Patent Application
20250145576
The present invention relates to the field of medicinal chemistry, specifically to a compound inhibiting the cathepsin K activity, a method for preparing same and use thereof in the preparation of a drug for treating a disease related with the cathepsin K activity or characterized by the cathepsin K activity, such as osteoporosis.
Osteoporosis is a metabolic disorder that reduces the bone mass of the whole body and bone density, and further increases the fracture risk of patients. With the aggravation of aging of population, the health problems related to the disease become more serious day by day and the fracture probability of the patients with osteoporosis is greatly increased, thereby bringing heavy burden to society and families and seriously influencing the life of people. The pathogenesis of the osteoporosis is caused by imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Common drugs for treating osteoporosis in the market at present comprise a bone resorption inhibitor such as diphosphate and a bone formation promoter, but both drugs have certain defects. The long-term use of the bone resorption inhibitor can influence the differentiation and proliferation of osteocytes, and further causes the occurrence of a low bone turnover state; the bone formation promoter increases the probability of osteosarcoma in the patients.
Since the drugs in the market at present all have certain defects, it is a current research direction on researching a novel drug for treating osteoporosis, which can treat the osteoporosis, does not influence the proliferation and differentiation of osteoblasts and osteoclasts, and avoids the occurrence of the low bone turnover state.
Cathepsin K (Cat K) belongs to a cysteine protease of the papain family, is abundant in osteoclasts, and becomes a new target for treating osteoporosis currently. The human bone matrix consists of 25% of water, 25% of an organic matrix and 50% of a mineral matrix, wherein 90% of the composition of the organic matrix is type I collagen. The degradation of the type I collagen is a key process of osteoclast-mediated bone resorption. The Cat K plays a leading role in the degradation of the type I collagen. A Cat K inhibitor inhibits the resorption of mature osteoclasts by blocking the degradation of matrix collagen, and simultaneously can maintain the survival number of osteoclasts, such that coupling signals of the osteoclasts and osteoblasts are kept intact, and the occurrence of the low bone turnover state is avoided. In addition, the Cat K plays an important role in the pathogenic process of thyroid diseases, cardiovascular diseases and gum diseases. Diseases characterized by abnormal expression or activation of the Cat K comprise thyroid diseases, cardiovascular diseases, bone diseases and gum diseases, specifically hyperthyroidism, atherosclerosis, cardiac hypertrophy, heart failure, osteoporosis, osteoarthritis, rheumatoid arthritis, gingivitis and periodontitis. In recent years, there are more and more researches on the Cat K. The Cat K secretion of endothelial cells can be increased under pathological conditions such as coronary atherosclerosis. The Cat K is closely related to the occurrence and development of myocardial hypertrophy and heart failure. Garg et al. (Garg G, Pradeep A R, Thorat M K, et al. Effect of nonsurgical periodontal therapy on crevicular fluid levels of cathepsin K in periodontitis. Arch Oral Biol, 54: 1046-1051) proved that after the basic treatment, along with the reduction of the clinical gingival index, the periodontal probing depth and the attachment loss of patients with periodontitis, the level of Cat K was reduced through change of the Cat K in a gingival crevicular fluid before and after the basic treatment of the patients with periodontitis. The Cat K is used as a marker of periodontitis bone absorption and needs to be given further attention and research in the periodontitis treatment.
Currently, the activity of Cat K inhibitors and various inhibitors reported in a clinical research stage are all greatly improved. However, no drug targeting this target is available on the market. The main reason is that a compound has poor selectivity and relatively high inhibitory activity against other subtypes such as B-type and S-type, resulting in the occurrence of side effects of the drug. Therefore, it is a main research direction at present in the development and research of a novel Cat K inhibitor with high efficiency and selectivity aiming at reducing side effects.
Since Cat B and Cat K belong to cysteine proteases, but Cat L is an eosinophilic protease and is difficult to obtain, the present invention designs and synthesizes a cyano-substituted pyrimidine compound, and the activities thereof against K, B and S are determined. It is found that the compound has good selectivity and thus is expected to be used for treating diseases related to the Cat K activity or characterized by the Cat K activity.
The present invention provides a novel ketone compound and/or a nitrile compound acting on Cat K and a pharmaceutically acceptable salt thereof, and a preparation method therefor and use thereof.
a) The present invention provides a nitrile compound whose structural general formula is shown as follows:
When t is 0 and R* is H, the compound of formula 0 is as shown in formula I:
Further, X is C;
Further, X is C;
In some examples, R1 is selected from neopentyl and cyclohexyl.
In some examples, Y is selected from the following groups:
phenyl, pyridyl, thiophenyl and thiazolyl.
In some preferred examples, Y is selected from
phenyl and thiazolyl.
In some examples, R5 is selected from H, F, Cl, cyano, methyl, methylthio, methoxy, methylsulfonyl, methylcarbonyl and methylpiperazinyl.
In some preferred examples, R5 is selected from methyl and methylpiperazinyl.
The present invention provides a ketone cathepsin K inhibitor whose structural general formula is shown as follows:
Further, R1 and R2 are each independently selected from H, halogen, C1-3 alkyl, and C1-3 alkoxy, and the C1-3 alkyl or C1-3 alkoxy is further substituted by at least one halogen.
Further, R1 and R2 are each independently selected from H, halogen, C1-3 alkyl and C1-3 alkoxy, and the C1-3 alkyl or C1-3 alkoxy is further substituted by 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 trifluoromethyl.
The C1-3 alkoxy is trifluoromethyl.
In some examples, R1 and R2 are each independently selected from H, F, Cl, Br, methoxy and trifluoromethyl.
In some preferred examples, R1 and R2 are each independently selected from H and F.
The halogen is selected from F, Cl, Br and I.
The compound provided by the present invention comprises the following specific structures:
Another objective of the present invention is to provide a method for preparing the compound and a pharmaceutically acceptable salt thereof. A method for preparing the compound of formula I comprises the following steps:
A method for preparing the compound of formula II comprises the following steps:
The present invention further provides a composition containing the cathepsin K inhibitor, a therapeutically effective amount of one or more of the compounds or a pharmaceutically acceptable salt thereof.
The present invention further provides use of the cathepsin K inhibitor in the preparation of a drug for treating a disease taking cathepsin K as a target.
Further, the disease taking cathepsin K as a target comprises a thyroid disease, a cardiovascular disease, a bone disease, a gum disease and a tumor.
Further, the thyroid disease comprises hyperthyroidism.
Further, the cardiovascular disease comprises atherosclerosis, cardiac hypertrophy and heart failure.
Further, the bone disease comprises osteoporosis, osteoarthritis and rheumatoid arthritis.
Further, the gum disease comprises gingivitis and periodontitis.
Further, the tumor comprises tumor invasion and tumor metastasis.
Further, the disease taking cathepsin K as a target is osteoporosis.
Compared with the prior art, the present invention has the following advantages and technical effects.
The compound of the present invention has a high inhibition rate on Cat K, a low IC50 value and the good inhibitory activity on the Cat K. The inhibition rate of the compound on the Cat K at 10 μM can reach 90% or more and the IC50 is less than 50 nM.
The compound of the present invention has good selectivity: at the levels of 1 μM and 10 μM, the inhibition rates of the compound on Cat B and Cat S are both <50%, and the IC50 values of the compounds on the Cat B and Cat S are both >10 μM, indicating that the compound of the present invention has good selectivity on the Cat K.
The compound of the present invention has good druggability.
The cathepsin K inhibitor can be used for treating diseases characterized by abnormal expression or activation of the Cat K. The diseases comprise thyroid diseases, cardiovascular diseases, bone diseases and oral diseases, specifically hyperthyroidism, atherosclerosis, cardiac hypertrophy, heart failure, osteoporosis, osteoarthritis, rheumatoid arthritis, gingivitis and periodontitis. The compound of the present invention can inhibit the formation of osteoclasts and can be used for treating osteoporosis.
The terms in the present invention:
The term “C1-10 alkyl” refers to a straight or branched chain hydrocarbyl group containing 1 to 10 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl and the like. The term “C1-6 alkyl” refers to any straight or branched chain group containing 1-6 carbon atoms.
The term “C1-10 alkoxy” comprises —O—C1-10 alkyl, referring that the C1-10 alkyl is attached to an oxygen atom.
The term “C1-6 alkoxy” comprises —O—C1-10 alkyl.
The term “C3-10 cycloalkyl” refers to a stable non-aromatic monocyclic or multicyclic hydrocarbon group consisting of only carbon and hydrogen atoms, may include fused, bridged or spiro ring systems, having 3 to 10 carbon atoms, and is saturated or unsaturated and may be attached to the rest of the molecule by a single bond via any suitable carbon atom. Unless otherwise specifically indicated in the description, a carbon atom in the cycloalkyl may be optionally oxidized. An example of the cycloalkyl includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
The term “C6-12 membered aromatic ring” indicates a planar ring system with conjugation, has the bonding between atoms being not a discontinuous single-double bond alternation, but covered by a delocalized π electron cloud, and has 6-12 atoms, which may be carbon, nitrogen, sulfur and oxygen atoms. Unless otherwise specifically indicated in the description, the aromatic ring may be a monocyclic, bicyclic, tricyclic, or more ring system.
The expressions of the terms “C1-10” and “C1-C10” indicate groups containing 1-10 carbon atoms.
The term “C6-12 heteroaromatic ring” is a monocyclic or bicyclic aromatic ring containing 6-12 ring atoms, wherein 1, 2, 3 or 4 ring atoms are selected from nitrogen, sulfur or oxygen, wherein the nitrogen or the sulfur in the ring may be oxidized. For the purpose of the present invention, heteroaryl is preferably a stable 5- to 10-membered aromatic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably, a stable 5- to 8-membered aromatic group containing 1 to 2 heteroatoms selected from nitrogen, oxygen and sulfur. An example of the heteroaryl includes but is not limited to thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl and pyridazinyl.
The term “C5-12 heterocyclic ring” is a saturated, unsaturated, or partially saturated monocyclic or bicyclic ring containing 5-12 ring atoms, wherein 1, 2 or 3 ring atoms are selected from nitrogen, sulfur or oxygen and the ring may be attached by carbon or nitrogen, wherein a —CH2- group in the ring is optionally substituted by a —C(O) group, wherein the nitrogen or sulfur atoms in the ring may be optionally oxidized to form a N-oxide or a S-oxide, and wherein —NH— in the ring is optionally substituted by acetyl, formyl, methyl or methanesulfonyl.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills belonging to the art of the subject matter of the claims.
It is to understanding understood that both the foregoing brief description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter of the present invention. Furthermore, the term “comprises” and other forms thereof, such as “contains”, “includes” and “including” are non-limiting.
The section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter. All documents or document portions cited in the present application include, but are not limited to patents, patent applications, articles, books, operation manuals and papers, are hereby incorporated herein by reference in their entirety.
The pharmaceutical composition of the present invention comprises the compound of formula I or formula II, or the optical isomer thereof, the pharmaceutically acceptable salt thereof or a prodrug thereof in the first aspect of the present invention.
In the present application, unless otherwise specified, the term “pharmaceutically acceptable salt” refers to a salt which is suitable for tissue contact of a subject without causing undue side effects. The salt in the present application is mainly a pharmaceutically acceptable acid addition salt and a pharmaceutically acceptable alkali addition salt.
The term “pharmaceutically acceptable acid addition salt” refers to a salt formed with inorganic or organic acids while maintaining the biological effectiveness of a free alkali without other side effects. The inorganic acid salt comprises hydrochloride, hydrobromide, sulfate, nitrate, phosphate and the like; and the organic acid salt comprises formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, caprates, undecenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbates, salicylates, 4-aminosalicylates, naphthalene disulfonates and the like. These salts can be prepared by methods known in the art.
The term “pharmaceutically acceptable alkali addition salt” refers to a salt formed with inorganic or organic alkalis while maintaining the biological effectiveness of a free acid without other side effects. The salts derived from the inorganic alkalis include but are not limited to sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, ferric salts, zinc salts, copper salts, manganese salts, aluminum salts and the like. The preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts. The salts derived from the organic alkalis include but are not limited to salts of primary amines, secondary amines and tertiary amines, and substituted amines including natural 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. The preferred organic alkalis comprise diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the art.
The present invention further comprises the prodrug of the compound. In the present application, the term “prodrug” indicates a compound that can be converted under physiological conditions or by solvolysis to the biologically active compound of the present invention. Therefore, the term “prodrug” refers to a pharmaceutically acceptable metabolic precursor of the compound of the present invention. The prodrug may not be active when administered to a subject in need thereof, but is converted in vivo to the active compound of the present invention. The prodrug is generally rapidly converted in vivo to generate a parent compound of the present invention, for example, by hydrolysis in blood. The prodrug compound generally has the advantages of solubility, histocompatibility or sustained release in mammalian organisms. A specific method for preparing the prodrug can refer to Saulnier, M. G., et al., Bioorg. Med. Chem. Lett. 1994, 4, 1985-1990; and Greenwald, R. B., et al., J. Med. Chem. 2000, 43, 475.
The term “pharmaceutically acceptable” used herein refers to a substance (e.g., a carrier or a diluent) that does not affect the biological activity or properties of the compound of the present invention. Besides, the substance is relatively non-toxic, i.e., the substance can be administered to an individual without causing an undesirable biological response or interacting in an undesirable manner with any component contained in the composition.
The term “treatment” and other similar synonyms used herein comprise the following meanings:
A compound M1 (500 mg, 2.47 mmol) was dissolved in 8 mL of anhydrous tetrahydrofuran, cooled to 0° C., NaH (178 mg, 7.41 mmol) was added portionwise while stirring, and moved to room temperature for reaction for 2 h. A compound M2 (732 mg, 2.72 mmol) was added while stirring and the mixture reacted at room temperature for 2 h. After the reaction was finished, 10 mL of water was added to quench the reaction, tetrahydrofuran was removed by distillation under reduced pressure, water was used for extraction twice, and the organic phases were combined and dried over anhydrous sodium sulfate. The dried organic phase was purified by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid, namely a compound K1, with the yield of 60.3%. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J=4.7 Hz, 1H), 7.12 (d, J=4.7 Hz, 1H), 7.01 (d, J=8.1 Hz, 2H), 6.84 (d, J=8.3 Hz, 2H), 4.80 (s, 2H), 3.47 (s, 2H), 3.07 (t, J=5.0 Hz, 4H), 2.41 (t, J=4.9 Hz, 4H), 2.20 (s, 3H), 0.94 (s, 9H); ESI-MS m/z: 379.5 [M+H]+.
Referring to the synthesis method of example 1, an intermediate M4 was prepared.
The intermediate M4 (291 mg, 0.81 mmol) was dissolved in a mixed solution of 1 mL of water and 9 mL of DMF, phenylborodiol (108 mg, 0.89 mmol) and potassium carbonate (223 mg, 1.616 mmol) were added successively while stirring, and the mixture was vacuumized and heated to 80° C. for reaction for 4 h under argon protection. After the reaction was finished, the reaction solution was returned to room temperature, added with 20 mL of water for dilution and extracted twice with ethyl acetate. The organic phases were combined, washed twice with a saturated salt solution and dried over anhydrous sodium sulfate. The dried organic phase was purified by column chromatography (petroleum ether:ethyl acetate=12:1) to obtain a white solid, namely a compound K2, with the yield of 54.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 7.69-7.56 (m, 4H), 7.45 (q, J=7.2 Hz, 2H), 7.36 (t, J=6.7 Hz, 1H), 7.30-7.15 (m, 3H), 4.96 (s, 2H), 3.58 (s, 2H), 0.99 (d, J=13.6 Hz, 9H); ESI-MS m/z: 357.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-methylthiophenylboron diol to obtain a white solid, namely a K3 compound, with the yield of 48.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.63-7.55 (m, 4H), 7.35-7.30 (m, 2H), 7.23 (d, J=7.9 Hz, 2H), 7.17 (d, J=4.7 Hz, 1H), 4.95 (s, 2H), 3.58 (s, 2H), 2.51 (d, J=1.9 Hz, 3H), 0.97 (s, 9H); ESI-MS m/z: 403.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-fluorophenyl bornanediol to obtain a white solid, namely a K4 compound, with the yield of 47.1%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.62 (d, J=20.0 Hz, 4H), 7.49 (s, 2H), 7.24 (s, 2H), 7.17 (s, 1H), 4.95 (s, 2H), 3.58 (s, 2H), 0.97 (s, 9H); ESI-MS m/z: 375.4[M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-methoxyphenyl borondiol to obtain a white solid, namely a K5 compound, with the yield of 44.6%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.55 (t, J=8.7 Hz, 4H), 7.20 (d, J=7.8 Hz, 2H), 7.16 (d, J=4.7 Hz, 1H), 7.00 (d, J=8.3 Hz, 2H), 4.93 (s, 2H), 3.78 (s, 3H), 3.56 (s, 2H), 0.97 (s, 9H); ESI-MS m/z: 387.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-methanesulfonyl phenylborodiol to obtain a white solid, namely a K6 compound, with the yield of 53.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.98 (d, J=8.5 Hz, 2H), 7.90 (d, J=8.2 Hz, 2H), 7.69 (d, J=7.9 Hz, 2H), 7.29 (d, J=7.8 Hz, 2H), 7.17 (d, J=4.7 Hz, 1H), 4.97 (s, 2H), 3.59 (s, 2H), 3.24 (s, 3H), 0.97 (s, 9H); ESI-MS m/z: 435.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 5-(dihydroxyboryl)thiophene-2-carbonitrile to obtain a white solid, namely a K7 compound, with the yield of 31.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.97 (d, J=4.0 Hz, 1H), 7.69 (d, J=7.9 Hz, 2H), 7.64 (d, J=4.0 Hz, 1H), 7.25 (d, J=7.9 Hz, 2H), 7.17 (d, J=4.7 Hz, 1H), 4.93 (s, 2H), 3.58 (s, 2H), 0.96 (s, 9H); ESI-MS m/z: 388.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by (2-chloropyridin-5-yl)bornanediol to obtain a white solid, namely a K8 compound, with the yield of 34.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.63 (s, 1H), 8.13 (d, J=8.8 Hz, 1H), 7.68 (d, J=7.9 Hz, 2H), 7.59 (d, J=8.4 Hz, 1H), 7.28 (d, J=7.8 Hz, 2H), 7.18 (d, J=4.7 Hz, 1H), 4.96 (s, 2H), 3.59 (s, 2H), 0.97 (s, 9H); ESI-MS m/z: 392.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by (1,2-dichlorophen-4-yl)borodiol to obtain a white solid, namely a K9 compound, with the yield of 52.9%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.89 (d, J=2.1 Hz, 1H), 7.67 (s, 1H), 7.65-7.61 (m, 3H), 7.24 (d, J=8.0 Hz, 2H), 7.16 (d, J=4.7 Hz, 1H), 4.94 (s, 2H), 3.58 (s, 2H), 0.96 (s, 9H); ESI-MS m/z: 425.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by (5-methylthiophen-2-yl)borodiol to obtain a white solid, namely a K10 compound, with the yield of 34.8%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.49 (d, J=8.0 Hz, 2H), 7.23 (d, J=3.6 Hz, 1H), 7.19-7.12 (m, 3H), 6.79 (dd, J=3.6, 1.2 Hz, 1H), 4.89 (s, 2H), 3.56 (s, 2H), 2.44 (d, J=1.1 Hz, 3H), 0.95 (s, 9H); ESI-MS m/z: 377.4 [M+H]+.
a) Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-(dihydroxyboryl)methyl benzoate to obtain a white solid, namely a K11 compound, with the yield of 44.7%. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.01 (d, J=8.4 Hz, 2H), 7.79 (d, J=8.4 Hz, 2H), 7.68 (d, J=7.9 Hz, 2H), 7.27 (d, J=7.9 Hz, 2H), 7.17 (d, J=4.7 Hz, 1H), 4.96 (s, 2H), 3.87 (s, 3H), 3.59 (s, 2H), 0.97 (s, 9H); ESI-MS m/z: 415.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by (1-chloro-2-fluorobenzen-4-yl)borodiol to obtain a white solid, namely a K12 compound, with the yield of 51.8%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.71 (dd, J=11.0, 2.1 Hz, 1H), 7.64 (t, J=8.2 Hz, 3H), 7.52 (dd, J=8.4, 2.1 Hz, 1H), 7.25 (d, J=7.9 Hz, 2H), 7.16 (d, J=4.7 Hz, 1H), 4.95 (s, 2H), 3.58 (s, 2H), 0.96 (s, 9H); ESI-MS m/z: 409.4 [M+H]+.
Referring to the synthesis method of example 2, the raw material phenylborodiol was replaced by 4-(4-methyl-1-piperazin)phenylborodiol to obtain a white solid, namely a K13 compound, with the yield of 68.7%. 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J=4.7 Hz, 1H), 7.47 (d, J=8.1 Hz, 4H), 7.17 (s, 2H), 6.98 (d, J=8.8 Hz, 2H), 6.74 (d, J=4.7 Hz, 1H), 4.99 (s, 2H), 3.58 (s, 2H), 3.29-3.23 (m, 4H), 2.59 (t, J=5.0 Hz, 4H), 2.36 (s, 3H), 1.01 (s, 9H); ESI-MS m/z: 455.6 [M+H]+.
Referring to the synthesis method of example 2, a white solid, namely a K14 compound, with the yield of 53.1% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 7.61 (m, 4H), 7.44 (m, 2H), 7.32 (m, 3H), 7.15 (m, 1H), 4.88 (s, 2H), 3.46 (m, 2H), 1.81 (s, 1H), 1.62 (m, 5H), 1.14 (m, 3H), 0.97 (m, 2H); ESI-MS m/z: 383.5 [M+H]+.
Referring to the synthesis method of example 2, a white solid, namely a K15 compound, with the yield of 60.7% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.51 (dd, J=15.1, 8.1 Hz, 4H), 7.23 (d, J=7.7 Hz, 2H), 7.14 (d, J=4.7 Hz, 1H), 6.99 (d, J=8.4 Hz, 2H), 4.85 (s, 2H), 3.16 (s, 4H), 2.45 (s, 4H), 2.22 (s, 3H), 1.81 (s, 1H), 1.62 (d, J=16.2 Hz, 5H), 1.15 (s, 3H), 0.97 (d, J=11.8 Hz, 2H); ESI-MS m/z: 481.7 [M+H]+.
Referring to the synthesis method of example 2, a white solid, namely a K16 compound, with the yield of 49.3% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 7.98 (d, J=8.5 Hz, 2H), 7.91 (d, J=8.3 Hz, 2H), 7.70 (d, J=7.8 Hz, 2H), 7.35 (d, J=7.9 Hz, 2H), 7.15 (d, J=4.7 Hz, 1H), 4.90 (s, 2H), 3.48 (d, J=7.4 Hz, 2H), 3.24 (s, 3H), 1.86-1.76 (m, 1H), 1.62 (d, J=15.1 Hz, 5H), 1.14 (d, J=8.0 Hz, 3H), 0.98 (d, J=11.8 Hz, 2H); ESI-MS m/z: 461.6 [M+H]+.
Referring to the synthesis method of example 2, a white solid, namely a K17 compound, with the yield of 32.9% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.62 (s, 1H), 8.09 (s, 1H), 7.91 (s, 2H), 7.22 (s, 3H), 4.93 (s, 2H), 3.57 (s, 2H), 0.96 (s, 9H); ESI-MS m/z: 364.3 [M+H]+.
Referring to the synthesis method of example 2, a white solid, namely a K18 compound, with the yield of 48.1% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.75 (t, J=8.6 Hz, 2H), 7.26-7.07 (m, 4H), 4.91 (d, J=8.2 Hz, 2H), 3.56 (s, 2H), 3.43 (s, 4H), 2.42 (s, 4H), 2.22 (s, 3H), 0.95 (s, 9H); ESI-MS m/z: 462.6 [M+H]+.
A compound 1 (530 mg, 2.34 mmol) and a compound 2 (300 mg, 1.95 mmol) were weighed and put into a 100 mL eggplant-shaped flask, dissolved in 25 mL of DMF, and stirred at room temperature for 12 h. After the reaction was finished, the reaction solution was diluted with DCM, washed with 10% hydrochloric acid, dried over anhydrous sodium sulfate, and separated by column chromatography (dichloromethane:methanol=50:1) to obtain a yellowish white solid, namely an intermediate 3.
The intermediate 3 (100 mg, 0.29 mmol) and HATU (121 mg, 0.32 mmol) were weighed and put into a 25 mL eggplant-shaped flask, 10 mL of acetonitrile was added, and DIPEA (191 μL, 1.16 mmol) and a compound 4 (33 mg, 0.32 mmol) were added for reaction for 10 min. After the reaction was finished, the reaction solution was dried and purified by column chromatography (dichloromethane:methanol=50:1) to obtain a yellowish white solid, namely an intermediate 5.
The intermediate 5 was placed into a 25 mL two-necked flask under N2 protection, 7 mL of anhydrous DCM and DIPEA (386 μL, 0.92 mmol) was added and dissolved, and the mixture was placed under a low-temperature cold trap and cooled to −15° C.; and a sulfur trioxide pyridine complex (146 mg, 0.88 mmol) was additionally weighed and dissolved in 1 mL of anhydrous DMSO to form a mixed solution, and the mixed solution was added into the reaction solution in the two-necked flask at −15° C. for reaction for 1 h at low temperature. After the reaction was finished, the reaction solution was purified by column chromatography (petroleum ether:ethyl acetate:methanol=16:4:1) to obtain a white solid, namely a compound K19, with the yield of 68%. M.P. 146-148° C., 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 7.75 (dd, J=16.0, 8.3 Hz, 1H), 7.39-7.09 (m, 2H), 6.85 (dtd, J=9.1, 6.1, 2.9 Hz, 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, 7.6 Hz, 1H), 1.97-1.54 (m, 7H), 1.53-1.07 (m, 6H); 13C NMR (101 MHz, DMSO) δ 215.55, 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]+.
The preparation method of the present example referred to the preparation steps of example 19. The compound 1 was replaced by 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-2-ium to finally obtain a white solid, namely a compound K20, with the yield of 71%. M.P. 137-140° C.; 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 8.30-8.01 (m, 1H), 7.48 (d, J=7.6 Hz, OH), 7.38 (d, J=7.7 Hz, 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); 13C NMR (101 MHz, CDCl3) δ 211.38, 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]+.
The preparation method of the present example referred to the preparation steps of example 19. The compound 4 was replaced by a compound 6 to obtain a white solid product, namely a compound K21, with the yield of 67%. 1H NMR (400 MHz, DMSO) δ 11.01 (s, 1H), 8.15-8.02 (m, 1H), 7.32-7.14 (m, 2H), 6.86 (tdd, J=9.2, 4.8, 2.6 Hz, 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid product, namely a compound K22, with the yield of 66%. 1H NMR (400 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid product, namely a compound K23, with the yield of 65%. 1H NMR (400 MHz, DMSO) δ 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, 1.9 Hz, 1H), 7.03 (ddt, J=7.9, 3.6, 1.9 Hz, 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a yellowish white solid, namely a compound K24, with the yield of 69%. 1H NMR (400 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid product, namely a compound K25, with the yield of 40%. 1H NMR (300 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K26, with the yield of 42%. 1H NMR (300 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K27, with the yield of 66%. 1H NMR (400 MHz, DMSO) δ 11.48 (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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K28, with the yield of 51%. 1H NMR (400 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K29, with the yield of 63%. 1H NMR (400 MHz, DMSO) δ 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, 5.7 Hz, 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K30, with the yield of 43%. 1H NMR (300 MHz, DMSO) δ 10.96 (s, 1H), 8.33-8.02 (m, 1H), 7.15-6.83 (m, 2H), 6.63 (dd, J=7.7, 4.2 Hz, 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid product, namely a compound K31, with the yield of 68%. 1H NMR (400 MHz, DMSO) δ 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, 2.0 Hz, 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).
The preparation method of the present example referred to the preparation steps of example 19 to obtain a white solid, namely a compound K32, with the yield of 58%. 1H NMR (400 MHz, DMSO) δ 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).
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 55% by using 4-chlorobenzyl bromide as a raw material. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.36 (d, J=8.4 Hz, 2H), 7.21-7.15 (m, 3H), 4.88 (s, 2H), 3.56 (s, 2H), 0.95 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.69, 159.34, 141.29, 136.28, 132.79, 128.68, 128.36, 116.23, 112.24, 57.96, 51.89, 34.75, 28.64. HRMS(ESI) for C17H19ClN4 [M+H]+: calcd, 315.1371; found, 315.1370.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 55% by using 4-bromobenzyl bromide as a raw material. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 7.48 (d, J=8.2 Hz, 2H), 7.17 (d, J=4.7 Hz, 1H), 7.11 (d, J=8.0 Hz, 2H), 4.85 (s, 2H), 3.55 (s, 2H), 0.94 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 162.68, 159.35, 141.29, 136.82, 131.63, 128.84, 120.85, 116.23, 112.26, 57.98, 51.96, 34.75, 28.63. HRMS(ESI) for C17H19BrN4 [M+H]+: calcd, 359.0866; found, 359.0865.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 50%. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.89-7.80 (m, 3H), 7.62 (s, 1H), 7.51-7.44 (m, 2H), 7.34 (d, J=8.5 Hz, 1H), 7.17 (d, J=4.7 Hz, 1H), 5.08 (s, 2H), 3.63 (s, 2H), 0.98 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.90, 159.35, 141.32, 135.21, 133.33, 132.64, 128.40, 127.70, 126.19, 125.72, 125.29, 116.33, 112.11, 57.91, 52.50, 34.81, 28.71. HRMS(ESI) for C21H22N4 [M+H]+: calcd, 331.1917; found, 331.1919.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 27%. 1H NMR (300 MHz, DMSO-d6) δ 8.75-8.44 (m, 1H), 8.28 (d, J=8.6 Hz, 1H), 7.94-7.86 (m, 2H), 7.77-7.66 (m, 1H), 7.60-7.50 (m, 1H), 7.31 (d, J=8.6 Hz, 1H), 7.15 (d, J=4.7 Hz, 1H), 5.12 (s, 2H), 3.71 (s, 2H), 0.99 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.93, 159.29, 158.69, 147.93, 141.24, 136.59, 129.57, 129.02, 127.59, 127.21, 126.15, 118.62, 116.28, 112.29, 59.41, 55.82, 34.71, 28.58. HRMS(ESI) for C20H21N5 [M+H]+: calcd, 332.1870; found, 332.1880.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 53%. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J=4.7 Hz, 1H), 7.56-7.45 (m, 4H), 7.20 (s, 2H), 7.00 (d, J=8.8 Hz, 2H), 6.77 (d, J=4.7 Hz, 1H), 5.02 (s, 2H), 3.75 (t, J=5.3 Hz, 2H), 3.60 (s, 2H), 3.38-3.27 (m, 4H), 3.13 (q, J=7.4 Hz, 2H), 2.86-2.77 (m, 4H), 2.72 (t, J=5.3 Hz, 2H), 1.03 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.87, 159.34, 150.14, 141.26, 139.61, 135.89, 132.25, 127.67, 127.30, 126.64, 116.33, 116.29, 112.03, 77.39, 59.58, 57.57, 52.90, 52.03, 48.64, 45.91, 34.76, 28.68. HRMS(ESI) for C29H36N6O [M+H]+: calcd, 485.3023; found, 485.3013.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 46%. 1H NMR (300 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.56-7.43 (m, 4H), 7.23-7.13 (m, 3H), 7.03-6.93 (m, 2H), 4.93 (s, 2H), 4.71 (d, J=4.2 Hz, 1H), 3.69-3.50 (m, 5H), 2.95-2.81 (m, 2H), 1.87-1.74 (m, 2H), 1.54-1.39 (m, 2H), 0.97 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.88, 159.33, 150.43, 141.28, 139.75, 135.74, 131.56, 127.62, 127.29, 126.59, 116.51, 116.34, 112.02, 67.88, 57.70, 52.03, 47.07, 34.77, 34.09, 28.69. HRMS(ESI) for C28H33N5O [M+H]+: calcd, 456.2758; found, 456.2747.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 54%. 1H NMR (300 MHz, CDCl3) δ 8.46 (d, J=4.7 Hz, 1H), 7.23 (d, J=8.7 Hz, 2H), 7.18-7.13 (m, 1H), 6.98 (d, J=8.7 Hz, 4H), 6.77 (d, J=4.7 Hz, 1H), 5.00 (s, 2H), 3.61 (s, 2H), 3.35-3.25 (m, 4H), 2.63 (t, J=5.0 Hz, 4H), 2.40 (s, 3H), 2.27 (s, 3H), 1.03 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 162.91, 159.20, 149.88, 141.20, 140.52, 136.01, 135.74, 132.76, 130.05, 129.97, 128.62, 124.12, 116.35, 115.39, 111.97, 57.80, 55.15, 51.90, 48.85, 46.13, 34.78, 28.71, 20.74. HRMS(ESI) for C29H36N6 [M+H]+: calcd, 469.3074; found, 469.3064.
The preparation method of the present example referred to the preparation steps of example 1 to obtain a white solid product with the yield of 51%. 1H NMR (300 MHz, CDCl3) δ 8.46 (s, 1H), 7.36 (d, J=8.2 Hz, 2H), 7.28-7.19 (m, 2H), 7.12-6.93 (m, 3H), 6.80 (d, J=4.6 Hz, 1H), 4.98 (s, 2H), 3.61 (s, 2H), 3.31 (s, 4H), 2.62 (s, 4H), 2.39 (s, 3H), 1.03 (s, 9H). HRMS(ESI) for C28H33ClN6 [M+H]+: calcd, 489.2528; found, 489.2519.
Referring to the preparation steps of example 1, a white solid product K41-1 with the yield of 64% was obtained. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (d, J=4.7 Hz, 1H), 7.11 (d, J=4.7 Hz, 1H), 7.04 (d, J=8.1 Hz, 2H), 6.91 (d, J=8.1 Hz, 2H), 4.81 (s, 2H), 3.44 (s, 2H), 3.37 (t, J=4.9 Hz, 4H), 3.14-3.04 (m, 4H), 1.42 (s, 9H), 0.94 (s, 9H).
The intermediate K41-1 (330 mg, 1.41 mg) was added to 10 mL of a cold 4 N hydrochloric acid/dioxane solution and reacted in an ice bath for 30 min. After TLC detected that the reaction was finished, the reaction solution was removed by distillation under reduced pressure, and a small amount of toluene was added until the reaction solution was completely removed. Then the residue was dissolved in 10 mL of DMF, potassium carbonate (487 mg, 3.53 mmol) and chloroacetone (195 mg, 2.11 mmol) were successively added, and the reaction solution was heated to 80° C. and reacted under heat preservation for 2 h. After the TLC detected that the reaction was finished, the reaction solution was cooled to room temperature. The reaction solution was added with 20 mL of water and extracted with ethyl acetate (20 mL×3). The organic phases were combined, successively washed with dilute hydrochloric acid (10 mL×2) and a saturated sodium chloride solution (20 mL×2), and dried over anhydrous sodium sulfate for 4 h. The organic phase was subjected to vacuum filtration. A filtrate was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=1:1) to obtain 249 mg of a faint yellow solid K41 with the total yield of 42% in two steps. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J=4.7 Hz, 1H), 7.13 (dd, J=4.7, 1.6 Hz, 1H), 7.02 (d, J=8.4 Hz, 2H), 6.85 (d, J=8.3 Hz, 2H), 4.81 (s, 2H), 3.52 (s, 2H), 3.28-3.21 (m, 2H), 3.16-3.03 (m, 4H), 2.59-2.52 (m, 4H), 2.16-2.02 (m, 3H), 0.95 (s, 9H). HRMS(ESI) for C24H32N6O [M+H]+: calcd, 421.2710; found, 421.2704.
Referring to the preparation steps of example 1, a white solid product with the yield of 46% was obtained. 1H NMR (300 MHz, DMSO-d6) δ 8.66 (s, 1H), 7.59-7.46 (m, 4H), 7.25 (d, J=7.9 Hz, 2H), 7.17 (d, J=4.6 Hz, 1H), 7.00 (d, J=8.6 Hz, 2H), 4.88 (s, 2H), 3.47-3.40 (m, 2H), 3.23-3.12 (m, 4H), 2.49-2.42 (m, 4H), 2.23 (s, 3H), 2.19-2.08 (m, 1H), 0.88 (d, J=6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 162.18, 159.56, 150.53, 141.56, 139.92, 135.68, 131.81, 127.95, 127.65, 126.64, 116.40, 116.05, 111.87, 55.10, 54.33, 50.61, 48.86, 46.20, 26.78, 20.24. HRMS(ESI) for C27H32N6 [M+H]+: calcd, 441.2761; found, 441.2753.
Referring to the preparation steps of example 1, a white solid product with the yield of 43% was obtained. 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 8.43 (d, J=4.7 Hz, 1H), 7.50 (d, J=8.5 Hz, 2H), 7.22-7.08 (m, 2H), 6.76 (d, J=4.7 Hz, 1H), 4.94 (s, 2H), 3.56 (s, 2H), 3.15 (s, 2H), 2.79-2.41 (m, 8H), 2.36 (s, 3H), 1.01 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 168.12, 162.76, 159.26, 141.22, 136.45, 133.68, 127.80, 119.67, 116.29, 112.03, 61.72, 57.71, 54.99, 52.95, 51.98, 45.63, 34.72, 28.64. HRMS(ESI) for C24H33N7O [M+H]+: calcd, 436.2819; found, 436.2811.
An intermediate K44-1 was dissolved in 10 mL of acetonitrile, DIPEA (2.13 g, 16.48 mmol) was added, and the reaction solution was heated to 80° C. and reacted under heat preservation for 16 h. After the TLC detected that the reaction was finished, the reaction solution was cooled to room temperature. The reaction solution was added with 20 mL of water and extracted with ethyl acetate (20 mL×3). The organic phases were combined, successively washed with dilute hydrochloric acid (10 mL×2) and a saturated sodium chloride solution (20 mL×2), and dried over anhydrous sodium sulfate for 4 h. The organic phase was subjected to vacuum filtration. A filtrate was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=12:1) to obtain a white solid K44-2 with the yield of 55%. 1H NMR (300 MHz, DMSO-d6) δ 8.59 (s, 1H), 7.89 (d, J=8.2 Hz, 2H), 7.28 (d, J=8.0 Hz, 2H), 7.18 (d, J=4.7 Hz, 1H), 4.96 (s, 2H), 3.83 (s, 3H), 3.60 (s, 2H), 0.96 (s, 9H).
The intermediate K44-2 (700 mg, 2.07 mmol) was added to 8 mL of a mixed solution of methanol and water (3:1), then lithium hydroxide (99 mg, 4.14 mmol) was added, and then the mixture reacted in an ice bath for 4 h. After the TLC detected that the reaction was finished, the reaction solution was concentrated, then the pH of the reaction solution was adjusted to about 2 by using a 1 N hydrochloric acid solution in an ice bath to generate a large amount of a white solid, suction filtration was performed, and a filter cake was washed by using ethyl acetate and a small amount of ethanol, and dried overnight in a vacuum environment to obtain a crude carboxylic acid product which was directly used for the next reaction. The obtained carboxylic acid was added to 10 mL of anhydrous tetrahydrofuran, and HATU (787 mg, 2.07 mmol), triethylamine (524 mg, 5.18 mmol) and N-methylpiperazine (249 mg, 2.48 mmol) were successively added and reacted at room temperature for 2 h. After the TLC detected that the reaction was finished, the reaction solution was diluted with 40 mL of ethyl acetate, successively washed with a saturated sodium carbonate solution (15 mL×2), a diluted hydrochloric acid solution (15 mL×2) and a saturated sodium chloride solution (15 mL×2), and dried over anhydrous sodium sulfate for 4 h The reaction solution was subjected to vacuum filtration. A filtrate was concentrated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=1:2) to obtain 480 mg of a white solid, namely a compound K44, with the yield of 57% in two steps. 1H NMR (300 MHz, CDCl3) δ 8.44 (s, 1H), 7.35 (d, J=7.8 Hz, 2H), 7.18 (d, J=7.8 Hz, 2H), 6.79 (d, J=4.7 Hz, 1H), 4.99 (s, 2H), 3.83 (s, 2H), 3.57 (s, 4H), 2.37 (s, 7H), 1.01 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 170.13, 162.74, 159.36, 141.25, 139.65, 134.38, 127.39, 127.24, 126.98, 116.22, 112.25, 58.07, 55.16, 54.67, 52.19, 47.50, 45.91, 41.93, 34.71, 28.62. HRMS(ESI) for C23H30N6O [M+H]+: calcd, 407.2554; found, 407.2547.
Referring to the preparation steps of example 44, a white solid product with the yield of 34% was obtained. 1H NMR (300 MHz, DMSO-d6) δ 8.72-8.47 (m, 2H), 7.77 (d, J=8.0 Hz, 2H), 7.23 (d, J=8.0 Hz, 2H), 7.18 (d, J=4.7 Hz, 1H), 4.94 (s, 2H), 3.59 (s, 2H), 3.18-3.02 (m, 6H), 2.95-2.80 (m, 2H), 2.70 (s, 3H), 1.89-1.69 (m, 3H), 0.96 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 167.71, 162.65, 159.41, 141.54, 141.10, 132.81, 127.62, 126.93, 116.25, 112.29, 58.21, 54.59, 52.36, 45.88, 44.51, 43.65, 34.67, 33.18, 28.56, 27.22. HRMS(ESI) for C25H34N6O [M+H]+: calcd, 435.2867; found, 435.2859.
Reagent information: Cat K inhibitor screening kit: batch number, 6L23K01500; supplier, BiVision. Drug preparation: compounds were dissolved in DMSO and prepared into 10 mM stock solutions. When in use, the stock solutions were prepared into the required concentrations by using a buffer.
Ref-03 was used as a comparative compound with the structural formula as follows:
Cathepsin K (CTSK, EC 3.4.22.38) is a lysosomal cysteine protease, participates in osteoclastic bone remodeling and resorption, and can further degrade collagen, gelatin and elastin. The cathepsin K inhibitor screening kit from Biovision uses the ability of the active cathepsin K to cleave a synthetic AFC-based peptide substrate to release AFC and the AFC can be easily quantified by using a fluorometer or fluorescent microplate reader. In the presence of a specific cathepsin K specific inhibitor, the cleavage of the substrate is reduced/eliminated, resulting in a reduction or complete loss of AFC fluorescence. The simple and high-throughput adaptive assay kit can be used to screen/study/characterize a potential inhibitor of the cathepsin K.
20 μL of a buffer, a Cat K inhibitor and test compounds (1 μM and 10 μM) were added to 96-well plates to serve as an EC well, an IC well and an S well, respectively; 50 μL of a Cat K enzyme solution was added into all the wells respectively and the mixture was incubated for 10-15 min at room temperature to establish an enzyme inhibitor complex; 30 μL of a Cat K substrate solution was added into all the wells and the mixture was incubated for 30-60 min at room temperature; and the final volume of the reaction was 100 μL. At 30-60 min, two time points T1 and T2 were selected to detect fluorescence absorption values (Ex/Em=400/505 nm), the fluorescence absorption values were recorded as RFU1 and RFU2, and the inhibition rates (%) of the test compounds on the Cat K were calculated.
The screening method was the same as 1.1, the concentrations of the test compounds were set between 0.1 nM and 10 μM, 4-5 concentrations were selected for detection, IC50 curves were drawn, and IC50 values were calculated.
Data processing: all the data were statistically analyzed using Graph pad.
The results were shown in Table 1.
The compounds of the present invention had a high inhibition rate on the Cat K, low IC50 values and good inhibition activity on the Cat K. A part of the compounds such as K13 and K15 had the IC50 values of less than 10 nM, and a part of the compounds such as K1 and K18-K22 had the IC50 values of all less than 100 nM, such that the compounds of the present invention had better inhibitory effect on the Cat K.
10 μL of a buffer, a Cat B inhibitor and test compounds (1 μM and 10 μM) were added to 96-well plates to serve as an EC well, an IC well and an S well, respectively; 50 μL of a Cat B enzyme solution was added into all the wells respectively and the mixture was incubated for 10-15 min at room temperature to establish an enzyme inhibitor complex; 40 μL of a Cat B substrate solution was added into all the wells and the mixture was incubated for 30-60 min at room temperature; and the final volume of the reaction was 100 μL. At 30-60 min, two time points T1 and T2 were selected to detect fluorescence absorption values (Ex/Em=400/505 nm), the fluorescence absorption values were recorded as RFU1 and RFU2, and the inhibition rates (%) of the test compounds on the Cat B were calculated.
The screening method was the same as above, the concentrations of the test compounds were set between 0.1 nM and 10 μM, 4-5 concentrations were selected for detection, IC50 curves were drawn, and IC50 values were calculated.
Data processing: all the data were statistically analyzed using Graph pad.
10 μL of a buffer, a Cat S inhibitor and test compounds (1 μM and 10 μM) were added to 96-well plates to serve as an EC well, an IC well and an S well, respectively; 50 μL of a Cat B enzyme solution was added into all the wells respectively and the mixture was incubated for 10-15 min at room temperature to establish an enzyme inhibitor complex; 40 μL of a Cat S substrate solution was added into all the wells and the mixture was incubated for 30-60 min at room temperature; and the final volume of the reaction was 100 μL. At 30-60 min, two time points T1 and T2 were selected to detect fluorescence absorption values (Ex/Em=400/505 nm), the fluorescence absorption values were recorded as RFU1 and RFU2, and the inhibition rates (%) of the test compounds on the Cat S were calculated.
The screening method was the same as above, the concentrations of the test compounds were set between 0.1 nM and 10 μM, 4-5 concentrations were selected for detection, IC50 curves were drawn, and IC50 values were calculated.
Data processing: all the data were statistically analyzed using Graph pad.
The ratios of the inhibitory activities of the compounds against the Cat S and the Cat B to those against the Cat K were shown in Table 2.
A part of the compounds showed 1,000-fold selectivity on the Cat K far more than that on the other two enzymes. Although the test values of a positive control Odanacatib in the experiment were slightly lower than those reported in the literature, compared with the positive control Odanacatib, the Cat K inhibitor of the present invention still obviously improved the selectivity on the Cat K and particularly made up for the defect of the selection capability on the Cat K and the Cat S.
1 mL of a dichloromethane solution was respectively added into centrifuge tubes filled with test substance powders K2 (molecular formula C23H27N3O4, 4.6 mg), K3 (molecular formula C23H26FN3O4, 5.0 mg) and K4 (molecular formula C23H25F2N3O4, 5.6 mg) for dissolving, and subjected to ultrasonic treatment for 30 min to obtain a K2 stock solution with the mass concentration of 4.6 mg/mL, a K3 stock solution with the mass concentration of 5.0 mg/mL and a K4 stock solution with the mass concentration of 5.6 mg/mL. The stock solutions were sealedly stored in a refrigerator at −20° C. for later use. Before use, the stock solutions were gradually diluted with methanol to corresponding mass concentration to respectively prepare 0.02, 0.05, 0.22, 0.49, 0.96, 2.37 and 4.90 ng/mL of K2 series standard sample working solutions, 0.02, 0.05, 0.10, 0.19, 1.00, 2.01 and 4.93 ng/mL K3 series standard sample working solutions, and 0.10, 0.20, 0.53, 0.96, 2.42, 5.10 and 10.10 ng/mL K4 series standard sample working solutions.
5 μL of the test drugs, 2 μL of rat liver microsomes and 468 μL of a PBS buffer solution were taken by using a pipette and prepared a premixed solution. The premixed solution was incubated for 5 min in a water bath kettle at 37° C., 47.5 μL of the premixed solution was taken after the incubation was finished, and the premixed solution, 100 μL of ice-cold acetonitrile and 2.5 μL of a PBS buffer solution were added to another new tube after to be gently mixed evenly to obtain a control group. The reaction was started by adding 22.5 μL of a NADPH solution to the remaining premixed solution, the reaction solution was gently pipetted, and the reaction was stopped by immediately taking 50 μL of the premixed solution and 100 μL of acetonitrile in a new tube. Then the incubation liquid was put into the water bath for incubation, 50 μL of the incubation liquid was respectively taken at 5 min, 10 min, 20 min, 30 min, 45 min, 60 min and 90 min, and the reaction was terminated by adding 100 μL of acetonitrile. The reaction solution was centrifuged at 12,000 r/min for 10 min, and the supernatant was taken and detected on a machine.
Electrospray ionization (ESI); ion spray voltage: 4,500 V; ion source temperature: 450° C.; air curtain pressure: 35 psi; and declustering voltage: 80 V.
Sample solutions were subjected to sample injection analysis under the chromatographic and mass spectrometry conditions, and the peak areas were recorded. Linear regression was performed by using the mass concentration of a test substance as an x-coordinate and the peak area of the test substance as a y-coordinate using a weighting method (weighting coefficient of 1/x2). The results showed that a linear range of K2 mass concentration detection was 0.02-4.90 ng/mL and a quantitative lower limit was 0.02 ng/mL; a linear range of K3 mass concentration detection was 0.02-4.93 ng/mL and a quantitative lower limit was 0.02 ng/mL; and a linear range of K4 mass concentration detection was 0.10-10.10 ng/mL and a quantitative lower limit was 0.10 ng/mL. Statistical analysis was performed on the basis of the results to calculate clearance rate (Clint) and half-life (T1/2).
The results were shown in Table 3.
Compared with Ref-03, the compounds had reduced intrinsic clearance rate of the rat liver microsomes, wherein the clearance rate of the compound K4 was reduced by 2 times, the clearance rates of other compounds were all less than that of Ref-03. The half-life of Ref-03 was 0.6 h, while the half-life of the compounds of the present invention was all greater than 0.6 h. Therefore, the compounds of the present invention were more metabolically stable.
RAW264.7 cells were inoculated at a density of 1×105/well in 96-well plates and cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin. The cells were cultured in a constant-temperature incubator containing 5% CO2 at 37° C. After 24 h of the culture, different concentrations of inhibitor working solutions were respectively added to administration groups, and an equal volume of a PBS working solution was added to a control group. After the cells were continuously cultured for 2 days, a culture supernatant was removed, the cells were washed with PBS 3 times, and the viability of the cells was detected with a CCK8 cell proliferation detection kit. 100 μL of PBS containing CCK8 was added to each well, the mixture was incubated at 37° C. in the dark for 2 h, the absorbance at 450 nm was detected by a microplate reader, and CCK8 was added to a culture medium containing free of cells and drug as a blank group.
The viability was calculated by the following equation:
The results were shown in Table 4.
It can be seen from table 5, the cell viabilities of RAW264.7 cells in a compound K29 at the concentrations of 1 μM and 10 μM were 96.06% and 98.89%, respectively; the cell viabilities of RAW264.7 cells in a compound K31 at the concentrations of 1 μM and 10 μM were 85.84% and 82.99%, respectively. It can be seen that the compound K29 had no significant cytotoxicity to RAW264.7 cells at the concentrations of 1 μM and 10 μM, and the compound K31 had cytotoxicity to RAW264.7 cells at the concentrations of 1 μM and 10 μM.
RAW264.7 cells were inoculated at a density of 1×105/well in 96-well plates and cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured in a constant-temperature incubator containing 5% CO2 at 37° C. 100 ng/mL of a RANKL inducing solution was added into each well of the administration groups and a model group to culture osteoclasts, the solution (containing RANKL) was changed every 2-3 days, inhibitor working solutions with different concentrations were added into the administration groups after the solution was changed for the 3rd time, a PBS working solution with the same volume was added into the model group, and the cells were continuously cultured for 24 h; and in the control group, the RANKL inducing solution and the inhibitor working solution were not added, and the PBS working solution with the same total volume was added. A culture supernatant was removed, the cells were rinsed 3 times with PBS and stained with a TRAP staining kit, and the multinucleated osteoclasts were observed under an optical microscope and counted.
The results were shown in Table 5.
It can be seen from Table 5 that compared with the model group, the compound K29 at the concentrations of 1 μM and 10 μM and the K31 at the concentrations of 100 nM and 500 nM both had a tendency to inhibit osteoclastogenesis, wherein the K29 obviously inhibited the osteoclastogenesis at the concentration of 10 μM. Combining Tables 4 and 5, the compound K29 was not cytotoxic and had a good osteoclastogenesis inhibitory effect.
Male ICR mice of 6-8 weeks were used as experimental subjects and randomly divided according to body weight after adaptive feeding for one week. Then compounds of different concentrations were prepared and used for single-administration and multiple-administration experiments by intraperitoneal injection (i.p), and a vehicle of the same component was used as a reference.
As shown in
The above results showed that the compound K18 had relatively low in-vivo toxicity and a certain safety.
An osteoporosis mouse model was established by injecting dexamethasone subcutaneously into male C57BL/6 mice, 8 days after the model establishment, the model mice were administered intraperitoneally at administration doses of 12 mg/kg, 24 mg/kg and 36 mg/kg, and a Cat K inhibitor Ballicaib was used as a positive control. After 6 weeks of continuous administration once a day, the mice were anesthetized and dissected, blood serum was retained, the content of a bone resorption biomarker CTX in the blood was detected by ELISA, and one side of the femur was taken for measuring the bone density. The therapeutic effects of the Cat K inhibitors on osteoporosis were evaluated by comparison with the vehicle group. The effects of the preliminary experiment were obvious. At present, formal experiments were performed.
The treatment effects of the compounds of the present invention on bone metastasis were further researched by establishing a prostate cancer bone metastasis model by injecting C4-2B cells into the tibia of NOD-SCID mice by puncture. After 4 weeks of molding, the model mice were administered intraperitoneally at administration doses of 25 mg/kg and 50 mg/kg, and a common anti-bone metastasis drug zoledronic acid was used as a positive control. After 4 weeks of continuous administration once a day, the changes of body weight and tumor volume of the mice were observed and recorded during the period. After the administration, the mice were sacrificed. The content of prostate specific antigen (PSA) in plasma was tested by ELISA, then the tibial bone density of the mice was tested, and pathological sections were made to test the pathological changes of bone tissues. From these test results, the inhibitory effect on the development of prostate cancer bone metastasis and the protection effect on the bone tissues by the Cat K inhibitors were evaluated.
The test results preliminarily showed that the compound K18 can inhibit the migration, invasion and adhesion processes of the C4-2B cells in a concentration-dependent manner, indicating that the Cat K inhibitors can effectively exert the antitumor activity by influencing the metastasis process of tumors.
Acquity ultra performance liquid chromatography (UPLC) system (Waters corporation); TSQ Endura triple quadrupole MS instrument (Thermo corporation); KQ-500E type ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd); 3K15 benchtop high-speed centrifuge (SIGMA, Germany); and XW-80A vortex mixer (Shanghai Jingke Industrial Co., Ltd.)
Test substances: K29 (molecular weight of 439.21 and mass of 27 mg) and K31 (molecular weight of 457.20 and mass of 14 mg+20 mg).
Reagents: HS-15 was analytically pure, sodium carboxymethylcellulose (CMC-Na) was medicinal grade, formic acid, methanol and acetonitrile were chromatographic grade, and water was purified water.
Electrospray ionization (ESI); ion spray voltage: 3,500 V; ion source temperature: 300° C.; auxiliary gas: 45 Arb; and sheath gas: 10 Arb.
Each administration was tested using 2 SD rats per compound.
The two compounds K29 and K31 were subjected to intravenous injection and intragastric administration respectively. The intravenous administration volume was 5 mL/kg. 0.2 mL of blood was collected in jugular veins 2 min, 10 min, 30 min, 1 h, 2 h, 4 h, 7.5 h and 24 h after the administration respectively. The volume was 10 mL/kg in the intragastric administration group. 0.2 mL of blood was collected in jugular veins 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h after the administration respectively. All blood samples were anticoagulated by heparin and centrifuged for 10 min at 4,000 g. The supernatant plasma was taken for cryopreservation to be tested.
50 μL of the plasma sample was taken, 150 μL of acetonitrile was added, the mixture was evenly mixed by vortex and centrifuged at 9,100 g for 10 min, and a supernatant was taken and 2 μL of the supernatant was injected and detected on a machine.
The experimental results were shown in Tables 6-10.
It can be seen from Table 10, the oral administration bioavailability of the compounds K29 and K31 was 62.1% and 51.0% respectively. The oral administration bioavailability was good.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210098531.3 | Jan 2022 | CN | national |
| 202210156862.8 | Feb 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/073612 | 1/28/2023 | WO |
