CN109485691B - Tanshinone compound and application thereof in treating hemangioma - Google Patents

Abstract

The invention provides tanshinone compounds and application thereof in treating hemangioma, and particularly provides compounds shown as a formula I below or application of pharmaceutically acceptable salts of the compounds, wherein definitions of groups are described in an instruction. The tanshinone compound has hemangioma cell inhibiting activity and can be used for preparing a pharmaceutical composition for treating hemangioma.

Description

Tanshinone compound and application thereof in treating hemangioma

Technical Field

The invention relates to the field of medicaments, and particularly provides a tanshinone compound and application thereof in treating hemangioma.

Background

Hemangioma (IH), the most common benign tumor in infants, is a true hemangioma resulting from abnormal proliferation of vascular endothelial cells. The incidence rate is about 5% -10%, and the proportion of men and women is 1. The disease is difficult to be found at birth, and a recent queue research suggests that the peak growth period of the infantile hemangioma is mostly 5.5-7.5 weeks, and about 4 weeks on average, then the focus gradually appears, and enters a rapid growth period, the regression is slow, few months are needed, and seven to ten years are needed. The angioma of a few children patients has large area, remains pigmentation, is accompanied by the signs of shallow scar, skin atrophy and sagging and even cannot be healed for life.

The pathogenesis of IH is now obscured. Early theories suggest that maternal behaviour during pregnancy leads to the development of hemangiomas or vascular malformations, and that strawberry hemangiomas were even once thought to be caused by maternal consumption of red fruit. At present, the mechanism of hemangioma is mainly described by the following theories: embryonic vascular dysplasia, angiogenic diseases, effects of estrogen, and the like. Recent studies of hemangiomas have mostly focused on the recruitment of progenitor cells, stromal tumor cells, abnormalities in the extracellular matrix, pro-angiogenic factors, and the like.

More than half of IH can be removed by self, generally advocates that the early stage of hemangioma is in a state of observation and palliative rest, but the occurrence and development of hemangioma may affect the life of a child patient, and as the self-consciousness is improved along with the increase of age, the child patient often shows lack of confidence, anxiety and feelings of guilt, and the like, and particularly when the growth of hemangioma endangers the life or organ functions of the child patient, the child patient should be treated in time. Current treatment modalities include: oral or topical drug therapy, surgical resection, cryotherapy, radionuclide therapy, laser therapy, and the like. Hormones and propranolol are currently the first choice for treating IH orally, and propranolol has become the first line drug for treating IH. The action mechanism of propranolol and hormone on IH is not clear, and the propranolol has obvious curative effect and also faces a plurality of side effects. For example, propranolol can induce bradycardia, different degrees of atrioventricular block, hypotension, cardiogenic shock, bronchospasm, asthma, pulmonary edema, etc.; hormones can cause fungal infections, hypertension, cushing's syndrome, and gastrointestinal upset, among others. Other drugs for external application to hemangioma are still used in trial cases, such as imiquimod, which induces hemangioma regression by producing large amounts of cytokines, but the actual efficacy needs to be observed and summarized. Therefore, the task of developing new anti-hemangioma drugs and developing more effective therapeutic means is urgent.

Disclosure of Invention

The invention aims to provide a novel drug compound with the activity of inhibiting hemangioma cell generation and application thereof in treating hemangioma.

In a first aspect of the invention, there is provided a use of a compound of formula I, or a pharmaceutically acceptable salt thereof:

wherein, ring A is selected from the following group: substituted or unsubstituted 3-to 10-membered carbocyclic ring, substituted or unsubstituted 3-to 10-membered heterocyclic ring, substituted or unsubstituted C ₆ -C ₁₀ An aromatic ring, a substituted or unsubstituted 5-12 membered heteroaromatic ring;

R ₁ selected from the group consisting of: H. oxygen atom (= O), O-R';

R ₂ selected from the group consisting of: H. oxygen atom, O-R';

r' is selected from the following group: H. substituted or unsubstituted C ₂ -C ₁₀ Acyl group of (2), fluorenylmethoxycarbonylacyl (Fmoc), or substituted or unsubstituted C ₁ -C ₁₀ An alkyl group; preferably, R' is H;

R ₃ selected from the group consisting of: H. halogen, oxygen atom, cyano, hydroxy, carboxy, C ₁ -C ₄ Alkoxy group of (C) ₁ -C ₄ Alkyl of (C) ₁ -C ₄ Haloalkyl, substituted or notSubstituted C ₂ -C ₁₀ Acyl, substituted or unsubstituted C ₂ -C ₁₀ Ester group of, -OC (O) -R, -NH-R; wherein R is selected from the group consisting of: H. c ₁ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl groups of (a);

R ₄ selected from the group consisting of: H. halogen, oxygen atom, cyano, hydroxy, carboxy, C ₁ -C ₄ Alkoxy group of (1), C ₁ -C ₄ Alkyl of (C) ₁ -C ₄ Haloalkyl, substituted or unsubstituted C ₂ -C ₁₀ Acyl, substituted or unsubstituted C ₂ -C ₁₀ Ester group of-OC (O) -R, -NH-R; wherein R is selected from the group consisting of: H. c ₁ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl groups of (a);

or R ₁ 、R ₂ 、R ₃ And R ₄ Any two of which, together with the adjacent carbon atom, form a group selected from: substituted or unsubstituted C ₃ -C ₁₀ Carbocyclic ring, substituted or unsubstituted 3-10 membered heterocycle, substituted or unsubstituted C ₆ -C ₁₀ An aromatic ring, a substituted or unsubstituted 5-12 membered heteroaromatic ring;

R ₅ selected from the group consisting of: H. halogen, cyano, hydroxy, C ₁ -C ₄ Alkoxy, -COOR, substituted or unsubstituted C ₁ -C ₁₀ Alkyl, substituted or unsubstituted C ₁ -C ₁₀ Haloalkyl, substituted or unsubstituted C ₂ -C ₁₀ Acyl, substituted or unsubstituted C ₂ -C ₁₀ Ester group of (a); wherein R is selected from the group consisting of: H. c ₂ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl groups of (a);

R ₆ selected from the group consisting of: H. halogen, oxygen atom, cyano, carboxyl, hydroxyl, C ₁ -C ₄ Alkoxy, -COOR, substituted or unsubstituted C ₁ -C ₁₀ Alkyl, substituted or unsubstituted C ₁ -C ₁₀ Haloalkyl, substituted or unsubstituted C ₂ -C ₁₀ Acyl, substituted or unsubstituted C ₂ -C ₁₀ An ester group of (a); wherein R is selected from the group consisting of: H. c ₂ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl groups of (a);

or R ₅ And R ₆ Together form-R ' -O-R ' -, wherein R ' is none, or substituted or unsubstituted C ₁ -C ₄ An alkylene group of (a);

R ₇ is a group on the A ring selected from the group consisting of: H. hydroxy, amino, substituted or unsubstituted C ₁ -C ₁₀ Alkoxy of, -COOR, substituted or unsubstituted C ₂ -C ₁₀ Acyl, substituted or unsubstituted C ₁ -C ₁₀ Alkyl, substituted or unsubstituted C ₂ -C ₁₀ An ester group of (a); wherein R is selected from the group consisting of: H. c ₂ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl groups of (a);

n =1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

in the above formulae, substituted means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: carboxy, phenyl, C ₃ -C ₆ Cycloalkyl of, C ₂ -C ₁₀ Ester group of (2), halogen, C ₁ -C ₁₀ Alkyl-oxy, C ₂ -C ₁₀ Acyl, hydroxy-C ₁ -C ₁₀ An alkylene group of (a);

the "" is a double bond or a single bond;

it is characterized by being used for preparing:

(a) Pharmaceutical compositions for the treatment of hemangiomas;

(b) Pharmaceutical compositions that inhibit the proliferative activity of hemangioma cells;

(c) A pharmaceutical composition for promoting hemangioma cell apoptosis;

(d) A pharmaceutical composition for inhibiting cell tube formation.

In another preferred embodiment, R is ₃ Selected from the group consisting of: H. halogen, oxygen atom, cyano, hydroxy, carboxyl, C ₁ -C ₄ Alkyl of (C) ₁ -C ₄ A haloalkyl group of (a);

R ₄ selected from the group consisting of: H. halogen, oxygen atom, cyano, hydroxy, carboxy, C ₁ -C ₄ Alkyl of (C) ₁ -C ₄ A haloalkyl group of (a);

or R ₃ And R ₄ Together form a group selected from: a substituted or unsubstituted 3-8 membered heterocyclic ring, a substituted or unsubstituted 5-7 membered heteroaromatic ring.

In another preferred embodiment, the a ring is selected from the group consisting of: a substituted or unsubstituted 5-7 membered carbocyclic ring, a substituted or unsubstituted 5-7 membered heterocyclic ring, a substituted or unsubstituted 6 membered aromatic ring, a substituted or unsubstituted 5-7 membered heteroaromatic ring.

In another preferred embodiment, R ₇ Is one or more groups selected from the group consisting of: H. hydroxy, amino, C ₁ -C ₄ Alkyl of (C) ₁ -C ₄ Halogenoalkyl of, C ₁ -C ₄ Alkoxy of, -COOR; wherein R is selected from the group consisting of: H. c ₁ -C ₄ Acyl, substituted or unsubstituted C ₁ -C ₄ Alkyl group of (1).

In another preferred embodiment, R ₁ Is = O.

In another preferred embodiment, R is ₃ Is C ₁ -C ₄ Alkyl or C of ₁ -C ₄ Haloalkyl of (a);

R ₄ selected from the group consisting of: H. an oxygen atom;

or R ₃ And R ₄ Together form a group selected from: a substituted or unsubstituted furan ring, a substituted or unsubstituted dihydrofuran ring, a substituted or unsubstituted tetrahydrofuran ring.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

in another preferred embodiment, the effective dose of the compound of formula I in the pharmaceutical composition is 0.1-50mg/kg body weight, preferably 1-20mg/kg body weight.

In another preferred embodiment, the pharmaceutical composition is in a dosage form selected from the group consisting of: oral dosage forms, and injection dosage forms.

In another preferred embodiment, the pharmaceutical composition is also used for inhibiting angiogenesis of hemangioma endothelial cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 to 3: under the corresponding drug concentration (3 uM and 50 uM) and fixed time (48 h), the cell apoptosis becomes time-dependent and concentration-dependent;

FIG. 4: protein result chart of dihydrotanshinone I and propranolol on apoptosis;

FIG. 5 is a schematic view of: a result chart of the influence of the dihydrotanshinone I and the propranolol on cell tube formation;

FIG. 6: the result chart of the effect of dihydrotanshinone I and propranolol on hemangioma in mice is shown.

Detailed Description

The inventor of the invention has long and intensive research and unexpectedly found that the tanshinone compound has good hemangioma cell inhibitory activity and the activity is superior to that of the conventional general medicament propranolol. The compounds can inhibit the proliferation of hemangioma cells in vivo or in vitro, both time-dependent and dose-dependent. Based on the above findings, the inventors have completed the present invention.

Term(s)

As used herein, the term "C ₁ -C ₄ Alkyl "or" C ₁ -C ₁₀ Alkyl "means a straight or branched chain alkyl group having 1 to 4 or 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

The term "C ₃ -C ₆ Cycloalkyl "means a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, methylcyclobutyl, cyclopentyl, or the like。

As used herein, the term "C ₁ -C ₁₀ Acyl radical "or" C ₁ -C ₄ Acyl "refers to a substituent such as a" straight or branched alkyl/cycloalkyl/aryl-carbonyl having 0 to 9 or 0 to 3 carbon atoms "structure, such as acetyl, propionyl, butyryl, or the like.

The term "C ₂ -C ₁₀ The "ester group" means a substituent such as "straight-chain or branched alkyl/cycloalkyl/aryl-carbonyl-oxygen atom having 1 to 9 carbon atoms" structure, such as acetyl group, propionyl group, butyryl group, or the like.

The term "C ₁ -C ₄ Alkylene "means a group formed after a C1-C4 alkyl group as described above has lost one hydrogen atom, e.g. -CH ₂ -、-CH ₂ -CH ₂ -, or the like.

The term "halogen" refers to F, cl, br and I.

The term "C ₆ -C ₁₀ Aryl "means an aryl group having 6 to 10 carbon atoms, such as phenyl, naphthyl, and the like.

The term "C ₁ -C ₁₀ Heteroaryl "refers to heteroaryl having 1 to 10 carbon atoms and one or more heteroatoms selected from O, S and/or N.

As used herein, the terms "comprising," "including," or "including" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an accurate effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.

Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C ₂ -C ₆ Acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, the term "compound of the invention" refers to a compound of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

Tanshinone compound and application thereof

Hemangioma of infants belongs to surgical hemangioma in traditional medicine. Blood tumor is growing in the skin, and it is mostly caused by blockage of meridians, disharmony between ying and wei and stagnation of qi and blood, so meridians are obstructed. The traditional medical science theory and method formula and medicines are integrated, and hemangioma is mostly caused by qi and blood blocking in channels and collaterals, and mainly takes blood circulation promotion and blood stasis removal as main purposes.

Salvia miltiorrhiza, which is a root component of labiate, was recorded in Shen nong Ben Cao Jing and is a commonly used Chinese medicine for promoting blood circulation, removing blood stasis and promoting tissue regeneration. A series of tanshinone compounds are obtained by separating from a salvia miltiorrhiza fat-soluble extract (tanshinone extract), and in-vitro activity screening finds that the tanshinone compounds have obvious inhibition effect on hemangioma cell proliferation (positive control: propranolol), wherein the effect of Dihydrotanshinone I (DHTS) is obviously superior to the inhibition effect of Propranolol on hemangioma cells. The research of action mechanism shows that DHTS and Propranol can obviously promote the apoptosis of hemangioma cells, and the apoptosis of the hemangioma cells is time-dependent and concentration-dependent under corresponding drug concentration (3 uM and 50 uM) and fixed time (48 h). Western Blot and tube format results show that DHTS acts on hemangioma cells through FAS/FASL and mitochondrial pathways to produce an apoptotic effect and an inhibitory effect on angiogenesis of hemangioma endothelial cells.

The tanshinone compound has the structure shown as the following formula:

wherein A, R1, R2, R3, R4, R5, R6, R7 are the corresponding groups in the specific compounds of the invention.

Preferred tanshinone compounds of the present invention include compounds selected from the group consisting of:

pharmaceutical compositions and methods of administration

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient can be used for treating, preventing and relieving diseases caused by hemangioma cell generation, such as fatty liver, and the like, because the compound of the present invention has excellent inhibitory activity against hemangioma cells.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-3000mg (active dose range 3-30 mg/kg) of a compound of the invention per dose, more preferably 10-2000mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, 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.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (e.g. tween, etc.)

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous 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, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if desired.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When using pharmaceutical compositions, safe and effective amounts of the compounds of the present invention are suitable for administration to a mammal (e.g., human) in need of treatment at dosages that are pharmaceutically considered to be effective for administration, typically from 1 to 2000mg, preferably from 6 to 600mg per day for a human having a body weight of 60 kg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Compared with the prior art, the invention has the main advantages that:

1. the tanshinone compound is found to have good hemangioma cell inhibitory activity for the first time, and the in vitro activity is superior to that of the known medicine propranolol in the prior art;

2. the tanshinone compound can effectively inhibit hemangioma cells to promote apoptosis and can also inhibit angiogenesis of hemangioma endothelial cells;

3. the compound has effective in vitro and in vivo activity and low active dose, can show tumor inhibition effect at a dose as low as 10mg/kg, and is far lower than the drug effect dose of propranolol.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions, or according to conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

1. Apparatus and equipment

The spectrum UV: SHIMADIU UV-2550 and Beckman DU-7 ultraviolet visible light spectrometers;

infrared spectrum IR: a Perkin-Elmer model 577 Infrared spectrophotometer;

low resolution mass spectroscopy LR-EIMS: finnigan MAT-95;

high resolution mass spectrometry HR-EIMS: a Kratos 1H spectrometer;

nuclear magnetic resonance spectroscopy: varian INOVA 600 NMR spectrometer, bruker AM-500, AM-400, AM-300 NMR spectrometer, delta (ppm), using TMS as an internal standard;

LC-MS: agilent 1100 liquid phase coupling to a Bruker equire mass spectrometer;

analytical HPLC: waters 2690 Separate model, waters PDA 996 Detector coupled to the Alltch ELSD 2000 detector, millennium 2000 operating System, waters RP18 column (5.0X 125mm,5 μm, waters), flow rate 1.0ml/min, CH3CN (Merck, germany), H2O (Leber); jasco HPLC (Chiralcel IA column,5 μm, 150X 4.6 mm), flow rate 0.6ml/min, hexane/ethanol (7.

HPLC-MS: waters 2695separator model, waters PDA 2998 Detector coupled to an Alltch ELSD 2424 Detector, 3100Ms Detector, sunFireTM C-18column (4.6X 100mm,3.5 μm, waters), flow rate 1.0ml/min, CH3CN (Merck, germany), H2O (Leber); jasco HPLC (Chiralcel IA column,5 μm, 150X 4.6 mm), flow rate 0.6ml/min, hexane/ethanol (7.

Preparative HPLC: varian SD1 instrument, varian 320 single wavelength detector, C18 column (220X 25nm,10 μm, waters), flow rate 15ml/min, CH3CN (Merck, germany), H2O (Leber);

the main biological experiment equipment comprises: a carbon dioxide cell incubator, a microplate reader, a centrifuge, a purification workbench and a chemiluminescence apparatus (Thermo Scientific, USA); optical microscope and photomicrography system Leica DM 4000B microscope (leiia, germany); inverted fluorescence microscopy (OLYMPUS, japan); flow cytometry (Beckman Coulter); horizontal shaking table (Beijing Ding Guosheng biotechnology Limited liability company); electrophoresis apparatus (Bio-Rad, USA); fully automatic autoclave (Tomy digital biology, japan); PVDF membranes (Millipore, USA);

2. reagents and materials

Column chromatography silica gel: silica gel H of 100-200 meshes and 200-300 meshes are produced in Qingdao ocean factories;

TLC thin layer preparation plate: HSGF254 is produced in a tobacco bench chemical plant;

MCI resin: CHP20P (75-150 μm) is produced by Mitsubishi corporation;

sephadex LH-20: pharmacia Biotech AB, uppsala, sweden.

Color developing agent: 10% sulfuric acid-vanillin solution, potassium permanganate solution;

plant material: salvia miltiorrhiza is purchased from the Bozhou medicinal material market, collected and identified by professor Shenjingui in specimen room of Shanghai pharmaceutical research institute, and the specimen is stored in the specimen room of Shanghai pharmaceutical research institute.

Biological main reagents: 1640 medium, fetal bovine serum (Gibco, USA); double antibody (Sigma, USA); antibody: caspase3, caspase 8, caspase 9, AIF, PARP, bax, FADD, cyst3 (Abclonal/China); cyclodextrins (Medchem, USA); flow apoptosis fluorescent reagent (FITC-Annexin, PI) (BD, USA); matrigel (Corning, USA); western Blot gel reagent, secondary antibody, CCK8 reagent, enhanced chemiluminescence agent, tween-20 and crystal violet (assist in san/China); protease inhibitors (Roche, switzerland); the experimental animals are nude mice, the weight of the experimental animals is 20 +/-5 g, the experimental animals are purchased from Xinhua hospital affiliated to Shanghai traffic university, and the experimental animals are stored in SPF animal rooms of the Xinhua hospital.

Example 1: separation and identification of tanshinone compounds

Pulverizing 30kg Saviae Miltiorrhizae radix, adding 95% industrial ethanol (50L), and soaking at room temperature for 5 days for 3 times. Mixing the ethanol extractive solutions, and concentrating under reduced pressure to obtain fluid extract. Suspending the fluid extract with 10L distilled water, extracting with 8L ethyl acetate for 3 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain total extract of tanshinone.

Taking 200g of total extract of tanshinone, dissolving and mixing with dichloromethane, purifying by a silica gel column (2kg, 200-300 meshes, qingdao ocean chemical production), eluting with petroleum ether-ethyl acetate 100, 100:3, 100. And recrystallizing the fraction S3 (5.46 g) by using petroleum ether-ethyl acetate to obtain the tanshinone IIA (3.2 g). The fraction S6 (4.32 g) was subjected to silica gel column chromatography (petroleum ether: ethyl acetate 100; subjecting the fraction S6A (152 mg) to Sephadex LH-20 gel column chromatography (chloroform: methanol 1; the fraction S6C (311 mg) was purified by ODS column chromatography (methanol-water, 65% -95%) to give the compound, wiener paraquinone A (10 mg). 800mg of fraction S10 (6.5 g) was subjected to ODS column chromatography (methanol-water, 65% -95%) to obtain fraction S10C, which was purified with preparative silica gel plate (dichloromethane) to obtain isopropyl phenanthrenequinone (10 mg). Fraction S13 (10.89 g) was recrystallized from petroleum ether-ethyl acetate to give tanshinone I (3.2 g). Fraction S14 (17.72 g) was recrystallized from petroleum ether-ethyl acetate to give cryptotanshinone (1.5 g). Fraction S15 (3.61 g) was separated by silica gel column chromatography (petroleum ether: acetone 30; after 25mg of S15D was subjected to Sephadex LH-20 (chloroform: methanol 1. The fraction S16 (20 g) was purified by a silica gel column (petroleum ether: acetone 100:3 to 100. Subjecting fraction S18 (950 mg) to Sephadex LH-20 gel column chromatography (chloroform: methanol 1: 1) to obtain fraction S18C, subjecting to ODS column chromatography (methanol-water, 65% -95%), and purifying with silica gel plate (dichloromethane) to obtain Saviae Miltiorrhizae radix alcohol B (3 mg) and tanshinone IIB (5 mg).

Tanshinone IIA, tanshinone IIA and cherry red needle crystals. EIMS m/z 284[ M ]] ⁺ ； ¹ H NMR(500MHz,CDCl ₃ )δ7.59(d,J＝8.0Hz,1H,H-6),7.10(d,J＝8.0Hz,1H,H-7),7.07(d,J＝1.0Hz,1H,H-14),3.17(t,J＝6.4Hz,1H,H-1),3.02(m,1H,H-15),1.79(m,2H,H-3),1.65(m,2H,H-2),1.29(s,3H,H-18,19),1.16(d,J＝6.19Hz,3H,H-16,17). ¹³ C NMR(125MHz,CDCl ₃ )δ183.6,175.8,161.7,150.2,144.3,141.3,133.6,127.4,126.6,121.1,120.2,38.0,34.7,31.9,29.8,19.1,8.7.

Salvianic acid neoketone, rosmariquinone, cherry red solid. EIMS m/z 284[ M ]] ⁺ ； ¹ H NMR(500MHz,CDCl ₃ )δ7.59(d,J＝8.0Hz,1H,H-6),7.10(d,J＝8.0Hz,1H,H-7),7.07(d,J＝1.0Hz,1H,H-14),3.17(t,J＝6.4Hz,1H,H-1),3.02(m,1H,H-15),1.79(m,2H,H-3),1.65(m,2H,H-2),1.29(s,3H,H-18,19),1.16(d,J＝6.19Hz,3H,H-16,17). ¹³ C NMR(125MHz,CDCl ₃ )δ182.6,181.7,149.9,145.2,144.7,140.1,134.6,134.0,128.4,128.1,38.0,34.7,32.0,30.1,27.1,21.7,19.3.

Grandma paraquinone A, sibiriquinone, cherry red solid; EIMS m/z 284[ M ]] ⁺ ； ¹ H NMR(400MHz,CDCl3)δ7.86(d,J＝9.8Hz,1H,H-1),7.50(d,J＝7.8Hz,1H,H-6),7.11(d,J＝7.9Hz,1H,H-7),7.08(s,1H),6.33(dt,J＝9.7,4.6Hz,1H,H-2),3.02(p,J＝6.9Hz,1H,H-15),2.27(dd,J＝4.6,1.9Hz,2H,H-3),1.28(s,6H,H-18,H-19),1.16(d,J＝6.9Hz,6H,H-16,H-17). ¹³ C NMR(126MHz,CDCl ₃ )δ183.38,181.71,148.15,145.13,140.09,137.42,134.61,134.41,130.74,129.37,124.86,124.84,38.16,34.22,28.52,27.06,21.68.

Isopropyl o-phenanthrenequinone, ro 09-0680, cherry red solid; EIMS m/z 264[ m ]] ⁺ ； ¹ H NMR(500MHz,CDCl ₃ )δ9.26(d,J＝9.0Hz,1H,H-6),8.30(d,J＝9.0Hz,1H,H-7),7.00-7.70(overlap,3H,H-1,2,3),7.14(s,1H,H-14),7.10(d,J＝8.0Hz,1H,H-7),7.07(d,J＝1.0Hz,1H,H-14),3.07(m,1H,H-15),2.70(s,3H,H-18),1.23(d,J＝6.0Hz,6H,H-16,17). ¹³ C NMR(125MHz,CDCl ₃ )δ182.5,181.7,139.6,136.9,136.7,135.1,132.9,132.9,130.9,128.5,126.7,125.4,125.4,125.0,27.3,21.7,21.7,19.9.

Tanshinone I, tanshinone I, cherry red solid, EIMS m/z 264[ Tanshinone I ], [ Yinghong solid ]] ⁺ ； ¹ H NMR(500MHz,CDCl ₃ )δ9.20(d,J＝9.0Hz,1H,H-6),8.23(d,J＝8.0Hz,1H,H-7),7.74(d,J＝9Hz,1H,H-1),7.51-7.54(m,1H,H-2),7.32(d,J＝9.0Hz,1H,H-3),7.27(d,J＝1.5Hz,1H,H-17),2.66(s,3H,H-18),2.27(d,J＝1.5Hz,1H,H-16). ¹³ C NMR(125MHz,CDCl ₃ )δ183.4,175.6,161.1,142.0,135.2,133.6,132.7,130.6,129.6,128.3,124.7,123.1,121.7,123.1,121.7,120.5,118.7,19.8,8.8.

Cryptotanshinone, cryptostanshinone, cherry red needle crystal, EIMS m/z 296[ M ], [] ⁺ ； ¹ H NMR(500MHz,CDCl ₃ )δ7.64(d,J＝8.0Hz,1H,H-6),7.51(d,J＝8.0Hz,1H,H-7),4.90and 4.37(dd,J＝6.0,9.6Hz,2H,17),3.59-3.65(m,1H),3.22(m,2H),1.77-1.83(m,2H),1.65-1.69(m,2H),1.36(d,J＝6.0Hz,3H,H-16),1.32(s,6H,H-18/19). ¹³ C NMR(125MHz,CDCl ₃ )δ184.0,175.5,170.8,152.3,143.6,132.5,128.8,126.1,122.5,118.2,81.5,38.7,34.7,34.6,31.7,29.6,19.3,18.7.

Tanshinone Methyl ester, methyl tanshinonate, red solid, EI MS m/z 338[ 2 ], [ M ]] ⁺ . ¹ H NMR(500MHz,CDCl ₃ )δ7.56(d,J＝8.3Hz,1H,H-6),7.48(d,J＝8.3Hz,1H,H-7),7.23(d,J＝1.4Hz,H-17),3.67(s,3H,-OCH ₃ ),3.24(m,2H,H-1),2.27(d,J＝1.4Hz,3H,H-16),2.26and 1.75(m,each 1H,H-3),1.82(m,2H,H-2),1.58(s,3H,H-18). ¹³ C NMR(125MHz,CDCl ₃ )δ183.6,177.3,175.8,161.5,144.6,143.3,141.8,135.2,128.6,126.7,121.5,120.5,52.8,47.4,34.2,29.2,27.8,19.4,9.0.

15,16-Dihydrotanshinone I, cherry red needle solid, EI MS m/z 278[ m ], [] ⁺ . ¹ H NMR(500MHz,CDCl ₃ )δ9.28(d,J＝9.0Hz,1H,H-6),8.30(d,J＝9.0Hz,1H,H-7),7.76(d,J＝9.2Hz,H-1),7.57(m,1H,H-2),7.40(d,J＝7.0Hz,1H,H-3),4.97and 4.43(t,J＝6.3,9.5Hz,each 1H,H-17),3.66(m,1H,H-17),2.70(s,3H,H-18),1.41(d,J＝6.5Hz,1H,H-16). ¹³ C NMR(125MHz,CDCl ₃ )δ184.3,175.8,170.6,135.0,134.6,132.2,131.9,130.4,128.9,128.2,126.2,125.0,120.5,118.3,81.6,34.7,19.8,18.8.

Salvianolic acid B, tanshinol B, cherry red solid, EIMS m/z 296[ sic ], [ M ] m] ⁺ . ¹ H NMR(500MHz,CDCl ₃ )δ7.95(d,J＝8.1Hz,1H,H-6),7.61(d,J＝8.3Hz,1H,H-15),7.25(m,H-7),3.28(m,1H,H-1),3.16(m,1H,H-1),2.27(d,J＝1.3Hz,3H,H-17),2.00(overlap,2H),1.83(m,1H),1.56(s,3H,H-18). ¹³ C NMR(126MHz,CDCl ₃ )δ183.35,175.60,161.39,147.29,143.95,141.74,133.68,129.04,126.09,121.46,120.80,120.49,71.03,38.75,31.18,29.14,20.17,8.94.

Tanshinone IIB, tanshinone IIB, cherry red solid, EIMS m/z 310M] ⁺ . ¹ H NMR(600MHz,CDCl ₃ )δ7.65(d,J＝8.1Hz,1H,H-6),7.52(d,J＝8.1Hz,1H,H-6),7.21(s,1H,H-16),3.79(d,J＝11.1Hz,1H,H-18b),3.62(d,J＝11.3Hz,1H,H-18a),3.18(m,2H,H-1),2.28(s,3H,H-17),1.98(m,1H,H-3b),1.88(m,1H,H-2b),1.75(m,1H,H-2a),1.58(m,1H,H-3a),1.29(s,3H,H-19). ¹³ C NMR(126MHz,CDCl ₃ )δ183.38,175.57,161.55,146.31,146.08,141.59,133.87,127.94,126.67,121.31,120.36,120.24,71.66,40.09,32.38,29.85,26.81,18.91,8.94.

Example 2: cell proliferation inhibitory activity of tanshinone compounds on hemangioma cells

1) Cell culture: the EOMA cell line originated from the 129/J line of rat-bearing spontaneous hemangioma cells. The cell culture was carried out in 1640 medium containing 10% fetal bovine serum, and 100mg/l of penicillin and 100ug/l of streptomycin were mixed. The cells were cultured at 37 ℃ in an incubator containing 5% of CO2. The medium was changed every other day and passaging was performed when the cell density reached 80%.

2) Dissolution of the drug: dissolving the medicine powder in DMSO, storing at a concentration of 10uM, and subpackaging at-20 degrees for storage.

3) CCK8 detection IC ₅₀ : before the medicine adding treatment, the cells are plated in a 96-well plate, the cell counting is carried out, the cell concentration of each well reaches 2000-3000/ul, after the cells adhere to the wall, the culture medium is sucked, and the dihydrotanshinone I with different concentration gradients is added. After 72h, the medium containing the drug was aspirated, cck8 was added, three blank control wells were set simultaneously, and the absorbance at 450 wavelengths was measured for about 2 h. According to the following steps: the percentage of cell proliferation at different concentrations was calculated [ (drug-added group-blank control group)./(control group-blank pair)./[ 100%)Lighting group)]The drug concentration, i.e., IC, corresponding to a half-reduction in cell proliferation rate was determined by nonlinear regression using Graphpad Prim software ₅₀ 。

The cell proliferation inhibitory activity of tanshinone compounds on hemangioma cells is shown in table 1 below. The activity test result shows that the tanshinone compound can obviously inhibit the proliferation of hemangioma cells.

Table 1: inhibitory activity of tanshinone compounds on hemangioma cell proliferation

Name of Compound	IC50(μM)
		Dihydrotanshinone I	2.63
Salvianic acid A neoketone	3.17
		All-grass of Begonia Pythioides A	3.19
Tanshinone methyl ester	4.33
		Salvianolic acid B	5.32
Isopropyl ortho phenanthrenequinone	7.83
		Cryptotanshinone	10.48
Positive control (Propranolol)	53.67

Example 3: effect of dihydrotanshinone I on apoptosis of hemangioma cells

1) Cell clone formation experiment: adding 1000/2 ml cells into a six-well plate, respectively adding different drug concentrations for treatment after the cells adhere to the wall, adding DMSO (solvent) into a blank group for comparison, changing to a fresh culture medium after treatment conditions are met (24 h and 48 h), performing crystal seed staining after about ten days, and taking a picture.

2) Flow cytometry and hochest33342 detected the percentage and morphology of apoptotic cells, respectively: preparing cells, paving the cells in a six-hole plate, carrying out drug treatment after the cells adhere to the wall, collecting the cells after the conditions are met (different concentrations and different time treatment), adding the cells into a centrifuge tube, centrifuging 800rmp for 5min, eppendorf precooling, then washing the cells by pbs, continuing centrifuging in a precooled centrifuge for 800rmp for 5min, sucking the pbs, adding a sample into 200ul binding buffer, then adding 5ul FITC-Annexin V for resuspension, keeping out of the sun for 10min, then adding 5ul PI, transferring into a flow tube, keeping out of the sun at 4 ℃, and loading on the machine after one hour.

3) Hochest33258 staining: and (3) carrying out cell plating in a 12-well plate, carrying out drug treatment after the cells are attached to the wall, fixing the cells after corresponding conditions are met, then washing away a fixing solution, adding hochest33258, and dyeing for 30min at 37 ℃. Dense, solid-condensed nuclei were observed.

The test results are shown in FIGS. 1-3 below: under the corresponding drug concentration (3 uM and 50 uM) and the fixed time (48 h), the apoptosis formation time and the concentration dependence of the dihydrotanshinone I and the propranolol, and the variance analysis result indicates that the difference has statistical significance.

4) western blot detection of apoptotic proteins: different protein expression levels in the cell can be detected by wb. After the cells are treated by the medicines (dihydrotanshinone I and propranolol) and the solvent, pbs washing and collection are carried out, then the cells are cracked for 30min by RIPA on ice, after cracking, the cells are centrifuged for 10min at the speed of 12000g under the condition of 4 ℃, and cell supernatant is taken. Protein concentration was quantified using BCA, and proteins were adjusted to the same concentration. Loading in sufficient electrophoresis solution to ensure the loading amount is 20-30ug, transferring the proteins on SDS-PAG gel to PVDF membrane at 300Ma current, blocking 5% BSA for 1h, adding primary antibody according to 1.

The Western Blot results are shown in FIG. 4: DHTS can act on hemangioma cells through FAS/FASL and mitochondrial pathways, and mainly acts on mitochondrial pathways at low concentration and FASL pathways at high concentration; propranolol z mainly has the FASL pathway at low concentrations and the mitochondrial pathway at high concentrations.

Example 4: tube format observation the effect of drugs on cell tubulation:

laying the unsolidified Matrigel on a 96-well plate in an amount of 10ul per well, placing the 96-well plate in an incubator for 1h to completely coagulate the colloid, adding EOMA cells (3 × 104/per well) into the 96-well plate, simultaneously laying a culture medium containing or not containing the drug on the surfaces of the colloid and the cells, continuously placing the culture medium in the incubator for 2h, observing under a microscope, and taking a picture for storage.

The tube-forming experiment reflects the angiogenesis of cells in vivo, and the processes include the action of Vascular Endothelial Growth Factor (VEGF), the degradation of intercellular matrix (mmp 9), and the migration of endothelial cells.

The results of Tube format and Western blot are shown in fig. 5, and the effect of the drug on cells is that the endothelial growth factor is reduced, the synthesis of the metal matrix enzyme is reduced, the endothelial cells cannot migrate, and the cells cannot carry out angiogenesis, which is enough to illustrate the capability of the drug in inhibiting blood vessels.

Example 5: animal in vivo experiment preliminary exploration on curative effect of drug on hemangioma

The mice used in the in vivo experiment are BALB/c nude mice, which are 6-week female mice in total 15 weeksOnly. After about 1X 107 cells were collected, they were suspended in 200ul of pbs, inoculated in the axilla of nude mice for about two weeks, and the average size of the tumor body reached 100mm3 (volume = length X width) ² And/2) carrying out random grouping. According to the literature, dihydrotanshinone I (10 mg/kg), propranolol (40 mg/kg) and a solvent (beta-cyclodextrin +20% DMSO) are injected into the abdominal cavity, changes of tumor bodies and the body weight of a naked mouse are detected three times a week, after 4 weeks, the naked mouse is killed, the tumor bodies are cut off and measured, meanwhile, partial tissues are subjected to immunohistochemistry, and changes of observation indexes such as CD31, CD34, vWF, glut1, caspase3, MMP9 and VEGFR2 are detected.

The test results are shown in fig. 6: both dihydrotanshinone I and Propranolol can inhibit hemangioma at corresponding concentrations, and the results of anova analysis indicate that the effects of the drugs have significant statistical significance (P = 0.0008), while the results of DHTS and Propranolol indicate no significant difference (P = 0.097).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (6)

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof:

wherein the compound of formula I is selected from the group consisting of:

；

characterized in that the pharmaceutical composition is used for preparing the pharmaceutical composition for inhibiting the proliferation activity of hemangioma cells.

2. The use according to claim 1, wherein the compound of formula I is selected from the group consisting of:

。

3. the use according to claim 1, wherein the effective dose of the compound of formula I in the pharmaceutical composition is 0.1-50mg/kg body weight.

4. The use according to claim 1, wherein the effective dose of the compound of formula I in the pharmaceutical composition is 1-20mg/kg body weight.

5. The use of claim 1, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of: oral dosage form and injection dosage form.

6. The use of claim 1, wherein said pharmaceutical composition is further used for inhibiting angiogenesis of hemangioma endothelial cells.

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