Compounds for treating connective tissue diseases and pharmaceutical compositions thereof

Abstract

The invention provides a compound for treating connective tissue diseases and a pharmaceutical composition thereof. Specifically, the invention provides application of a compound shown as a formula I or pharmaceutically acceptable salt thereof in preparation of a pharmaceutical composition for preventing or treating connective tissue diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a compound for treating connective tissue disease and a pharmaceutical composition thereof. BACKGROUND

[0002] Broadly, connective tissue disease includes a group of genetic connective tissue diseases, i.e. diseases caused by congenital defects in the biosynthesis or degradation of certain components (such as collagen, elastin or glycosaminoglycan) in connective tissue. Connective tissue disease includes Marfan syndrome, Ehlers-Danlos syndrome, tuberous polyarteritis, Wegener's granuloma, giant cell arteritis, skeletal dysplasia, aortic ectasia and Sjogren's syndrome, etc.

[0003] Marfan syndrome (MFS) is a very representative genetic connective tissue disease, which is autosomal dominant inheritance. The characteristics of the disease are long and uneven limbs, fingers and toes, significantly taller than the average person, accompanied by cardiovascular and eye abnormalities. It is caused by the synthesis disorder of collagen fibers due to the mutation of extracellular matrix protein FBN1 gene, and is also called collagen fiber disease. A similar mutation (Cys1039Tyr) in humans can cause typical manifestations of human Marfan syndrome. Heterozygous mice have proximal aortic aneurysm, mitral valve thickening, alveolar septal defect, mild thoracic kyphosis and skeletal muscle disease. In a small number of cases, Marfan syndrome patients may also be relatively short, but due to the mutation of FBN1 gene, the aortic root is significantly dilated, and is still diagnosed as Marfan syndrome.

[0004] FBN1 gene mutations can induce a variety of diseases. The main manifestations of MASS syndrome include myopia, mitral valve prolapse, mild aortic dilatation, skeletal abnormalities, and skin stripes. The skeletal abnormalities are similar to Marfan syndrome, but are relatively mild, and the skin stripes appear as white or red stripes similar to stretch marks. Patients with Weill-Marchesani syndrome are short in stature, short in limbs, short in hands and feet, limited in joint movement, and muscular, which is contrary to the physical characteristics of Marfan syndrome. Patients with Weill-Marchesani syndrome also have lens dislocation, but unlike Marfan syndrome, they may also have other eye problems such as small and thick corneas and shallow anterior chambers. In severe cases, vision and intraocular pressure may be affected, leading to eye diseases such as glaucoma. Geleophysic dysplasia, acromicric dysplasia, and Myhre syndrome all have common characteristics such as short stature, short limbs, and limited joint movement, but each has its own unique clinical manifestations. Stiff skin syndrome is mainly characterized by thickening and hardening of the skin, which reduces skin elasticity and limits movement, similar to the symptoms of scleroderma. It can affect the skin of multiple parts of the body and severely affect the patient's normal life and activities. These diseases cover the main conditions that FBN1 gene mutations can cause, and have different clinical characteristics and severity.

[0005] Ehlers-Danlos syndrome (EDS) is also known as systemic elastofibrodysplasia. It is characterized by excessive stretching of the skin and joints, easy tissue damage, increased fragility, difficulty healing from trauma, increased vascular fragility, eye abnormalities, and internal organ abnormalities. It is a congenital metabolic disorder of collagen, one of the main proteins of connective tissue.

[0006] Therefore, there is a need in the art to provide a drug that can prevent / treat connective tissue disease and the multiple tissue and organ lesions caused by it. SUMMARY

[0007] The purpose of the present application is to provide a new drug that can be used to prevent / treat connective tissue disease.

[0008] In the first aspect of the present application, a compound of formula I or a pharmaceutically acceptable salt thereof is provided for use in the preparation of a pharmaceutical composition for preventing or treating connective tissue disease.

[0009]

[0010] wherein R1, R2are each independently selected from the group consisting of OH and C1-C8 alkoxy; and

[0011] R3, R4and R5are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted C1-C8 alkyl;

[0012] R6is selected from the group consisting of H, halogen and OH;

[0013] R7is selected from the group consisting of H, halogen, substituted or unsubstituted phenyl; is a double bond;

[0014] R8is -(C=0)-R9;

[0015] R9is selected from the group consisting of hydroxyl, and -0-R 11 ;

[0016] R 11 is selected from the group consisting of substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C2-C8 alkenyl, substituted or unsubstituted C2-C8 alkynyl, and substituted or unsubstituted C3-C10 cycloalkyl; and

[0017] wherein said "substituted" means substituted with one or more substituents selected from the group consisting of halogen, deuterium and C1-C6 alkyl.

[0018] In another preferred embodiment, the connective tissue disease is Marfan syndrome, Ehlers-Danlos syndrome, Takayasu arteritis, MASS syndrome, Weill-Marchesani syndrome, geleophysic dysplasia, acromicric dysplasia, Myhre syndrome, stiff skin syndrome, Wegener's granulomatosis, giant cell arteritis, skeletal dysplasia, aortic ectaisa or Sjogren's syndrome.

[0019] In another preferred embodiment, the connective tissue disease is Marfan syndrome, Ehlers-Danlos syndrome, Takayasu arteritis, Wegener's granulomatosis, giant cell arteritis, skeletal dysplasia, aortic ectaisa or Sjogren's syndrome; preferably Marfan syndrome.

[0020] In another preferred embodiment, the connective tissue disease is a connective tissue disease resulting from a mutation in FBN1.

[0021] In another preferred embodiment, one of R1and R2is C1-C8 alkoxy; preferably one of R1and R2is C1-C3 alkoxy.

[0022] In another preferred embodiment, R1is OH.

[0023] In another preferred embodiment, R1is OH and R2is C1-C8 alkoxy.

[0024] In another preferred embodiment, R2 is C1-C6 alkoxy, preferably R2 is C1-C3 alkoxy.

[0025] In another preferred embodiment, R3, R4 and R5 are each independently H or halogen, preferably H.

[0026] In another preferred embodiment, R6 is H or halogen, preferably H.

[0027] In another preferred embodiment, R7 is H or halogen, preferably H.

[0028] In another preferred embodiment, R7 is substituted or unsubstituted C6 aryl, and said "substituted" means substituted with one or more groups selected from the group consisting of halogen, C1-C6 alkyl.

[0029] In another preferred embodiment, R9 is OH or C1-C3 alkoxy, preferably R9 is OH or methoxy.

[0030] In another preferred embodiment, R1 and R9 are both OH.

[0031] In another preferred embodiment, R 11 is C1-C8 alkyl, preferably C1-C3 alkyl or methyl.

[0032] In another preferred embodiment, the compound is selected from the group consisting of:

[0033]

[0034] In another preferred embodiment, the salt of the compound is selected from the group consisting of: sodium salt, potassium salt, magnesium salt, lithium salt or piperazine salt.

[0035] In another preferred embodiment, the prevention or treatment of connective tissue disease comprises prevention or treatment of a pathology selected from the group consisting of: eye, bone, lung, heart, blood vessel, skin or a combination thereof.

[0036] In another preferred embodiment, the prevention or treatment of connective tissue disease is for one or more uses selected from the group consisting of:

[0037] Improving lens ectopia;

[0038] Improving bone structure (increasing bone density, bone thickness);

[0039] Improving lung injury (such as alveolar fusion, absence of lung septa);

[0040] Improving heart injury (such as myocardial fibrosis, decreased cardiac function);

[0041] Improving aortic pathology (such as aortic valve granulomata deposition, aortic dilation, improving aortic wall sclerosis).

[0042] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0043] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid formulation (e.g. solution, emulsion, suspension), solid formulation (e.g. lyophilized formulation).

[0044] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of injection (e.g. injection solution or powder injection), oral formulation (e.g. capsule, tablet, pill, powder, granule, syrup, oral solution or tincture), more preferably, the dosage form is oral formulation.

[0045] In a second aspect of the present application, a method for preventing and / or treating connective tissue disease is provided, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0046] In another preferred embodiment, the preventing or treating connective tissue disease comprises preventing or treating a pathology selected from the group consisting of eye, bone, lung, heart, blood vessel, skin or a combination thereof.

[0047] In another preferred embodiment, the connective tissue disease is Marfan syndrome, Ehlers-Danlos syndrome, Takayasu arteritis, MASS syndrome, Weill-Marchesani syndrome, geleophysic dysplasia, acromicric dysplasia, Myhre syndrome, stiff skin syndrome, Wegener's granulomatosis, giant cell arteritis, skeletal dysplasia, aortic root dilatation or Sjogren's syndrome.

[0048] In another preferred embodiment, the connective tissue disease is a connective tissue disease caused by FBN1 mutation.

[0049] In another preferred embodiment, the connective tissue disease is Marfan syndrome.

[0050] In another preferred embodiment, the subject is a mammal.

[0051] In another preferred embodiment, the subject is a human, a mouse or a rat.

[0052] In another preferred embodiment, the subject has a connective tissue disease, preferably Marfan syndrome.

[0053] It should be understood that, within the scope of the present application, all the technical features described above and in the following (e.g. in the examples) of the present application can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 A simple diagram of the eyeball;

[0055] Figure 2 Representative graph of the relative position of ciliary muscle and lens

[0056] Figure 3 Representative graph of femoral histology HE staining;

[0057] Figure 4 Representative graph of lung tissue HE staining;

[0058] Figure 5 Representative graph of myocardial picrosirius staining;

[0059] Figure 6 Representative graph of aortic valve picrosirius staining;

[0060] Figure 7 Representative graph of aortic valve Prussian blue staining;

[0061] Figure 8 Representative graph of aortic ultrasound;

[0062] Figure 9 Representative graph of mouse femoral trochanter trabecular bone and trabecular bone morphology quantitative results;

[0063] Figure 10 CT scan results of male mouse lung, (A) lung cross-sectional representative graph; (B) lung coronal representative graph; (C) left lung sagittal representative graph; (D) right lung sagittal representative graph;

[0064] Figure 11 CT scan results of female mouse lung, (A) lung cross-sectional representative graph; (B) lung coronal representative graph; (C) left lung sagittal representative graph; (D) right lung sagittal representative graph;

[0065] Figure 12 Lung HE staining representative graph of male and female mice;

[0066] Figure 13 Oxygen saturation quantification graph. DETAILED DESCRIPTION

[0067] The present inventors, through extensive and in-depth research, through a large number of screening and testing, provide a drug for treating connective tissue disease. The present application first found that the compound of formula I and its pharmaceutically acceptable salt can treat connective tissue disease and the lung, eye, heart, bone, and vascular lesions caused thereby. On this basis, the present application is completed.

[0068] TERMS

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0070] As used herein, the term "containing" or "including" can be open, semi-closed and closed. In other words, the term also includes "consisting essentially of or "consisting of.

[0071] As used herein, the term "alkyl" by itself or as part of another substituent, means a straight or branched chain hydrocarbon group having the indicated number of carbon atoms (e.g., Ci-C8, Ci-C6, or Ci-C3, where Ci-C8means from 1 to 8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0072] As used herein, the term "alkenyl" includes straight chain or branched chain alkenyl groups. For example, C2-C8alkenyl means a straight chain or branched chain alkenyl group having from 2 to 8 carbon atoms, e.g., C2-C6alkenyl or C2-C4alkenyl, specifically, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0073] As used herein, the term "alkynyl" includes straight chain or branched chain alkynyl groups. For example, C2-C8alkynyl means a straight chain or branched chain alkynyl group having from 2 to 8 carbon atoms, e.g., C2-C6alkynyl or C2-C4alkynyl, specifically, such as ethynyl, propynyl, butynyl, or the like.

[0074] As used herein, the term "C3-C10cycloalkyl" means a cycloalkyl group having from 3 to 10 carbon atoms. It can be monocyclic, e.g., C4-C7cycloalkyl or C5-C6cycloalkyl, specifically, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It can also be in the form of a bicyclic ring, e.g., a bridged or spirocyclic ring.

[0075] As used herein, the term "C1-C8alkoxy" means a straight chain or branched chain alkoxy group having from 1 to 8 carbon atoms; for example, methoxy, ethoxy, propyloxy, isopropyloxy, butoxy, isobutyloxy, t-butyloxy, and the like.

[0076] As used herein, the term "plurality" means 2 or more, e.g., 2, 3, 4, 5, or 6, preferably 2 or 3.

[0077] Active ingredient

[0078] As used herein, "compounds of the invention" refer to compounds of Formula I and pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" refer to salts of the compounds of the invention with acids or bases that are suitable for use in medicine. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is salts of the compounds of the invention with acids. Suitable acids for salt formation include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, nitric, phosphoric, and the like, organic acids such as formic, acetic, propionic, oxalic, malonic, succinic, fumaric, maleic, lactic, malic, tartaric, citric, picric, methanesulfonic, benzenesulfonic, benzenesulfonic, and the like, and acidic amino acids such as aspartic acid, glutamic acid, and the like. One preferred class of salts is salts of the compounds of the invention with bases. Suitable bases for salt formation include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, and the like, organic bases such as ammonia, triethylamine, diethylamine, piperazine, and the like.

[0079] The present invention is also intended to include stereoisomers, crystalline forms, hydrates, or solvates of the compounds of Formula I.

[0080] Connective tissue disease

[0081] Connective tissue disease refers to diseases in which connective tissue is affected. Connective tissue diseases often involve multiple tissue organs, such as skin, skeleton, lungs, heart valves, aorta, eyeball, etc., and have significant heterogeneity. Interstitial lung disease is a common pulmonary manifestation of connective tissue disease, leading to significant morbidity and mortality. Connective tissue disease is manifested in the skeleton as osteoporosis, peripheral and vertebral bone fractures, changes in bone length, etc.

[0082] Common connective tissue diseases include, but are not limited to, Marfan syndrome, Ehlers-Danlos syndrome, Takayasu's arteritis, MASS syndrome, Weill-Marchesani syndrome, geleophysic dysplasia, acromicric dysplasia, Myhre syndrome, stiff skin syndrome, Wegener's granulomatosis, giant cell arteritis, skeletal dysplasia, aortic ectasia, Sjogren's syndrome.

[0083] Preferably, the connective tissue disease of the present invention is a connective tissue disease caused by mutation of the FBN1 gene, such as Marfan syndrome, MASS syndrome, Weill-Marchesani syndrome, geleophysic dysplasia, acromicric dysplasia, Myhre syndrome, stiff skin syndrome. Preferably, the connective tissue disease is caused by mutation of the Cys1039Tyr site of the human FBN1 gene.

[0084] Marfan syndrome

[0085] Marfan syndrome (MFS) is an autosomal dominant genetic disease with high penetrance and significant clinical variability, symptoms can become more apparent as the age increases. MFS is caused by pathogenic variants in FBN1, which encodes fibrillin-1, a major structural component of the extracellular matrix that provides support to connective tissues, particularly in the arteries, perichondrium, and ocular structures. The most prominent manifestations of Marfan syndrome are asymptomatic aortic root aneurysm, lens subluxation (ectopia lentis), and skeletal abnormalities characterized by overgrowth of long bones, and in some patients, short stature.

[0086] Marfan syndrome can cause ocular complications such as lens subluxation, ocular vascular abnormalities, macular degeneration, retinal detachment, and reduced vision. Lens subluxation refers to the displacement of the lens from its normal anatomical position, which can be related to abnormalities in ocular structures caused by genetic factors. It can cause phenomena such as blurred vision, black shadows in front of the eyes, and in severe cases, permanent vision loss.

[0087] As used herein, the term "treatment" or "treatment" includes disease-modifying treatment and symptomatic treatment, either of which can be prophylactic (i.e., before the onset of symptoms, to prevent, delay, or reduce the severity of symptoms) or therapeutic (i.e., after the onset of symptoms, to reduce the severity and / or duration of symptoms).

[0088] Use

[0089] It has been unexpectedly found that the compound of formula I of the present application can prevent or treat connective tissue disease.

[0090] Experiments have shown that the compound of the present application can significantly improve lung, eye, heart, bone, and vascular lesions caused by connective tissue disease. In particular, in the presence of FBN-1 gene mutations (Marfan syndrome), the compound of formula I can also significantly improve multi-organ lesions. It is very suitable as a drug for preventing or treating connective tissue disease.

[0091] Pharmaceutical composition and administration method

[0092] The present application also provides a pharmaceutical composition for preventing or treating connective tissue disease, said pharmaceutical composition containing the compound of formula I of the present application, or a pharmaceutically acceptable salt as an active ingredient.

[0093] As used herein, "a safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present application per dose, more preferably 10-500 mg of the compound of the present application per dose. Preferably, the "dose" is a capsule or tablet.

[0094] "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler substances or gel materials, which are suitable for human use and which are of sufficient purity and sufficiently low toxicity. By "compatible" it is meant that the carrier is not chemically reactive with the compound of the formula I in a deleterious way. Examples of suitable pharmaceutically acceptable carriers are celluloses and their derivatives (e.g., sodium carboxymethylcellulose, ethylcellulose sodium, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., lecithin), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0095] The mode of administration of the pharmaceutical compositions of the present application is not particularly limited, and representative modes of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

[0096] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as sodium citrate or dicalcium phosphate, or with such ingredients as (a) fillers or extenders, e.g., starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) absorbents, e.g., kaolin and bentonite clay; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, buffering agents can be included.

[0097] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They can contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0098] ​Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, or elixirs. In addition to the active ingredient, the liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, or combinations thereof.

[0099] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0100] Suspensions, in addition to the active ingredient, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar, or mixtures thereof, and the like.

[0101] The compositions for parenteral injection can comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyol, and suitable mixtures thereof.

[0102] Dosage forms for topical administration of a compound of this application include ointments, powders, sprays, and inhalers. The active component is admixed with a carrier, which can be a sterile powder, a sterile non-fluid, or a sterile fluid. Carriers, in some cases, can be a propellant under pressure.

[0103] The active ingredients of this application can be administered alone, or in combination with other pharmaceutically acceptable compounds.

[0104] In the present application, the therapeutically effective dosage of a compound of Formula I will generally be in the range of about 1-2000 mg / day, about 10 to about 1000 mg / day, about 10 to about 500 mg / day, about 10 to about 250 mg / day, or about 10 to 100 mg / day. The therapeutically effective dosage will be administered in one or more doses. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the patient's age, size, body surface area, general health, diet, individual response, time of administration, severity of the disease being treated, the activity of the specific compound employed, the mode of application, and the like. The therapeutically effective amount for a given situation will be determined by routine experimentation and is within the competence of the skilled clinician or physician. In any case, the active ingredient will be administered on the basis of the individual patient's needs and in a manner that will deliver a therapeutically effective amount.

[0105] The principal advantages of the present application include:

[0106] 1. The present application first discovers that the compound of formula I and its pharmaceutically acceptable salts can treat connective tissue disease and have significant therapeutic effect on multiple organ lesions caused by connective tissue disease.

[0107] 2. The compound of the present application is safe in use and has small toxic and side effects.

[0108] The present application will be further described in conjunction with specific implementations. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions or according to the conditions suggested by the manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0109] I. Experimental animals

[0110] B6.129-Fbn1 tm1Hcd / J Marfan mice, referred to as Fbn1 C1041G / + mice, referred to as Fbn1 C1039G / + mice, purchased from The Jackson Laboratory (Jackson ID: 012885). The Cys1041Gly site mutation of the mice is equivalent to the Cys1039Tyr site mutation of human, which is the main mutation site leading to human Marfan. Male Fbn1C 1041G / + mice were crossed with wild type female mice to obtain Fbn1C 1041G / + and wild type offspring for subsequent studies. The mice of 6-7 months old were used in this experiment. They were bred in the SPF level animal room of the Experimental Animal Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, at a constant temperature of 22±2℃, with 12h light, fed with ordinary feed, and free water.

[0111] II. Experimental materials and reagents

[0112] 1. Experimental instruments

[0113] Fluorescence microscope (BX51, OLYMPUS, Japan)

[0114] Gel electrophoresis instrument (AD1603E, Takara, Japan)

[0115] Gel imaging instrument (Tanon-2500, Shanghai Tianyi Instrument and Equipment Company)

[0116] Small animal ultrasound instrument (Vevo3100, FUJIFILM VisualSonics, Japan)

[0117] PCR instrument (MX3000P, Agilent, USA)

[0118] 2. Experimental reagents

[0119] acepromazine (8535895249, vikunl, france)

[0120] celazine (M0413A, melon biotech co., ltd.)

[0121] isopentane anesthetic (shenzhen ruivode life science co., ltd.)

[0122] hematoxylin staining solution (HS0920Y, shanghai yifan biological technology co., ltd.)

[0123] eosin staining solution (HS0920Y, shanghai yifan biological technology co., ltd.)

[0124] cortex staining solution (shanghai national pharmaceutical group chemical reagent co., ltd.)

[0125] sodium chloride injection (S210429E25, jimin health management co., ltd.)

[0126] formaldehyde (20211207, shanghai national pharmaceutical group chemical reagent co., ltd.)

[0127] concentrated hydrochloric acid (20190617, shanghai national pharmaceutical group chemical reagent co., ltd.)

[0128] neutral resin (20220324, shanghai national pharmaceutical group chemical reagent co., ltd.)

[0129] paraffin (39601006, leica, germany)

[0130] mouse tail rapid DNA extraction identification kit (D7283M, biyun tian biological technology co., ltd.)

[0131] PCR primer (shanghai biosun biological engineering co., ltd.)

[0132] 3. test object

[0133] SF (A0213A, melon biotech co., ltd.)

[0134] MHMA (20220903, commercial commission synthesis)

[0135] The structural formula of 3-methoxy-4-hydroxycinnamic acid sodium (SF) is:

[0136]

[0137] The structural formula of α-[(3-hydroxy-4-methoxyphenyl) methylene]-2-methyl phenylacetic acid methyl ester (MHMA)

[0138]

[0139] Example 1

[0140] Experimental methods

[0141] 1. Drug preparation

[0142] Solution Name Procedure SF SF was dissolved at 5 mg / ml in 0.5% CMC and mixed by vortexing on a vortex mixer. MHMA MHMA was dissolved at 5 mg / ml in 0.5% CMC and mixed by vortexing on a vortex mixer.

[0143] 2. Animal grouping

[0144] 6-7 month old Marfan mice were randomly divided into three groups, namely the solvent control group, the SF group and the MHMA group (n = 10).

[0145] 3. Experimental animal administration

[0146] 3.1 Solvent control group: mice were administered with 0.5% CMCNa 10 ml / kg by gavage five times a week, with a 90-day administration period

[0147] 3.2 SF group: mice were administered with SF at a dose of 50 mg / kg, with a volume of 10 ml / kg by gavage five times a week, with a 90-day administration period

[0148] 3.3 MHMA group: mice were administered with MHMA at a dose of 50 mg / kg, with a volume of 10 ml / kg by gavage five times a week, with a 90-day administration period

[0149] 4. Efficacy evaluation

[0150] 4.1 After the completion of the administration period, the mice were evaluated by aortic ultrasound to evaluate the ascending aorta diameter and pulse wave velocity (PWV) of the mice.

[0151] (1) Anesthesia: Mice were anesthetized with isoflurane in an anesthetic machine (RWD Life Science Co., Ltd., China; 5% vol / vol for induction, 2% vol / vol for maintenance of anesthesia), and were fixed as soon as they lost the righting reflex.

[0152] (2) Fixation: The mice were supine on an ultrasound heating platform, with their noses placed in a breathing tube, and were maintained under anesthesia with 2% isoflurane. The limbs of the mice were attached to four electrocardiogram (ECG) electrodes on the platform for measuring heart rate, ECG, and respiratory rate. Body temperature was monitored by a rectal probe, and the heating platform was used to maintain the body temperature at 36-38°C.

[0153] (3) Ultrasound measurement

[0154] Probe type: MX550D ultrasound probe (25-55 MHz)

[0155] Aortic diameter measurement: Ultrasound measurement was performed under B mode on the right side of the chest of the mice to obtain an ultrasound image of the aorta from the root of the aorta to the beginning of the descending aorta.

[0156] PWV measurement: Pulse Doppler signals were recorded at the aortic root and at the beginning of the descending aorta in PW mode. The Doppler waveforms were measured from the corresponding Doppler images, with the R-wave peak of the electrocardiogram as the starting point. The time of blood arrival at the aortic root (t1) and the beginning of the descending aorta (t2) was measured (t1and t2values were the average of 10 cardiac cycles). The aortic arch length (d) was the distance of the propagation trajectory between the two points where the Doppler signals were recorded. Finally, the PWV was calculated by dividing the aortic arch length (d) by the blood transit time (t2-t1) (PWV = d / (t2-t1)).

[0157] 4.2 Histological evaluation

[0158] 4.2.1 Tissue fixation, dehydration and sectioning method

[0159] Standardized sampling was performed for each tissue site. Fresh tissues were cut into approximately 3-4 mm size tissue blocks using a blade. The cut tissue blocks were fixed in 4% neutral formaldehyde for 48 hours. The femur tissue was placed in EDTA decalcification agent after fixation was completed, and fresh solution was replaced daily until the tissue was softened. After fixation was completed, the tissue blocks were rinsed with running water for 3 times, 30 minutes each. The tissue blocks were placed in embedding boxes and gradient dehydration was performed using a fully automatic dehydration instrument (Leica, TP1020). The dehydrated tissue blocks were placed in a mold containing molten wax, and an embedding machine was used to embed them into tissue paraffin blocks for subsequent sectioning. A paraffin sectioning machine (Leica, RM2016) was used to cut the tissue paraffin blocks into 4 μm thick paraffin sections for subsequent histological staining.

[0160] 4.2.2 Histological staining method

[0161] (1) Hematoxylin-eosin staining (HE staining)

[0162] The 4 μm thick paraffin sections were placed on a sectioning machine and sectioned (65°C, 60 min), and then dehydrated to the water phase (xylene 15 min→ anhydrous ethanol 5 min→ 95% ethanol 5 min→ 75% ethanol 5 min→ running water for 5 min); after staining in hematoxylin staining solution for 15 min, rinse with running water for 5 min; differentiate with 1% hydrochloric acid ethanol for 3-6 s (the differentiation time is adjusted according to the time the differentiation solution is placed), and rinse with running water for 5 min; after staining in eosin staining solution for 1 min, dehydrate and permeabilize with xylene (75% ethanol 5 min→ 95% ethanol 5 min→ anhydrous ethanol 5 min→ xylene 15 min), and seal with neutral balsam.

[0163] (2) Sirius red staining

[0164] The paraffin section with a thickness of 4 μm was placed on a slice machine for slice baking (65 °C, 60 min), and then dewaxed to an aqueous phase (xylene 15 min→ absolute ethanol 5 min→ 95% ethanol 5 min→ 75% ethanol 5 min→ distilled water washing 5 min); placed in PBS for 2 min, then the slice was placed in the Picrosirius staining solution for 8 min, after staining, quickly placed in absolute ethanol for 1 s, and then permeated with xylene for 15 min, and the slice was sealed with neutral balsam.

[0165] (3) Prussian blue staining

[0166] The paraffin section with a thickness of 4 μm was placed on a slice machine for slice baking (65 °C, 60 min), and then dewaxed to an aqueous phase (xylene 15 min→ absolute ethanol 5 min→ 95% ethanol 5 min→ 75% ethanol 5 min→ distilled water washing 5 min); placed in PBS for 2 min, then the slice was placed in the Picrosirius staining solution for 8 min, after staining, quickly placed in absolute ethanol for 1 s, and then permeated with xylene for 15 min, and the slice was sealed with neutral balsam.

[0167] 4.2.3 Histopathological evaluation;

[0168] (1) Evaluation of lens dislocation

[0169] We observed the pathological changes of eyeballs by HE staining method, and the ectopic degree of mouse eyeball lens was evaluated by measuring the distance from ciliary muscle to lens. After taking photos of mouse eyeballs, the axial length, width and anterior chamber depth of eyeballs were measured according to the general diagram of eyeballs, and the measurement method was as shown in Figure 1 .

[0170] (2) Evaluation of femur tissue structure

[0171] After taking photos of mouse femurs, the pathological changes of femurs were characterized by measuring the thickness of cortical bone.

[0172] (3) Evaluation of lung tissue structure

[0173] The pathological changes of lung were observed by HE staining method. The pathological changes of mouse lung were measured by investigating the number of alveoli per unit area and the area ratio of alveolar septum.

[0174] (4) Evaluation of heart tissue structure

[0175] HE staining was used to observe cardiac lesions. The degree of cardiac fibrosis was characterized using Sirius red staining, assessed by measuring the amount of Sirius red in cardiac tissue and the proportion of Sirius red in coronary vessels. Furthermore, the proportion of hemosiderin was measured by calculating the percentage of black material on the valve surface. Finally, Prussian blue staining was used to observe for the presence of hemorrhagic lesions in the heart.

[0176] 5. Statistical Analysis

[0177] All experimental data are expressed as mean ± standard error (mean ± SEM). Grubbs' method was used for preliminary detection of spillover values. For comparisons between two groups, if both normality and homogeneity of variance were satisfied, Student's t-test was used; otherwise, Mann-Whitney U test was used. For comparisons among multiple groups, if both normality and homogeneity of variance were satisfied, one-way ANOVA with completely randomized design was used; otherwise, Kraskal-Wallis rank-sum test was used. P < 0.05 was considered statistically significant.

[0178] IV. Experimental Results

[0179] 1. Improves the ocular structure of Marfan mice, especially inhibits lens dislocation (lens ectopic displacement).

[0180] like Figure 2 As shown, the degree of lens displacement was characterized by measuring the distance from the ciliary muscle to the lens. Marfan mice showed a significantly higher degree of lens displacement compared to age-matched wild-type mice. SF administration significantly improved lens displacement.

[0181] 2. Improves bone structure in Marfan mice

[0182] Measurements of femur length revealed that the femurs of Marfan mice were longer than those of age-matched wild-type mice, and SF treatment showed a tendency to normalize femur length. Assessment of cortical bone thickness showed that the femoral cortex of Marfan mice was thinner than that of age-matched wild-type mice. Both MHMA and SF showed an effect on improving cortical bone thickness in Marfan mice, with SF showing a more pronounced effect on improving cortical bone thickness compared to MHMA. Figure 3 As shown.

[0183] 3. Improve lung tissue structure

[0184] Based on HE staining analysis of the lungs, compared with wild-type mice, Marfan mice exhibited pathological changes in their lungs, including alveolar fusion and missing pulmonary septa. Both MHMA and SF showed ameliorative effects on the lung lesions in Marfan mice, with SF showing a more significant improvement compared to MHMA. Figure 4 As shown.

[0185] 4. Improvement of cardiac tissue structure

[0186] According to the investigation of myocardial fibrosis by Picrosirius red staining, SF and MHMA both showed significant effects in improving myocardial fibrosis, as shown in Figure 5 .

[0187] According to the observation of HE staining, black particle deposits were observed in the aortic valve, and no black particle was observed in the rest of the tissue such as myocardial cells, indicating that the presence of particles on the valve has location specificity. The deposition of particles on the aortic valve of Marfan mice is very obvious, and SF significantly improves the deposition of particles on the aortic valve, and MHMA also shows the ability to improve, as shown in Figure 6 .

[0188] Through the Prussian blue results, it was found that Prussian blue was positive in the aortic ring of Marfan mice, indicating bleeding. There was basically no area of Prussian blue positive around the aortic ring of Marfan mice treated with SF and MHMA Figure 7 .

[0189] 5. Improvement of aortic wall hardening in Marfan mice

[0190] In order to explore the therapeutic effect of SF on aortic lesions in Marfan mice, we used a small animal ultrasound instrument Vevo3100 to measure the PWV of the ascending aorta of mice, and measured the diameter of the ascending aorta of mice after dissection Figure 8 .

[0191] Among them, the PWV of the wild type solvent control group was 2.82±0.45m / s, relative to the PWV of the Marfan mouse solvent control group(4.59±0.32m / s), SF significantly down-regulated the PWV(2.89±0.41m / s, p<0.001). It can be seen that SF not only inhibits the expansion process of the aorta of Marfan mice, but also significantly improves the process of aortic hardening.

[0192] Example 2

[0193] I. Experimental instruments

[0194] Small animal CT scanner(Inveon, Siemens)

[0195] Pathological section fluorescence scanner(NanoZoomer 2.0HT, Hamamatsu Photonics Corporation)

[0196] PowerLab eight-channel physiological recorder(PowerLab8 / 30, ADInstruments)

[0197] II. Experimental methods

[0198] 1. Drug formulation

[0199] Solution Name Procedure 50 mg / kg SF SF was dissolved at 2.5 mg / ml in ultrapure water and mixed by vortexing on a vortex mixer. 30 mg / kg SF SF was dissolved at 1.5 mg / ml in ultrapure water and mixed by vortexing on a vortex mixer. 10 mg / kg SF SF was dissolved at 0.5 mg / ml in ultrapure water and mixed by vortexing on a vortex mixer.

[0200] 2. Animal grouping

[0201] 6-7 month old mice were randomly divided into four groups, namely solvent control group, 10 mg / kg SF group, 30 mg / kg SF group, 50 mg / kg SF group (n = 12)

[0202] 3. Experimental animal administration

[0203] 3.1 Solvent control group: mice were given 20 ml / kg of ultrapure water by gavage five times a week, and the administration period was 90 days

[0204] 3.2 10 mg / kg SF group: mice were given 10 mg / kg of low-dose by gavage five times a week, with a volume of 20 ml / kg, and the administration period was 90 days

[0205] 3.3 30 mg / kg SF group: mice were given 30 mg / kg of medium-dose by gavage five times a week, with a volume of 20 ml / kg, and the administration period was 90 days

[0206] 3.4 50 mg / kg SF group: mice were given 50 mg / kg of high-dose by gavage five times a week, with a volume of 20 ml / kg, and the administration period was 90 days.

[0207] 4. Efficacy evaluation

[0208] 4.1 Histopathological evaluation

[0209] (1) Evaluation of lung tissue structure

[0210] The lung lesions were observed by HE staining method. The number of alveoli per unit area and the area ratio of alveolar septum were used to measure the lung lesions of mice. The slides were read according to the scoring standard, and the lung injury of each sample was scored according to the following five items: cell infiltration, hemorrhage, alveolar septum swelling, lung injury and injury range. 0 points (no injury), 1 point (mild injury), 2 points (moderate injury), 3 points (severe injury), record the lung tissue injury score of each mouse, and statistically analyze.

[0211] 4.2 After the completion of the administration period, the mice were subjected to whole body CT scanning

[0212] (1) Data acquisition: use 50 (Vic Inc, France) and Xylazine (Tirex (Shanghai) Chemical Industry Development Co., Ltd.) mixed solution as anesthetic, intraperitoneal injection of 10 uL / g mixed solution for anesthesia, loss of righting reflex. CT scan conditions: current: 500 uA; exposure time: 950 ms; voltage: 60 kV; bed length: 3 bed. Before starting the scan, calibrate according to the bed length, after calibration, cut off the ear tag of the mouse, put it into the small animal CT scanner (Siemens Inveon), and scan at medium resolution.

[0213] (2) Bone density and trabecular bone morphology analysis: analyzed using Inoveon Research Workplace (IRW) software, one layer by one layer, the femoral trochanter bone marrow and trabecular bone area were selected, the threshold was used to extract the bone marrow and trabecular bone, and the bone density and trabecular bone morphology data were calculated using the software function. The formula for calculating the bone volume fraction is bone volume fraction = total bone volume / (total bone volume + bone marrow volume); bone surface area bone volume ratio = bone surface area / bone volume; trabecular bone thickness = 2 / (bone surface area / bone volume). For cortical bone thickness, manually measure the cortical bone thickness of the greater trochanter and the junction of the greater trochanter and the femoral neck at 4 or more positions, and take the average.

[0214] (3) Lung injury analysis: analyzed using IRW software, the analysis area was selected to the maximum transverse, coronal and sagittal planes of the left and right lungs in the analysis interface, and each plane was compared and analyzed.

[0215] 4.3 Blood oxygen saturation evaluation

[0216] The PowerLab eight-channel physiological recorder was connected to the blood oxygen sensor transducer and the blood oxygen probe, and the infrared probe of the blood oxygen probe was clamped on the left hind foot of the animal. After waiting for a period of time, the blood oxygen saturation data of the animal could be collected.

[0217] Four, experimental results

[0218] 1. SF improves bone structure in Marfan mice

[0219] It was found by CT measurement that the trabecular bone volume fraction of the femoral trochanter of Marfan mice decreased significantly, and the cortical bone thickness had a downward trend. After giving SF, the trabecular bone volume fraction and cortical bone thickness of the greater trochanter were significantly increased. In terms of trabecular bone surface area bone volume ratio, due to the significant decrease in bone volume of Marfan mice, the bone area fraction increased significantly, indicating that the trabeculae were broken. This change decreased significantly after administration of 10 mg / kg SF and returned to normal level. In terms of trabecular bone thickness, the trabecular bone thickness increased significantly to the level of control mice after administration of 10 mg / kg SF, such asFigure 9 Quantitative results of each group are shown in Table 1.

[0220] Table 1. CT quantitative data of femur greater trochanter of mice

[0221]

[0222] 2. SF improves lung tissue structure

[0223] From the CT scan results, the male mice of the Marfan group showed loss of normal lung structure due to alveolar fusion, and thus presented low-density shadows without lung texture, showing the image of early pneumothorax. After treatment with SF, an improvement trend was shown, as shown in Figure 10 A-D.

[0224] For female mice, the left and right lungs of the Marfan mice showed low-density shadows without lung texture in the transverse and coronal planes, and there was a recovery trend by giving SF, as shown in Figure 11 A-D.

[0225] In summary, the Marfan mice showed loss of lung structure compared with normal mice. In male mice, SF could improve the right lung. In female mice, administration could improve both the left and right lungs.

[0226] The lung structure of mice was evaluated by histological staining. For male mice, the Marfan lung damage was greater, and the administration improved in a dose-dependent manner; for female mice, administration of any dose could improve lung structure changes, as shown in Figure 12 .

[0227] Further analysis of the blood oxygen saturation of mice found that the blood oxygen saturation of Marfan mice was significantly reduced, and administration could significantly improve the blood oxygen saturation, as shown in Figure 13 and Table 2.

[0228] Table 2. Quantitative data of blood oxygen saturation

[0229]

[0230]

[0231] In summary, the effective dose of SF in the treatment of Marfan mice can be further reduced (≤10 mg / kg), and shows a certain dose-dependent manner, which shows that SF has a very excellent therapeutic effect on Marfan syndrome.

[0232] V. Conclusion

[0233] The marfan mouse animal model and wild type mice of the same month are adopted. Through 90-day cycle of intragastric administration, CMCNa or ultrapure water is used as solvent control, ultrasonic detection analysis, histological evaluation, daily health detection, biochemical index determination and organ coefficient determination and other methods are adopted to comprehensively evaluate the treatment effect of SF and MHMA and other series of compounds on the organ pathological changes of marfan mice and long-term drug toxicity.

[0234] Experiments prove that the compound of the application can significantly improve the pathological process of the eyes, lungs, hearts, bones and blood vessels of marfan mice, has very excellent treatment effect on connective tissue diseases represented by marfan, has the advantages and characteristics of multi-organ protection. In addition, long-term administration of SF does not cause damage to the liver, kidney, heart and other organs, and no significant effect of SF on physiological and biochemical indexes is found, and the safety is very clear.

[0235] All the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for the prevention or treatment of a connective tissue disease; wherein R1, R2 are each independently selected from the group consisting of OH and C1-C8 alkoxy; and R3, R4 and R5 are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted C1-C8 alkyl; R6 is selected from the group consisting of H, halogen and OH; R7is selected from the group consisting of H, halogen, substituted or unsubstituted phenyl; is a double bond; R8 is -(C=0)-R9; R9is selected from the group consisting of hydroxyl, and -O-R 11 ; R 11 is selected from the group consisting of substituted or unsubstituted Ci-Cs alkyl, substituted or unsubstituted C2-C8 alkenyl, substituted or unsubstituted C2-C8 alkynyl, and substituted or unsubstituted C3-C10 cycloalkyl; and wherein, said "substituted" means substituted with one or more substituents selected from the group consisting of halogen, deuterium and C1-C6 alkyl.

2. Use according to claim 1, characterized in that, said connective tissue disease is Marfan syndrome, Ehlers-Danlos syndrome, Takayasu arteritis, MASS syndrome, Weill-Marchesani syndrome, geleophysic dysplasia, acromicric dysplasia, Myhre syndrome, stiff skin syndrome, Wegener's granulomatosis, giant cell arteritis, skeletal dysplasia, aortic root dilatation or Sjogren's syndrome.

3. Use according to claim 1, characterized in that, said connective tissue disease is a connective tissue disease resulting from a mutation in FBN1.

4. The use according to claim 1, characterized in that, one of R1 and R2 is C1-C8 alkoxy.

5. The use according to claim 1, characterized in that, R1 is OH and R2 is C1-C8 alkoxy.

6. The use according to claim 1, characterized in that, R7 is H or halogen.

7. The use according to claim 1, characterized in that, R7 is substituted or unsubstituted C6 aryl, and said "substituted" means substituted with one or more groups selected from the group consisting of halogen, C1-C6 alkyl.

8. The use according to claim 1, characterized in that, R9 is OH or C1-C3 alkoxy.

9. The use according to claim 1, characterized in that, said connective tissue disease is Marfan syndrome.

10. The use according to claim 1, characterized in that, said compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of: or a sodium, potassium, magnesium, lithium or piperazine salt thereof.

11. Use according to claim 1, characterized in that, said prevention or treatment of a connective tissue disease comprises prevention or treatment of a pathology selected from the group consisting of ocular, skeletal, pulmonary, cardiac, vascular, cutaneous or a combination thereof.

12. The use according to claim 1, characterized in that, said prevention or treatment of a connective tissue disease is for one or more uses selected from the group consisting of: improving lens ectopia; improving bone structure (increasing bone density, bone thickness); improving lung damage (such as alveolar fusion, absence of lung septa); improving cardiac damage (such as myocardial fibrosis, decreased cardiac function); improving aortic pathology (such as aortic valve granulomata deposition, aortic dilatation, improving aortic wall stiffness).

13. The use according to claim 1, characterized in that, said pharmaceutical composition is in a dosage form selected from the group consisting of a liquid formulation, a solid formulation, a semi-solid dosage form, a gaseous dosage form.

14. The use according to claim 1, characterized in that, said pharmaceutical composition is in a dosage form selected from the group consisting of an injection, a suspension, an eye drop, a spray, an oral formulation.

15. The use according to claim 1, characterized in that, said pharmaceutical composition is in a dosage form selected from the group consisting of a solution (such as a syrup, an injection), a suspension, an emulsion, a tablet, a capsule, a granule, a powder, an ointment, a cream, a gel, a suppository, an aerosol, a spray, a powder spray, an injection, a powder injection, a capsule, a tablet, a pill, a powder, a granule, a syrup, a patch, an oral solution or a tincture.

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