HK1080473B - Bicyclic inhibitors of hormone sensitive lipase - Google Patents

Description

Bicyclic hormone sensitive lipase inhibitors

Benzotriazoles are well known in numerous fields, for example for use photochemically (US4,255,510, kodak) or as orexin antagonists (WO02/090355, SKB). Furthermore, a synthesis for the preparation of benzotriazole has been described by Katritzky et al J.org.chem.1997, 62, 4155-4158. Carbamates which are also known to be useful as Lipase inhibitors are described, for example, by ShamkantPatkar et al in Paul Woolley, Steffen B. Petterson (ed), Lipase (1994)207-227 or WO03/051842 Red.

Surprisingly, it has been shown that the benzotriazoles of the present invention can show activity with respect to HSL, a hormone sensitive lipase.

The present invention relates to benzotriazoles of formula I:

wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent: br, Cl, CH₃、CN、NH₂、NO₂、CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl, CO-OCH₃(ii) a Or

The two substituents of the series are: r1 ═ Cl and R3 ═ CF₃Or R2 ═ F and R3 ═ Cl;

n is an integer 0, 1 or 2; and is

One of the substituents R6 or R7 may represent: r6 is CH₃(ii) a R7 is CH₃、C₂H₅、CH(CH₃)₂、C(CH₃)₃、CF₃Br, Cl, benzyl or CO-OC₂H₅(ii) a Or

R6 and R7 are both CH₃(ii) a Or

The ring may contain a double bond in place of R6 and R7; or

R5 and R6 or R6 and R7 may together with the carbon atoms bearing them represent a fused benzene ring or, if n ═ 0, a cyclohexanediyl group; wherein in case R6/R7 is a closed ring, the substituent may optionally be substituted by NH₂Or NO₂Mono-substituted or substituted OCH₃Mono-or di-substituted; and is

R7 and R8 together are cyclopentyl, diaza * or ═ CH₂；

Wherein R1 to R5 and R8 ═ H, n ═ 1 and R6/R7 are fused benzenes and R1, R3-R8 ═ H, R2 ═ CH₃And n is 1.

The invention relates to racemates, racemic mixtures and pure enantiomeric forms of the compounds of formula I, as well as diastereomers and mixtures thereof.

The alkyl group may be linear or branched. Halogen is fluorine, chlorine or bromine, in particular fluorine or chlorine.

Benzotriazoles of formula I are preferred, wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent:

r2 is Br, Cl, CN, NO₂、CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl, CO-OCH₃；

R3 is CH₃、CN、Br、Cl、NH₂、NO₂And a benzoyl group.

Benzotriazoles of the formula I are particularly preferred, wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent:

r2 is Br, Cl, NO₂、OCH₃Phenoxy, CO-OCH₃；

R3 is NH₂(ii) a Or

The two substituents of the series are: r2 ═ F and R3 ═ Cl;

n is an integer of 1 or 2; and is

One of the substituents R5 or R6 may represent: r6 is CH₃(ii) a R7 is CH₃、CF₃Or Br; or the ring may contain a double bond in place of R6 and R7, or

R6 and R7 may, together with the carbon atoms bearing them, represent a fused benzene ring, which may optionally be substituted by NH₂Mono-substituted or substituted OCH₃Mono-or di-substituted; and is

R7 and R8 together are cyclopentyl, or

n is an integer of 0; and is

R6 and R7 may represent, together with the carbon atom bearing them, a fused benzene ring or a cycloadilyl group; or a benzotriazole of formula I wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent:

r2 is Br, CN, CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl; r3 is CN, Br, Cl, NO₂A benzoyl group; or

The two substituents of the series are: r1 ═ Cl and R3 ═ CF₃；

n is an integer of 1; and is

One of the substituents R6 and R7 may represent: r6 is CH₃(ii) a R7 is CH₃、C₂H₅、CH(CH₃)₂、C(CH₃)₃Benzyl or CO-OC₂H₅(ii) a Or

R6 and R7 are both CH₃(ii) a Or

The ring may contain a double bond in place of R6 and R7, or

R5 and R6 or R6 and R7 may represent, together with the carbon atoms which carry them, a fused benzene ring;

with the exception of compounds in which R1 to R5 and R8 ═ H, n ═ 1 and R6/R7 ═ fused benzenes.

Benzotriazoles of the following structure are very particularly preferred:

or a benzotriazole of the structure:

benzotriazoles of the following structure are also very particularly preferred:

and a benzotriazole of the structure:

pharmaceutically acceptable salts are particularly suitable for medical applications because they have a greater solubility in water than the starting or base compound. These salts must possess a pharmaceutically acceptable anion or cation. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention are salts of inorganic acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and salts of organic acids such as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic, isethionic, lactic, lactobionic, maleic, malic, methanesulfonic, succinic, p-toluenesulfonic and tartaric acids. Suitable pharmaceutically acceptable base salts are the ammonium, alkali metal (e.g. sodium and potassium), alkaline earth metal (e.g. magnesium and calcium), tromethamine (2-amino-2-hydroxymethyl-1, 3-propanediol), diethanolamine, lysine or ethylenediamine salts.

Salts with pharmaceutically unacceptable anions, such as trifluoroacetate, are likewise within the scope of the invention, and are useful intermediates for the preparation or purification of pharmaceutically acceptable salts and/or for non-therapeutic applications, such as in vitro applications.

The term "physiologically functional derivative" as used herein refers to any physiologically tolerated derivative of a compound of formula I according to the invention, for example an ester, which on administration to a mammal such as a human is capable of forming (directly or indirectly) a compound of formula I or an active metabolite thereof.

Physiologically functional derivatives also include prodrugs of the compounds of the invention, for example as described in chem.pharm.Bull.1994, 42, 57-61, H.Okada et al. Such prodrugs can be metabolized in vivo to the compounds of the invention. These prodrugs may or may not be active themselves.

The compounds of the present invention may also exist in various polymorphic forms, such as amorphous and crystalline polymorphic forms. All polymorphic forms of the compounds of the invention are within the scope of the invention and constitute a further aspect of the invention.

All references hereinafter to "compounds of formula I" refer to compounds of formula I as described above, as well as salts, solvates, and physiologically functional derivatives thereof as described herein.

The compounds of formula (I) may also be administered in combination with other active ingredients.

The amount of a compound of formula I required to achieve a desired biological effect depends on a variety of factors, such as the particular compound selected, the intended use, the mode of administration, and the clinical condition of the patient. The daily dose is usually 0.3mg to 100mg (usually 3mg to 50mg) per kg body weight per day, for example 3-10 mg/kg/day. The intravenous dose may be, for example, from 0.3mg/kg to 1.0mg/kg, which may suitably be administered as an infusion of from 10ng to 100ng per kg per minute. Suitable infusion solutions for these purposes may contain, for example, from 0.1ng to 10mg, usually from 1ng to 10mg, per ml. Single doses may contain, for example, from 1mg to 10g of active ingredient. Thus, ampoules for injection may contain, for example, from 1mg to 100mg of active ingredient, and orally administrable single-dose preparations, such as capsules or tablets, may contain, for example, from 1.0mg to 1000mg, usually from 10mg to 600mg, of active ingredient. In the treatment of the above conditions, the compounds of formula I may be used as such, but they are preferably in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with the other ingredients of the composition and not deleterious to the health of the patient. The carrier may be a solid, a liquid, or both, and is preferably formulated with the compound as a single dose, e.g., a tablet, which may contain from 0.05% to 95% by weight of the active ingredient. Other pharmaceutically active substances may also be present, including other compounds of formula I. The pharmaceutical compositions of the present invention may be prepared by any of the well-known pharmaceutical methods which essentially comprise admixing the ingredients with pharmacologically acceptable carriers and/or excipients.

The pharmaceutical compositions of the invention are suitable for oral, rectal, topical, peroral (e.g. sublingual) and parenteral (e.g. subcutaneous, intramuscular, intradermal or intravenous) administration, but the most suitable mode of administration depends in each individual case on the nature and severity of the condition to be treated and on the nature of the compound of the formula I used in each case. Coated formulations and coated sustained release formulations are also within the scope of the invention. Acid and gastric juice resistant formulations are preferred. Suitable coatings resistant to gastric juices include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethyl cellulose phthalate and anionic polymers of methacrylic acid and methyl methacrylate.

Suitable pharmaceutical compositions for oral administration may be in the form of discrete units, such as capsules, wafers, buccal tablets or tablets, each of which contains a defined amount of a compound of formula I; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water or water-in-oil emulsion. As mentioned above, these compositions may be prepared by any suitable pharmaceutical method which includes the step of bringing into contact the active ingredient and the carrier, which may consist of one or more additional ingredients. Compositions are generally prepared by uniformly or homogeneously mixing the active ingredient and/or finely divided solid carrier, and then, if necessary, shaping the product. Thus, for example, a tablet may be prepared by compressing or molding a powder or granules of the compound, as appropriate, with one or more additional ingredients. Compressed tablets may be prepared by mixing the compound in free-flowing form, for example in powder or granular form, with a binder, glidant, inert diluent, and/or surfactant/dispersant(s), as appropriate, and compressing in a suitable machine. Molded tablets (molded tablets) may be prepared by molding the compound in powder form and moistened with an inert liquid diluent in a suitable machine.

Pharmaceutical compositions suitable for oral (sublingual) administration include buccal tablets, which contain a compound of formula I in combination with a flavouring agent, usually sucrose and acacia or tragacanth, and lozenges comprising the compound in an inert base such as gelatin and glycerol or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration preferably comprise sterile aqueous preparations of a compound of formula I, which are preferably isotonic with the blood of the intended recipient. These formulations are preferably administered intravenously, but may also be administered by subcutaneous, intramuscular or intradermal injection. These formulations can preferably be prepared by mixing the compound with water, rendering the resulting solution sterile and isotonic with blood. The injectable compositions of the present invention generally contain from 0.1% to 5% by weight of the active compound.

Pharmaceutical compositions suitable for rectal administration are preferably in the form of single-dose suppositories. They may be prepared by mixing a compound of formula I with one or more conventional solid carriers, for example cocoa butter, and shaping the resulting mixture.

Pharmaceutical compositions suitable for topical application to the skin are preferably in the form of ointments, creams, lotions, pastes, sprays, aerosols or oils. Carriers which may be used are petrolatum, lanolin, polyethylene glycols, alcohols and combinations of two or more of the foregoing. The active ingredient is typically present at a concentration of from 0.1% to 15%, for example from 0.5% to 2%, by weight of the composition.

Transdermal administration is also possible. Pharmaceutical compositions suitable for transdermal use may be in the form of a single plaster which is suitable for prolonged intimate contact with the epidermis of the patient. Such plasters suitably contain the active ingredient in an aqueous solution, dissolved and/or dispersed in an adhesive or dispersed in a polymer, which is buffered as appropriate. Suitable concentrations of the active ingredient are about 1% to 35%, preferably about 3% to 15%. In particular, the active ingredient may also be released by electromigration or iontophoresis, as for example pharmaceutical research2 (6): 318 (1986).

Other active ingredients suitable for use in the combination product are all antidiabetic agents mentioned in Rote Liste 2001, chapter 12. They can be combined with the compounds of the formula I according to the invention, in particular for synergistically increasing the therapeutic effect. The administration of the active ingredient combination can be carried out by separate administration of the active ingredients to the patient or in the form of a combination product in which a plurality of active ingredients are present in one pharmaceutical preparation. Most of the following active ingredients are disclosed in USP Dictionary and International drug names, USAN, US pharmacopoeia, Rockville 2001.

Antidiabetic agents include insulin and insulin derivatives, e.g. Lantus^®(seewww.lantus.com) or HMR1964, rapid acting insulins (see US6,221,633), GLP-1 derivatives, such as those disclosed in WO98/08871 to Novo Nordisk A/S, and oral administrationEffective hypoglycemic active components.

Orally active hypoglycemic active ingredients preferably include sulfonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, glucosidase inhibitors, glucagon antagonists, GLP-1 agonists, potassium channel openers such as those disclosed in WO 97/26265 and WO 99/03861 of Novo Nordisk a/S, insulin sensitizers, liver enzyme inhibitors involved in the stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, compounds that alter lipid metabolism such as antihyperlipidemic active ingredients and antilipidemic active ingredients, compounds that reduce food intake, PPAR and PXR agonists, and active ingredients that act on ATP-dependent potassium channels of beta cells.

In one embodiment of the invention, the compound of formula I is administered in combination with an HMGCoA reductase inhibitor, e.g. simvastatin, fluvastatin, pravastatin, lovastatin, atorvastatin, cerivastatin, rosuvastatin.

In one embodiment of the invention, the compounds of the formula I are administered in combination with cholesterol absorption inhibitors, for example ezetimibe (ezetimibe), tiqueside, pamabrin (pamaquide).

In one embodiment of the invention, the compounds of the formula I are administered in combination with a PPAR γ agonist, for example rosiglitazone, pioglitazone, JTT-501, GI 262570.

In one embodiment of the invention, the compounds of formula I are administered in combination with a PPAR α agonist, e.g. GW9578, GW 7647.

In one embodiment of the invention, the compounds of the formula I are administered in combination with a mixed PPAR α/γ agonist, for example GW1536, AVE8042, AVE8134, AVE0847 or as described in WO00/64888, WO00/64876, WO 03/020269.

In one embodiment of the invention, the compounds of formula I are administered in combination with a fibrate, e.g. fenofibrate, clofibrate, bezafibrate.

In one embodiment of the invention, the compounds of formula I are administered in combination with an MTP inhibitor, e.g., implipide, BMS-201038, R-103757.

In one embodiment of the invention, the compounds of formula I are administered in combination with a bile acid absorption inhibitor (see e.g. US6,245,744 or US6,221,897), such as HMR 1741.

In one embodiment of the invention, the compound of formula I is administered in combination with a CETP inhibitor, such as JTT-705.

In one embodiment of the invention, the compounds of formula I are administered in combination with a polymeric bile acid adsorbent, e.g. cholestyramine, colesevelam.

In one embodiment of the invention, the compound of formula I is administered in combination with an LDL receptor inducing agent (see US6,342,512), e.g. HMR1171, HMR 1586.

In one embodiment of the invention, the compounds of the formula I are administered in combination with an ACAT inhibitor, for example avasimibe (avasimibe).

In one embodiment of the invention, the compounds of the formula I are administered in combination with an antioxidant, for example OPC-14117.

In one embodiment of the invention, the compounds of the formula I are administered in combination with a lipoprotein lipase inhibitor, for example NO-1886.

In one embodiment of the invention, the compounds of formula I are administered in combination with an ATP-citrate lyase inhibitor, such as SB-204990.

In one embodiment of the invention, the compound of formula I is administered in combination with a squalene synthetase inhibitor, e.g. BMS-188494.

In one embodiment of the invention, the compounds of formula I are administered in combination with a lipoprotein (a) antagonist, such as CI-1027 or nicotinic acid.

In one embodiment of the invention, the compounds of the formula I are administered in combination with a lipase inhibitor, for example orlistat.

In one embodiment of the invention, the compounds of the formula I are administered in combination with insulin.

In one embodiment, the compounds of the formula I are administered in combination with sulfonylureas, for example tolbutamide, glyburide, glipizide or glimepiride.

In one embodiment, the compound of formula I is administered in combination with a biguanide, for example, metformin.

In another embodiment, the compound of formula I is administered in combination with a glinide drug, such as repaglinide.

In one embodiment, the compound of formula I is administered in combination with a thiazolidinedione drug, such as troglitazone, ciglitazone, pioglitazone, rosiglitazone or a compound disclosed in WO97/41097 of the dr. reddy's research foundation, especially 5- [ [4- (3, 4-dihydro-3-methyl-4-oxo-2-quinazolinylmethoxy) phenyl ] methyl-2, 2, 4-thiazolidinedione.

In one embodiment, the compounds of the formula I are administered in combination with an α -glucosidase inhibitor, for example miglitol or acarbose.

In one embodiment, the compounds of the formula I are administered in combination with an active ingredient which acts on the ATP-dependent potassium channel of the beta cells, for example tolbutamide, glibenclamide, glipizide, glimepiride or repaglinide.

In one embodiment, the compound of formula I is administered in combination with more than one of the above compounds, for example in combination with sulfonylurea and metformin, with sulfonylurea and acarbose, with repaglinide and metformin, with insulin and sulfonylurea, with insulin and metformin, with insulin and troglitazone, with insulin and lovastatin, and the like.

In another embodiment, the compound of formula I is administered in combination with: CART modulators (see, "cocaine-amphetamine-regulated transcription affects energy metabolism, anxiety, and gastric emptying in mice," Asakawa, A et al, M.: Hormone and Metabolic Research (2001), 33 (9); 554-); NPY antagonists such as naphthalene-1-sulfonic acid {4- [ (4-aminoquinazolin-2-ylamino) methyl ] -cyclohexylmethyl } amide hydrochloride (CGP 71683A); MC4 agonists (e.g. 1-amino-1, 2, 3, 4-tetrahydronaphthalene-2-carboxylic acid [2- (3 a-benzyl-2-methyl-3-oxo-2, 3, 3a, 4, 6, 7-hexahydropyrazolo [4, 3-c ] pyridin-5-yl) -1- (4-chlorophenyl) -2-oxoethyl ] -amide (WO 01/91752)); anorectic antagonists (e.g., 1- (2-methylbenzoxazol-6-yl) -3- [1, 5] naphthyridin-4-ylurea hydrochloride (SB-334867-A)); h3 agonists (3-cyclohexyl-1- (4, 4-dimethyl-1, 4, 6, 7-tetrahydroimidazo [4, 5-c ] pyridin-5-ylprop-1-one oxalate (WO 00-63208))), TNF agonists, CRF antagonists (e.g., [ 2-methyl-9- (2, 4, 6-trimethylphenyl) -9H-1, 3, 9-triazafluoren-4-yl ] dipropylamine (WO 00/66585))), CRF BP antagonists (e.g., urocortin), urocortin agonists, beta 3 agonists (e.g., 1- (4-chloro-3-methanesulfonylmethyl-phenyl) -2- [2- (2, 3-dimethyl-1H-indol-6-yloxy) ethylamino ] ethanol hydrochloride (WO01/83451)), MSH (prokinetic Black hormone) agonist; CCK-A agonists (e.g. {2- [4- (4-chloro-2, 5-monomethoxyphenyl) -5- (2-cyclohexylethyl) thiazol-2-ylcarbamoyl ] -5, 7-dimethylindol-1-yl } acetic acid trifluoroacetate (WO 99/15525)); 5-hydroxytryptamine reuptake inhibitors (e.g., dexfenfluramine); a mixed form of 5-hydroxytryptamine and norepinephrine (e.g., WO 00/71549); 5HT agonists, such as 1- (3-ethylbenzofuran-7-yl) -piperazine oxalate (WO 01/09111); bombesin agonists; a galanin antagonist; growth hormone (e.g., human growth hormone); a growth hormone releasing compound (6-benzyloxy-1- (2-diisopropylaminoethylcarbamoyl) -3, 4-dihydro-1H-isoquinoline-2-carboxylic acid-3-tert-butyl ester (WO 01/85695)); TRH agonists (see, e.g., EP 0462884); uncoupling protein 2 or 3 modulators; leptin agonists (see, e.g., Lee, Daniel W; Leinung, Matthew C.; Rozhavskaya-Arena, Marina; Grasso, Patricia. "leptin agonists as a potential means of treating obesity", Drugs of the future (2001), 26(9), 873-); DA agonists (bromocriptine, Doprexin); lipase/amylase inhibitors (see WO 00/40569); PPAR modulators (e.g., WO 00/78315); RXR modulators or TR-beta agonists.

In one embodiment of the invention, the other active ingredient is leptin, see for example "therapeutic application prospects for leptin", Salvador, Javier; Gomez-Ambrosi, Javier; fruhbeck, Gema, Expert Opinion on Pharmacotherapy (2001), 2(10), 1615-.

In one embodiment, the other active ingredient is dextroamphetamine or amphetamine.

In one embodiment, the other active ingredient is fenfluramine or dexfenfluramine.

In another embodiment, the other active ingredient is sibutramine.

In one embodiment, the other active ingredient is orlistat.

In one embodiment, the other active ingredient is mazindol or phentermine.

In another embodiment, the compounds of formula I are administered in combination with a bulking agent, preferably an insoluble bulking agent (see, e.g., carob/Caromax)^®(Zunft H J et al, "Carob pulp formulation for treatment of hypercholesterolemia", ADVANCES IN THERAPY (9-10 months 2001), 18(5), 230-6.). Caromax is a product containing carob, produced by Nutriova, Nutrition specialties&Food Ingredients' GmbH, Industriaparkk H ö chst, 65926 Frankfurt/Main)). And Caromax^®The combination may be in one formulation or by separate administration of the compound of formula I and Caromax^®The process is carried out. In this respect, Caromax^®It can also be administered in the form of a food product such as a bakery product or muesli (muesli bar).

It is to be understood that every suitable combination of a compound of the invention with one or more of the compounds described above and optionally one or more other pharmacologically active substances is to be considered as falling within the scope of the present invention.

The benzotriazoles of the formula I of the present invention are prepared by methods known per se, for example by acylating substituted or unsubstituted benzotriazole 2 with carbamoyl chloride 3 (method A), or in two steps by reacting the benzotriazole with phosgene and further reacting the resulting benzotriazole carbonyl chloride with an amine or aniline (method B).

Since these reactions usually release acid, it is advisable to add a base such as pyrimidine, triethylamine, sodium hydroxide solution or alkali metal carbonate to increase the reaction rate. The reaction can be carried out over a wide temperature range. It has generally proven advantageous to operate at temperatures from 0 ℃ to the boiling point of the solvent used. Examples of solvents used are dichloromethane, THF, DMF, toluene, ethyl acetate, n-heptane, dioxane, diethyl ether.

The compounds of formula I according to the invention have a surprising inhibitory effect on the hormone sensitive lipase HSL, an allosteric enzyme in adipocytes, which is inhibited by insulin and is responsible for the breakdown of fat in adipocytes, and thus for the transfer of fat components into the bloodstream. The inhibitory effect of the compounds of the invention on this enzyme is therefore equivalent to an insulin-like effect, which ultimately leads to a reduction in free fatty acids in the blood and also to a reduction in blood glucose. They are therefore useful for metabolic disorders such as non-insulin dependent diabetes mellitus, diabetic syndrome, syndrome X and direct pancreatic injury.

Inhibition of HSL in beta cells should result in direct recovery of insulin release (M.Winzell et al, Diabetes, Vol52, 8 months 2003, 2057-2065). The compounds of formula I according to the invention can therefore also be used for insulin release.

The effect of the compounds of formula I of the present invention was tested in the following enzyme assay system:

preparation of a substrate:

NAG (NBD monoacylglycerol) substrate preparation

6mg of phosphatidylcholine and 6mg of phosphatidylinositol were dissolved in 1ml of chloroform, respectively. 10mg of NAG was dissolved in 1ml of chloroform. Two portions of phosphatidylinositol solution (e.g., 83.5. mu.l) and one portion of phosphatidylcholine solution (e.g., 41.5. mu.l) and 100. mu.l of NAG solution were pipetted into a plastic scintillation container (final assay concentration of 0.0375mg phospholipid/ml; 0.05 mg/NAG/ml). Introduction of N₂Chloroform (total volume 225. mu.l) was completely removed by flow. The dried substrate can be stored at 4 ℃ for no more than three days. To prepare phospholipid vesicles/micelles with NAG interlayers (on the day of the assay), the dried substrate was placed in 20ml assay buffer (25mM Tris/HCl, pH 7.4; 150mM NaCl) and then sonicated twice with a sonication probe (Branson Sonifier type II, standard microtip): a first treatment set at 2, 2 × 1 minutes, 1 minute on ice between each time; second treatment, set at 4,2 × 1min, 1min on ice between each. In this procedure, the color of the substrate solution changed from yellow (481 nm maximum extinction) to red (550 nm maximum extinction) due to the intercalation of NAG between phospholipid molecules in the lipid vesicles/capsules. The solution was incubated on ice for a further 15 minutes before being used as substrate (within the next two hours).

Indirect NAG assay

The assay was carried out in 1.5ml Eppendorf vessels or 96-well plates at 30 ℃ for 60 minutes. To detect the HSL inhibitors, 10. mu.l of the test substance were introduced into an assay buffer (25mM Tris/HCl, pH 7.4; 150mM NaCl) containing 16.6% DMSO. Mu.l of substrate solution (20. mu.g/ml phosphatidylcholine, 10. mu.g/ml phosphatidylinositol, 50. mu.g/ml NAG in assay buffer) was added. After a preincubation at 30 ℃ for 15 minutes, 20. mu.l of an enzyme solution diluted 1 to 4 times with test buffer are pipetted and the extinction is immediately determined at 480nm in a colorimeter (0.5ml cuvette) or microtiter plate reader. After incubation at 30 ℃ for 60 minutes, the extinction values were again determined. The increase in extinction at 480nm was taken as a measure of the enzyme activity. Under standard conditions, 20 μ g of partially purified HSL resulted in a change of 0.4, which is equivalent to an extinction value of 4000arb.

Direct NAG assay

As an alternative to measuring the change in extinction value of the substrate solution, the products of the HSL reaction were investigated by phase separation/thin layer chromatography. For this purpose, 1.3ml of methanol/chloroform/heptane (10: 9: 7) and then 0.4ml of 0.1M NaOH were added to the culture mixture in a 2ml Eppendorf vessel (total volume 200. mu.l, see Indirect NAG test method). After vigorous mixing (10 seconds), phase separation was initiated by centrifugation (800 Xg, 20 min, room temperature). An equal volume (e.g., 0.4ml) of the solution was removed from the upper aqueous phase and the extinction at 481nm was measured in a luminometer. For thin layer chromatography, the aqueous phase is dried (SpeedVac) and then placed in 50 μ l tetrahydrofuran. Mu.l of the sample was spotted on a silica gel Si-60 plate (Merck). Thin layer chromatography was performed using 78ml diethyl ether/22 ml petroleum ether/1 ml glacial acetic acid as mobile phase. The amount of fluorescing NBD fatty acid released was determined by phosphoimaging (molecular dynamics, Storm 840 and ImageQuant Software) with an excitation wavelength of 460nm and an emission wavelength of 540-560 nm.

Preparation of enzymes

Preparation of partially purified HSL

Ex vivo rat adipocytes were obtained from epididymal adipose tissue of untreated male rats (Wistar, 220-250g) according to published collagenase treatment methods (e.g., S.Nilsson et al, anal.biochem.158, 1986, 399-407; G.Fredrikson et al, J.biol.chem.256, 1981, 6311-6320; H.Tornquist et al, J.biol.chem.251, 1976, 813-819). Adipocytes from 10 rats were subjected to flotation washing three times using 50ml of homogenization buffer (25ml Tris/HCl, pH7.4, 0.25M sucrose, 1mM EDTA, 1mM DTT, 10. mu.g/ml leupeptin, 10. mu.g/ml antipain, 20. mu.g/ml pepstatin) each time and finally placed in 10ml of homogenization buffer. The adipocytes were homogenized in a Teflon-in-glass homogenizer (Braun-Melsungen) under conditions of 15 ℃ at 1500rpm for 10 strokes. The homogenate was centrifuged (Sorvall SM24 tube, 5000rpm, 10 min, 4 ℃). The supernatant between the top fat layer and the pellet was removed and centrifugation repeated. The thus obtained supernatant was centrifuged again (Sorvall SM24 tube, 20000rpm, 45 min, 4 ℃). The supernatant was removed and 1g of heparin-Sepharose (Pharmacia-Biotech, CL-6B, 5 washes with 25mM Tris/HCl, pH7.4, 150mM NaCl) was added. After incubation at 4 ℃ for 60 minutes (shaking well at 15 minute intervals), the mixture was centrifuged (Sorvall SM24 tube, 3000rpm, 10 minutes, 4 ℃). The supernatant was adjusted to pH5.2 by adding glacial acetic acid and incubated at 4 ℃ for 30 minutes. The pellet was collected by centrifugation (Sorvall SS34, 12000rpm, 10 minutes, 4 ℃) and suspended in 2.5ml of 20mM Tris/HCl, pH7.0, 1mM EDTA, 65mM NaCl, 13% sucrose, 1mM DTT, 10. mu.g/ml leupeptin/pepstatin/antipain. The suspension was dialyzed at 4 ℃ overnight against 25mM Tris/HCl, pH7.4, 50% glycerol, 1mM DTT, 10. mu.g/ml leupeptin/pepstatin/anti-protease, and then loaded onto a hydroxiapatite column (0.1 g per ml suspension, equilibrated with 10mM potassium phosphate, pH7.0, 30% glycerol, 1mM DTT). The column was washed with 4 volumes of equilibration buffer at a flow rate of 20-30 ml/h. HSL was eluted with a volume of equilibration buffer containing 0.5M potassium phosphate at 4 ℃ followed by dialysis (see above) and concentration 5-10 fold using an ultrafiltration apparatus (Amicon Diaflo PM10 Filter). The semi-purified HSL can be stored at-70 deg.C for 4-6 weeks.

And (3) determination:

for the preparation of the substrate, 25-50. mu. Ci [3H ] is added]Triolein (in toluene), 6.8. mu. mol unlabelled triolein and 0.6mg phospholipid (phosphatidylcholine/phosphatidylinositol 3: 1w/v) were mixed and purified N₂Dried and then dissolved in 2ml of 0.1M KPi (pH7.0) by sonication (Branson 250, microtip, setting 1-2, 2X 1min, interval 1 min). After addition of 1ml of Kpi and re-sonication (4X 30 seconds on ice, 30 second intervals), 1ml of 20% BSA (in Kpi) (final concentration of triolein 1.7mM) was added. In the reaction, 100. mu.l of the substrate solution was pipetted into 100. mu.l of HSL solution (HSL prepared as described above, diluted in 20mM KPi, pH7.0, 1mM EDTA, 1mM DTT, 0.02% BSA, 20. mu.g/ml pepstatin, 20. mu.g/ml leucin)Aprotinin) and incubated at 37 ℃ for 30 minutes. 3.25ml of methanol/chloroform/heptane (10: 9: 7) and 1.05ml of 0.1M K were added₂CO₃0.1M boric acid (pH10.5), then mixed well and finally centrifuged (800 Xg, 20 min). After phase separation, one equivalent of the upper phase (1ml) was removed and the radioactivity was determined by liquid flash measurement.

Evaluation:

testing of the substance is typically performed in four separate mixtures. The inhibition of HSL enzyme activity by the test substance is determined by comparison with an uninhibited control reaction. Calculating IC from inhibition curves containing at least 10 concentrations of test substance₅₀. Data processing was performed using the GRAPHIT, Elsevier-BIOSOFT software package.

In this test, the compounds of examples 1 to 55 show an inhibitory effect, IC₅₀In the range of 0.04-5. mu.M.

Example (b):

the following examples were prepared according to the methods described subsequently.

The method A comprises the following steps:

to a solution of 2mmol of 1H-benzotriazole in pyridine (5ml) and dichloromethane (10ml) was added a solution of the corresponding carbamoyl chloride (1mmol) in dichloromethane (10 ml). The reaction mixture was stirred at RT for 16 h, then mixed with EtOAc (15ml) and filtered through silica gel, then the filtrate was concentrated. The product was purified by preparative HPLC and lyophilized.

Method B embodiment:

a) preparation of benzotriazole-1-carbonyl chloride solution

A solution of benzotriazole (6g, 50.4mmol) in THF (100ml) was added dropwise to a phosgene solution (20% in toluene; 90 ml; 182mmol) under ice-cooling. The ice bath was removed and the solution was then stirred at RT for a further 2 hours. The solvent was distilled off, and the residue was taken up in THF to a total volume of 25 ml.

b) Benzotriazole carbonyl chloride reaction to give the corresponding benzotriazole-1-carboxamide and anilide in each case 10 amines or anilines (2mmol) were introduced into THF (1ml) and pyridine (0.2ml) was added. The mixture was incubated with benzotriazole-1-carbonyl chloride solution (1ml,. about.2 mmol) and stirred at RT for 16 h. The mixture was then diluted with ethyl acetate (5ml) and filtered through silica gel and the filtrate evaporated to dryness in vacuo. The crude product was purified by flash chromatography.

Example 1: 3- (4-methylpiperidine-1-carbonyl) -3H-benzotriazole-5-carboxylic acid methyl ester

M+H+：303.14

Example 2: (8-aza-spiro [4.5] decan-8-yl) -benzotriazol-1-ones

M+H+：285.16

Example 3: benzotriazol-1-yl- (6, 7-dimethoxy-3, 4-dihydro-1H-isoquinolin-2-yl) -methanone

M+H+：339.13

Example 4: (5-Phenoxybenzotriazol-1-yl) - (4-trifluoromethylpiperidin-1-yl) methanone

M+H+：391.13

Example 5: (6-chloro-5-fluorobenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanone

M+H+：297.74

Example 6: benzotriazol-1-yl- (4-bromopiperidin-1-yl) methanones

M+H+：310.3

Example 7: benzotriazol-1-yl- (4-trifluoromethylpiperidin-1-yl) methanones

M+H+：299.18

Example 8: benzotriazol-1-yl- (1, 3-dihydroisoindol-2-yl) methanones

M+H+：265.0

Example 9: 1- (3, 6-dihydro-2H-pyridine-1-carbonyl) -1H-benzotriazole-5-carboxylic acid methyl ester

M+H+：287.04

Example 10: (3, 6-dihydro-2H-pyridin-1-yl) - (5-nitrobenzotriazol-1-yl) methanone

M+H+：296.21

Example 11: (3, 4-dihydro-1H-isoquinolin-2-yl) - (5-nitrobenzotriazol-1-yl) methanone

M+H+：324.10

Example 12: (5-bromobenzotriazol-1-yl) - (3, 6-dihydro-2H-pyridin-1-yl) methanone

M+H+：306.98

Example 13: (5-bromobenzotriazol-1-yl) - (3-trifluoromethylpiperidin-1-yl) methanone

M+H+：377.30

Example 14: (5-bromobenzotriazol-1-yl) - (3, 4-dihydro-1H-isoquinolin-2-yl) methanone

M+H+：357.04

Example 15: benzotriazol-1-yl- (octahydroisoindol-2-yl) methanones

M+H+：271.15

Example 16: (7-amino-3, 4-dihydro-1H-isoquinolin-2-yl) benzotriazol-1-yl methanone

M+H+：294.0

Example 17: (3, 4-dihydro-1H-isoquinolin-2-yl) - (5-methoxybenzotriazol-1-yl) methanone

M+H+：309.04

Example 18: (5-methoxybenzotriazol-1-yl) - (3-methylpiperidin-1-yl) methanones

M+H+：275.5

Example 19: (6-aminobenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanones

M+H+：260.1

Example 20: (5-chlorobenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanones

M+H+：279.6

Example 21: benzotriazol-1-yl- (1, 2, 6-triaza-spiro [2.5] oct-1-en-6-yl) methanones

M+H+：279.19

Example 22: benzotriazol-1-yl- (4-ethylpiperidin-1-yl) methanones

M+H+：259.04

Example 23: (4-methylpiperidin-1-yl) - (6-nitrobenzotriazol-1-yl) methanones

M+H+：290.4

Example 24: 1- (benzotriazole-1-carbonyl) piperidine-4-carboxylic acid ethyl ester

M+H+：303.13

Example 25: benzotriazol-1-yl- (4-methylpiperidin-1-yl) methanones

M+H+：245.0

Example 26: 3- (4-methylpiperidine-1-carbonyl) -3H-benzotriazole-5-carbonitrile

M+H+：270.12

Example 27: benzotriazol-1-yl- (3, 4-dihydro-1H-isoquinolin-1-yl) methanones

M+H+：279.11

Example 28: benzotriazol-1-yl- (3, 4-dihydro-2H-quinolin-1-yl) methanones

M+H+：279.2

Example 29: (6-methylbenzotriazol-1-yl) -pyrrolidin-1-yl-methanones

M+H+：231.11

Example 30: benzotriazol-1-yl- (3-methylpiperidin-1-yl) methanones

M+H+：245.13

Example 31: benzotriazol-1-yl- (3, 4-dimethylpiperidin-1-yl) methanones

M+H+：259.14

Example 32: [1- (4-methylpiperidine-1-carbonyl) -1H-benzotriazol-5-yl ] phenyl methanone

M+H+：349.15

Example 33: (4-methylpiperidin-1-yl) - (5-trifluoromethylbenzotriazol-1-yl) methanone

M+H+：313.5

Example 34: (6-bromobenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanone

M+H+：324.0

Example 35: benzotriazol-1-yl- (4-tert-butylpiperidin-1-yl) methanones

M+H+：287.17

Example 36: [5- (Hydroxyphenylmethyl) benzotriazol-1-yl ] - (4-methylpiperidin-1-yl) -methanone

M+H+：350.17

Example 37: (4-methylpiperidin-1-yl) - (5-phenoxybenzotriazol-1-yl) methanones

M+H+：337.3

Example 38: (5-Cyclohexylsulfanylbenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanone

M+H+：359.17

Example 39: benzotriazol-1-yl- (4-isopropylpiperidin-1-yl) methanones

M+H+：273.3

Example 40: (6-chlorobenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanones

M+H+：279.5

Example 41: benzotriazol-1-yl- (6-methoxy-3, 4-dihydro-1H-isoquinolin-2-yl) methanone

M+H+：309.3

Example 42: benzotriazol-1-yl- (4-benzylpiperidin-1-yl) methanones

M+H+：321.1

Example 43: (5-bromobenzotriazol-1-yl) - (3-methylpiperidin-1-yl) methanone

M+H+：325.31

Example 44: 1- (3, 4-dihydro-1H-isoquinoline-2-carbonyl) -1H-benzotriazole-5-carbonitrile

M+H+：270.12

Example 45: (4-chloro-6-trifluoromethylbenzotriazol-1-yl) - (3, 4-dihydro-1H-isoquinolin-2-yl) methanone

M+H+：381.06

Example 46: 1- (3, 4-dihydro-2H-quinoline-1-carbonyl) -1H-benzotriazole-5-carbonitrile

M+H+：304.11

Example 47: (4-chloro-6-trifluoromethylbenzotriazol-1-yl) - (3, 6-dihydro-2H-pyridin-1-yl) -methanone

M+H+：331.04

Example 48: benzotriazol-1-yl- (7-methoxy-3, 4-dihydro-1H-isoquinolin-2-yl) methanone

M+H+：309.1

Example 49: (5-methoxybenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanones

M+H+：275.3

Example 50: (3, 4-dihydro-1H-isoquinolin-2-yl) - (6-nitrobenzotriazol-1-yl) methanone

M+H+：324.3

Example 51: (6-benzoylbenzotriazol-1-yl) - (4-methylpiperidin-1-yl) methanone

M+H+：349.15

Example 52: benzotriazol-1-yl- (7-nitro-3, 4-dihydro-1H-isoquinolin-2-yl) methanone

M+H+：324.1

Example 53: azepan-1-yl-benzotriazol-1-yl-methanones

M+H+：245.3

Example 54: benzotriazol-1-yl- (4-chloropiperidin-1-yl) methanones

M+H+：265.7

Example 55: benzotriazol-1-yl- (3, 6-dihydro-2H-pyridin-1-yl) methanones

M+H+：229.2

Claims (12)

1. A benzotriazole of the formula (I),

wherein:

r1 to R8 are H, or

Wherein one of the radicals R2 or R3 may represent Br, Cl, CH₃、CN、NH₂、NO₂、CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-ringHexyl, CO-OCH₃(ii) a Or

The two substituents of the series are: r1 ═ Cl and R3 ═ CF₃Or R2 ═ F and R3 ═ Cl;

n is an integer 0, 1 or 2; and is

One of the substituents R6 or R7 may represent: r6 is CH₃(ii) a R7 is CH₃、C₂H₅、CH(CH₃)₂、C(CH₃)₃、CF₃Br, Cl, benzyl or CO-OC₂H₅(ii) a Or

R6 and R7 are both CH₃(ii) a Or

The ring may contain a double bond in place of R6 and R7; or

R5 and R6 or R6 and R7 may, together with the carbon atoms bearing them, represent a fused benzene ring or, if n ═ 0, a cycloadipyl group; wherein in the case where R6/R7 is a fused benzene ring or a cycloadilyl group, the fused benzene ring or the cycloadilyl group may be optionally substituted with NH₂Or NO₂Mono-substituted or substituted OCH₃Mono-or di-substituted; or

R7 and R8 together are cyclopentyl or ═ CH₂Or if R1 to R6 are H and n is 1, R7 and R8 together may also be diazacyclopropenyl;

wherein R1 to R5 and R8 ═ H, n ═ 1 and R6/R7 ═ fused benzenes, and R1, R3-R8 ═ H, R2 ═ CH₃And n is 1.

2. Benzotriazole of formula I according to claim 1,

wherein:

r1 to R8 are H,

wherein one of the radicals R2 or R3 may represent: r2 is Br, Cl, CN, NO₂、CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl, CO-OCH₃(ii) a R3 is CH₃、CN、Br、Cl、NH₂、NO₂And a benzoyl group.

3. Benzotriazole of formula I according to claim 1 wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent: r2 is Br, Cl, NO₂、OCH₃Phenoxy, CO-OCH₃(ii) a R3 is NH₂(ii) a Or

The two substituents of the series are: r2 ═ F and R3 ═ Cl;

n is an integer of 1 or 2; and is

One of the substituents R6 or R7 may represent: r6 is CH₃(ii) a R7 is CH₃、CF₃Or Br; or

The ring may contain a double bond in place of R6 and R7, or

R6 and R7 may, together with the carbon atoms bearing them, represent a fused benzene ring, which may optionally be substituted by NH₂Mono-substituted or substituted OCH₃Mono-or di-substituted; and is

R7 and R8 together are cyclopentyl, or

n is an integer of 0; and is

R6 and R7 together with the carbon atom bearing them represent a fused benzene ring or a cycloadilyl group.

4. Benzotriazole of formula I according to claim 1 wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent: r2 is Br, CN, CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl; r3 is CN, Br, Cl, NO₂A benzoyl group; or

The two substituents of the series are: r1 ═ Cl and R3 ═ CF₃；

n is an integer of 1; and is

One of the substituents R6 and R7 may represent: r6 is CH₃(ii) a R7 is CH₃、C₂H₅、CH(CH₃)₂、C(CH3)₃Benzyl or CO-OC₂H₅(ii) a Or

R6 and R7 are both CH₃(ii) a Or

The ring may contain a double bond in place of R6 and R7, or

R5 and R6 or R6 and R7 may represent, together with the carbon atoms which carry them, a fused benzene ring;

with the exception of compounds in which R1 to R5 and R8 ═ H, n ═ 1 and R6/R7 ═ fused benzenes.

5. Benzotriazole of formula I as defined in claim 1 selected from the group of compounds having the following structures:

6. benzotriazole of formula I as defined in claim 1 selected from the group consisting of compounds having the structure

7. The compound of claim 6, selected from compounds having the following structures,

8. the compound of claim 7, selected from compounds having the following structures,

9. a process for the preparation of a compound of formula I according to any one of claims 1 to 4, which comprises:

a) acylation of benzotriazole 2 with carbamoyl chloride 3, or

b) First of all, benzotriazole 2 is reacted with phosgene and then the resulting benzotriazole carbonyl chloride 5 is reacted with an amine to give the compound of the formula I,

wherein the substituents have the meaning as defined in any one of the preceding claims 1 to 4.

10. Use of a benzotriazole of the formula I in the preparation of a medicament having an inhibitory effect on hormone sensitive lipase HSL,

wherein:

r1 to R8 are H;

wherein one of the radicals R2 or R3 may represent: br, Cl, CH₃、CN、NH₂、NO₂、CF₃、OCH₃Phenoxy, benzoyl, CH (OH) -phenyl, S-cyclohexyl, CO-OCH₃(ii) a Or

The two substituents of the series are: r1 ═ Cl and R3 ═ CF₃Or R2 ═ F and R3 ═ Cl;

n is an integer 0, 1 or 2; and is

One of the substituents R6 or R7 may represent: r6 is CH₃(ii) a R7 is CH₃、C₂H₅、CH(CH₃)₂、C(CH₃)₃、CF₃Br, Cl, benzyl or CO-OC₂H₅(ii) a Or

R6 and R7 are both CH₃(ii) a Or

The ring may contain a double bond in place of R6 and R7; or

R5 and R6 or R6 and R7 may, together with the carbon atoms bearing them, represent a fused benzene ring or, if n ═ 0, a cycloadipyl group; wherein in the case where R6/R7 is a fused benzene ring or a cycloadilyl group, the fused benzene ring or the cycloadilyl group may be optionally substituted with NH₂Or NO₂Mono-substituted or substituted OCH₃Mono-or di-substituted; or

R7 and R8 together are cyclopentyl or ═ CH₂Or if R1 to R6 are H and n is 1, R7 and R8 together may also be diazacyclopropenyl.

11. The use according to claim 10 for the preparation of a medicament for the treatment of non-insulin dependent diabetes mellitus, diabetic syndrome or syndrome X.

12. A medicament comprising at least one benzotriazole of formula I as claimed in any one of claims 1 to 8 for the treatment of non-insulin dependent diabetes mellitus or diabetic syndrome.