Menthol medicine for treating respiratory system diseases as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a medicine for treating respiratory system diseases. Specifically, the invention relates to an L-menthol substance which plays a role in inhibiting ROCK, a preparation method of the L-menthol substance, a pharmaceutical composition containing the L-menthol substance and application of the L-menthol substance to treatment of respiratory system diseases, inflammations and pulmonary fibrosis diseases. And in the # imgabs0 #, each substituent group is shown in the specification.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an L-menthol substance that acts by inhibiting ROCK for treating respiratory diseases, a preparation method thereof, a pharmaceutical composition containing the same, and uses thereof. Background Art

[0002] As a key member of the Rho effector family, serine / threonine protein kinase ROCK exists in two subtypes, ROCK1 (ROKβ) and ROCK2 (ROKα), which are encoded by the human genome loci 18q12.1 and 12q24, respectively. Although the catalytic domains of both show a high degree of conservation (sequence similarity > 90%), the N-terminal regulatory region only retains about 35% homology, and this structural difference may explain their functional specificity. It is worth noting that ROCK1 is significantly enriched in the myocardium, lung parenchyma, and skeletal muscle, while ROCK2 dominates in the central nervous system, suggesting its involvement in tissue-specific signal transduction.

[0003] The activation mechanism of this kinase presents multi-level regulatory characteristics: RhoA GTPase, as an upstream regulatory factor, relieves the auto-inhibited conformation by binding to the RBD domain; meanwhile, the interaction between the carboxyl-terminal PH domain and lipid mediators such as arachidonic acid can independently trigger conformational rearrangement. Under pathological conditions, caspase-mediated proteolytic cleavage generates constitutively active fragments, and this dual activation mode provides potential targets for disease-specific intervention.

[0004] At the cellular physiological level, the ROCK signaling network mediates smooth muscle tension by regulating the phosphorylation state of MLCP, and simultaneously integrates the dynamic assembly of the actin-myosin contractile unit, affecting cell migration, polarity establishment, and morphology maintenance. Its pathological significance extends to the regulation of the inflammatory microenvironment, promoting the release of pro-inflammatory factors such as IL-6 and TNF-α through the NF-κB signaling axis, and promoting collagen deposition through the TGF-β / Smad pathway during the process of pulmonary fibrosis.

[0005] Clinical translational research reveals that ROCK inhibitors exhibit multi-dimensional therapeutic advantages in airway hyperresponsiveness diseases: in addition to directly relaxing bronchial smooth muscle, they can also inhibit E-selectin-mediated eosinophil infiltration, block PDGF-induced fibroblast proliferation, and enhance the nuclear translocation efficiency of glucocorticoid receptors. It is worth noting that the local administration strategy can avoid the risk of systemic hypotension. For example, topical eye administration can specifically reduce the outflow resistance of the trabecular meshwork, and inhalation preparations can target and improve airway remodeling.

[0006] Current clinical needs highlight the limitations of existing treatment options: β2 agonists exhibit rapid tolerance, prostaglandin analogs are limited by side effects such as conjunctival congestion, and glucocorticoids have limited efficacy against neutrophil-predominant inflammatory responses. The development of novel ROCK inhibitors requires consideration of tissue-selective distribution and multi-pathway regulation capabilities, especially for pathological processes such as smooth muscle contraction, mucus hypersecretion, and epithelial barrier damage coexisting in respiratory diseases.

[0007] The L-menthol derivatives in this study, through a novel structure, aim to provide a new drug for treating respiratory and inflammatory diseases and achieve pharmacokinetic characteristics of local high-concentration accumulation and rapid systemic clearance, providing a chemical entity for breaking through existing treatment bottlenecks. Summary of the Invention

[0008] The present invention provides a ROCK inhibitor having the general formula I.

[0009] The present invention also provides a method for preparing a compound having the general formula I.

[0010] Another object of the present invention is to provide a pharmaceutical composition containing a compound having the general formula I as an active ingredient, and one or more pharmaceutically acceptable carriers, excipients or diluents, and its applications in treating respiratory diseases, inflammatory diseases, pulmonary fibrosis, etc.

[0011] Now, the content of the present invention will be specifically described in connection with the objects of the present invention.

[0012] The compound of the present invention having the general formula (I) has the following structural formula:

[0013]

[0014] Wherein, R 1 is selected from alkyl groups of C 1 -C 10 , cycloalkyl groups of C 3 -C 10 , phenyl, phenyl substituted by F, Cl, Br, I, NO 2 , CN and alkyl groups of C 1 -C 10 , benzyl and benzyl substituted by F, Cl, Br, I, NO 2 , CN and alkyl groups of C 1 -C 10 ; R 2 is selected from alkyl groups of C 1 -C 10 .

[0015] Preferably, the following compounds of the general formula (I)

[0016] Wherein, R1 Selected from C 1 -C 4 alkyl, C 3 -C 4 cycloalkyl, phenyl, substituted by F, Cl, NO 2 , CN and C 1 -C 4 alkyl-substituted phenyl, benzyl and substituted by F, Cl, NO 2 , CN and C 1 -C 4 alkyl-substituted benzyl; R 2 Selected from C 1 -C 4 alkyl.

[0017] More preferably, the compounds of general formula (I) are as follows,

[0018]

[0019]

[0020] The compounds of general formula (I) according to the present invention can be synthesized by the following route:

[0021]

[0022] Cyanuric chloride II reacts with L-menthol III in the presence of a base to obtain compound IV; compound IV reacts with sodium thiolate V to obtain compound VI; compound VI reacts with amine VII to obtain compound I; wherein R 1 and R 2 are as defined above.

[0023] This technical solution relates to the pharmaceutical formulation engineering application of the compound of formula I, and its therapeutic formulation can achieve multi-channel drug delivery through the synergistic combination with a suitable pharmaceutical excipient system. According to the characteristics of clinical needs, dosage forms including but not limited to the following can be developed: the oral administration system includes immediate-release / sustained-release tablets, orally disintegrating tablets, pellets capsules and dry suspensions; the parenteral administration system includes intravenous injections, sterile freeze-dried preparations and pre-filled syringes; special dosage forms can extend to inhaled powder aerosols and mucosal administration preparations.

[0024] The pharmaceutical excipient system is selectively formulated according to functional requirements and is mainly divided into the following categories: ① Functional excipients: including but not limited to microcrystalline cellulose (diluent), croscarmellose sodium (disintegrant), hypromellose (binder); ② Process excipients: such as colloidal silicon dioxide (glidant), magnesium stearate (lubricant); ③ Stability regulators: disodium edetate (chelating agent), nitrogen replacement (antioxidant protection); ④ Palatability improvers: sucralose (sweetener), peppermint essence (flavoring agent). For liquid preparations that need to be stored for a long time, sodium benzoate (concentration 0.1 - 0.5% w / v) can be added as an anti-corrosion system.

[0025] The compound of general formula I described in the present invention has ROCK inhibitory effect and can be used as an active ingredient for preparing therapeutic drugs for diseases such as respiratory diseases, inflammatory diseases, and pulmonary fibrosis diseases. Detailed implementation mode

[0026] The present invention will be further described below in conjunction with the embodiments. It should be noted that the following embodiments are only for illustration and not for limiting the present invention. All changes made by those of ordinary skill in the art according to the teachings of the present invention should be within the protection scope required by the claims of this application.

[0027] Synthesis of Compound I-1 in Example 1

[0028]

[0029] Compound III (1.56 g, 10 mmol) was added to a 100 mL round-bottom flask, dissolved in 30 mL of THF, stirred under an ice-water bath, and NaH (0.40 g, 10 mmol, 60%) was added in batches. After addition, the reaction mixture was stirred at room temperature for another half hour. Then cyanuric chloride II (1.84 g, 10 mmol) was added, and after addition, the reaction mixture was stirred at room temperature for 1 hour. Sodium methyl mercaptide V-1 (1.10 g, 10 mmol) was added, and the reaction mixture was stirred at room temperature overnight. Finally, an aqueous solution of methylamine VII-1 (36%, 3 mL) and DIPEA (1 mL) were added. After addition, the reaction mixture was heated to reflux overnight.

[0030] The reaction mixture was carefully poured into 200 mL of ice water, stirred, and extracted with 50 mL × 3 CH 2 Cl 2 The extraction phases were combined, washed with 100 mL of 5% brine, and dried over anhydrous sodium sulfate. The desiccant was removed by suction filtration, and the filtrate was evaporated to dryness on a rotary evaporator. The residue was purified by silica gel column chromatography to obtain a white solid, 1.91 g (combined yield 63%). ESI-MS, m / z = 311 ([M+H] + )。 11H-NMR (in DMSO, Brucker 300 MHz) δ ppm: 3.54 (m, 1H), 7.94 (br, 1H), 1.74 (m, 1H), 1.54 (m, 1H), 1.89 (t, 1H), 1.62 ((m, 3H), 1.37 (m, 2H), 2.50 (s, 3H), 2.92 (s, 3H), 1.41 (t, 1H), 0.89 (s, 3H), 0.82 (s, 6H). That is, Compound I-I.

[0031] Examples 2 - 12

[0032] Following the operating steps of Reference Example 1, the compounds listed in the following table were synthesized.

[0033]

[0034]

[0035] In vitro ROCK inhibition analysis of the compound of Example 13

[0036] The enzyme inhibition activity was determined using the IMAP detection system based on fluorescence polarization technology. The core components of the experiment included: ROCK IMAP detection kit (Molecular Devices, R8093), fluorescently labeled substrate (F1)-AKRRRLSSLRA (Molecular Devices, R7184), and ROCKII recombinant enzyme (Upstate Biotechnology, 14 - 451). The detection system was constructed in a 384-well plate. The compound concentration gradient was set from 100 μM to 0.1 nM. A dose-response curve was established by three-fold serial dilution, and Y-27632 (Tocris) was used as a positive control (working concentration 0.4 μM).

[0037] The reaction system construction was divided into three stages: First, 1 μL of the test compound (dissolved in DMSO) was premixed with 2 μL of the enzyme working solution (containing 10 mM Tris-HCl, 10 mM MgCl2, 0.1% BSA, 0.05% NaN3, pH 7.2) in the well plate. The final enzyme concentration was 26 nM, and it was equilibrated at room temperature for 30 minutes; then 2 μL of the substrate-ATP mixture (final concentrations were 0.2 μM and 10 μM respectively) was added to initiate the enzymatic reaction for 60 minutes; finally, 12 μL of the IMAP binding reagent (prepared according to the kit instructions) was added to immobilize the phosphorylated product through molecular size exclusion effect to form a stable detection complex.

[0038] Fluorescence signal collection was performed using an Envision 2100 high-throughput detection system (PerkinElmer), equipped with a FITC FP480 excitation filter and a dual-channel polarization detection module (P-pol535 / S-pol535 emission filters). The data were fitted with a four-parameter logistic equation using XL-Fit software to calculate the half-maximal inhibitory concentration (IC50) of each compound. It should be noted that all liquid transfer steps must strictly control the incubation time, and the addition of NaN3 to the reaction buffer system can effectively inhibit microbial growth, ensuring long-term experimental stability.

[0039] The test results are as follows:

[0040] Compound <![CDATA[IC 50 (nM)]]> Compound <![CDATA[IC 50 (nM)]]> Compound of Example 1 2.4 Compound of Example 7 21 Compound of Example 2 1.7 Compound of Example 8 3.6 Compound of Example 3 45 Compound of Example 9 8.1 Compound of Example 4 0.96 Compound of Example 10 42 Compound of Example 5 4.8 Compound of Example 11 57 Compound of Example 6 8.3 Compound of Example 12 149

[0041] The data in the above table show that the compounds of the present invention have a strong inhibitory effect on ROCK and can be used to prepare drugs for treating diseases such as respiratory system and inflammation.

Claims

1. A compound of the general formula I, in, R 1 Selected from C1-C 10 Alkyl, C3-C 10 Cycloalkyl, phenyl, F, Cl, Br, I, NO2, CN and C1-C 10 Alkyl-substituted phenyl, benzyl and F, Cl, Br, I, NO2, CN and C1-C 10 Alkyl substituted benzyl; R 2 Selected from C1-C 10 of alkyl.

2. A compound of formula I as defined in claim 1, wherein: R 1 is selected from C1-C4 alkyl, C3-C4 cycloalkyl, phenyl, phenyl substituted by F, Cl, NO2, CN and C1-C4 alkyl, benzyl and benzyl substituted by F, Cl, NO2, CN and C1-C4 alkyl; R 2 An alkyl group selected from C1-C4.

3. A compound of formula I as defined in claim 2. Selected from:

4. A method for synthesizing a compound of formula I according to any one of claims 1 to 3: Cyanuric chloride II reacts with L-menthol III in the presence of a base to obtain compound IV; compound IV reacts with sodium thiolate V to obtain compound VI; compound VI reacts with amine VII to obtain compound I; wherein R 1 and R 2 The definition as described in any one of claims 1 to 3.

5. Use of the compound of general formula I according to any one of claims 1 to 3 in the preparation of ROcK inhibitor drugs.

6. The use according to claim 5, characterized in that Used in the preparation of drugs for treating respiratory system, inflammation and pulmonary fibrosis.

7. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 3, and a suitable carrier or excipient.

8. The pharmaceutical composition according to claim 7, wherein The composition is a solid oral preparation, a liquid oral preparation or an injection.

9. The solid and liquid oral preparation according to claim 8 comprises: Dispersible tablets, enteric-coated tablets, chewable tablets, orodisintegrating tablets, capsules, granules, oral solutions; the injection preparations include water injection, freeze-dried powder injection, large infusion, and small infusion.

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