 -yl- [ 1 ,2,4]triazolo [4,3 -b]ρyridazine; 3-(4-Chloroρhenyl)-6-(3,5-dimethylpiρeridin- 1 -yl)- [l,2,4]triazolo[4,3-b] pyridazine; Cyclohexyl-[l,2,4]triazolo[4,3-b]pyridazm~6-ylamine; and (2-Methylcyclohexyl)-[l,2,4]triazolo[4,3-b]pyridazin-6-ylamine.
The compound of formula (I) of the present invention may be used in the form of a pharmaceutically acceptable salt derived using an inorganic or organic acid, and the preferred are an inorganic acid salt such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and acetic acid, and an organic acid salt such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.
The compound of the present invention may be chemically synthesized by the procedure shown in Reaction Schemes (A) to (E), but these are not intended to limit the scope of the invention in any way.
Reaction Scheme CA)
Figure imgf000009_0001
wherein,
R5 and R6 are each independently hydrogen, C1J7 alkyl, or C3.8 cycloalkyl optionally substituted with hydroxy or C 1.7 alkyl; or fused to each other together with the nitrogen atom they are attached to, to form
Figure imgf000009_0002
Q11(J ^ wherein, Z and Rj have the same meanings as
Figure imgf000009_0003
described above. As shown in Reaction Scheme (A), i) dichloropyridazine is brought to react with aroylhydrazine in an organic solvent in the presence of a base to obtain 6-chloro-3-aryl-[l,2,4]triazolo[4,3-b]pyridazine (I.Collins et al.5 Tetrahedron Letters, 41, 781, 2000); and ii) the compound thus obtained is allowed to react with NHR5R6 amine to obtain 6-alkylamino-3-aryl- [l,2,4]triazolo[4,3-b]ρyridazine (Ia).
In this reaction, the organic solvent may be para-xylene, toluene or benzene, and the base may be triethylamine hydrochloride.
Reaction Scheme (B)
Figure imgf000010_0001
wherein, R7 is hydrogen or Cμ5 alkyl.
As shown in Reaction Scheme (B), the compound obtained by reacting arylamine (ArNH2) with dichloropyridazine in a solvent is treated with aroylhydrazine in a solvent in the presence of a base to obtain 6- aryollamino-3 -aryl- [ 1 ,2,4]triazolo [4,3 -bjpyridazine (Ib) .
The solvent in the first step of the above reaction may be ethanol or acetonitrile, and the other solvent used in the second step, para-xylene, toluene or benzene together with a base such as triethylamine hydrochloride.
Reaction Scheme (C)
Figure imgf000010_0002
wherein, R8 is hydrogen or Q.5 alkyl.
As shown in Reaction Scheme (C)3 6-chloro-[l,2,4]triazolo[4,3- b]pyridazine obtained by the reaction of (6-chloropyridazin-3-yl)hydrazine with the oxime derivative in isopropyl alcohol (I.Kolenc et al., Acta CMm. Slov. 46(2), 281, 1999) is subjected to a reaction with a cyclohexane amine derivate to obtain the compound of formula (Ic).
Reaction Scheme (D)
Figure imgf000011_0001
wherein, R9 is phenyl optionally substituted with hydroxy, C 1.5 alkyl, Ci_5 alkoxy, trifluoromethyl, C1.5 alkoxycarbonyl or halogen; hydroxy cyclohexyl; or benzdioxolane.
As shown in Reaction Scheme (D), 6-chloro-3-ayl- [l,2,4]triazolo[4,3-b]pyridazine is subjected to a cross-coupling reaction with an alkyne under Sonogashira reaction condition to obtain the compound of formula (Id). (L.R Lemiere et al., Tetrahedron 57, 10009, 2001).
Reaction Scheme (E)
Figure imgf000011_0002
As shown in Reaction Scheme (E), 6-chloro-3-aryl- [l,2,4]triazolo[4,3-b]pyridazine is subjected to Suzuki reaction with arylboronic acid to obtain the compound of formula (Ie). The compounds used in the above reactions as starting materials may be easily prepared according to the conventional methods or purchased commercially.
The inventive triazolopyridazine derivative of formula (I) inhibiting the activity of ACC2 may prevent or treat obesity, diabetes, dyslipidemia
(e.g., hypercholesterolemia, hyperlipemia) and diseases related to metabolic syndrome (e.g., arteriosclerosis, hypertension, hyperlipemia) by increasing the fatty acids oxidation.
Therefore, the inventive triazolopyridazine derivative of formula (I) can be used for an ACC2 activity inhibitor.
Further, the present invention provides a pharmaceutical composition for inhibiting the activity of ACC2 comprising the compound of formula (I) or the pharmaceutically acceptable salt thereof as an active ingredient.
The compound of formula (I) as an active ingredient may be employed in an amount of 0.01 to 10 weight%, preferably 0.1 to 5 weight% based on the total weight of the inventive pharmaceutical composition.
The pharmaceutical composition of the present invention may be formulated for administration orally or parenterally. The formulation for oral administration may include tablets, powder, soft and hard gelatin capsules, aqueous solutions, suspensions, emulsions, syrups and granules, and additionally include conventional additives such as a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose glycine), lubricant (e.g., silica, talc, stearic acid or magnesium or calcium salt thereof, and polyethyleneglycol) and the like. In the case of the tablet form, the composition may further comprise a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally include a disintegrant such as starch, agar, alginic acid or a sodium salt thereof, boiling mixture, absorbent, colorant, flavoring agent, and sweetener. Further, the preferred formulation for parenteral administration may include injection formulations such as isotonic aqueous solutions and suspensions.
The composition may be sterilized and/or contain an adjuvant such as a preservative, stabilizer, wetting agent, emulsifier, a salt for controlling an osmotic pressure and/or a buffer solution, and other pharmaceutically effective materials, and formulated in accordance with conventional mixing, granulating or coating methods.
The inventive compound of formula (I) may be administered orally or parenterally as an active ingredient in an effective amount ranging from about 0.01 to 500 mg/kg, preferably from about 0.5 to 100 mg/kg body weight per day in case of mammals including human in a single dose or in divided doses.
The following Examples are intended to further illustrate the present invention without limiting its scope.
Example 1: Preparation of cyclohexyl-(3-phenyl-[l,2,4]triazolo[4,3-b] pyridazin-6-yl)amine
(1-1) Preparation of 6-chloro-3-phenyl-[l,2,4]triazolo[4,3-b]pyridazine
Figure imgf000013_0001
A mixture of 3,6-dichloropyridazine (10.0 g, 67.1 mmol), benzoylhydrazine (10.2 g, 74.6 mmol) and triethylamine hydrochloride (10.3 g, 74.6 mmol) was dissolved in para-xylene (40 ml) and refluxed for 24 hours. The solvent was removed under a reduced pressure, and the resulting residue was extracted with dichloromethane and water. The aqueous layer was basified with potassium carbonate, and extracted twice with dichloromethane. The organic layers were combined and the solvent was removed therefrom. The resulting residue was recrystalized from ethyl acetate to obtain the title compound as a yellow solid (15.0 g, 96%).
1H NMR (SOO MHz, CDCl3) 8.35(2H5 d), 8.12(1H5 d), 7.58-7.40(3H5 m), 7.08(1H, d).
(1-2) Preparation of cyclohexyl-(3 -phenyl- [I5254]triazolo [4,3 -b]pyridazin-6- yl)amine
Figure imgf000014_0001
A mixture of the 6-chloro-3-phenyl-[l,2,4]triazolo[4,3-b]pyridazine obtained in (1-1) (50 mg, 0.22 mmol) and cyclohexylamine (44 mg, 0.44 mmol) was refluxed at 130 °C for 24 hours. The resulting mixture was diluted with water (1 ml), and extracted twice with dichloromethane. The solvent was removed, and the resulting residue was purified by column chromatography (ethyl acetate) to obtain the title compound as a white solid (40 mg, 62%).
Examples 2 to 10
The procedure of Example 1 was repeated except for reacting the following compounds as shown in Table 1 as a starting material to obtain the compounds of Examples 2 to 10. Table 1
Figure imgf000015_0001
Figure imgf000016_0001
Example 11: Preparation of 3-phenyl-6-piperidin-l-yl- [1 ,2,4] triazolo [4,3-b] py ridazine
Figure imgf000017_0001
6-Chloro-3-ρhenyl-[l,2,4]triazolo[4,3-b]ρyridazine (200 mg, 0.87 mmol) thus obtained in (1-1) was mixed with piperidine (89 mg, 1.0 mmol) and diisopropylethylamine (225 mg, 1.7 mmol) in acetonitrile (2 ml), and heated at 85 °C for 24 hours. The resulting mixture was diluted with water (1 ml), and extracted twice with dichloromethane. The solvent was removed, and the resulting residue was purified by column chromatography (hexane:ethyl acetate = 1:2) to obtain the title compound as a white solid (192 mg, 80%).
Examples 12 to 36
The procedure of Example 11 was repeated except for reacting the following compounds as shown in Table 2 as a starting material to obtain the compounds of Examples 12 to 36.
Table 2
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Example 37: Preparation of phenyl-(3-thiophen-2-yl-[l,2,4]triazolo[4,3- b] pyridazin-6-yl)amine
(37-1) Preparation of (6-chloro-pyridazin-3-yl)-phenylamine
Figure imgf000023_0001
A mixture of 3,6-dichloropyridazine (200 mg, 1.3 mmol) and aniline
(125 mg, 1.3 mmol) was dissolved in ethanol (2 ml), and refluxed for 24 hours. The resulting mixture was cooled to room temperature, and the produced precipitate was filtered to obtain the title compound as a white solid (250 mg, 93%).
(37-2) Preparation of ρhenyl-(3-thiophen-2-yl-[l,2,4]triazolo[4,3- b]pyridazin-6-yl)amine
Figure imgf000023_0002
(6-Chloropyridazin-3-yl)phenylamine thud obtained in (37-1) (200 mg, 0.97 mmol), 2-thiophene carboxylic acid hydrazide (184 mg 1.07 mmol) and triethylamine hydrochloride (147 mg, 1.1 mmol) were dissolved in para- xylene (4 ml), and refluxed for 24 hours. The solvent was removed under a reduced pressure, and the resulting residue was extracted with dichloromethane and water. The separated water layer was basified with potassium carbonate, and extracted twice with dichloromethane. The solvent was removed, and the resulting residue was crystallized with ethyl acetate to obtain the title compound as a solid (270 mg, 95%).
Example 38
The procedure of Example 37 was repeated except for reacting the following compound as shown in Table 3 as a starting material to obtain the compound of Example 38.
Table 3
Figure imgf000024_0001
Example 39: Preparation of cyclohexyl-[l,2,4]triazoϊo[4,3-b]pyridazin-6- yl-amimne (39-1) Preparation of 6-chloro-[l,2,4]1xiazolo[4,3-b]pyridazine (Ic)
Figure imgf000025_0001
A mixture of (6-chloro-pyridazin-3-yl)-hydrazine (1.0 g, 7.0 mmol) and cyclohexanone (9-diethoxymethyloxinie (1.5 g, 7.0 mmol) was refluxed in isopropyl (10 ml) for 3 hours, and cooled to room temperature. The produced precipitate was filtered, and recrystalized with dichloromethane to obtain the title compound as a white solid (720 mg, 71%).
(39-2) Preparation of cyclohexyl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl- amimne
Figure imgf000025_0002
A mixture of 6-chloro-[l,2,4]triazolo[4,3-b]pyridazine (100 mg, 0.65 mmol) and cyclohexylamine (129 mg, 1.3 mmol) was refluxed at 130°C for
24 hours. The resulting mixture was diluted with water (1 ml), and extracted twice with dichloromethane. The solvent was removed under a reduced pressure, and the resulting residue was purified by column chromatography to obtain the title compound as a brown solid (128 mg, 91%).
Example 40
The procedure of Example 39 was repeated except for reacting the following compound as shown in Table 4 as a starting material to obtain the compound of Example 40. Table 4
Figure imgf000026_0002
Example 41: Preparation of 6-phenyIethynyl-3-thiophen-2-yI- [l,2,4]triazolo[4,3-b]pyridazine
(41-1) Preparation of 6-chloro-3-thiophen-2-yl-[l,2,4]triazolo[4,3- b]pyridazine
Figure imgf000026_0001
3.6-Dichloropyridazine (5.0 g, 33.6 mmol), 2-thioρhene carboxylic acid hydrazide (6.4 g, 37.3 mmol) and triethylamine hydrochloride (5.2 g, 37.3 mmol) were refluxed in para-xylene (20 ml) for 24 hours. The solvent was removed under a reduced pressure, and the resulting residue was extracted with dichloromethane and water. The separated water layer was basified with potassium carbonate, and extracted twice with dichloromethane. The organic layer was removed, and the resulting residue was recrystalized with ethyl acetate and purified to obtain the title compound as a yellow solid (6.7 g, 67%).
(41-2) Preparation of 6-phenylethynyl-3-thiphen-2-yl-[l,2,4]triazolo[4,3- b]pyridazine
Figure imgf000027_0001
A mixture of 6-chloro-34hiophen-2-yl-[l,2,4]triazolo[4,3- b]pyridazine obtained in (41-1) (100 mg, 0.42 mmol), phenylacetylene (52 mg5 0.50 mmol), bis[triphenylphosphine]palladium dichloride (9.0 mg, 0.013 mmol), copper iodide (I) (4.2 mg, 0.022 mmol) and triethylamine (85 mg, 0.84 mmol) were dissolved in tetrahydrofuran (1 ml), and heated in a pressure tube at 60 °C for 12 hours. The resulting mixture was diluted with water (1 ml), and extracted three times with ethyl acetate. The solvent was removed, and the resulting residue was purified by column chromatography (hexane:ethyl acetate = 1 :1) to obtain the title compound as a brown solid (112 mg, 88%).
Examples 42 to 46
The procedure of Example 41 was repeated except for reacting the following compounds as shown in Table 5 as a starting material to obtain the compounds of Examples 42 to 46. Table 5
Figure imgf000028_0001
Example 47: Preparation of 6-(3-butoxyphenyl)-3-phenyl- [l,2,4]triazolo[4,3-b]pyridazine
Figure imgf000029_0001
6-Chloro-3-thiophen-2-yl-[l,2,4]triazolo[4,3-b]pyτidazine thus obtained in (41-1) of Example 47 (100 mg, 0.42 mmol), 3- butoxyphenylboronic acid (98 mg, 0.5 mmol), tetrakis[triphenylphosphine]palladium ((PPh3)4Pd, 15 mg, 0.013 mmol) and 2M sodium carbonate (2 mmol) were dissolved in 1,2-dimethoxyethane in a pressure tube at 80 °C for 12 hours. The resulting mixture was diluted with water (1 ml), and extracted three times with ethyl acetate. The solvent was removed, and the resulting residue was purified by column chromatography (hexane:ethyl acetate = 1 :1) to obtain the title compound as a white solid (105 mg, 71%).
Example 48 to 51 The procedure of Example 47 was repeated except for reacting the following compounds as shown in Table 6 as a starting material to obtain the compounds of Examples 48 to 51.
Table 6
Figure imgf000030_0001
Test Example 1: Assay for inhibiting the activity of human ACC2
Step 1) Cloning and expression of ACC2 gene cDNA cloning of human ACC2 (hACC2) without N-terminus, and the expression thereof in HEK293 cell (ATCC, #CRL- 1573) were carried out as follows.
Human ACC2 gene was cloned by PCR using cDNA library of human skeletal muscle (Clontech) as a template, and primers of SEQ ID NO.: 1 (hACC2F) and SEQ ID NO.: 2 (hACC2B). The primers were prepared from a sequence of human ACC2 (hACC2; GenBank accession No.: BC028417) by adding Nhel/Xhol restriction site, and the primer sequence are as shown in Table 7.
Table 7
Figure imgf000031_0001
PCR was carried out using BD Advantage2 PCR Enzyme System (Clontech, #S1798), and the expression and activity was confirmed by inserting the amplified DNA fragment into Nhel/Xhol restriction site of pcDNA3.1-Flag vector (Invitrogen, #V790-20) and transforming thereof into
293T (ATCC) cell.
The hACC2 confirmed its activity was treated with Nhel/Xhol restriction enzyme, and subcloned into Flp-In™-293 cell with pcDNA5/FRT vector (Invitrogen, #D6020-01) and pOG44 vector (Flp-restriction enzyme expression plasmid, Invitrogen, #V6005-20) to prepare a stable cell line continuously and stably expressing hACC2.
In this test, 100 /zg/ml of hygromycin (Invitrogen, #10687-010) as an antibiotics for selection. Step 2) Isolation of hACC2 protein
The Flp-In-293 cell lines stably expressing hACC2 was cultured in a
150mm culture dish with DMEM (Delbecco's modified eagle medium) containing 10% FBS (fetal bovine serum), 1% Antibiotic- Antimycotic
(Invitrogen, #15240-062) and 100 /zg/ml hygromycin at 37 °C, 5% CO2 for about 7 days.
The culture solution was centrifuged at l,000xg for 5 min to obtain the hACC2 expressing cell. The cell was washed with PBS (CGXINC)5 centrifuged under a same condition as described above, and cryopreserved at -70 TC .
The cell was melted at 4 °C , and Complete Protease Inhibitor (Roche, #1873580) was suspended in 50 niM HEPES (2-[4-(2-hydroxyethyl)-l- piperazinyl] ethanesulfonic acid) buffer (pH 7.5) containing 250 mM sucrose, 2 mM EDTA, 5% glycerol and 2 mM dithiothreitol (DTT) per 50 ml cell. The suspension was subjected to a sonicator (Fisher Scientific), centrifuged at 30,000χg for 60 min, and filtered with a 0.45 μm filer. The supernatant was fractioned 3%, 5% and 10% concentration (w/v) using PEG8000 (Polyethylene glycol 8000), and centrifuged at 30,000χg, 4 °C for 60 min to obtain a supernatant and precipitate. The precipitate was dissolved in salt free buffer (5OmM HEPES, pH7.5, 2mM DTT, 2mM EDTA, 5% glycerin, and protease inhibitor), and the samples expressing the enzyme activity were separated by the protein size using Superdex 200 (Pharmarcia, #17-1069-01) column. In this step, a buffer containing 5OmM HEPES, pH 7.5, 2mM DTT, 5% glycerol, protease inhibitor and 125 mM NaCl was used, and the separated hACC2 protein was cryopreserved at -70 °C .
Step 3) Determination of ACC2 (hACC2) inhibitory activity
The obtained hACC2 protein was melted, and preincubated in a buffer containing 50 mM Tris (pH7.5), 10 mM potassium citrate, 8 mM MgSO4, 1 mM DTT and fatty acid-free BSA at ) at 37 °C for 20 min.
The compounds prepared in Examples were dissolved in DMSO to the final concentration of 3 mM, 1 μi of each compounds was added to the polypropylene tube with 79 fΛ of the preincubated hACC2 solution. In this step, the control group contained only 1 μi of DMSO (the final concentration of DMSO was 1%). A substrate mixture containing 0.25 mM ATP, 0.2 mM acetyl-CoA and 0.5 mM NaHCO3 (2.4 μCi) was put into a test tube of the test group and control group to the final volume of 100 μl, and reacted at 37 °C for 15 min. 50 μi of 6N HCl was added thereto to complete the reaction, and the resulting mixture was centrifuged at l,000*g for 2 min. The supernatant was dried by evaporation on GF/C filter for more than 1 hour, the dried filter was put in a bial, and 2 ml of a liquid scintillation fluid was added thereto. Then, the radioactivity was measured by using a liquid scintillation counter, and the inhibition rate (%) of hACC2 was calculated according to the Formula 1. The results are shown in Table 8.
Formula 1
% Inhibition = {l-(cpm of the compound treated sample - cpm of Blank)/(cpm of Control - cpm of Blank)}xlOO wherein, Blank is treated with an equal amount of buffer instead of hACC2 protein, and Control is only treated with an equal amount of DMSO instead of the compound.
Table 8
Figure imgf000034_0001
As shown in Table 8, the triazolopyridazine derivatives of formula (I) showed maximum 84% inhibition against hACC2 on 30 μM.
Test Example 2: Assay for inhibiting the activity of rodent ACC2
Step 1) Preparation of ACC2
C3H mouse myoblast C2C12 (ATCC #CRL-1772) was cultured in a culture dish with DMEM containing 105 FBS until the cultured cells are filled up to 70% of the dish. In order to differentiate the cell into a muscle cell, the medium was replace with DMEM containing 1% FBS, and the cell was cultured for 6 days again. The medium was removed, and the cell was washed with PBS. 50 ml of the cell was lysed in a mixture of cell lysis buffer (a mixture of 50 mM Tris (pH7.5), 1 mM EDTA, 1 mM PMSF5 0.25% sucrose, 0.4 mg/ml Digitonin, 0.5 mM Na3VU4 and 50 mM NaF) and protease inhibitor, and centrifuged at l,000xg for 5 min. The obtained supernatant was used for the following test.
Step 2) Determination of rACC2 inhibitory activity
The C2C12 cell extract obtain in step 1) is diluted with 50 mM Tris (pH7.5), 10 mM potassium citrate, 8 mM MgSO4, 1 mM DTT and fatty acid- free BSA) to the final concentration of 0.38 mg/ml, and preincubated at 37 °C for 20 min.
The procedure of step 3) of Test Example 1 was repeated except for using the preincubated C2C12 cell extract to determine the rACC2 inhibitory activity. (%) Inhibition of rACC2 was calculated according to the above formula 1, and IC50 was calculated therefrom. The results are shown in
Table 9.
Table 9
Figure imgf000036_0001
As shown in Table 9, the triazolopyridazine derivatives of formula (I) showed the inhibitory activity ranging from 1 to 20 μM of IC50 against rACC2.
While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made and also fall within the scope of the invention as defined by the claims that follow.

Claims

What is claimed is:
1. A triazolopyridazine derivative of formula (I) or a pharmaceutically acceptable salt thereof:
Figure imgf000037_0001
wherein:
X is hydrogen, pyridyl, thiophenyl, furanyl, or phenyl optionally substituted with Ci_5 alkyl, Ci_5 alkoxy, hydroxy, or halogen; and
Y is pyridine, thiophene,
Figure imgf000037_0004
NHR2,
Figure imgf000037_0002
in which,
Z is O5 S, NH, methylene, ethylene, or -CH(CH3)-; Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl. I is 1, 2, or 3, and when I is 2 or 3, two R1 5S may be fused together to form a phenyl or cyclohexane ring; R2 is hydrogen, C 1.7 alkyl, hydroxy, C3.8 cycloalkyl optionally substituted with C^7 alkyl, or phenyl optionally substituted with Ci_5 alkyl;
R3 is selected from the group consisting of hydroxy, C1-5 alkyl, C1-5 alkoxy and halogen, and m is 1, 2, or 3;
R4 is selected from the group consisting of hydroxy, Cμ5 alkyl, Ci_5 alkoxy, trifluoromethyl, Ci_5 alkoxycarbonyl and halogen, n is 1, 2, or 3, and when n is 2 or 3, two R4 5S may be fused together to form a dioxolane ring; and
W is Ci_2 alkylene, alkenylene, alkynylene or a bond that directly
links to the triazolopyridazine ring.
Figure imgf000037_0003
2. The derivative or salt of claim I5 wherein:
X is hydrogen, thiophenyl, furanyl, or phenyl optionally substituted with methyl, hydroxy, bromo or chloro; and
Y is pyridine, thiophene,
Figure imgf000038_0004
, <
Figure imgf000038_0005
, NHR25
Figure imgf000038_0001
in which;
Z is O, S, NH, methylene, ethylene or -CH(CH3)-; Ri is selected from the group consisting of methyl, hydroxy and hydroxymethyl, and I is 1, 2, or 3, and when I is 2 or 3, two Ri 's may be fused together to form a phenyl ring; R2 is hydrogen, C6.7 a.kyl, C6_7 cycloalkyl optionally substituted with hydroxy or methyl, or phenyl optionally substituted with methyl;
R4 is selected from the group consisting of hydrogen, hydroxy, methyl, Q.4 alkoxy, trifluoromethyl, methoxycarbonyl, fluoro, chloro and bromo, n is 1, 2, or 3 and when n is 2 or 3, two Rj's may be fused together to form a dioxolane ring; and
W is Ci_2 alkylene, alkenylene, alkynylene or a bond that directly
links to the triazolopyridazine ring.
Figure imgf000038_0002
3. The derivative or salt of claim 2, wherein:
X is hydrogen, thiophen-2-yl, furan-2-yl, phenyl optionally substituted with methyl, bromo or chloro; and
Figure imgf000038_0003
Figure imgf000039_0001
in which;
Z is O, methylene or ethylene; Ri is selected from the group consisting of methyl, hydroxy or hydroxymethyl, and & is 1, 2, or 3, and when i is 2 or 3, two Ri 's may be fused together to form a phenyl ring;
R2 is hexyl. heptyl, cyclohexyl, cycloheptyl, methylcyclohexyl or hydroxycyclohexyl; R4 is selected from the group consisting of hydrogen, methoxy, butoxy, trifluoromethyl, methoxycarbonyl and fluoro, n is 1, 2, or 3, and n is 2 or 3, two R4 5S may be fused together to form a dioxolane ring; and
W is -CH=CH-, -C=C- or a bond that directly links
Figure imgf000039_0002
to the triazolopyridazine ring.
4. The derivative or salt of claim 3, which is selected from the group consisting of:
Cyclohexyl-(3 -phenyl- [ 1 ,2,4]triazolo [4,3 -b]pyridazin-6-yl)-amine; Cyclohexyl-(3-furan-2-yl-[l52,4]triazolo[4,3-b]pyridazin-6-yl)- amine;
Cyclohexyl-(3-thioρhen-2-yl-[l,2,4]triazolo[4?3-b]ρyridazin-6-yl)- amine;
(2-Methylcyclohexyl)-(3-thioρhen-2-yl-[l,2,4]triazolo[4,3- b]pyridazin-6-yl)-amine;
2-(3 -Thioρhen-2-yl- [ 1 ,2,4]triazolo[4,3 -b]pyridazin-6-ylamino)- cyclohexanol;
{l-[3-(4-Chlorophenyl)-[l,2,4]triazolo[4,3-b]ρyridazin-6-yl]- piperidin-2-yl}methanol; 3-(4-Chlorophenyl)-6-morρholin-4-yl-[l52,4]triazolo[4,3- b]pyridazine;
3 -(3 -Bromoρhenyl)-6-ρiρeridin- 1 -yl-[ 1 ,2,4]triazolo[4,3 -b]pyridazine;
[2-(3-Phenyl-[l ,2,4]triazolo[4,3-b]pyridazin-6-yl)-l ,2,3,4- tetrahydroisoquinolin-3-yl]methanol; 6-Piperidin-l-yl-3-para-tolyl-[l,2,4]triazolo[4,3-b]pyridazine;
[l-(3-Para-tolyl-[l,2,4]triazolo[4,3-b]pyridazin-6-yl)-piperidin-3-yl]- methanol;
3-(4-Chlorophenyl)-6-(3,5-dimethylpiperidin- 1 -yl)- [l,2,4]triazolo[4,3-b] pyridazine; Cyclohexyl-[1.2,4]triazolo[4,3-b]pyridazin-6-ylamine; and
(2-Methylcyclohexyl)-[l,2,4]triazolo[4,3-b]pyridazin-6-ylamine.
5. The derivative or salt of claim 4, which is selected from the group consisting of: Cyclohexyl-(3-furan-2-yl-[ 1 ,2,4]triazolo[4,3-b]pyridazin-6-yl)- amine;
Cyclohexyl-(3-thiophen-2-yl-[l,2J4]triazolo[4,3-b]pyridazin-6-yl)- amine;
(2-Methylcyclohexyl)-(3-thiophen-2-yl-[l,2,4]triazolo[4,3- b]pyridazin-6-yl)-amine;
2-(3 -Thiophen-2-yl- [ 1 ,2,4]triazolo[4,3 -b]pyridazin-6-ylamino)- cyclohexanol;
3 -(4-Chlorophenyl)-6-morpholin-4-yl- [ 1 ,2,4]triazolo [4,3 - b]pyridazine; 3-(3-Bromophenyl)-6-piperidin-l-yl-[l,2,4]tiazolo[4,3-b]pyridazine;
3-(4-Chlorophenyl)-6-(3,5-dimethylpiperidin- 1 -yl)- [l,2,4]triazolo[4,3-b] pyridazine;
Cyclohexyl-[l52,4]triazolo[4,3-b]pyridazin-6-ylamine; and (2-Methylcyclohexyl)-[l,2,4]triazolo[4,3-b]pyridazin-6-ylamine.
6. An Acetyl-CoA Carboxylase2 (ACC2) activity inhibitor comprising the triazolopyridazine derivative or salt of claim 1 as an active ingredient.
7. A pharmaceutical composition for preventing or treating obesity, diabetes, dyslipidemia and diseases related to metabolic syndrome comprising the triazolopyridazine derivative or salt of claim 1 as an active ingredient.
PCT/KR2007/006136 2006-12-07 2007-11-30 Triazolopyridazine derivatives having inhibitory activity against acetyl-coa carboxylase WO2008069500A1 (en)

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CN107474047A (en) * 2017-08-29 2017-12-15 浙江工业大学 synthesis method of 1,2, 4-triazolo heterocyclic compound
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US8188083B2 (en) 2007-06-28 2012-05-29 Abbott Laboratories Triazolopyridazines
EP2170337A4 (en) * 2007-06-28 2013-12-18 Abbvie Inc Novel triazolopyridazines
EP2170337A1 (en) * 2007-06-28 2010-04-07 Abbott Laboratories Novel triazolopyridazines
WO2011067306A1 (en) * 2009-12-03 2011-06-09 Novartis Ag Cyclohexane derivatives as inhibitors of acetyl-coa carboxylase (acc)
WO2011157827A1 (en) 2010-06-18 2011-12-22 Sanofi Azolopyridin-3-one derivatives as inhibitors of lipases and phospholipases
CN102020648A (en) * 2011-01-14 2011-04-20 南京英派药业有限公司 3-aryl-6-aryl-[1,2,4] triazol [4,3-b] pyridazine taken as cell proliferation inhibitor and application of cell proliferation inhibitor
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CN102020648B (en) * 2011-01-14 2012-11-07 南京英派药业有限公司 3-aryl-6-aryl-[1,2,4] triazol [4,3-b] pyridazine taken as cell proliferation inhibitor and application of cell proliferation inhibitor
EP2844660A4 (en) * 2012-05-02 2015-09-23 Southern Res Inst Triazolopyridazine compounds, use as inhibitors of the kinase lrrk2, and methods for preparation thereof
US9187484B2 (en) 2012-05-02 2015-11-17 Southern Research Institute Triazolopyridazine compounds, use as inhibitors of the kinase LRRK2, and methods for preparation thereof
EP2818471A1 (en) * 2013-06-27 2014-12-31 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Nitrogen bicyclic compounds as inhibitors for Scyl1 and Grk5
WO2015036892A1 (en) 2013-09-12 2015-03-19 Pfizer Inc. Use of acetyl-coa carboxylase inhibitors for treating acne vulgaris
CN106188068A (en) * 2016-07-26 2016-12-07 沈阳药科大学 3,6 diaryl [1,2,4] triazole also [4,3 b] pyridazine compound and purposes
CN106188069A (en) * 2016-07-26 2016-12-07 沈阳药科大学 3,6 diaryl 1H pyrazolo [5,1 c] [1,2,4] triazole compounds are as the purposes of tumor cell proliferation inhibitor
CN106188069B (en) * 2016-07-26 2018-05-08 沈阳药科大学 Purposes of 3,6- diaryl -1H- pyrazolos [5,1-c] [1,2,4] triazole compound as tumor cell proliferation inhibitor
CN107474047A (en) * 2017-08-29 2017-12-15 浙江工业大学 synthesis method of 1,2, 4-triazolo heterocyclic compound
WO2023150793A1 (en) * 2022-02-07 2023-08-10 Cornell University Inhibitors of malic enzyme 1

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