TWI362388B - Pyrrolidine compound - Google Patents

Description

IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to. In particular, it is a method for inhibiting dipeptide peptidase IV or treating type 2 diabetes by using a beta piroxime compound. [Prior Art] Glycoglycan peptide-1 (GLP-1) is a gut hormone produced by nutrient uptake by enteroendocrine gland L cells. GLP-1 inhibits glycemic secretion and promotes the release of glucose-dependent insulin from the pancreas. It is known that administration of GLP-1 is effective in reducing the blood glucose level of type 2 diabetic patients (Zander M, et al. Lancet 2002, 359: 824-830). However, GLP-1 administered either in vitro or in vivo degrades rapidly (Kieffer TJ et al. Endocrinology 1995, 136: 3585-3596; and Mentlein R, et al. Eur. J. Biochem. 1993, 214 : 829-839). This degradation can be attributed to dipeptidyl peptidase IV (DPP-IV), a member of the prolyl peptidase family. Clinical data indicate that inhibition of DPP_IV will occur. Lead to increased insulin secretion 'lower blood glucose levels and improved pancreatic cell function (Pederson RA, et al. Diabetes 1998, 47: 1253-1258; and

Ahren B, et al· £) Secretly c (10) 2002, 25: 869-875). Therefore, DPP-IV inhibitors are potential candidates for η diabetes.

HRI-P060063-TW SUMMARY OF THE INVENTION The present invention is based on an unexpected discovery that a group of diced amine compounds are effective in inhibiting the activity of DPP-IV. One aspect of the present invention is directed to a diamine compound having the formula: R4 Rfi \ .R3

Wherein τ is c(R9Rl.), NH, oxygen or sulfur; x is (CRiiRi2)n, and each of iWγ and z is hydrogen, alkyl, cycloalkyl, hetero, aromatic, heterocyclic, fragrant, or a pyridyl group, a r group or a hydroxy group, t, z and the I atom to which they are attached "to form a five-six hexavalent oxime, a ring can be found, a base", an aryl group, a self group, a hydroxyl group, Alumina, fluorenyl, alkoxycarbonyl or carboxy substituent; and =, = can be 舆 - a three-to-membered aromatic ring or a non-aromatic ring slightly miscellaneous,) and == aroma or non-aromatic ring Containing a small 2 or 3 alkyl group, cyanide <, nitrate 1 and 2, R6, R7 and 1^ are each hydrogen, an aromatic group; r3, anthracene ring, heterocycloalkyl, aryl or hetero The cycloalkyl group, from the base 3, "AR9, RlG, .Rl1 and R" are each hydrogen, alkyl, aryl cyano, nitro, alkoxy hydroxy, cycloalkyl, heterocyclic two: = Aromatic base. The (subset) feature is a diamine compound of the formula: in a combination of compounds, γ is CH2, CH2CH2 or C(CH3)2. In these °'s are stupid, alkaryl or cycloalkyl, or gamma, z

HRI-P060063-TW and the nitrogen atom to which it is attached may be an isoindolyl group or a 2,3,4•tetrahydroisoquinoline, the isoindolyl group or 1,2,3,4-tetra Hydroisoquinoline may have alkyl, aryl, dentate, hydroxy, alkoxy, nitro, amino, alkoxycarbonyl or carboxy substituent; τ may be oxygen, sulfur or CH2;

Ri can be a cyano group, and R2, R3 'r4, r5, r6, &, Rg, &, Ri. ,

Rn and Rn may be hydrogen; r3 may be fluorine, and the heart may be hydrogen or fluorine, and R2, R3, R4, R5, R6, R7, r8, r9, Ri. And Ru may be hydrogen; or R7 is methyl', then Rs is hydrogen or methyl; or R11, and heart 2 is methyl. The following are illustrative compounds of the invention:

7 HRI-P060063-TW

1362388 "Hetero atom" means a non-carbon atom. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur. "Alkyl" as used herein refers to a straight chain or branched hydrocarbon containing from 1 to 10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and neobutyl. "Alkoxy" as used herein means -0-alkyl. "Alkoxyalkyl" as used herein refers to an alkyl group having one or more alkoxy substituents. By "dentylalkyl" is meant herein an alkyl group having one or more dentate substituents. "Hydroxyalkyl" as used herein refers to an alkyl group having one or more hydroxy substituents. "Aromatic group" herein means a 6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic aromatic ring system in which each ring may have 1 to 4 substituents. Examples of the aryl group include, but are not limited to, phenyl, naphthyl, and anthracenyl. Fang

HRI-P060063-TW 8 aryloxy is used herein to mean 〇-aryl. "Aromatic aralkyl" as used herein refers to an alkyl group having an aryl substituent. "Cycloalkyl" as used herein refers to a saturated or partially unsaturated cyclic hydrocarbon having from 3 to 12 carbons. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl. "Heterocyclic aromatic group" as used herein means an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclic system having one or more heteroatoms (e.g., nitrogen, oxygen or sulfur). Examples of heterocyclic aromatic groups include, but are not limited to, pyridinyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, sinyl, quinolyl, decyl and thiazolyl. "Heteroaralkyl" as used herein means an alkyl group having a heterocyclic aryl substituent. "Heterocycloalkyl" as used herein means a non-aromatic 5-8 membered monocyclic ring, 8-12 membered bicyclic ring or 11-14 membered bicyclic ring having one or more heteroatoms (eg nitrogen, oxygen or sulfur). . Examples of heterogeneous groups include, but are not limited to, piperazinyl, pyrrolinyl, dioxo, morpholinyl and tetrahydrofuranyl. The alkyl, cycloalkyl, heterocyclic, aryl, heterocyclic aryl, aralkyl, heterocyclic aralkyl, alkoxy and aryloxy groups mentioned herein include substituted and unsubstituted Examples of the substituents include, but are not limited to, halo, hydroxy, amino, cyano, nitro, thiol, alkoxycarbonyl, amine, carboxyl, alkylthio, alkylcarbonyl, carbamid Sentence, carbamyl, enemy, thioureido, thiocyanato, sulfonamide, alkyl, alkenyl, fast

9 HRI-P060063-TW 136.2388

, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl, phenyl, alkoxy, aryl, heterocyclic aryl wherein alkyl, alkenyl, alkynyl, alkoxy, molybdenum, heterocycle The alkyl group may have a substituent.

The aforementioned bisamine compound comprises a pharmaceutically acceptable salt thereof and a prodrug (pnxlmg), if appropriate. This salt can be formed between a positively charged ionic group (e.g., an ammonium group) and a negative ion counter (e.g., trifluoroacetic acid) of the amine compound. Similarly, a negatively charged ionic group (e.g., ruthenium) of the arylamine compound can also form a salt with a positively charged counterion (e.g., sodium, potassium, thief magnesium). The diamine compound may comprise a non-aromatic (n()n-af double bond, or comprise - one asymmetric center. Shame, they may be present in the following form: a acemic mixture), a single mirror image isomer ( Enantiomer), individual diastereomers (extracted (10) Li), diastereomeric complexes Qianshun _ _ _ lion style. All of these isoforms are foreseen.

Another aspect of the present invention relates to a method of inhibiting Dpp_IV by contacting Dpp_IV with the aforementioned compound of Effective I. Surprisingly, these diamine compounds are biased towards inhibiting Dpp_IV activity (more than Dmi, DPP-vm or fibroblast activated protein). The inhibition of DPP-IV f leads to a decrease in blood glucose and an increase in the secretion of tensin, and the compound can also be used for the treatment of type 2 diabetes. Thus, the present invention further contemplates a method of treating type 2 diabetes by administering an effective amount of one or more of the foregoing compounds to a patient in need thereof. 3 having one or more declarative compounds and a pharmaceutically acceptable

HRI-P060063-TW 10 Carrier = Topical Composition, and the use of the composition to manufacture a therapeutic Type II diabetes drug is within the scope of the present invention. The details of many embodiments of the invention are described in the following description. Other features, objects, and advantages of the invention will be apparent from the description and appended claims. [Embodiment] The bisamine compound of the present invention can be synthesized by a synthetic method known in the art. Two exemplary synthetic pathways are shown in Schemes J and 2 below.

5 6

Scheme 1.(4) THF, NaOH IN, 0.5 equiv, 30 min, 0〇C then 1 h, rt; (b) DCC, HOSa CH2CI2, l, 4*dioxane, 16 h, rt; (c) πσ, NaOH m , 1.5 equiv, 30 min, 0 ° C ^en 16 h, rt; (6) isobutyl chlorofonnate, 1-methylpiperidine, CH202i' 30 min, -15 ° C then 3 h, rt; (e) HBr/CH3COOH, 15 min, Rt. 11

HRI-P060063-TW 1362388 In Scheme W, the starting compound is the compound (Compound i), the amino group of which has been protected, which can be prepared by slending cw i99i from the procedure described in 717. Partial hydrolysis of this compound produces a monoacid (compound 2) which is similar to the monoacid (compound 2). The primer reaction gives a guanamine intermediate (compound 3). The intermediate product contains a monoester group. Hydrolysis of the ester group provides another monoacid (compound 4) which is combined with a pyrrolopridine compound to form a diamine (compound 5). The ammonia protecting group is removed to provide the desired diamine (compound 6).

In Scheme 2, the starting step can be prepared as described in (10) 1995, 5/, 8545. This compound is partially hydrolyzed, followed by amino group protection to give the monoacid compound 8, and the monoacid compound 8 is reacted with pyrroline pyridine-2-carboxylic acid decylamine to give the decylamine intermediate (compound 9). Hydrolysis of another ester group to provide another monoacid, which is combined with 2-(3·dioxamethyl-phenyl)-cyclopropylamine to form tridecylamine (combination

HRI-P060063-TW 12 1362388 Object 10). Dehydration converts the primary guanamine group to a cyano group. The ammonia protecting group is removed to form the dimethylamine compound 11. The synthesized amine compound can be further purified by column chromatography, performance liquid chromatography or crystallization.

The foregoing schemes demonstrate the synthesis of two specific diamine compounds of the present invention. Those skilled in the art, in view of this example, will be able to modify the process to incorporate other diamine compounds of the invention. Moreover, those skilled in the art can synthesize the thiamine compounds of the present invention by other methods known in the art.

Synthetic chemical conversion and protecting group methods (protection and removal protection) suitable for the synthesis of useful compounds are known in the art and include, for example, the following, R. Larock, Compre/ze (d) 've OgOm.c Transformations, VCH Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fiesers Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.;, Encyclopedia of for Organic Synthesis, John. Wiley and Sons (1995) and subsequent versions. The diamine compound of the present invention shows an effective inhibition against DPP-IV. Accordingly, the present invention is directed to a method of inhibiting DPP_IV by contacting Dpp_iv with an effective amount of one or more diamine compounds. The present invention also encompasses the treatment of type 2 diabetes by administering an effective amount of one or more of the aforementioned diamine compounds to a patient in need thereof

HRI-P060063-TW

13 law. / σ treatment refers to the administration or administration of a diamine compound to a patient with η-induced urinary disease, a symptom of type 11 diabetes, or a type 2 diabetes, with the aim of treating, curing, alleviating, alleviating, changing, healing, Improve, improve or affect the disease, the condition or the tendency. By "effective amount" is meant an amount of a bisamine compound sufficient to impart a desired effect to a patient. As is well known to those skilled in the art, effective amounts will vary depending on the route of administration, the amount of excipient, and the likelihood of using other treatments (e.g., using other active agents). To practice the method of the present invention, a composition having one or more of the aforementioned bantam compounds can be administered parenterally, orally, nasally, rectally, topically or orally. The method of the present invention is to systemically administer a composition containing a G-CSFR agent via a parenteral or rectal route. "Parenteral" as used herein refers to subcutaneous, intracutaneous, intravenous 'intramuscuiar', intraarticular, intraarterial (intraarterial). ), intrasynovial, intrasternal, intrathecal, intralesional or intracrania injection, and any suitable infusion technique. The sterile injectable composition can be a solution or suspension (e.g., a solution of 1,3-butanediol) in a non-toxic parenterally acceptable diluent or solvent. Mannitol and water can be used in acceptable carriers and solvents. In addition, it is known to use a fixed oil as a solvent or a suspending medium (for example, synthesizing mono- or diacids).

14 HRI-P060063-TW Glyceride). Fatty acids, such as oleic acid and its glycoside, Θ 'ester derivatives, can be used for the preparation of injections, natural pharmaceutically acceptable, also set human oil (such as eucalyptus oil or E sesame oil (especially in the form of polyoxyethylene) It can also be used to prepare injections. These oil solutions or stock solutions may also contain a cleavage agent, a carboxymethyl cellulose or a similar dispersing agent. For the purposes of formulation, other commonly used surfactants such as Tw_ or Spans' or other similar emulsifiers, or bioavailability enhancers, are commonly used in the manufacture of pharmaceutically acceptable solid liquids or other dosage forms. , can also be used. The oral composition can be any orally acceptable dosage form comprising a capsule, a key, an emulsion, and an aqueous suspension, dispersion plaque solution. In the case of a key agent, a commonly used carrier comprises lactose and jade powder. A lubricant such as magnesium stearate is added. In the case of oral administration in a capsule form, it is useful to contain lactose and dried corn powder. When it is necessary to use water (4) float or milk, the ingredients are suspended or dissolved in a emulsifier or a suspension-oil base. Add specific sweetness, flavor or toner as needed. Nasal or inhalation compositions can be prepared according to conventional techniques in the art of pharmaceutical formulation. For example, 'such a composition can be prepared as a brine solution, 'using a clauded alcohol or other suitable preservative known in the art, an absorption enhancer that is used for bioavailability, gas carbide (4) sinking (4) Or other dissolving or dispersing agent. The composition of the active diamine compound can also be administered via a rectal suppository formulation. The carrier in the pharmaceutical composition must be "acceptable" in

HRI-P060063-TW 15 1362388 is compatible with the main component of the composition (beneficially and preferably can stabilize the main component) and is not harmful to the patient to be treated. One or a smear/decomposition agent can be used as a pharmaceutical excipient for delivering an active diamine compound. Examples of other carriers include colloidal cerium oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow#1〇. The diamine compound of the present invention is initially screened by in vitro testing of the desired activity = (e.g., inhibition of DPP_IV). In the initial screening, compounds with high activity were confirmed to be screened for their potency by _ test. For example, the compound to be tested can be administered to an animal having n-type diabetes (e.g., a mouse model) and then evaluated for therapeutic effects. Based on these results, the appropriate dosage and route of administration can be determined. The specific embodiments described hereinafter are merely illustrative and are not intended to limit the remainder of the present invention. Without further elaboration, it is apparent that those skilled in the art will be able to make full use of the present invention based on the description herein. All publications, including patents, cited herein are hereby incorporated by reference in their entirety. Compounded into 4-(2-phenyl-cyclopropylamine fluorenyl)_2_(pyrroline bit-1-yl-pyrroline pyridine hydrobromic acid (i) clothing cm-N-(stupyloxycarbonyl) _4_carboxy-proline decyl ester (Compound 2) An aqueous solution of sodium hydroxide (1 N, 10 mL) was added to #·(phenoxycarbonyl>4_carboxy-L-valine di-f-ester (3.2 g, 10 mm 〇 1) MTI^ (2 〇 mL) in a solution, stirred at 〇〇C for 60 minutes. After 〇〇c 3 〇 minutes and room temperature-hours, the reaction mixture was concentrated and added to Et0Ac.

HRJ-P060063-TW 16 136.2388 with 6 N gasification hydrogen (5 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by using a chloroform/column (95:5) as a solvent to obtain the desired compound (colorless oil) (2.1 g, 68%) 〇(ii). 4-(2-Phenyl-cyclopropylaminoindenyl)-n-Bilolin bite _1,2·di-baric acid-1-phenyl ester-2-methyl ester (Compound 3)

00:(:(454 11^, 2.2 111111〇1) of dichlorohydrazine (2 1111 〇 solution was added to the stirred (phenoxycarbonyl)-4·carboxy-proline decyl ester (615 mg, 2 mmol) and HOSu (173 mg, 3 mmol) in dioxane (2 mL). After 16 hours, the resulting hydrazine, hydrazine-dicyclohexylurea was filtered off. The filtrate was taken with saturated sodium bicarbonate. The solution (5 mL), 1 柠檬酸 citric acid (5 mL), water (5 mL) was rinsed with MgSO4 and concentrated. Res. Purification of the extract to give the desired compound (yield oil) (633 mg, 75%).

(iii) Preparation of 4-(2-phenyl-cyclopropylaminoindenyl)-indolyl pyridine-I,2-dicarboxylic acid-1-phenyl ester (Compound 4), stirred at 〇 ° C, Add 1N sodium hydroxide (1.5 mL) to 4-(2-phenyl-cyclopropylaminecarbamyl)-π-pirolene bite-1,2-didecanoate-1-phenyl ester-2-methyl ester (422 mg, 1 mmol) in THF (2 mL). The reaction mixture was concentrated at EtOAc (EtOAc) (EtOAc) The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by using a silica gel column eluted with dichloromethane (1:5:5) to give the desired compound (yield oil).

17 HRI-P060063-TW mg, 84%). (iv) Preparation of 4-(2-phenyl-cyclopropylaminecarbamyl)-2-(pyrroline pyridine-1-carbonyl-pyrroline pyridine-1-carboxylic acid phenyl ester (Compound 5) Propyl ester (103 mg, 0.75 mmol) was added to -15 °C of "-(2-phenyl-cyclopropylaminemethanyl)-pyrroline pyridine-1,2-dicarboxylic acid-1-benzene vinegar (306) Mg, 0.75 mmol) and a solution of (piperidine) (75 mg, 0.75 mmol) in (15 mL) dichloromethane (15 mL). After two minutes at -15 °C, the mixture was added.嘻琳 bite (57 mg, 0.8 mmol). After two minutes at -15 °C, the reaction was saturated with sodium bicarbonate (5 mL), 1 N citrate (5 mL), water. (5 mL) was washed with MgSO4, EtOAc (EtOAc m. 187 mg, 54%). (v) Preparation of 4-(2-phenyl-cyclopropylaminecarbamimidyl)-2-(pyrroline pyridine-1-carbonylpyranidine occluded hydrobromic acid (Compound 6) 4-(2-Phenyl-cyclopropylaminecarbamimidyl)-2-(pyrroline pyridine-l-carbonyl-indole 11 linaldine-1-phenylate (115 mg, 0.25 mmol) at room temperature Under 33% hydrobromic acid ( Treatment with glacial acetic acid (1 mL). After 15 minutes, the reaction mixture was concentrated under reduced pressure to <RTI ID=0.0></RTI> <RTIgt; Mg, 80%). NMR (CDC13, 300 MHz, δ): 7.70 (brs, NH), 7.26-7.21 (m, 2 Η), 7.17-7.11 (m, 3 Η), 3.93 (brs, 1 Η), 3.55- 3.15 (m, 7H), 3.00-2.91 (m, 2H), 2.34 (dt, J = 12.9, 8.4 Hz, 1H), 2.09-1.84 (m, 6H), 1.21-1.16 (m, 2H);

18 HR1-P060063-TW MS (ES+) m/z calcd. for Q9H25N3O2: 327.42; foimd:328.2 (M+H), 350.2 (M+Na). Example 2 Synthesis of (3S,5S)-5-(pyroline bit-1-carbonyl)-N-((lS,2R)-2-(3-(trifluoromethyl)phenyl)cyclopropyl ). The specific compound of the pyroline pyridine-3-carboguanamine was synthesized in a manner similar to the compound 6. lR NMR (CDCI3, 300 MHz, δ,): 7.97 (brs, NH), 7.39-7.31 (m, 4H), 3.99 (t, J = 7.5 Hz, 1H), 3.59-3.16 (m, 7H), 3.08 -2.93 (m, 2H), 2.40 (dt, J = 13.2, 9.0 Hz, 1H), 2.15-1.84 (m, 6H), 1.32-1.19 (m, 2H); MS (ES+) m/z calcd. for C20H24F3N3O2: 395.42; found: 396.1 (M+H), 418.1 (M+Na). Example 3: Synthesis of (3S,5S)-5-(pyrrolidinium-1-carbonyl)-N-((lS,2R)-2-(4-(trifluoromethyl)phenyl)cyclopropyl)indole The desired compound of the porphyrin pyridine-3-carboguanamine was synthesized in a manner similar to the compound 6. NMR (CDCI3, 300 MHz, δ,): 7.89 (brs, ΝΗ), 7.48 (d, 7 = 8.0 Hz, 2H), 7.22 (d, 8.0 Hz, 2H), 4.02 (dd, */= 7.2, 8.4 HZ, 1H), 3.61-3.18 (m,7H), 3.09-2.91 (m,2H), 2.38 (ddt, /= 1.8, 13.2, 9.0 Hz, 1H), 2.15-1.85 (m, 6H), 1.35- 1.18 (m, 2H); MS (ES+) m/z calcd. for C20H24F3N3O2: 395.42; found:

19 HRI-P060063-TW 396.1 (M+H), 418.1 (M+Na). Example 4: Synthesis of (3S,5S)-N-((lS,2R)-2-(4-chlorophenyl)cyclopropyl)-5-(0 than 洛 1 -carbyl) Lolidine Bite - The desired compound of the 3-terminal acid amine is synthesized in a manner similar to the compound 6. H NMR (CDC13, 300 MHz, δ,): 7.76 (brs, NH), 7.20 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 3.94 (dd, J = 6.9, 8.1 Hz, 1H), 3.59-3.35 (m, 3H), 3.27-2.93 (m, 5H), 2.88-2.85 (m, 1H), 2.33 (ddt, J= 1.5, 13.2, 9.0 Hz, 1H) , 2.05-1.85 (m, 6H), 1.25-1.11 (m, 2H); MS (ES+) m/z calcd. for C19H24ClN3 〇2: 361.87; found: 362.5 (M+H). Example 5: Synthesis of (3S,5S)-N-((lS,2R)-2-(3-fluorophenyl)cyclopropyl)-5-(ntb sulphide.-1 -domain)D The compound of Bilolin's bite 3-carbonamine is synthesized in a manner similar to Compound 6. ]H NMR (CDCI3, 300 MHz, δ): 7.78 (brs, ΝΗ), 7.23-7.16 (m, 1Η), 6.92-6.80 (m, 3H), 3.94 (dd, J= 6.6, 8.4 Hz, 1H) , 3.62-3.36 (m, 3H), 3.29-2.88 (m, 6H), 2.34 (dt, 13.2, 9.0 Hz, 1H), 2.08-1.85 (m, 6H), 1.27-1.14 (m, 2H); MS (ES+) m/z calcd. for C19H24FN302: 345.41; found: 346.5 (M+H), 368.5 (M+Na).

20 HRI-P060063-TW : Synthesis (2S,4S,5S)_ l-{5-Methyl-4-[2-(3-trifluoromethyl-phenyl)-cyclopropanecarbonyl]-pyrroline- 2-carbonyl}-pyrroline pyridine (Compound 11) (1) Preparation of (2S,4S,5S)-5-mercapto-nonylpyridinium-tris-acidic neobutyl ester 4-decyl ester (Compound 8 ) (2S,4S,5S)-5-mercaptopirin bite-2,4-di-acid 2-ethyl butyl 4-methyl ester (730 mg, 3 mmol) of TFA (5 mL) The solution was stirred at room temperature for thirty minutes. The reaction product was concentrated in vacuo. The resulting residue was dissolved in dichloromethane (8 mL). Then triethylamine (6 〇 7 mg, 6 mmol) was added followed by di-n-butyl succinate (980 mg, 4.5 mmol) in dioxane (3 mL). After stirring at 0 ° C for thirty minutes and at room temperature for eight hours, the reaction product was concentrated in vacuo, dissolved in dioxane (50 mL), and extracted with 1 N citric acid (i 〇mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified using a silica gel column eluting with dichloromethane (1:5:5) to give compound 8 (517 mg, 1.8 mmol, 60%). (ϋ) Preparation of (2S,4S,5S)-5-(2-carboguanamine-°pyrrolinepyridin-1-carbonyl)-2-methyl ratio 11 each bite-1,3-diwei acid New butyl hydrazine 3-mercapto vinegar (9) A solution of DCC (454 mg, 2 - 2 mmol) in dichloromethane (3 mL) was added to stirring compound 8 (517 mg, i.8 mmol), 4-DMAP (439 Mg, 3.6 mmol) and HOBt (316 mg, 2.3 mmol) in 1,4-dioxane (Pioxane) (3 mL). After 10 minutes at room temperature, (S)-pyrroline pyridine-2-carbonium was added to dichloromethane (2 mL) with stirring.

21 HRI-P060063-TW Amine (251 mg, 2.2 mmol). The reaction mixture was stirred for 16 hours and then filtered. The filtrate was washed with a saturated aqueous solution of sodium hydrogensulfate (5 mL), 1N citric acid (5 mL), and water (5 mL) and dried over magnesium sulfate and concentrated. The residue was purified by using a gas kiln column with two gas crater/sterol (95.5) as the extract to obtain compound 9 (586 mg, 1.5 mmol, 85%) and y (iii) (2S, 4S,5S)- 5-(2-Carboguanamine-pyrroline pyridine+carbonyl)_2-indolyl-3-[2-(3-trifluorodecyl-phenyl)cyclopropanylcarbonyl]-pyrroline </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> <RTIgt; The reaction mixture was concentrated after EtOAc (EtOAc)EtOAc. The organic layer was dried over magnesium sulfate and concentrated. The residue was purified by using a silica gel column with dichloromethane (yield: 95:5) to give an acid compound (553 mg, 1.5 mmol, 99%). A solution of DCC (454 mg, 2.2 mmol) in dichloromethane (2 mL) was added to 4-DMAP (366 mg, 3.0 mmol), HOBt (275 mg, 2.0 mmol) and the acid compound (553 mg, 1.5 mmol) The 1,4-dioxane (2 mL) was stirred in the solution. After ten minutes at room temperature, 2-(3-(trifluoromethyl)phenyl)cyclopropaneamine was added to dichloromethane (2 mL) to hydrogenate hydrogen (380 mg, 1.6 mmol) and triethylamine ( 162 mg, 1.6 mmol) The reaction mixture was allowed to mix for a period of sixteen hours. The sputum was saturated with sodium bicarbonate solution (5 mL), 1 N citric acid solution (5 mL), water (5 mL)

22 HRI-P060063-TW Rinse 'Dry with sulphuric acid and concentrate. The residue was purified using a hydrazine column using dioxane / methanol (95:5) to give compound 10 (607 mg, 1.1 mmol, 73%). (iv) Preparation of (2S,4S,5S)-1_{5_mercapto-4-[2-(3-trifluorodecyl-phenyl)·cyclopropanone carbonyl] than 嘻 咬 bit -2-yl} ·^Lorraine bite _2_carbonitrile (Compound 11) Slowly added phosphonium chloride (674 mg, 4 4 πιπιοί) to compound 1〇 (607 mg, 1.1 mmol) in -30 °C for five minutes. Imidazole (l 12 mg, 1.6 mmol) and. More than a mixture of pyridine (5 mL). The resulting hazy white reaction mixture was stirred at _3 〇 〇 c for one hour. The resulting bright yellow opaque mixture was concentrated in vacuo and treated with dichloromethane and 1N citric acid (5 mL). The organic layer was dried over magnesium sulfate and concentrated. The residue was purified using a silica gel column eluting with dichloromethane (EtOAc) (1:1) to afford crude product (506 mg, 0.9 mmol, 82%). The crude product was stirred in TFA (5 mL) EtOAc. The reaction mixture was concentrated in vacuo to give the desired compound. NMR (CD3OD, 300 MHz, δ): 7.47-7.39 (m, 4Η), 4.87-4.80 (m, 1H), 4.62 (dd, J = 9.0, 9.3 Hz, 1H), 3.99-3.90 (m, 1H) , 3.61 (t, J = 6.6 Hz, 2H), 3.20-3.13 (m, 1H), 2.97-2.79 (m, 2H), 2.36-2.08 (m, 6H), 1.44 (d, J= 1.5, 6.9 Hz , 3H), 1.34-1.26 (m, 2H); MS (ES) m/z calcd. for C22H25F3N4O2: 434.45; found: 435.3 (M+H).

23 HRI-P060063-TW Example 7: Inhibition of DPP-IV and DPP-VIII activity DPP-IV is de Meester et al. (de Meester et al. (1996) J. Immun. Method 189: 99-105) The method described (with minor modifications) was purified from human semen. Briefly, semen was diluted in 5 mL mL of phytic acid saline and centrifuged at 900 xg for ten minutes. The supernatant was again centrifuged at 105,000 Xg for one hundred and twenty minutes to separate the prostasome from the seminal plasma. The prostatic body, i.e., the pellet, and the seminal plasma, i.e., the supernatant, were used for further purification of dpp-FV. The pellet was washed twice with 20 mM Tris-HCl (pH 7.4), and then incubated at 20 C in 20 mM Tris-HCl (pH 7.4), 1% Triton X-100 for one hour. Prior to dialyzing with 20 mM Tris-HCl (pH 7.4), 70 mM NaCl and 0.1% Triton X-100, the previously produced solution was centrifuged at 40,000 xg for ten minutes to remove the prostatic body (prostasome) ) Pills. The solution was then passed through a DEAE-Sepharose fast flow tube (2.6 x 10 cM) equilibrated with 20 mM Tris-HCl (pH 7.4) and 0.1% Triton X-100 at a flow rate of 2 mL/min. The column was then eluted with a linear gradient flow rate of 3 mL/min at 300 mL NaCl (70 to 350 mM). The positive fractions were concentrated and adjusted to pH 8.0 with 0.5 M Tris-HCl (pH 8.0) and added to the adenosine deaminase-Sepharose column. The column was prepared as described in deMeester et al. The column was rinsed with ten column volumes of equilibration buffer and an equivalent amount of 50 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl and 0.1% Triton X-100.

After 24 HRI-P060063-TW, DPP-IV was flushed out with 2 mM Tris-HCl (pH 8.0) containing 〇·1% Triton X-100. The supernatant was mutated at 4 〇c in 20 mM Tris-HCl (pH 7.4) in 1% Tris X-100 for one hour. The resulting solution was treated as above to give purified DPP-IV. DPP-VIII was also expressed and purified. Briefly, the full-length human DPP-VIII cDNA was amplified from the human liver cDNA library by RT-PCR using the primers 5'-AAGCTTCCAT GGC AGC AGC AATGGAAAC A-3, and 5'-GCGGCCGCTTATATCACTTTTAGAGCAGCAATA-3'. The resulting fragment was cloned into pCR®_Blunt II- Topo vector (Invitrogen). This full-length human DPP-VIII cDNA fragment was added/cut out with the blunt and iV and then ligated into the baculovirus expression vector pBac-PAC-His2 (Clontech). The plasmid was transfected into Sf9 cells to obtain a recombinant virus. Perform further amplification of the virus. In short, the virus titer is determined by the endpoint dilution test. The baculovirus infection was carried out as follows: Sf9 cells were cultured in a six-well plate to a concentration of 1 〇 6 cells per well. The medium was removed and replaced with an inoculum (multiplicity of infection (MOI) of 0.1 TCID5 〇 /cell (TCID5 〇 50% tissue culture infection amount)). After removal of the media containing unadsorbed virus, fresh medium was added and incubated at 27 ° C for 72 to 96 hours. Sf9 cells up to 0.5 TCID50/cell viral infection (MOI) were harvested 72 hours after transfection for subsequent protein purification.

25 HRI-P060063-TW

The purification of DPP·VIII was carried out by a Ni-NTA column. Sf9 cells expressing DPP-VIII were pelleted and resuspended in ligation buffer containing 5 mM sodium phosphate buffer (pH 7.6) and 300 mM sodium hydride. The super-chopper spreads the cells and passes the clarified cytosol (lySate) through the Ni-afflnity camp. The column was carried out in a bed volume (be (jv〇iume) three to five times the amount of the binding buffer containing 10 mM imidazole, the binding buffer containing 3 mM mM imidazole, and the binding buffer containing 1 mM mM imidazole. Flushing. Note that the performance of DPP-VIII is based on fluorescent eGFP expression or protein shell activity assay. The activities of DPP-IV and DPP-VIII are tested by SDS-PAGE (commassie blue stain or silver stain). DPP-IV The concentration of DPP-VIII was measured by Bradford's method using BSA as a standard (Bradford, MM (1976) 72, 248-254). The biological activity of DPP-IV and DPP-vm was measured by measuring the enzyme dynamic constant. For example, the dynamic constant of DPP-IV is measured as follows: All reactions are carried out in P竺S, and HGlyPro~^N〇2 is used as a receptor in the presence of 1〇nM DPP-IV. The reaction was monitored at D 4 〇 5 nm. The initial rate was measured when less than 10% of the receptor was used up. The steady state parameter Arcat (=Vmax/[E]^ Km was originally determined by the 0.5-5 Km receptor concentration. The 300 second starting rate measurement depends on the Km value.

Lineweaver-Burk plots (which are obtained using linear regression of the classical Michaelis-Menten equation (Equation 1)). ^^ is calculated from Vmax/[E], and the molecular weight of DPP-IV is set at 85,000. V〇=Vmax[S]/(Km+[S]) (Equation 1)

HR1-P060063-TW 26 where V〇 is the initial rate, [S] is the acceptor concentration, Vmax is the maximum rate, and Km is the Michaelis constant. From beginning to end, the correlation coefficient is better than 〇99〇. The compounds of the present invention have been tested for their inhibitory effects against DPP-IV and DPP-VIII. There are eight to twelve serial dilutions per compound to generate the data points from which the ic50 can be determined based on Sigma plot. All tested compounds showed inhibitory activity against DPP-IV with low IC5G values (eg 2 ca to 1 _). Surprisingly, all tested compounds were biased to inhibit DPP-IV more than DPP-VIII. Other Embodiments All of the features disclosed in the 5th book may be grouped in any manner. Each of the features disclosed in the book can be replaced by the same, "phase == change feature. Therefore, unless there is another = two each feature is only - the average or similar base of the total family 'cooked artisans can be surely known to the present invention: the spirit and the norm of =: r, and the examples of the following changes are also expected. For example, the compound of other compounds can also be used. Shishi clothes are selected by the teacher for their DPP-IV, so the other implementation of the financial person's application within the scope of the wire TI_ urine Lai was secretly implemented

HR1-P060063-TW 27

Claims (1)

1362388 公'丁.3⁄43⁄4. iOi. 2· 3 95131385 ' ιοί年月月修正页 Φ where: τ is C(R9R1()) or sulfur; X is (CRllRl2)n ' η is 1 or 2. Υ It is a C3_]2 ring hospital base, and 2 rabbits are 虱, Ci-i 〇 alkyl, C3.12 cycloalkyl group which is left-substituted as needed, and 5.8-membered heterocyclic ring containing more than one gas oxygen sulfone hetero atom. Silk, C6.1G aromatic group, ^5,8-membered heterocyclic aromatic group of ship nitrogen, oxygen or hetero atom, or C6·10 aryl CM0 炫 base; and f 卜, patent application scope: 1. a compound, The structure is like a squat. Ri R2 R5, R6, r7 and Rs are each a gas filament, a cyano group, a nitro group, a C3 i2 ring filament, a 5- to 8-membered heterocyclic group containing a diazo, oxygen or sulfur hetero atom, or a fragrant group or 5_8 = a cyclic aryl group containing more than one nitrogen, oxygen or sulfur hetero atom, and each of - respectively a gas, a % alkyl group, a dentate group, a cyano group, an exact group, a Ci i methoxy group, a thiol group, ' A cycloalkyl group, a fluorene heterocyclic fluorenyl group containing more than one nitrogen, oxygen or sulfur hetero atom, C6·. An aromatic group or a 5-8 membered heterocyclic aryl group containing one or more nitrogen or sulfur heteroatoms, &S; s Coll. 9. 2 3 Rl 〇, R&quot; and Rl2 are each hydrogen, Ci-ioalkyl , halo, cyano, nitro, alkoxy or hydroxy. For example, the compound described in claim 1 of the patent range, 1 CH2. , A X is a compound as described in claim 2, or CH2. , A T is a compound according to item 3 of the patent scope of the application, wherein &amp; is cyano, and R2, r3, r4, r5, r6, r7, ^, &amp;, ho, Rn and Rl2 are hydrogen . 9 A compound according to claim 3, wherein the gas 'R4 is hydrogen or fluorine, and R], r2, r5, R6, ^, R9, R10, Rn and r12 are hydrogen. The compound of claim 3, wherein r7 is methyl 'R8 is hydrogen or fluorenyl. The compound of claim 3, wherein τ is sulfur or Cl*!. ^ ' The compound of claim 1, wherein τ is CH, β 9 ', as defined in claim 1, wherein Y is a C3-u cycloalkyl group and hydrazine is hydrogen. , Cl 1 decylalkyl, optionally substituted C 3 ·] 2 cycloalkyl, containing more than one nitrogen, oxygen or thiane = 5-8 membered heterocycloalkyl 'C6_1 fluorene aryl group, containing one nitrogen A 5-8 membered heterocyclic aromatic group of an oxygen or sulfur hetero atom, a bite rr ^ 匕 6-10 square 1丨-1〇. 10. The compound of claim i, wherein x C(CH3)2. U. The compound of claim 10, wherein γ is 〇3·ηcycloalkyl' and ζ is hydrogen, Cm〇alkyl, optionally substituted C3_u cycloalkyl, and more than one a 5-8 membered heterocyclic alkyl group of a nitrogen, oxygen or sulfur hetero atom, a C6_1Q aryl group, a 5-8 membered heterocyclic aromatic group containing more than one murine, gas or sulfur hetero atom, or a q-square C丨-丨. Burning base. 12. The compound of claim 11, wherein τ is sulfur or CH2. 13. The compound of claim 1, wherein the compound is: 1362388 S8. 9. 2 3 A pharmaceutical composition for inhibiting dipeptide peptidase IV, which comprises an effective amount of a compound of 31 1362388 as described in claim 1 of the patent application and a pharmaceutically acceptable carrier, excipient or dilution Agent. 15. The pharmaceutical composition according to claim 14, wherein Y is C 3 -1 2 Yuyi Hyun* base and is a mouse, C 1 _ 1 〇 base, as needed C8"2-cycloalkyl group to be substituted, 5-8 membered heterocyclic alkyl group containing more than one nitrogen, oxygen or cesium atom, Cm aryl group, 5-8 of more than one nitrogen, oxygen or sulfur hetero atom A heterocyclic aromatic group, or a c6_1q aromatic Cm. alkyl. 16. The pharmaceutical composition as described in claim 4, wherein γ is a cyclopropyl group. 17. The pharmaceutical composition of claim 14, wherein the compound is: 32 S North 2388 18. The compound of claim 9, wherein γ is cyclopropyl. 19. The compound of claim 18, wherein τ is sulfur or CH2. 2. A compound according to claim 19, wherein the compound is an aryl group and R2, R3, R4, R5, R6, R7, R8, R9, R10, R&quot; and R12 are hydrogen. A compound according to claim 19, wherein 33 is fluorine, the rule 4 is hydrogen or fluorine, and Ri, R2, R5, R6, r7, R8' R9, R10, Rn and r12 are hydrogen. • A compound according to claim 19, wherein r7 is methyl and r8 is hydrogen or fluorenyl. 23 The compound of claim 18, wherein the parent is CH2. 24. A compound as claimed in claim 22, wherein τ is CH. 34