Compound for treating connective tissue diseases, and pharmaceutical composition comprising same

Abstract

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

Description

Compounds for treating connective tissue disease and pharmaceutical compositions thereof TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a compound for treating connective tissue disease and pharmaceutical compositions 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, and 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 few cases, Marfan syndrome patients may also be relatively short in stature, but due to the mutation of FBN1 gene leading to significant expansion of the aortic root, they are 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 the degree is relatively light, and the skin stripes are 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 body characteristics of Marfan syndrome. Patients with Weill-Marchesani syndrome also have lens dislocation, but unlike Marfan syndrome, the eye disease may also be accompanied by small and thick cornea, shallow anterior chamber, and other characteristics. In severe cases, it can affect vision and intraocular pressure, and cause glaucoma and other eye diseases. Geleophysic dysplasia, acromicric dysplasia, and Myhre syndrome have common characteristics such as short stature, short limbs, and limited joint movement, but there are also certain differences in their respective clinical manifestations. Stiff skin syndrome is mainly manifested as thickening and hardening of the skin, resulting in reduced skin elasticity and limited movement, similar to the manifestations of scleroderma. It can affect the skin of multiple parts of the body, and in severe cases, it can affect the normal life and activities of patients. These diseases cover the main symptoms that FBN1 gene mutations can cause, and have different clinical characteristics and severity.

[0005] Ehlers-Danlos syndrome (EDS) is also known as systemic elastofibrodysplasia. Clinically, it is characterized by excessive stretching of the skin and joints, easy tissue damage, increased fragility, difficult wound healing, increased vascular fragility, eye abnormalities, and internal organ abnormalities. It is a congenital metabolic abnormality 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 thereby. SUMMARY

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

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

[0009] wherein R1, R2 are each independently selected from the group consisting of OH and C1-C8 alkoxy; and

[0010] R3, R4, and R5 are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted Ci-C8alkyl;

[0011] R6 is selected from the group consisting of H, halogen, and OH;

[0012] R7 is selected from the group consisting of H, halogen, substituted or unsubstituted phenyl; is a double bond;

[0013] R8 is -(C=0)-R9;

[0014] R9 is selected from the group consisting of hydroxyl, and -0-R 11 ;

[0015] R 11 is selected from the group consisting of substituted or unsubstituted Ci-C8alkyl, substituted or unsubstituted C2-C8alkenyl, substituted or unsubstituted C2-C8alkynyl, and substituted or unsubstituted C3-C10cycloalkyl; and

[0016] wherein said "substituted" is substituted with one or more substituents selected from the group consisting of halogen, deuterium, and Ci-C6alkyl.

[0017] 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 ectaisia, or Sjogren's syndrome.

[0018] 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 ectaisia, or Sjogren's syndrome; preferably Marfan syndrome.

[0019] In another preferred embodiment, the connective tissue disease is a connective tissue disease resulting from a mutation in FBN1.

[0020] In another preferred embodiment, one of R1and R2is Ci-C8alkoxy; preferably one of R1and R2is Ci-C3alkoxy.

[0021] In another preferred embodiment, R1is OH.

[0022] In another preferred embodiment, R1is OH, and R2is Ci-C8alkoxy.

[0023] In another preferred embodiment, R2 is C1-C6 alkoxy, preferably R2 is C1-C3 alkoxy.

[0024] In another preferred embodiment, R3, R4 and R5 are each independently H or halogen, preferably H.

[0025] In another preferred embodiment, R6 is H or halogen, preferably H.

[0026] In another preferred embodiment, R7 is H or halogen, preferably H.

[0027] 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.

[0028] In another preferred embodiment, R9 is OH or C1-C3 alkoxy, preferably R9 is OH or methoxy.

[0029] In another preferred embodiment, R1 and R9 are both OH.

[0030] In another preferred embodiment, R 11 is C1-C8 alkyl, preferably C1-C3 alkyl or methyl.

[0031] In another preferred embodiment, the compound is selected from the group consisting of:

[0032] 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.

[0033] 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.

[0034] In another preferred embodiment, the prevention or treatment of connective tissue disease is for one or more uses selected from the group consisting of:

[0035] Improving lens ectopia;

[0036] Improving bone structure (increasing bone density, bone thickness);

[0037] Improving lung injury (such as alveolar fusion, lung septum absence);

[0038] Improving heart injury (such as myocardial fibrosis, heart function decline);

[0039] Improving aortic disease (such as aortic valve granular material deposition, aortic dilation, improving aortic wall sclerosis).

[0040] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid preparations (such as solutions, emulsions, suspensions), solid preparations (such as lyophilized preparations).

[0042] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of injections (such as injection solutions or powder injections), oral preparations (such as capsules, tablets, pills, powders, granules, syrups, oral solutions or tinctures), more preferably, the dosage form is an oral preparation.

[0043] 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.

[0044] In another preferred embodiment, the prevention or treatment of connective tissue disease comprises prevention or treatment of a lesion selected from the group consisting of eye, bone, lung, heart, blood vessel, skin or a combination thereof.

[0045] 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.

[0046] In another preferred embodiment, the connective tissue disease is a connective tissue disease caused by FBN1 mutation.

[0047] In another preferred embodiment, the connective tissue disease is Marfan syndrome.

[0048] In another preferred embodiment, the subject is a mammal.

[0049] In another preferred embodiment, the subject is a human, a mouse or a rat.

[0050] In another preferred embodiment, the subject has a connective tissue disease, preferably Marfan syndrome.

[0051] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a simple structure diagram of eyeball;

[0053] Figure 2 is a representative diagram of the relative position of ciliary muscle and lens

[0054] Figure 3 is a representative image of femur histology HE staining;

[0055] Figure 4 is a representative image of lung histology HE staining;

[0056] Figure 5 is a representative image of myocardium Sirius red staining;

[0057] Figure 6 is a representative image of aortic valve Sirius red staining;

[0058] Figure 7 is a representative image of aortic valve Prussian blue staining;

[0059] Figure 8 is a representative image of aortic ultrasound;

[0060] Figure 9 is a representative image of mouse femur greater trochanter trabecular bone and the results of trabecular bone morphology quantification;

[0061] Figure 10 is a result image of male mouse lung CT scan, (A) a representative image of lung cross section; (B) a representative image of lung coronal section; (C) a representative image of left lung sagittal section; (D) a representative image of right lung sagittal section;

[0062] Figure 11 is a result image of female mouse lung CT scan, (A) a representative image of lung cross section; (B) a representative image of lung coronal section; (C) a representative image of left lung sagittal section; (D) a representative image of right lung sagittal section;

[0063] Figure 12 is a representative image of lung HE staining of male and female mice;

[0064] Figure 13 is a quantitative image of blood oxygen saturation. DETAILED DESCRIPTION

[0065] 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.

[0066] Terminology

[0067] 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.

[0068] 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".

[0069] As used herein, the term "alkyl" by itself or as part of another substituent group refers to 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 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.

[0070] As used herein, the term "alkenyl" includes straight chain or branched chain alkenyl groups. For example, C2-C8alkenyl refers to straight chain or branched chain alkenyl groups having from 2 to 8 carbon atoms, for example, C2-C6alkenyl or C2-C4alkenyl, specifically, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0071] As used herein, the term "alkynyl" includes straight chain or branched chain alkynyl groups. For example, C2-C8alkynyl refers to straight chain or branched chain alkynyl groups having from 2 to 8 carbon atoms, for example, C2-C6alkynyl or C2-C4alkynyl, specifically, such as ethynyl, propynyl, butynyl, or the like.

[0072] As used herein, the term "C3-C10cycloalkyl" refers to cycloalkyl groups having from 3 to 10 carbon atoms. It can be monocyclic, for example, 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, for example, a bridged or spirocyclic form.

[0073] As used herein, the term "C1-C8alkoxy" refers to straight chain or branched chain alkoxy groups having from 1 to 8 carbon atoms; for example, methoxy, ethoxy, propyloxy, isopropyloxy, butoxy, isobutyloxy, t-butyloxy, and the like.

[0074] As used herein, the term "plurality" means 2 or more, such as 2, 3, 4, 5, or 6, preferably 2 or 3.

[0075] Active ingredient

[0076] 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.

[0077] The present invention is also intended to include stereoisomers, crystalline forms, hydrates, or solvates of the compounds of Formula I.

[0078] Connective tissue disease

[0079] 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.

[0080] 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.

[0081] 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.

[0082] Marfan's syndrome

[0083] 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 FBNl, 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.

[0084] 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.

[0085] 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).

[0086] Use

[0087] It has been unexpectedly found that the compound of formula I of the present application can prevent or treat connective tissue disease.

[0088] 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.

[0089] Pharmaceutical composition and administration method

[0090] 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.

[0091] 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.

[0092] "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. ), wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

[0093] 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.

[0094] 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 excipients 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.

[0095] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings and shells 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 desired, with one or more of the excipients described above.

[0096] 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.

[0097] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0098] 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, and agar-agar, or combinations thereof.

[0099] Compositions for parenteral injection can contain 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.

[0100] 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 of a physiologically acceptable excipient, such as lactose or starch, or a sterile oil, such as olive oil or sesame oil, or a combination of both, in an amount sufficient to carry the active component.

[0101] The active ingredients of the present application can be administered alone, or in combination with other pharmaceutically acceptable compounds.

[0102] 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 dosages. It will be appreciated that the specific dose level and frequency of dosage for any particular patient will depend on a variety of factors, including the age, size, general health, diet, individual response to the active ingredients of the present application, time of administration, severity of the disease being treated, the particular compound being administered, the dosing regimen and the mode of administration. The therapeutically effective amount for a given situation will be determined by routine experimentation and is within the competence and judgment of the clinician or physician. In any case, the active ingredients will be administered on the basis of the individual needs of the patient and in a manner to deliver a therapeutically effective amount.

[0103] The principal advantages of the present application include:

[0104] 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.

[0105] 2. The compound of the present application is safe in use and has small toxic and side effects.

[0106] 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.

[0107] I. Experimental animals

[0108] 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.

[0109] II. Experimental materials and reagents

[0110] 1. Experimental instruments

[0111] Fluorescence microscope (BX51, OLYMPUS, Japan)

[0112] Gel electrophoresis instrument (AD1603E, Takara, Japan)

[0113] Gel imaging instrument (Tanon-2500, Shanghai Tianyi Instrument and Equipment Company)

[0114] Small animal ultrasound instrument (Vevo3100, FUJIFILM VisualSonics, Japan)

[0115] PCR instrument (MX3000P, Agilent, USA)

[0116] 2. Experimental reagents

[0117] Xylazine (M0413A, Meridian Biotech, Inc.)

[0118] Xylazine (M0413A, Meridian Biotech, Inc.)

[0119] Isoflurane (Shenzhen Ruivode Life Science Co., Ltd.)

[0120] Hematoxylin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0121] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0122] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0123] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0124] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0125] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0126] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0127] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0128] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0129] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0130] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0131] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0132] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0133] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.) Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0134] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0135] Eosin staining solution (HS0920Y, Shanghai Yifan Biological Technology Co., Ltd.)

[0136] Experimental methods

[0137] 1. Drug preparation

[0138] 2. Animal grouping

[0139] 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).

[0140] 3. Experimental animal administration

[0141] 3.1 Solvent control group: the mice were administered with 0.5% CMCNa 10 ml / kg by gavage five times a week, and the administration period was 90 days

[0142] 3.2 SF group: the 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, and the administration period was 90 days

[0143] 3.3 MHMA group: the 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, and the administration period was 90 days

[0144] 4. Efficacy evaluation

[0145] 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.

[0146] (1) Anesthesia: the 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 fixed as soon as the righting reflex was lost.

[0147] (2) Fixation: the mice were supine on the ultrasound heating platform, with the nose placed in the breathing tube, and maintained under anesthesia with 2% isoflurane. The limbs of the mice were attached to the four electrocardiogram (ECG) electrodes on the platform for measuring heart rate, ECG and respiratory rate. The rectal probe was used to monitor the body temperature, and the heating platform was used to maintain the body temperature at 36-38℃.

[0148] (3) Ultrasound measurement

[0149] Probe type: MX550D ultrasound probe (25-55 MHz)

[0150] Aortic diameter measurement: ultrasound measurement was performed on the right side of the mouse chest under B mode to obtain the ultrasound image from the root of the mouse aorta to the beginning of the descending aorta.

[0151] PWV measurement: Under PW mode, pulse Doppler signals were recorded at the aortic root and the beginning of the descending aorta. The Doppler waveforms were measured according to the corresponding Doppler images, with the R-wave peak of the electrocardiogram as the starting point to measure the time of blood to the aortic root (t1) and the beginning of the descending aorta (t2) (t1and t2values are the average of 10 cardiac cycles); the length of the aortic arch (d) is the distance of the propagation trajectory between the two points where the Doppler signals are recorded. Finally, the PWV is calculated by dividing the length of the aortic arch (d) by the blood passing time (t2-t1) (PWV=d / (t2-t1)).

[0152] 4.2 Histological evaluation

[0153] 4.2.1 Tissue fixation, dehydration and sectioning method

[0154] Standardized sampling was performed for each tissue site, and fresh tissues were cut into approximately 3-4 mm tissue blocks using a blade. The cut tissue blocks were fixed in 4% neutral formaldehyde for 48 hours. After fixation was completed, the femur tissue was placed in EDTA decalcification agent, and fresh solution was replaced daily until the tissue was softened. After fixation was completed, the tissue was rinsed with running water for 3 times for 30 minutes. 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.

[0155] 4.2.2 Histological staining method

[0156] (1) Hematoxylin-eosin staining (HE staining)

[0157] The 4 μm thick paraffin sections were placed on a sectioning machine and sectioned (65°C, 60 min), and then dehydrated to 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, running water was used for 5 min; 1% hydrochloric acid ethanol differentiation was performed for 3-6 s (the differentiation time was adjusted according to the time of differentiation solution), and running water was used for 5 min; after staining in eosin staining solution for 1 min, the sections were placed in water, dehydrated, and then permeabilized with xylene (75% ethanol 5 min→ 95% ethanol 5 min→ anhydrous ethanol 5 min→ xylene 15 min), and then sealed with neutral balsam.

[0158] (2) Sirius red staining

[0159] 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.

[0160] (3) Prussian blue staining

[0161] 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.

[0162] 4.2.3 Histopathological evaluation;

[0163] (1) Evaluation of lens dislocation

[0164] We observed the pathological changes of the eyeball by HE staining method, and the ectopic degree of the mouse eyeball lens was evaluated by measuring the distance from the ciliary muscle to the lens. After taking a photo of the mouse eyeball, the axial length, width and anterior chamber depth of the eyeball were measured according to the general diagram of the eyeball, and the measurement method is shown in Figure 1.

[0165] (2). Evaluation of femur tissue structure

[0166] After taking a photo of the mouse femur, the pathological changes of the femur were characterized by measuring the thickness of the cortical bone.

[0167] (3) Evaluation of lung tissue structure

[0168] The pathological changes of the lung were observed by HE staining method. The pathological changes of the mouse lung were measured by investigating the number of alveoli per unit area and the area ratio of alveolar septum.

[0169] (4) Evaluation of heart tissue structure

[0170] HE staining method was used to observe the pathological changes of the heart. The degree of fibrosis in the heart was characterized by Sirius red staining, and the content of Sirius red in the heart tissue and the proportion of Sirius red in the coronary vessels were measured to evaluate the effect. In addition, the proportion of hemosiderin was measured by calculating the percentage of valve black substance area. Finally, the presence of hemorrhagic lesions in the heart was observed by Prussian blue staining technique.

[0171] 5. Statistical analysis

[0172] All experimental data were expressed as mean ± standard error (mean ± SEM). The Grubbs method was used for preliminary detection of outliers. Comparison of data between two groups, if normal distribution and homogeneity of variance were met at the same time, Student's t test method was used, otherwise Mann whitney U test method was used; comparison of data among multiple groups, if data met normal distribution and homogeneity of variance at the same time, one-way ANOVA was used, otherwise Kraskal Wallis rank sum test was used; P<0.05 was considered statistically significant.

[0173] Four. Experimental results

[0174] 1. Improve the eye structure of Marfan mice, especially inhibit lens dislocation (ectopia lentis).

[0175] As shown in Figure 2, the degree of lens dislocation was characterized by measuring the distance from the ciliary muscle to the lens. Compared with the same age wild type mice, the degree of lens dislocation in Marfan mice was significantly improved by SF administration.

[0176] 2. Improve the bone structure of Marfan mice

[0177] By measuring the length of the femur, it was found that the femur of Marfan mice was longer than that of the same age wild type mice, and SF treatment could make the femur length tend to normal. By evaluating the cortical bone thickness, it was found that the femur cortical bone of Marfan mice was thinner than that of the same age wild type mice, and MHMA and SF showed an improvement effect on the cortical bone thickness of Marfan mice, and compared with MHMA, SF showed a more obvious trend in improving the cortical bone thickness, as shown in Figure 3.

[0178] 3. Improve the structure of lung tissue

[0179] According to the analysis of lung HE staining, compared with wild type mice, Marfan mice showed pathological changes of alveolar fusion and interstitial septum absence, and MHMA and SF showed an improvement effect on the lung lesions of Marfan mice, and compared with MHMA, SF showed a more obvious improvement effect, as shown in Figure 4.

[0180] 4. Improve the structure of heart tissue

[0181] According to the investigation of myocardial fibrosis by Picrosirius staining, SF and MHMA both showed significant effects in improving myocardial fibrosis, as shown in Figure 5.

[0182] According to the observation of black particle deposits in the aortic valve during HE staining, no black particles were observed in the remaining tissues 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.

[0183] Through the Prussian blue results, it was found that Prussian blue was positive in the aortic ring of Marfan mice, indicating bleeding, and there was no area of Prussian blue positive around the aortic ring of Marfan mice treated with SF and MHMA (Figure 7).

[0184] 5. Improving the hardening of the aortic wall of Marfan mice

[0185] In order to explore the therapeutic effect of SF on the aortic lesions of 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).

[0186] 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.

[0187] Example 2

[0188] I. Experimental instruments

[0189] Small animal CT scanner (Inveon, Siemens)

[0190] Pathological section fluorescence scanner (NanoZoomer 2.0HT, Hamamatsu Photonics Corporation)

[0191] PowerLab eight-channel physiological recorder (PowerLab8 / 30, ADInstruments)

[0192] II. Experimental methods

[0193] 1. Drug preparation

[0194] 2. Animal grouping

[0195] 6-7 month old Marfan 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)

[0196] 3. Experimental animal administration

[0197] 3.1 Solvent control group: Mice were given 20 ml / kg of ultrapure water by gavage five times a week, with a drug administration period of 90 days

[0198] 3.2 10 mg / kg SF group: Mice were given 20 ml / kg of low-dose 10 mg / kg by gavage five times a week, with a drug administration period of 90 days

[0199] 3.3 30 mg / kg SF group: Mice were given 20 ml / kg of medium-dose 30 mg / kg by gavage five times a week, with a drug administration period of 90 days

[0200] 3.4 50 mg / kg SF group: Mice were given 20 ml / kg of high-dose 50 mg / kg by gavage five times a week, with a drug administration period of 90 days

[0201] 4. Efficacy evaluation

[0202] 4.1 Histopathological evaluation

[0203] (1) Evaluation of lung tissue structure

[0204] 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 statistical analysis.

[0205] 4.2 After the completion of the drug administration period, the mice were subjected to whole body CT scanning

[0206] (1) Data acquisition: Use 50 (Vic Inc, France) and Xylazine (Tirehai (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.

[0207] (2) Bone density and trabecular bone morphology analysis: analyzed using Inoveon Research Workplace (IRW) software, one layer by one layer to circle the femoral trochanter bone marrow and trabecular bone area, use threshold to pull the threshold value to extract bone marrow and trabecular bone, and use the software's own function to calculate the bone density and trabecular bone morphology data. 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 locations, and take the average.

[0208] (3) Lung injury analysis: analyzed using IRW software, and the analysis area was selected to the maximum transverse, coronal and sagittal planes of the left and right lungs in the analysis interface. Each plane was taken for comparative analysis.

[0209] 4.3 Blood oxygen saturation evaluation

[0210] The PowerLab eight-channel physiological recorder was connected with 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.

[0211] Four, experimental results

[0212] 1. SF improves bone structure in Marfan mice

[0213] CT scans revealed a significant decrease in trabecular bone volume fraction and a decreasing trend in cortical bone thickness in the greater trochanter of the femur in Marfan mice. Administration of SF significantly restored both trabecular bone volume fraction and cortical bone thickness in the greater trochanter. Regarding the trabecular bone surface area to bone volume ratio, the significant decrease in bone volume in Marfan mice led to a significant increase in bone surface area fraction, indicating trabecular bone fracture. This change significantly decreased after administration of 10 mg / kg SF, returning to normal levels. As for trabecular bone thickness, administration of 10 mg / kg SF significantly increased trabecular bone thickness compared to Marfan mice, reaching levels comparable to control mice (Figure 9). Quantitative results for each group are shown in Table 1.

[0214] Table 1. Quantitative CT data of mouse femoral greater trochanter.

[0215] 2. SF improves lung tissue structure

[0216] CT scans showed that male mice in the Marfan group exhibited low-density shadows without lung markings due to alveolar fusion causing loss of normal lung structure, indicating early-stage pneumothorax. Treatment with SF (sulfate-free lung tissue) showed an improving trend, as shown in Figures 10A-D.

[0217] In female mice, both lungs of Marfan mice showed low-density shadows without lung markings in both transverse and coronal sections. The lungs showed a tendency to recover after administration of SF, as shown in Figures 11A-D.

[0218] In summary, Marfan mice exhibited lung structure loss compared to normal mice. In male mice, SF treatment improved the right lung. In female mice, administration improved both lungs simultaneously.

[0219] Histological staining was used to evaluate the lung structure of mice. In male mice, Marfan lung damage was greater, and the drug showed dose-dependent improvement. In female mice, the drug improved lung structure regardless of the dosage, as shown in Figure 12.

[0220] Further analysis of blood oxygen saturation in mice revealed that blood oxygen saturation was significantly reduced in Marfan mice, and that administration of the drug significantly improved blood oxygen saturation, as shown in Figure 13 and Table 2.

[0221] Table 2 Quantitative data on blood oxygen saturation

[0222] In summary, it can be seen that the effective dose of SF in treating Marfan mice can be further reduced (≤10mg / kg) and shows a certain dose-dependent effect, which indicates that SF has a very good therapeutic effect on Marfan syndrome.

[0223] V. Conclusion

[0224] 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.

[0225] 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 is found on physiological and biochemical indexes, and has very clear safety.

[0226] 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: ###0001### 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, R2are each independently selected from the group consisting of OH and C1-C8alkoxy; and R3, R4and R5are each independently selected from the group consisting of H, OH, halogen, substituted or unsubstituted C1-C8alkyl; R6is 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; R8is -(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-C6alkyl.

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 R1and R2is C1-C8alkoxy.

5. The use according to claim 1, characterized in that, R1is OH and R2is C1-C8alkoxy.

6. The use according to claim 1, characterized in that, R7is H or halogen.

7. The use according to claim 1, characterized in that, R7is substituted or unsubstituted C6aryl, and said "substituted" means substituted with one or more groups selected from the group consisting of halogen, C1-C6alkyl.

8. The use according to claim 1, characterized in that, R9is OH or C1-C3alkoxy.

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 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 eye, bone, lung, heart, blood vessel, skin 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 injury (such as alveolar fusion, absence of lung septa); improving heart injury (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 liquid or a tincture.

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