CN116514804A

CN116514804A - Cathepsin K inhibitor, preparation method and application thereof

Info

Publication number: CN116514804A
Application number: CN202210098531.3A
Authority: CN
Inventors: 王进欣; 张贵民; 江凯璇; 姚景春; 王可
Original assignee: Shandong New Time Pharmaceutical Co Ltd
Current assignee: Shandong New Time Pharmaceutical Co Ltd
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2023-08-01

Abstract

The invention provides a cathepsin K inhibitor, the structure of which is shown in formula I, and (1R, 2R) -cyclohexane-1, 2-dimethylformamide is taken as a baseThe parent nucleus has good inhibition effect on cathepsin K by taking ketone groups as electrophilic groups as key groups. The invention also provides a preparation method and application thereof. The cathepsin K inhibitor provided by the invention has higher inhibition effect and selectivity, and is expected to be prepared for treating diseases including thyroid diseases, cardiovascular diseases, bone diseases and gum diseases.

Description

Cathepsin K inhibitor, preparation method and application thereof

Technical Field

The present invention is in the field of pharmaceutical chemistry, in particular to novel compounds which inhibit cathepsin K kinase activity, a process for their preparation and their use in the preparation of a medicament for diseases associated with or characterised by cathepsin K activity, such as osteoporosis.

Technical Field

Osteoporosis, simply referred to as osteoporosis, is a metabolic disorder disease that reduces overall bone mass and bone density, thereby leading to increased risk of fracture in patients. Along with the aggravation of the aging population, the health problem related to the disease is increasingly serious, the fracture probability of the osteoporosis patient is greatly increased, and the fracture probability brings heavy burden to society and families and seriously influences the life of people. Osteoporosis is caused by imbalance of osteoclast-mediated bone resorption and osteoblast-mediated bone formation, and currently commercially available osteoporosis treatment drugs have bone resorption inhibitors such as bisphosphate and bone formation promoters, but both drugs have certain drawbacks. Long-term use of bone resorption inhibitors affects the differentiation and proliferation of bone cells, further leading to the occurrence of "low bone turnover states"; bone formation promoters increase the probability of a patient suffering from osteosarcoma.

Because the medicines existing in the current market have certain defects, the research can not influence proliferation and differentiation of osteoblasts and osteoclasts while playing the role of osteoporosis treatment, and the novel osteoporosis treatment medicine for avoiding the occurrence of a low bone transition state is the current research direction.

Cathepsin K (Cat K), a cysteine protease belonging to the papain family, is present in large numbers in osteoclasts and is now a new target for osteoporosis treatment. The bone matrix of human body is composed of 25% of water, 25% of organic matrix and 50% of mineral matrix, wherein 90% of the organic matter is collagen type I, degradation of collagen type I is a key process of bone resorption mediated by osteoclasts, and Cat K plays a dominant role in degradation of collagen type I, and Cat K inhibitor inhibits resorption of mature osteoclasts by blocking degradation of collagen matrix, and at the same time, cat K inhibitor can maintain survival number of osteoclasts, which keeps coupling signals of osteoclasts and osteoblasts intact, thereby avoiding occurrence of 'low bone transition state'. Cat K also plays an important role in the pathogenesis of thyroid disorders, cardiovascular disorders and gum disease. Diseases characterized by abnormal expression or activation of Cat K include thyroid diseases, cardiovascular diseases, bone diseases and gum diseases, in particular hyperthyroidism, atherosclerosis, cardiac hypertrophy, heart failure, osteoporosis, osteoarthritis, rheumatoid arthritis, gingivitis and periodontitis. More and more researches on Cat K are carried out in recent years, and the secretion of Cat K by endothelial cells can be increased under pathological conditions such as coronary atherosclerosis and the like. Cat K has very close relation with the occurrence and development of cardiac hypertrophy and heart failure. Garg et al (Garg G, praadeep AR, thorat MK, et al effect of nonsurgical periodontal therapy on crevicular fluid levels of Cathepsin K in periontitis. Arch Oral Biol, 54:1046-1051) demonstrate that following basic treatment, with the clinical gum index of periodontitis patients, periodontal probe depth and loss of attachment decrease, and the levels of Cat K decrease as well, cat K is a marker of periodontitis bone resorption, and should be further paid attention to and studied in periodontitis treatment, by the change of Cat K in gingival crevicular fluid before and after the basic treatment.

Current cathepsin K inhibitors are predominantly non-covalent binding inhibitors depending on their mode of binding to proteins. Early synthetic covalent binding inhibitors are mostly irreversible inhibitors, and are not drug-resistant because of their irreversible covalent binding to proteins and their high toxicity. To solve the above problems, reversible inhibitors have been reported successively, wherein the cathepsin K inhibitor balcatib developed by northwest corporation, switzerland, has been reported to enter the clinical research stage, however, clinical studies have found that adverse reactions such as rashes (including scleroderma-like syndrome) and pruritus are generated while bone protection is provided, resulting in stopping the development. In intensive studies, balicatib was characterized by high selectivity for human cathepsin K, B, which selectivity was significantly reduced in cell assays, presumably due to aggregation of Balicatib in human dermal fibroblast lysosomes highly expressed by cathepsin B.

In order to solve the problems, the invention designs and synthesizes a novel ketone cathepsin K inhibitor which has good selectivity and is expected to be used for treating thyroid diseases, cardiovascular diseases, bone diseases and gum diseases.

Disclosure of Invention

The invention provides a novel ketone cathepsin K inhibitor and pharmaceutically acceptable salt thereof, and a preparation method and application thereof.

The invention aims to provide a ketone cathepsin K inhibitor, which has the following structural general formula:

wherein the said

R ₁ 、R ₂ Each independently selected from H, halogen, cyano, amino, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, said substituted C1-6 alkyl or substituted C1-6 alkoxy being further substituted with at least one halogen or hydroxy;

t is a covalent bond; optionally (optionally)One of them.

Further, wherein R is ₁ 、R ₂ Each independently selected from H, halogen, C1-3 alkyl, C1-3 alkoxy, said C1-3 alkyl or C1-3 alkoxy being further substituted with at least one halogen.

Further, wherein R is ₁ 、R ₂ Each independently selected from H, halogen, C1-3 alkyl, C1-3 alkoxy, said C1-3 alkyl or C1-3 alkoxy being further substituted with at least one F.

The C1-3 alkyl is methyl, ethyl, propyl or isopropyl.

The C1-3 alkoxy is methoxy, ethoxy, propoxy, isopropoxy, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

The C1-3 alkoxy is methoxy or trifluoromethoxy.

The C1-3 alkoxy is trifluoromethoxy.

In some embodiments, R ₁ 、R ₂ Each independently selected from H, F, cl, br, methoxy, trifluoromethoxy.

In some preferred embodiments, R ₁ 、R ₂ Each independently selected from H and F.

The halogen is selected from F, cl, br, I.

Another object of the present invention is to provide a process for preparing the above-mentioned compounds and pharmaceutically acceptable salts thereof, comprising the steps of:

step 1) reacting the compound P1 with (3 aR,7 aS) -hexahydroisobenzofuran-1, 3-dione to generate a compound P2;

reaction conditions: in a solvent; the solvent is a polar solvent selected from at least one of water, methanol, ethanol, glycerol, propylene glycol, formamide, acetonitrile, n-butanol, dioxane, dichloromethane, acetone, dimethyl sulfoxide, dimethylformamide, ethyl acetate and tetrahydrofuran;

step 2) reacting the compound P2 with 4-aminotetralin-3-ol to generate a compound P3;

reaction conditions: in a polar solvent containing a base; wherein the base is at least one selected from pyridine, 2-methylpyridine, 2, 6-dimethylpyridine, 4-dimethylaminopyridine, triethylamine, diethylamine, N-diisopropylethylamine, dimethylisopropylamine, piperidine, 1-methylpiperidine and 1-methylpyrrolidine; the polar solvent is as defined in step 1);

step 3) the compound P3 is subjected to oxidation reaction to generate a compound of the formula I;

reaction conditions: under the condition of an oxidizing agent; the oxidant is an inorganic oxidant or an organic oxidant or a mixed oxidant; wherein the inorganic oxidant is at least one selected from sodium dichromate, chromic acid, manganese dioxide, ammonium cerium nitrate, potassium permanganate, potassium ferrate, bromine simple substance, iodine simple substance, sodium hypochlorite, sodium chlorite, sodium bromate, sodium periodate and diiodide; the organic oxidant is at least one selected from 2-iodized benzoic acid, tetramethyl piperidine oxide, benzoyl peroxide and cyclohexanone peroxide; the mixed oxidant is at least one selected from pyridinium chlorochromate, pyridinium chromic anhydride, pyridinium dichromate and pyridinium sulfur trioxide;

further, the oxidizing agent is preferably a pyridinium chlorochromate, a pyridinium chromic anhydride, and a pyridinium sulfur trioxide.

The invention also provides compositions containing said cathepsin K inhibitors.

The invention also provides application of the cathepsin K inhibitor in preparing medicines for treating diseases targeting cathepsin K.

Further, the diseases targeted by cathepsin K include thyroid diseases, cardiovascular diseases, bone diseases and gum diseases.

Further, the thyroid disorder includes hyperthyroidism.

Further, the cardiovascular diseases include atherosclerosis, cardiac hypertrophy and heart failure.

Further, the bone diseases include osteoporosis, osteoarthritis, rheumatoid arthritis.

Further, the ungulates include gingivitis and periodontitis.

Further, the disease targeted to cathepsin K is osteoporosis.

In another aspect, the invention provides the use of a pharmaceutical composition comprising a cathepsin K inhibitor alone or in combination for the manufacture of a medicament for treating a disease targeted by cathepsin K.

Compared with the prior art, the invention has the advantages and technical effects that:

inhibition of Cat K enzyme by the Compounds of the inventionHigh rate, IC ₅₀ The value is low, and the inhibition activity to Cat K enzyme is good. The inhibition rate of the compound to Cat K enzyme at 10 mu M can reach more than 90%, and IC ₅₀ Less than 50nM.

The compound of the invention has good selectivity: at levels of 1. Mu.M and 10. Mu.M, inhibition rates for Cat B enzyme and Cat S enzyme were both high<50%, IC for Cat B enzyme and Cat S enzyme ₅₀ All values are>10. Mu.M, the compounds of the invention show a very good selectivity for Cat K enzyme.

The cathepsin K inhibitors are useful in the preparation of a medicament for the treatment of diseases characterized by abnormal expression or activation of cathepsin K, including thyroid diseases, cardiovascular diseases, bone diseases and oral diseases, in particular hyperthyroidism, atherosclerosis, cardiac hypertrophy, heart failure, osteoporosis, osteoarthritis, rheumatoid arthritis, gingivitis and periodontitis.

In the present invention, the terms:

1. abbreviations and definitions

DMF: n, N-dimethylformamide

DMSO: dimethyl sulfoxide

DMAP: 4-dimethylaminopyridine

DCM: dichloromethane (dichloromethane)

HATU:2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate

DIPEA: n, N-diisopropylethylamine

2. Other terms

The term "C1-6 alkyl" refers to any straight or branched chain group containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, tert-pentyl, n-hexyl, and the like.

The term C1-6 alkoxy includes-O-C1-6 alkyl.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the inventive subject matter. Furthermore, the terms "include" and other forms, such as "comprising," "including," and "containing," are not limiting.

The section headings used herein are for purposes of organizing articles only and should not be construed as limiting the subject matter. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals, and treatises, are hereby incorporated by reference in their entirety.

The pharmaceutical composition comprises the compound of the formula I in the first aspect of the invention, or optical isomer, pharmaceutically acceptable salt and prodrug thereof.

In the present application, unless specifically indicated otherwise, the term "pharmaceutically acceptable salt" refers to salts suitable for tissue contact in a subject without undue adverse effects, the salts in this application being primarily pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, and the like; organic acid salts include formate, acetate, 2-dichloroacetate, trifluoroacetate, propionate, hexanoate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacate, adipate, glutarate, malonate, oxalate, maleate, succinate, fumarate, sprinkle, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, naphthalenedisulfonate, and the like. These salts can be prepared by methods known in the art.

By "pharmaceutically acceptable base addition salt" is meant a salt with an inorganic or organic base that is capable of maintaining the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Preferred organic bases include iso-large amine diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

The present invention also includes prodrugs of the above compounds, and in this application the term "prodrug" means a compound that can be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the invention. Prodrugs may not be active when administered to an individual in need thereof, but are converted in vivo to the active compounds of the present invention. Prodrugs are typically rapidly converted in vivo to the parent compounds of the present invention, for example, by hydrolysis in blood. Prodrug compounds generally provide solubility, histocompatibility, or sustained release advantages in mammalian organisms. Specific methods of prodrug preparation can be found in saunnier, m.g., et al, biorg. Med. Chem. Lett.1994,4,1985-1990; greenwald, r.b., et al, j.med.chem.2000,43,475.

The term "pharmaceutically acceptable" as used herein refers to a material (e.g., carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic in that the material may be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition.

The term "treatment" and other similar synonyms as used herein include the following meanings:

(i) Preventing the occurrence of a disease or disorder in a mammal, particularly when such mammal is susceptible to the disease or disorder, but has not been diagnosed as having the disease or disorder;

(ii) Inhibiting the disease or disorder, i.e., inhibiting its progression;

(iii) Alleviating a disease or condition, i.e., causing the state of the disease or condition to subside; or alternatively

(iv) Alleviating symptoms caused by the disease or condition.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1 (1R, 2R) -2- (8-fluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide (K1)

Compound 1 (530 mg,2.34 mmol) and Compound 2 (300 mg,1.95 mmol) were weighed into a 100mL eggplant-shaped bottle, dissolved in 25mL DMF and stirred at room temperature for 12h. After the reaction was completed, the reaction mixture was diluted with DCM, washed with 10% hydrochloric acid, dried over anhydrous sodium sulfate and separated by column chromatography (dichloromethane: methanol=50:1) to give an off-white solid, intermediate 3.

Intermediate 3 (100 mg,0.29 mmol) and HATU (121 mg,0.32 mmol) were weighed into a 25mL eggplant-shaped bottle, 10mL acetonitrile was added, and DIPEA (191. Mu.L, 1.16 mmol) and compound 4 (33 mg,0.32 mmol) were added and reacted for 10min. At the end of the reaction, drying and column chromatography purification (dichloromethane: methanol=50:1) gave an off-white solid, intermediate 5.

Intermediate 5 was placed in a 25mL two-necked flask, N ₂ 7mL of anhydrous DCM and DIPEA (386. Mu.L, 0.92 mmol) were added for protection, and the mixture was cooled to-15℃in a cryotrap; sulfur trioxide pyridine (146 mg,0.88 mmol) was additionally weighed and dissolved in 1mL anhydrous DMSO to form a mixture, which was added to a two-necked flask reaction solution at-15℃for reaction at low temperature for 1h. Reaction completionDrying and column chromatography purification (petroleum ether: ethyl acetate: methanol=16:4:1) gave compound K1 as a white solid in 68% yield. Its M.P.146-148 deg.C, its preparation method ¹ H NMR(400MHz,DMSO-d ₆ )δ10.98(s,1H),7.75(dd,J＝16.0,8.3Hz,1H),7.39–7.09(m,2H),6.85(dtd,J＝9.1,6.1,2.9Hz,1H),4.84–4.32(m,2H),4.14–3.73(m,3H),3.12–2.89(m,2H),2.86–2.63(m,2H),2.18(dt,J＝17.4,7.6Hz,1H),1.97–1.54(m,7H),1.53–1.07(m,6H)； ¹³ C NMR(101MHz,DMSO)δ215.55,175.04,173.87,170.82,158.33,156.03,135.42,132.87,126.01,112.14,108.84,106.89,103.17,102.58,60.23,56.09,49.07,46.42,43.18,42.55,41.83,41.43,39.37,35.69,30.44,29.57,29.16,25.70,24.61,23.49,21.22,18.18,14.55；MS(ESI):426.4[M+H] ⁺ 。

Example 2 (1R, 2R) -2- (2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K2)

The preparation method of example 2 refers to the preparation procedure of example 1,2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ]]The indol-2-onium replaces compound 1 to finally obtain a white solid, namely compound K2, with a yield of 71%. Its M.P.137-140 deg.C; which is a kind of ¹ HNMR(400MHz,DMSO-d ₆ )δ10.88(s,1H),8.30–8.01(m,1H),7.48(d,J＝7.6Hz,0H),7.38(d,J＝7.7Hz,1H),7.32–7.24(m,1H),7.08–6.91(m,2H),4.78–4.46(m,2H),4.26–3.52(m,7H),3.09–2.81(m,2H),2.74–2.54(m,2H),1.86–1.61(m,4H),1.40–1.14(m,4H)； ¹³ C NMR(101MHz,CDCl3)δ211.38,211.31,175.85,175.79,174.54,174.16,136.00,131.28,125.61,121.72,119.67,117.50,110.85,106.62,70.07,69.90,69.71,69.49,54.76,54.69,46.33,46.17,43.39,42.96,40.07,39.87,29.27,28.86,25.27,24.49,23.29；MS(ESI):410.4[M+H] ⁺ 。

Example 3 (1R, 2R) -2- (8-fluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide (K3)

The preparation method of this example refers to the preparation procedure of example 1, wherein compound 6 replaces compound 4 to give the product as a white solid, compound K3, in 67% yield. 1H NMR (400 MHz, DMSO). Delta.11.01 (s, 1H), 8.15-8.02 (m, 1H), 7.32-7.14 (m, 2H), 6.86 (tdd, J=9.2, 4.8,2.6Hz, 1H), 4.74-4.42 (m, 2H), 4.34-3.38 (m, 7H), 3.05-2.53 (m, 4H), 1.92-1.57 (m, 4H), 1.36-1.23 (m, 4H).

Example 4 (1R, 2R) -2- (6, 8-difluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide (K4)

The preparation method of this example refers to the preparation procedure of example 1 to give the product as a white solid, compound K4, in 66% yield. 1H NMR (400 MHz, DMSO). Delta.11.48 (s, 1H), 8.30-8.02 (m, 1H), 7.21-7.05 (m, 1H), 6.88 (d, J=12.6 Hz, 1H), 4.70 (q, J=13.9 Hz, 2H), 4.51-3.36 (m, 7H), 3.06-2.86 (m, 2H), 2.74-2.57 (m, 2H), 1.71-1.27 (m, 8H)

Example 5 (1R, 2R) -2- (8-chloro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide (K5)

The preparation method of this example refers to the preparation procedure of example 1 to give the product as a white solid, compound K5, in 65% yield. 1H NMR (400 MHz, DMSO). Delta.11.12 (d, J=4.3 Hz, 1H), 8.30-8.05 (m, 1H), 7.63-7.42 (m, 1H), 7.30 (dd, J=8.6, 1.9Hz, 1H), 7.03 (ddt, J=7.9, 3.6,1.9Hz, 1H), 4.78-4.49 (m, 2H), 4.48-3.39 (m, 7H), 3.07-2.82 (m, 2H), 2.79-2.57 (m, 2H), 1.86-1.11 (m, 8H)

Example 6 (1R, 2R) -2- (8-bromo-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide (K6)

The preparation method of this example refers to the preparation procedure of example 1 to give a yellowish white solid, compound K6, in 69% yield. 1H NMR (400 MHz, DMSO). Delta.11.14 (d, J=5.1 Hz, 1H), 8.38-7.94 (m, 1H), 7.80-7.51 (m, 1H), 7.38-7.05 (m, 3H), 4.88-4.43 (m, 2H), 4.35-3.45 (m, 7H), 3.20-2.86 (m, 2H), 2.87-2.58 (m, 2H), 1.83-1.66 (m, 4H), 1.36-1.19 (m, 4H)

Example 7 (1R, 2R) -2- (6- (trifluoromethoxy) -2,3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K7)

The preparation method of this example refers to the preparation procedure of example 1 to give the product as a white solid, compound K7, in 40% yield. 1H NMR (300 MHz, DMSO). Delta.11.49 (s, 1H), 8.32-8.05 (m, 1H), 7.57-7.39 (m, 1H), 7.04 (d, J=5.4 Hz, 2H), 4.83-4.55 (m, 2H), 4.40-3.44 (m, 7H), 3.06-2.82 (m, 2H), 2.78-2.54 (m, 2H), 1.81 (d, J=15.7 Hz, 4H), 1.26 (d, J=12.3 Hz, 4H)

Example 8 (1R, 2R) -2- (6- (trifluoromethoxy) -2,3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K8)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K8, in 42% yield. 1HNMR (300 MHz, DMSO). Delta.11.49 (s, 1H), 8.29-8.03 (m, 1H), 7.56-7.39 (m, 1H), 7.04 (d, J=5.3 Hz, 2H), 4.82-4.64 (m, 2H), 4.57-3.39 (m, 7H), 3.07-2.89 (m, 2H), 2.78-2.54 (m, 2H), 1.72 (s, 4H), 1.33-1.23 (m, 4H) thereof

Example 9 (1R, 2R) -2- (6-fluoro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K9)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K9, in 66% yield. 1HNMR (400 MHz, DMSO) δ11.48 (s, 1H), 8.30-8.05 (m, 1H), 7.56-7.41 (m, 1H), 7.04 (d, J=6.5 Hz, 2H), 4.80-4.54 (m, 2H), 4.54-3.38 (m, 7H), 3.06-2.53 (m, 4H), 1.91-1.25 (m, 8H)

Example 10 (1R, 2R) -2- (6-fluoro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K10)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K10, in 51% yield. 1H NMR (400 MHz, DMSO). Delta.11.37 (s, 1H), 8.34-8.04 (m, 1H), 7.38-7.20 (m, 1H), 6.96-6.88 (m, 2H), 4.78-4.52 (m, 2H), 4.51-3.44 (m, 7H), 3.08-2.90 (m, 2H), 2.81-2.57 (m, 2H), 1.73 (m, 4H), 1.25 (m, 4H)

Example 11 (1R, 2R) -2- (6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K11)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K11, in 63% yield. 1H NMR (400 MHz, DMSO). Delta.10.96 (d, J=4.1 Hz, 1H), 8.31-7.99 (m, 1H), 7.12-6.83 (m, 2H), 6.63 (dd, J=7.6, 5.7Hz, 1H), 4.77-4.40 (m, 2H), 4.37-3.92 (m, 3H), 3.89 (s, 3H), 3.87-3.42 (m, 4H), 3.04-2.54 (m, 5H), 1.91-1.61 (m, 5H), 1.35-1.13 (m, 4H)

Example 12 (1R, 2R) -2- (6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K12)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K12, in 43% yield. 1H NMR (300 MHz, DMSO). Delta.10.96 (s, 1H), 8.33-8.02 (m, 1H), 7.15-6.83 (m, 2H), 6.63 (dd, J=7.7, 4.2Hz, 1H), 4.74-4.50 (m, 2H), 4.49-3.88 (m, 7H), 3.87-3.38 (m, 3H), 3.13-2.76 (m, 2H), 2.75-2.54 (m, 2H), 1.91-1.72 (m, 4H), 1.30 (m, 4H)

Example 13 (1R, 2R) -2- (8-fluoro-6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K13)

The preparation method of this example refers to the preparation procedure of example 1 to give the product as a white solid, compound K13, in 68% yield. 1H NMR (400 MHz, DMSO). Delta.11.07 (d, J=3.9 Hz, 1H), 8.30-8.02 (m, 1H), 6.94-6.72 (m, 1H), 6.56 (ddt, J=11.5, 3.9,2.0Hz, 1H), 4.78-4.46 (m, 2H), 4.45-3.88 (m, 7H), 3.87-3.46 (s, 3H), 3.16-2.52 (m, 4H), 2.05-1.45 (m, 4H), 1.44-1.09 (m, 4H)

Example 14 (1R, 2R) -2- (8-fluoro-6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide (K14)

The preparation method of this example refers to the preparation procedure of example 1 to give a white solid, compound K14, in 58% yield. 1H NMR (400 MHz, DMSO). Delta.11.07 (s, 1H), 8.30-8.03 (m, 1H), 6.91-6.75 (m, 1H), 6.60-6.52 (m, 1H), 4.71-4.45 (m, 2H), 4.45-3.88 (m, 7H), 3.87-3.44 (s, 3H), 3.04-2.75 (m, 2H), 2.70-2.54 (m, 2H), 1.82-1.26 (m, 8H)

EXAMPLE 15 inhibition of Cat K enzyme by Compounds

Reagent information: cat K inhibitor screening kit: lot number, 6L23K01500; vendor, biVision. Preparation of the medicine: the compound was dissolved in DMSO to prepare a 10mM stock solution. The composition is formulated into desired concentration with Buffer.

Ref-03 is taken as a comparison compound, and the structural formula is as follows:

experimental method

1. Medicine primary screening

Cathepsin K (CTSK, EC 3.4.22.38) is a lysosomal cysteine protease involved in osteoclast bone remodeling and resorption and also degrades collagen, gelatin and elastin. The ability of the Biovision's cathepsin K inhibitor screening kit to cleave synthetic AFC-based peptide substrates to release AFC using active cathepsin K can be easily quantified using a fluorometer or fluorescent microplate reader. In the presence of cathepsin K specific inhibitors, cleavage of this substrate is reduced/eliminated, resulting in a reduction or complete loss of AFC fluorescence. This simple and high throughput adaptive assay kit can be used to screen/study/characterize potential inhibitors of cathepsin K.

Adding 20 mu L of buffer, cat K inhibitor and a compound to be tested (1 mu M and 10 mu M) into a 96-well plate to be respectively used as an EC well, an IC well and an S well; 50. Mu.L of Cat K Enzyme Solution was added to each well and incubated at room temperature for 10-15min to establish enzyme inhibitor complexes; all wells were added with 30 μl Cat K Substrate Solution and incubated at room temperature for 30-60min; the final volume of the reaction was 100. Mu.L. At 30-60min, two time points T1, T2 were selected to detect fluorescence absorbance values (Ex/em=400/505 nm), which were recorded as RFU1, RFU2, and the inhibition (%) of the test compound against Cat K enzyme was calculated.

Slope= (RFU 2-RFU 1)/(T2-T1)

Inhibition ratio (%) = (EC slope-S slope)/EC slope×100

2.IC ₅₀ Measurement

Screening method is same as 1.1, the concentration of the compound to be tested is set between 0.1nM and 10 mu M, 4-5 concentrations are selected for detection, and IC is drawn ₅₀ Curve and calculate IC ₅₀ Values.

And (3) data processing: all data were statistically analyzed using Graph pad.

Experimental results

The results are shown in Table 1.

Compound inhibition ratio and IC of table 1 ₅₀ (nM)

The compound of the invention has high inhibition rate to Cat K enzyme and IC ₅₀ The value is low, and the inhibition activity to Cat K enzyme is good. The inhibition rate of partial compounds to Cat K enzyme at 1 mu M level can reach more than 90%, the inhibition rate of all compounds to Cat K enzyme at 10 mu M level can reach more than 97%, and IC ₅₀ The values are less than 50nM, so the compound of the invention has better inhibition effect on Cat K enzyme.

Example 16 inhibition of Cat B enzyme and Cat S by Compounds

Cathepsin B screening

Adding 10 mu L of buffer, cat B inhibitor and a compound to be tested (1 mu M and 10 mu M) into a 96-well plate to be respectively used as an EC well, an IC well and an S well; 50. Mu.L of Cat B Enzyme Solution was added to each well and incubated at room temperature for 10-15min to establish enzyme inhibitor complexes; all wells were added with 40 μl Cat B Substrate Solution and incubated at room temperature for 30-60min; the final volume of the reaction was 100. Mu.L. At 30-60min, two time points T1, T2 were selected to detect fluorescence absorbance values (Ex/em=400/505 nm), which were recorded as RFU1, RFU2, and the inhibition (%) of the test compound against Cat B enzyme was calculated.

Slope= (RFU 2-RFU 1)/(T2-T1)

Inhibition ratio (%) = (EC slope-S slope)/EC slope×100

IC ₅₀ Measurement

The screening method is the same as above, the concentration of the compound to be tested is set between 0.1nM and 10. Mu.M, 4-5 concentrations are selected for detection, and the IC is drawn ₅₀ Curve and calculate IC ₅₀ Values.

And (3) data processing: all data were statistically analyzed using Graph pad.

Cathepsin S screening

Adding 10 mu L of buffer, cat S inhibitor and a compound to be tested (1 mu M and 10 mu M) into a 96-well plate to be respectively used as an EC well, an IC well and an S well; 50. Mu.L of Cat B Enzyme Solution was added to each well and incubated at room temperature for 10-15min to establish enzyme inhibitor complexes; all wells were added with 40 μl Cat S Substrate Solution and incubated at room temperature for 30-60min; the final volume of the reaction was 100. Mu.L. At 30-60min, two time points T1, T2 were selected to detect fluorescence absorbance values (Ex/em=400/505 nm), which were noted as RFU1, RFU2, and the inhibition (%) of the test compound against Cat S enzyme was calculated.

Slope= (RFU 2-RFU 1)/(T2-T1)

Inhibition ratio (%) = (EC slope-S slope)/EC slope×100

IC ₅₀ Measurement

And (3) data processing: all data were statistically analyzed using Graph pad.

The results are shown in Table 2.

Table 2 inhibition of compounds and IC ₅₀ (μM)

Note that: experimental data for Ref-03 are from the literature

The inhibition of Cat S enzyme by the above compounds was less than 50% at both 1. Mu.M and 10. Mu.M levels, and represents IC for Cat S enzyme inhibition ₅₀ All values are>10 mu M, and the inhibition rate of Cat B enzyme is less than 50% at 1 mu M and 10 mu M levels, and IC for inhibiting Cat B enzyme ₅₀ All values are>10. Mu.M. And Ref-03 compound IC for Cat S enzyme and Cat B enzyme ₅₀ The values were 3.36. Mu.M and 1.42. Mu.M, respectively, compared with the compounds of the invention, which have better selectivity for Cat K enzyme.

Intrinsic clearance and half-life of the compound of example 17 on rat liver microparticles

1. Preparing a sample solution

Taking centrifuge tubes filled with powder K2 (molecular formula C23H27N3O4,4.6 mg), K3 (molecular formula C23H26FN3O4,5.0 mg) and K4 (molecular formula C23H25F2N3O4,5.6 mg) of the test substances, respectively adding 1mL of dichloromethane to dissolve, and performing ultrasonic treatment for 30min to obtain K2 stock solution with mass concentration of 4.6mg/mL, K3 stock solution with mass concentration of 5.0mg/mL and K4 stock solution with mass concentration of 5.6mg/mL, and sealing and storing in a refrigerator at-20deg.C for later use. Before use, the K2 series standard sample working solutions of 0.02, 0.05, 0.22, 0.49, 0.96, 2.37 and 4.90ng/mL, the K3 series standard sample working solutions of 0.02, 0.05, 0.10, 0.19, 1.00, 2.01 and 4.93ng/mL and the K4 series standard sample working solutions of 0.10, 0.20, 0.53, 0.96, 2.42, 5.10 and 10.10ng/mL are respectively prepared by gradually diluting the K2 series standard sample working solutions with methanol to corresponding mass concentrations.

2. Test method

5 mu L of the drug to be detected is taken by a pipetting gun, 2 mu L of rat liver microsomes and 468 mu L of PBS buffer solution are prepared into premix, the premix is incubated for 5min in a water bath kettle at 37 ℃, 47.5 mu L of premix, 100 mu L of ice-cold acetonitrile and 2.5 mu L of PBS buffer solution are taken to a new tube after incubation is completed, and the mixture is gently mixed to obtain a control group. The reaction was started by adding 22.5. Mu.L of NADPH solution to the remaining premix, gently swirled, and immediately 50. Mu.L of premix was taken and 100. Mu.L of acetonitrile was stopped in another fresh tube. Then the incubation liquid is put into a water bath kettle for incubation, 50 mu L of acetonitrile is respectively added into the incubation liquid for termination at 5min, 10min, 20min, 30min, 45min, 60min and 90min, and the incubation liquid is centrifuged for 10min at 12000r/min, and the supernatant is taken and detected by a machine.

Chromatographic conditions

Chromatographic column: hypersil GOLD 100 x 2.1mm 3 μm liquid chromatographic column

Mobile phase: 0.1% formic acid aqueous solution (A) 100% acetonitrile (B)

Gradient elution (0-1 min:50% B, 1-5 min:50% B-90% B, 5-7 min:90% B, 7-7.1 min:50% B, 7.1-10 min:50% B)

Flow rate: column temperature 0.3 ml/min: sample injection amount at 40 ℃): 5 mu L

Mass spectrometry conditions

Electrospray ion source (ESI); ion spray voltage: 4500V ion source temperature: curtain gas pressure at 450 ℃): 35psi; the declustering voltages were 80V.

3. Investigation of the lower limit of the drawing and quantification of the standard curve

And (3) carrying out sample injection analysis on the sample solution under the conditions of the chromatograph and the mass spectrum, and recording the peak area. And (3) carrying out linear regression by taking the measured substance concentration as an abscissa (x) and the peak area of the measured substance as an ordinate (y) and adopting a weighting method (the weighting coefficient is 1/x 2). The result shows that the linear range of K2 mass concentration detection is 0.02-4.90 ng/mL, the lower limit of quantification is 0.02ng/mL, the linear range of K3 mass concentration detection is 0.02-4.93 ng/mL, and the lower limit of quantification is 0.02ng/mL; the linear range of the K4 mass concentration detection is 0.10-10.10 ng/mL, and the lower limit of the quantification is 0.10ng/mL. Based on the results, statistical analysis was performed to calculate the clearance (Cl) _int ) And half-life (T) _1/2 )。

The results are shown in Table 3.

Intrinsic clearance (Cl) of the compounds of Table 3 on rat liver particles _int ) And half-life (T) _1/2 )

Note that: ref-03 clearance and half-life data from literature

The internal clearance rate of the compound on liver particles of rats is reduced compared with Ref-03, wherein the clearance rate of the compound K4 is reduced by 2 times, other compounds are smaller than the clearance rate of Ref-03 at 250, and the half-life of Ref-03 is 0.6 hour, and the half-life of the compound is larger than 0.6 hour. Therefore, the compound of the invention has better metabolic stability.

Claims

1. The general structure of the compound shown in the formula I or the isomer of the t position or the pharmaceutically acceptable salt thereof is as follows:

wherein,,

R ₁ 、R ₂ each independently selected from H, halogen, cyano, amino, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, said substituted C1-6 alkyl or C1-6 alkoxy being further substituted with at least one halogen or hydroxy.

2. The compound of claim 1, or an isomer or pharmaceutically acceptable salt thereof at position t, wherein R ₁ 、R ₂ Each independently selected from H, halogen, substituted or unsubstituted C1-3 alkyl, substituted or unsubstituted C1-3 alkoxy, said substituted C1-3 alkyl or C1-3 alkoxy being further substituted with at least one F.

3. The compound of claim 1, or an isomer or pharmaceutically acceptable salt thereof at position t, wherein R ₁ 、R ₂ Each independently selected from H, F, cl, br, methyl, methoxy, trifluoromethoxy.

4. The compound of claim 1, or an isomer or pharmaceutically acceptable salt thereof at position t, wherein R ₁ 、R ₂ Each independently selected from H and F.

5. The compound of claim 1, or an isomer or pharmaceutically acceptable salt thereof at position t, wherein said compound is selected from the group consisting of:

(1) (1 r,2 r) -2- (8-fluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide;

(2) (1 r,2 r) -2- (2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(3) (1 r,2 r) -2- (8-fluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide;

(4) (1 r,2 r) -2- (6, 8-difluoro-2, 3,4, 9-tetrahydro-1H-pyrido [3,4-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide;

(5) (1 r,2 r) -2- (8-chloro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide;

(6) (1 r,2 r) -2- (8-bromo-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- ((S) -4-oxotetrahydrofuran-3-yl) cyclohexane-1-carboxamide;

(7) (1 r,2 r) -2- (6- (trifluoromethoxy) -2,3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(8) (1 r,2 r) -2- (6- (trifluoromethoxy) -2,3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(9) (1 r,2 r) -2- (6-fluoro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(10) (1 r,2 r) -2- (6-fluoro-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(11) (1 r,2 r) -2- (6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(12) (1 r,2 r) -2- (6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(13) (1 r,2 r) -2- (8-fluoro-6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (S) - (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide;

(14) (1 r,2 r) -2- (8-fluoro-6-methoxy-2, 3,4, 5-tetrahydro-1H-pyrido [4,3-b ] indole-2-carbonyl) -N- (4-oxotetrahydrofuran-3-yl) -cyclohexane-1-carboxamide.

6. A process for the preparation of a compound of formula i according to claim 1, comprising the steps of:

step 1) the compound P2 and 4-amino tetrahydrofuran-3-alcohol are subjected to condensation reaction;

oxidizing the product obtained in the step 2) in the step 1 by an oxidant to obtain the compound shown in the formula I.

7. The method of claim 6, wherein step 1) is performed in a polar solvent comprising a base. Preferably, the base is a nitrogen-containing organic base selected from at least one of pyridine, 2-methylpyridine, 2, 6-dimethylpyridine, 4-dimethylaminopyridine, triethylamine, diethylamine, N-diisopropylethylamine, dimethylisopropylamine, piperidine, 1-methylpiperidine and 1-methylpyrrolidine; the polar solvent is at least one selected from water, methanol, ethanol, glycerol, propylene glycol, formamide, acetonitrile, n-butanol, dioxane, dichloromethane, acetone, dimethyl sulfoxide, dimethylformamide, ethyl acetate and tetrahydrofuran. Preferably, the oxidant in the step 2) is an inorganic oxidant or an organic oxidant or a mixed oxidant; wherein the inorganic oxidant is at least one selected from sodium dichromate, chromic acid, manganese dioxide, ammonium cerium nitrate, potassium permanganate, potassium ferrate, bromine simple substance, iodine simple substance, sodium hypochlorite, sodium chlorite, sodium bromate, sodium periodate and diiodide; the organic oxidant is selected from 2-iodized benzoic acid, tetramethyl piperidine oxide, benzoyl peroxide and cyclohexanone peroxide; wherein the mixed oxidant is preferably at least one of pyridinium chlorochromate, pyridinium chromic anhydride, pyridinium dichromate and pyridinium sulfur trioxide.

8. The method of claim 6, wherein the compound P2 is synthesized by the following method:

the reaction of compound P1 with (3 ar,7 as) -hexahydroisobenzofuran-1, 3-dione in a solvent to yield compound P2, wherein the solvent is selected from at least one of the group defined in claim 7.

9. A composition comprising a compound of any one of claims 1-5.

10. The use of a compound according to claim 9 for the manufacture of a medicament for the treatment of a disease targeted to cathepsin K.

11. The use according to claim 10, wherein the diseases targeted to cathepsin K include thyroid diseases, cardiovascular diseases, bone diseases and gum diseases.

12. The use of claim 11, wherein the thyroid disorder comprises hyperthyroidism.

13. The use according to claim 11, wherein the cardiovascular disease comprises atherosclerosis, cardiac hypertrophy, heart failure.

14. The use according to claim 11, wherein the bone disease comprises osteoporosis, osteoarthritis, rheumatoid arthritis.

15. The use according to claim 11, wherein the unguicide disease comprises gingivitis and periodontitis.

16. The use according to claim 11, wherein the disease targeted to cathepsin K is osteoporosis.