CN110358078A - Epimedium aglucone derivative and its preparation method and application - Google Patents

Epimedium aglucone derivative and its preparation method and application Download PDF

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CN110358078A
CN110358078A CN201810254006.XA CN201810254006A CN110358078A CN 110358078 A CN110358078 A CN 110358078A CN 201810254006 A CN201810254006 A CN 201810254006A CN 110358078 A CN110358078 A CN 110358078A
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epimedium aglucone
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formula
acid
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王进欣
关永霞
梁红宝
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Lunan Pharmaceutical Group Corp
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
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    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33344—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing carbamate group

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Abstract

The invention belongs to field of medicinal chemistry, and in particular to epimedium aglucone derivative, as formula W-5A and its pharmacologically acceptable salt with and preparation method thereof and its treat asthma in preparation, the purposes of drug in terms of bone marrow suppression.

Description

Epimedium aglucone derivative and its preparation method and application
Technical field
The invention belongs to field of medicinal chemistry, and in particular to epimedium aglucone derivative and its pharmacologically acceptable salt With and its preparation method and application.
Background technique
Epimedium aglucone (icaritin, ICT) is one of Berberidaceae barrenwort Herba Epimedii polyhydroxy flavones Class monomer component, structural formula are as follows:
Epimedium aglucone can isolated from the cylinder metabolism-ure of epimedium herb or icariin (Sun Pengyue, Xu Grain husk, text is bright etc., the chemical component of korean epimedium herb, Chinese Plants The Chemicals, 1998,8 (2): 122-125；Liu strong determined person, king It is firm, Wu Lijun etc., metabolic conversion of the intestines bacterium metabolism research I. intestinal bacterium of icariin to icariin, 2000,31 (11): 834-837), or by icariin through digest it is isolated (Ye Haiyong, Liu Jian, Lou Yijia, the preparation of two derivatives from icariin and investigation and Its estrogen-like effects, journal of Zhejiang university, 2005,34 (2): 131-136).
There is document report epimedium aglucone and causes rat primary cultured nerve cell apoptosis (Zhang Xiang with anti-A β peptide South, Wang Huanhuan, Wang Zhiqiang etc., the anti-A β peptide of epimedium aglucone cause the effect of rat primary cultured nerve cell apoptosis, Zhejiang University Journal, 2007,36 (3): 224-226).It is raw with antineoplastic vascular that Chinese patent CN101836976A discloses epimedium aglucone At effect.Chinese patent CN101428015A, which discloses epimedium aglucone, has the function for the treatment of endotoxemia.Chinese patent CN101284000A, which discloses epimedium aglucone, to be had the function of preventing and treating obesity or fatty liver.Chinese patent CN1869204A is public Purposes of the epimedium aglucone in terms of inducing stem cell in vitro directed differentiation is opened.
Epimedium aglucone has extensive pharmacological activity, but its dissolubility is poor, slightly molten in methylene chloride and ethyl acetate, It is almost insoluble in methanol, dehydrated alcohol and water, it is almost insoluble or insoluble in different pH buffers.The oral life of epimedium aglucone Object availability is low.
Summary of the invention
The present invention provides a kind of epimedium aglucone derivative, and structure is as shown in formula W-5A:
Or the pharmaceutically acceptable salt of formula W-5A compound；
Wherein, the integer of the m, n independently selected from 0-6；
The molecular weight of the PEG is selected from 400-20000；It is preferred that 1000-10000；
In certain embodiments, PEG be selected from PEG 400, PEG600, PEG1000, PEG2000, PEG3000, PEG4000, PEG6000, PEG10000 or PEG20000.
Further, the integer of the m, n independently selected from 0-4.
In certain embodiments, formula W-5A compound is specially Formula W-5A-22:
Above-mentioned formula W-5A compound can be, but not limited to be synthesized by following scheme, and steps are as follows:
1) Boc-ICT is reacted with isopropyl isocyanate, products therefrom reacted with halohydrin 2；
2) mPEG reacts to obtain compound 3 with compound 1；
The m is selected from the integer of 0-6；
3) compound 2 reacts production 5 with compound 3 in the presence of condensing agent:
Wherein, the condensation course can use condensing agent；Condensing agent is selected from 1- (3- dimethylamino-propyl) -3- ethyl carbon Diimmonium salt hydrochlorate/4-dimethylaminopyridine (EDCI/DMAP), 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride Salt/I-hydroxybenzotriazole/N, N- diisopropylethylamine (EDCI/HOBT/DIPEA), 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester/N, N- diisopropylethylamine (HATU/DIPEA), dicyclohexylcarbodiimide/4- diformazan At least one of aminopyridine (DCC/DMAP)；
The condensing agent dosage is 0.1-25eq；
The reaction temperature of the condensation reaction is 0-70 DEG C.
In some reactions, it is desirable to provide alkaline condition, the alkali be selected from triethylamine, pyridine, sodium carbonate, sodium hydroxide, At least one of sodium bicarbonate, potassium carbonate, sodium sulphate；Dosage is 0.05-10eq；
In some reactions, reaction carries out in a solvent, and the solvent is selected from but not limited to methylene chloride, chloroform, tetrahydro Furans, acetone, acetonitrile or toluene.
The present invention also provides a kind of pharmaceutical compositions, wherein the epimedium aglucone of the present invention containing therapeutically effective amount Derivative or its pharmacologically acceptable salt.Specifically, salt is pharmaceutically acceptable salt.Term is " pharmaceutically acceptable " include substance or composition must be suitble to chemistry or toxicology, with composition preparation other components and for the food in one's mouth for the treatment of Newborn animal is related.The salt of the compound of the present invention further include be used to prepare or purifying compound (formula W-VI) and its intermediate (such as Or the salt of the enantiomter (such as intermediate 2) of compound separation intermediate 2,4), but is not necessarily pharmaceutically acceptable salt.
If the compound of the present invention be it is alkaline, conceivable salt can be by provided in the literature any suitable Method is prepared, for example, using inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid etc..Or using organic Acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, malic acid, Lactic acid citric acid, Oxalic acid, glycolic acid and salicylic acid；Pyrans saccharic acid, such as glucuronic acid and galacturonic acid；Alpha-hydroxy acid, such as citric acid and winestone Acid；Amino acid, such as asparatate and glutamic acid；Aromatic acid, such as benzoic acid and cinnamic acid；Sulfonic acid, such as p-methyl benzenesulfonic acid, benzene Sulfonic acid, methanesulfonic acid, ethanesulfonic acid, trifluoromethanesulfonic acid etc. or their combination.
If the compound of the present invention be it is acid, conceivable salt can be prepared by suitable method, e.g., Using inorganic base or organic base, such as ammonia (primaquine, parahelium, tertiary ammonia), alkali metal hydroxide, ammonium, the salt and alkaline earth of N+ (R14) 4 Metal hydroxides, etc..Suitable salt includes, but is not limited to, organic salt obtained from amino acids, such as glycine and smart ammonia Acid, ammonia, such as primaquine, parahelium and tertiary ammonia, the salt of N+ (R14) 4, if R14 is H, C1-4 alkyl, C6-10 aryl, C6-10 aryl C1- 4 alkyl etc. and ring-type ammonia, such as piperidines, morpholine and piperazine etc., and nothing is obtained from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminium and lithium Machine salt.It also include appropriate, nontoxic ammonium, the amine cation that quaternary ammonium salt and gegenions are formed, such as halide, hydroxide Object, carboxylate, hydrosulphate, phosphoric acid compound, nitric acid compound, C1-8 sulphonic acid compound and aromatic sulphonic acid compound.Used in invention " pharmaceutically acceptable salt " refers to the organic salt and inorganic salts of the compound of the present invention.Pharmaceutically acceptable salt is in affiliated neck Domain is known to us, such as document: S.M.Berge et al., descr5Aepharmaceutically Recorded in acceptable salts in detail in J.Pharmaceutical Sciences, 66:1-19,1977. 's.The salt that pharmaceutically acceptable nontoxic acid is formed includes, but is not limited to, inorganic acid salt formed by reacting with amino groups to form There are hydrochloride, hydrobromate, phosphate, sulfate, perchlorate and an acylate such as acetate, oxalates, maleate, Tartrate, citrate, succinate, malonate, or handed over by other methods described in the books or literature such as ion Method is changed to obtain these salt.Other pharmaceutically acceptable salts include adipate, malate, 2 hydroxy propanoic acid salt, alginic acid Salt, ascorbate, aspartate, benzene sulfonate, benzoate, bisulphate, borate, butyrate, camphor hydrochlorate, camphor tree Brain sulfonate, cyclopentyl propionate, digluconate, lauryl sulfate, esilate, formates, fumaric acid Salt, gluceptate, glycerophosphate, gluconate, Hemisulphate, enanthate, caproate, hydriodate, 2- hydroxyl-second Sulfonate, lactobionate, lactate, laruate, lauryl sulfate, malate, malonate, mesylate, 2- Naphthalene sulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulfate, 3- phenylpropionic acid salt, Picrate, pivalate, propionate, stearate, rhodanate, tosilate, undecylate, valerate, etc.. Salt obtained by an appropriate base includes alkali metal, alkaline-earth metal, the salt of ammonium and N+ (C1-4 alkyl) 4.
Pharmaceutical composition.The agent such as solid orally ingestible, liquid oral medicine, injection can be made in the pharmaceutical composition Type.For example, pharmaceutical composition can be administered orally in the form of tablet, capsule, pill, powder, sustained release agent, solution, suspension； Pharmaceutical composition parenteral injection can also be administered in the form of sterile liquid, suspension or lotion；Pharmaceutical composition can also be with The form local administration of ointment or creams；Pharmaceutical composition can also in the form of suppository rectally.Pharmaceutical composition can be with Unit dosage forms are made into, the single administration of exact dose is suitable for.The solid and liquid oral medicine include: tablet, dispersible tablet, Enteric coatel tablets, chewable tablets, oral disintegrating tablet, capsule, syrup, granule, oral solution, injection.Non-intestinal drug delivery agent can be with The dosage forms such as injection, freeze-dried powder are made, such as creme, ointment, patch, spray can be made in local administration.But not It is limited to this.Oral preparation used can contain for example one or more colorants, sweetener, corrigent and/or preservative.
The carrier of lactose or starch as the solid orally ingestible can be used；Use gelatin, methylcellulose, hydroxypropyl first Cellulose, polyvinylpyrrolidone, starch slurry etc. are used as adhesive；Using starch, sodium carboxymethylcellulose, carboxyrnethyl starch sodium, Low-substituted hydroxypropyl methylcellulose, crospovidone, microcrystalline cellulose are as disintegrating agent；Use talcum powder, superfine silica gel powder, stearic acid Glyceride, calcium stearate or magnesium are as antiadhesives and lubricant.Suitable pharmaceutically acceptable excipient for tablet formulation includes For example, inert diluent such as lactose, sodium carbonate, calcium phosphate or calcium carbonate；Granulation and disintegrating agent such as cornstarch and alginic acid； Adhesive such as starch；Lubricant such as magnesium stearate, stearic acid or talcum powder；Preservative such as ethyl-para-hydroxybenzoate or Propyl ester and antioxidant, such as ascorbic acid.Tablet formulation can be it is uncoated, can also use coating to change its disintegration The subsequent absorption effect of effect and active constituent in gastrointestinal tract, or its stability and/or appearance are improved, in any situation In, conventional coating agents known to the field and method can be used.
The preparation method of the solid orally ingestible the following steps are included: by active constituent and carrier and selectively with Portion disintegration additive composition mixture, then makes the aqueous solution of the mixture and adhesive, alcohol or aqueous alcohol solution Wet process or dry granulation are carried out in suitable equipment, and other disintegrating agents, lubricant and antiplastering aid is then added and is made suitably Preparation.
Pharmaceutical composition of the present invention can also use the form of oil-in-water emulsion.Oil mutually can be vegetable oil, such as olive Olive oil or peanut oil perhaps mineral oil such as atoleine or their mixture.Emulsifier appropriate can be for example, Natural gum such as Arabic gum or tragacanth, natural phospholipid such as soybean lecithin, and it is derived from fatty acid and hexitol The ester or partial ester (such as E494) of acid anhydride and the condensation product of the partial ester and ethylene oxide, for example, Series compound of the invention can also be administered by non-bowel form.Preferred parenteral administration is injection administration. Syrup and elixir can be prepared with sweetener (such as glycerol, propylene glycol, D-sorbite, aspartame or sucrose), and can also Contain moderator, preservative, corrigent and/or colorant.
Described pharmaceutical composition can also be that Injectable sterile is aqueous or the form of Oil suspensions, can be according to known Method is prepared using one or more suitable dispersions or wetting agent and suspending agent, these reagents are as described above.Aseptic injection Preparation is also possible to that the Injectable sterile in the acceptable diluent of nontoxic parenteral or solvent is aqueous or Oil suspensions, example Solution such as in 1,3 butylene glycol.Can according to the difference of treated host and specific administration route, come determine with it is a kind of or A variety of excipient are mixed to prepare the amount of single dose form active constituent.For example, for general to the preparation of oral administration in human Activating agent containing such as 0.5mg-2g and appropriate and convention amount excipient (5-98% for accounting for about composition gross weight).It is single It is general about containing the active constituent of 1mg-500mg in the preparation of position.
The form of PEG+ number in the present invention, meaning are the PEG of the number molecular weight, such as PEG1000, i.e. molecule The PEG that amount is 1000.
Yet another object of the invention is that a kind of pharmaceutical composition is provided, wherein containing epimedium aglucone of the present invention Derivative or its salt pharmacologically allowed.
Yet another object of the invention is that offer epimedium aglucone derivative and its pharmacologically acceptable salt are being made The purposes of drug in terms of standby treatment asthma or bone marrow suppression.
Epimedium aglucone derivative of the present invention is significantly better than epimedium aglucone for the therapeutic effect of asthma.The excessive sheep of the present invention Leaves of pulse plants aglycone derivative is significantly better than epimedium aglucone for the therapeutic effect of bone marrow suppression caused by radiotherapy or chemotherapy.The present invention is excessive The bioavilability of sheep leaves of pulse plants aglycone derivative is substantially better than epimedium aglucone, this shows the compound in the present invention in druggability side Face has apparent technical advantage.
Epimedium aglucone derivative of the present invention is significantly better than type I compound for the therapeutic effect of asthma.The excessive sheep of the present invention Leaves of pulse plants aglycone derivative is significantly better than type I compound for the therapeutic effect of bone marrow suppression caused by radiotherapy or chemotherapy.The present invention is excessive The bioavilability of sheep leaves of pulse plants aglycone derivative is substantially better than type I compound, this shows the compound in the present invention in druggability side Face has apparent technical advantage.More importantly long poison is experimental studies have found that type I compound has certain liver kidney poison Property leads to the side effects such as the female rats proliferation of mammary gland, and the compounds of this invention, compared to type I compound, faces without overt toxicity Bed is safe to use.
Type I compound is document (Dell'Agli M, Galli G V, Dal C E, et al.Potent inhibition of human phosphodiesterase-5by icariin derivatives.[J].Journal of Natural Products, 2008,71 (9): 1513-1517.) in compound 5.Type I compound structural formula is as follows:
Specific embodiment
The contents of the present invention are described in further detail below by way of the embodiment of specific embodiment.But it does not answer Embodiment is interpreted as limitation of the present invention.In the case where not departing from above-mentioned thought of the invention, those skilled in the art The various replacements or change made according to ordinary skill knowledge and conventional means, are all contained within the present invention.
The synthesis of 1 intermediate 2 of embodiment
The boc-protected epimedium aglucone of raw material (Boc-ICT) 500mg (1.1mmol) is weighed to be placed in 100ml eggplant-shape bottle, 15ml methylene chloride is added as solvent, lower addition isopropyl isocyanate 0.14ml (1.65mmol) and triethylamine 32 is stirred at room temperature μ l (0.22mmol), back flow reaction.After fully reacting, column chromatographic purifying (petroleum ether: ethyl acetate=4:1) obtains yellow solid Product 450mg, yield 90%.
450mg (0.8mmol) product is placed in 100ml eggplant-shape bottle, 15ml methylene chloride is added as solvent, in ice bath It is cooling, trifluoroacetic acid 5ml is added, after fully reacting, column chromatography for separation (petroleum ether: ethyl acetate=4:1) obtains yellow solid 340mg, yield 91%.
Above-mentioned gained yellow solid 340mg (0.75mmol) product is placed in 100ml eggplant-shape bottle, 20ml acetone is added and makees For solvent, ethylene bromohyrin 0.1ml (1.5mmol) and potassium carbonate 207mg (1.5mmol) is added, is reacted in 60 DEG C.Fully reacting Afterwards, it filters, filtrate is spin-dried for, and column chromatography for separation (petroleum ether: ethyl acetate=4:1) obtains 2 290mg of yellow solid intermediate, receives Rate 78%.
The synthesis of 2 intermediate 3 of embodiment
It weighs mPEG1000 1g (1mmol) to be placed in 100ml eggplant-shape bottle, 10ml chloroform is added as solvent, is added Succinic anhydride 1g (10mmol) and pyridine 4ml is reacted in 70 DEG C.After complete reaction, it is spin-dried for solvent, saturated sodium bicarbonate is added Solution is added salt acid for adjusting pH to acidity, is extracted with dichloromethane 3 times, merges dichloromethane layer, drying is spin-dried for, ether is tied again Crystalline substance obtains 3 600mg of white solid intermediate, yield 54%.
The synthesis of 3 compound W-5 of embodiment
It weighs 2 180mg of intermediate (0.36mmol) and 3 220mg of intermediate (0.2mmol) is placed in 100ml eggplant-shape bottle, 20ml dry methylene chloride is added as solvent, lower addition DCC 82mg (0.4mmol) and DMAP 49mg is stirred at room temperature Reaction is stirred at room temperature in (0.4mmol).It after fully reacting, filters, washing, organic phase is dry with anhydrous sodium sulfate, column chromatography for separation (methylene chloride: acetone=40:1-10:1 gradient elution), obtains yellow solid W-5 230mg, yield 72%.
IR(KBr),cm-1:3587,3153,2867,1736,1698,1658,1589,1510,1490,1455,1351, 1327,1301,1259,1217,1107,1033,984,947,841,815,727,650,588,520。
Embodiment 4-11
By the PEG in embodiment 2 be respectively adopted PEG400, PEG600, PEG1000, PEG3000, PEG4000, PEG6000, PEG10000 or PEG20000.Other steps are repeated, product is obtained respectively, is shown in Table 1:
1 embodiment 4-11 of table and its product
Influence of 12 the compounds of this invention of embodiment to bone marrow suppression caused by mice with tumor chemotherapy
1. model preparation and grouping administration: choosing Balb/C mouse 84, weighing is randomly divided into 7 groups, respectively normally Group, blank control group, icariin tuple, type I compound group, each administration group of the compounds of this invention, every group 12.Mouse is daily 50mg/kg cyclophosphamide is injected intraperitoneally, platelet counts can be made to significantly reduce within continuous one week.Modeling starts the last week each treatment group Following therapeutic agents are given respectively:
Normal group: giving isometric sodium carboxymethylcellulose；
Blank control group: isometric sodium carboxymethylcellulose is given；
Icariin tuple: 5mg/kg epimedium aglucone is given in stomach-filling；
I group of formula: 5mg/kg type I compound is given in stomach-filling；
W-5A-22-400 group: 0.5mg/kg compound W-5A-22-400 is given in stomach-filling；
W-5A-22-3000 group: 0.5mg/kg compound W-5A-22-3000 is given in stomach-filling；
W-5A-22-20000 group: 0.5mg/kg compound W-5A-22-20000 is given in stomach-filling；
Each treatment group is administered once a day, normal to feed.After successive administration 3 weeks, anesthetized mice takes blood, detects leucocyte And platelet counts, investigate influence of the compounds of this invention to leucocyte and blood platelet.
2. experimental result
Each compound of the invention causes the influence (table 2) of murine interleukin and blood platelet to send out cyclophosphamide through this embodiment Existing, each compound of the present invention has the effect of the bone marrow suppression that unexpected alleviation chemotherapy generates.
Compared with model control group, the total white blood cells and total number of blood platelet of each compound group of the present invention, which have, to be increased, and There is extremely significant sex differernce.
Compared with icariin tuple, the total white blood cells and total number of blood platelet of each compound group of the present invention increase, and have aobvious Write sex differernce.
Compared with I group of formula, the total white blood cells and total number of blood platelet of each compound group of the present invention increase, and have conspicuousness poor It is different.
Influence of each compound of table 2 to caused by cyclophosphamide leucocyte and decrease of platelet
Compared with blank control group,$P < 0.05,$$p<0.01；
Compared with icariin tuple,#P < 0.05,##p<0.01；
With I group of ratio of formula,&P < 0.05,&&p<0.01。
13 the compounds of this invention pair of embodiment60The influence of Co radiation mouse haemocyte quantity
1. model preparation and grouping administration: choosing kunming mice 100.In addition to normal group (10), all other each groups are small Mouse carries out 4Gy irradiation60Co radiation, using disposable full-body exposure, absorbed dose 4Gy, absorbed dose rate 0.88Gy/min. After radiation the 3rd, 7,10d difference socket of the eye venous blood sampling detect whole blood cells number.It incites somebody to action white thin in whole blood cells measurement twice in succession Born of the same parents' quantity is lower than 3.0 × 109/ L or platelet counts are less than 500 × 109The mouse of/L is picked out, and remaining mouse is to test to use Mouse.
The mouse for meeting requirement of experiment after irradiation is randomly divided into following model group, icariin tuple, type I compound Group, W-5A-22-400 group, W-5A-22-3000 group, W-5A-22-20000 group, every group 10 groups, half male and half female.Each group is pressed respectively Such as under type processing is administered.
Normal group: giving isometric sodium carboxymethylcellulose；
Blank control group: isometric sodium carboxymethylcellulose is given；
Icariin tuple: intraperitoneal injection 1mg/kg epimedium aglucone；
I group of formula: intraperitoneal injection 1mg/kg type I compound；
W-5A-22-400 group: intraperitoneal injection 0.1mg/kg compound W-5A-22-400；
W-5A-22-3000 group: intraperitoneal injection 0.1mg/kg compound W-5A-22-3000；
W-5A-22-20000 group: intraperitoneal injection 0.1mg/kg compound W-5A-22-20000；
Each treatment group is administered once a day, normal to feed.After successive administration 10 days, anesthetized mice takes blood, detects leucocyte And platelet counts, investigate influence of the compounds of this invention to leucocyte and blood platelet.
2. experimental result
Each compound of the invention is to receiving through this embodiment60The influence (table 3) of Co radiation murine interleukin and blood platelet It was found that each compound of the present invention has the effect of the unexpected bone marrow suppression alleviating radiotherapy and generating.
Compared with model control group, the total white blood cells and total number of blood platelet of each compound group of the present invention, which have, to be increased, and is had Significant difference.
Compared with icariin tuple, the total white blood cells and total number of blood platelet of each compound group of the present invention increase, and have aobvious Write sex differernce.
Compared with I group of formula, the total white blood cells and total number of blood platelet of each compound group of the present invention increase, and have conspicuousness poor It is different.
Each compound pair of table 360The influence of Co radiation mouse haemocyte quantity
Compared with blank control group,$P < 0.05,$$p<0.01；
Compared with icariin tuple,#P < 0.05,##p<0.01；
With I group of ratio of formula,&P < 0.05,&&p<0.01。
Inhibiting effect of 14 the compounds of this invention of embodiment to airway smooth muscle
Airway Remodeling is the important pathological characters of bronchial asthma, and airway smooth muscle cells (ASMC) are to cause Airway Remodeling Main effects cell.Severe asthma patient is compared with normal people, and ASM C amount increased significantly, this phenomenon is mainly by smooth Caused by myocyte's hyperplasia.
1.1 material
150~200g SD rat；D M EM culture medium (Gibico company)；Fetal calf serum (the limited public affairs of Hangzhou Chinese holly Department)；Trypsase (Sigma company).
1.2 experimental method
1.2.1 the culture of rat ASMC
By the method for pertinent literature: aseptically, vertical shape splits tracheae, careful to remove outer membrane and gently strike off interior Tracheae section is carefully cut into lmm × 1mm × 1mm small tissue blocks with eye scissors, is affixed on the culture bottle bottom surface of 5cm × 5cm by film, Equidistant arrangement is added D M EM (high sugar) culture medium 2mL containing 25% fetal calf serum, culture solution is not made to contact tissue block.It will training Feeding bottom of bottle faces upward, and absolutely stands about 3h in the incubator of 37 DEG C and 5% carbon dioxide, turns over tissue block gently close to dry Turn culture bottle, so that culture solution was not had tissue block surface just, add culture solution to 5mL after the absolute stationary culture 3d of semi open model, Liquid is changed after 6d entirely, hereafter 3d is changed liquid 1 time.About 7d covers with rear secondary culture, and the 4th~5 generation cell is selected in experiment.The rat of culture ASM C is identified through morphological method.
1.2.2 the proliferation of CCK-8 method detection rat ASMC
The 4th generation rat ASMC for taking culture, is made single cell suspension, by 1 × 104A/hole is inoculated in 96 well culture plates, and 37 DEG C and 5% carbon dioxide under conditions of cultivate for 24 hours, when cell growth is in plocoid, the culture for containing 0.1% fetal calf serum is added Liquid (making cell growth arrest in 0 phase of G) continues culture for 24 hours.
It uses the culture solution containing 1% fetal calf serum instead, is randomly divided into:
DMEM is only added in control group, every hole；
Icariin tuple, epimedium aglucone concentration are 1 × 10-6mol/L；
I group of formula: type I compound concentration is 1 × 10-6mol/L；
W-5A-22-400 group: compound W-5A-22-400 concentration is 1 × 10-6mol/L；
W-5A-22-3000 group: compound W-5A-22-3000 concentration is 1 × 10-6mol/L；
W-5A-22-20000 group: compound W-5A-22-20000 concentration is 1 × 10-6mol/L；
5 multiple holes of every group of setting, while every group is respectively provided with blank group and (is free of cell, but DMEM concentration and respective sets are kept Unanimously).It giving after cultivating 48h under conditions of 37 DEG C and 5% carbon dioxide, 10 μ L of CCK-8 reagent is added in every hole, continue to cultivate 4h, At wavelength 450nm, each hole OD value is detected.According to the growth inhibition ratio of formula calculating group of cells: inhibitory rate of cell growth= 1- [(the corresponding blank group OD value of each administration group OD value -)/(the corresponding blank group OD value of control group OD value -)] × 100%.
2 results
The identification of 2.1 ASMC
It is observed with inverted phase contrast microscope, it is in shuttle shape that discovery ASMC, which does not converge preceding, and partial region cell is in after cell confluency Pencil arrangement, in typical " peak valley " shape.
The influence of 2.2 couples of ASMC proliferation
Each compound effects of the present invention are after ASMC 48h, and compared with icariin tuple, each compound group of the present invention is thin The OD value of born of the same parents is substantially reduced, and difference has conspicuousness.
Compared with icariin tuple cell, the inhibiting rate of the compounds of this invention group of cells is increased significantly, difference There is conspicuousness.
Compared with I group of cell of formula, the inhibiting rate of the compounds of this invention group of cells is increased significantly, and difference has significantly Property.(being shown in Table 4 and 5)
Each compound effects of table 4 are in the OD value of ASMC 48h
Compared with Normal group,$P < 0.05,$$p<0.01；
Compared with icariin tuple,#P < 0.05,##p<0.01；
With I group of ratio of formula,&P < 0.05,&&p<0.01。
Each compound effects of table 5 are in the inhibiting rate of ASMC 48h
Compared with icariin tuple,$P < 0.05,$$p<0.01；
With I group of ratio of formula,&P < 0.05,&&p<0.01。
The measurement of 15 the compounds of this invention bioavilability of embodiment
1. animal packet and administration
60 Wistar rat (270 ± 30) g, half male and half female, by Shandong Xinshidai Pharmaceutical Industry Co., Ltd. experimental animal The heart provides, production licence number: SCXK (Shandong) 20060019.At 20~22 DEG C of temperature, relative humidity 45%~65%, illumination/ It is raised under the conditions of dark 12h/12h, free diet, drinking-water.
The compounds of this invention gastric infusion group: female by healthy Wistar rat 30 of 12 hours free waters of fasting It is male fifty-fifty, it is divided into 5 groups, icariin tuple (epimedium aglucone is given in stomach-filling), I group of formula (type I compound is given in stomach-filling), W- 5A-22-400 group (compound W-5A-22-400 is given in stomach-filling), (compound W-5A-22- is given in stomach-filling to W-5A-22-3000 group 3000), W-5A-22-20000 group (compound W-5A-22-20000 is given in stomach-filling).The administration of each group single oral gavage, dosage For 3mg/kg.12h fasting, free water before being administered.Before administration (0h), administration after 0.083,0.25,0.5,1,1.5, 2,3,4,6,8,12 and retroorbital venous clump takes 300 μ L of blood or so for 24 hours, anticoagulant heparin, it is centrifuged under the conditions of 4 DEG C of 12000rpm 5min, separated plasma are stored in -20 DEG C of low temperature refrigerators.Free water during experiment, 2h is fed after stomach-filling.
The compounds of this invention intravenously administrable group: female by healthy Wistar rat 30 of 12 hours free waters of fasting It is male fifty-fifty, it is divided into 5 groups, icariin tuple (epimedium aglucone is given in injection), I group of formula (type I compound is given in injection), W- 5A-22-400 group (compound W-5A-22-400 is given in injection), (compound W-5A-22- is given in injection to W-5A-22-3000 group 3000), W-5A-22-20000 group (compound W-5A-22-20000 is given in injection).The administration of each group tail vein injection, gives medicament Amount is 3mg/kg.0.033,0.083,0.25,0.5,1,1.5,2,3,4,6,8,12 and after (0h), administration before administration Retroorbital venous clump takes 300 μ L of blood or so for 24 hours, anticoagulant heparin, and 5min is centrifuged under the conditions of 4 DEG C of 12000rpm, and separated plasma saves In -20 DEG C of low temperature refrigerators.Feed food and water freely during experiment.
2. plasma sample measures
UPLC-MS/MS quantitative analysis is carried out through processed plasma sample by all, measures plasma drug level.
3. the calculating of bioavilability
By the blood concentration-time data of measurement DAS software (Drug and Statistics, Chinese Mathematics pharmacology Association, the establishment such as Sun Ruiyuan) calculate pharmacokinetic parameters.The absolute bioavailability of each compound is calculated according to formula, t is last The sampling time of drug concentration can be surveyed.
4. the absolute bioavailability of each compound
The bioavilability of each compound of table 6
As can be seen from the above table, the bioavilability of each compound in the present invention is obviously higher than epimedium aglucone, formula I Compound, this shows that the compound in terms of the druggability in the present invention has apparent technical advantage.
Embodiment 16 the compounds of this invention W-5A-22-400, W-5A-22-3000, W-5A-22-20000 and type I compound Toxicity test research is administered in rat duplicate injection
SD rat is divided into five groups: Normal group, type I compound group (are injected intravenously type I compound 100mg/kg/d), W- 5A-22-400 group (intravenous injection W-5A-22-400100mg/kg/d), W-5A-22-3000 group (intravenous injection W-5A-22- 3000100mg/kg/d), W-5A-22-20000 group (intravenous injection W-5A-22-20000100mg/kg/d).
It groups of animals successive administration 28 days, is administered once a day, is discontinued and restores 4 weeks.At the end of the administration phase, abdomen cardinal vein is adopted Collect blood sample, carries out the detection of the indexs such as hematology, clotting time, blood biochemical analysis and electrolyte, carry out Systematic anatomy later, greatly It causes to observe each organs and tissues form, it is (female to brain, spleen, thymus gland, heart, kidney (bilateral), liver, adrenal gland (bilateral), mammary gland Property rat), prostate (male rat) weighed and calculates organ coefficient.
Experiment discovery, type I compound group rat creatinine, alkaline phosphatase and NSC 334200 transfer enzyme level increase.Disease The visible different degrees of renal damage of histological examination is managed, apparent Nephrotoxicity occurs.The female rats proliferation of mammary gland.Formula I compound shows certain toxic side effect.
Under this experimental condition, do not find that abnormal change relevant to W-5A-22-400 toxicity, W-5A-22-400 are administered to For medicine without overt toxicity, clinical use safety is higher.
Under this experimental condition, abnormal change relevant to W-5A-22-3000 toxicity, W-5A-22-3000 injection are not found Without overt toxicity, clinical use safety is higher for administration.
Under this experimental condition, abnormal change relevant to W-5A-22-20000 toxicity, W-5A-22-20000 note are not found Administration is penetrated without overt toxicity, clinical use safety is higher.

Claims (10)

1. a kind of epimedium aglucone derivative, structure is as shown in formula W-5A:
Or the pharmaceutically acceptable salt of formula W-5A compound；
Wherein, the integer of the m, n independently selected from 0-6；
The molecular weight of the PEG is selected from 400-20000.
2. epimedium aglucone derivative as described in claim 1, which is characterized in that described m, n are independently selected from the whole of 0-4 Number.
3. epimedium aglucone derivative as described in claim 1, which is characterized in that the molecular weight of the PEG is selected from 1000- 10000。
4. epimedium aglucone derivative as described in claim 1, which is characterized in that the molecular weight of the PEG is selected from PEG 400, PEG600, PEG1000, PEG2000, PEG3000, PEG4000, PEG6000, PEG10000 or PEG20000.
5. epimedium aglucone derivative as described in claim 1, which is characterized in that formula W-5A compound is specially Formula W-5A-22:
6. epimedium aglucone derivative as described in claim 1, which is characterized in that formula W-5A compound specifically:
7. epimedium aglucone derivative as described in claim 1, which is characterized in that the synthesis step of formula W-5A compound is such as Under:
1) Boc-ICT is reacted with isopropyl isocyanate, products therefrom reacted with halohydrin 2；
2) mPEG reacts to obtain compound 3 with compound 1；
The m is selected from the integer of 0-6；
3) compound 2 reacts production W-5A with compound 3 in the presence of condensing agent:
8. epimedium aglucone derivative as claimed in claim 7, which is characterized in that the condensation course can use condensation Agent；Condensing agent is selected from 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride/4-dimethylaminopyridine (EDCI/ DMAP), 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride/I-hydroxybenzotriazole/N, N- diisopropylethylamine (EDCI/HOBT/DIPEA), 2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester/N, N- bis- are different At least one of propylethylamine (HATU/DIPEA), dicyclohexylcarbodiimide/4-dimethylaminopyridine (DCC/DMAP).
9. a kind of pharmaceutical composition includes epimedium aglucone derivative or its medicine described in any one of claim 1-6 claim Acceptable salt and pharmaceutically acceptable adjuvant in Neo-Confucianism.
10. the purposes of pharmaceutical composition as claimed in claim 9 drug in terms of preparation treatment asthma or bone marrow suppression.
CN201810254006.XA 2018-03-26 2018-03-26 Epimedium aglucone derivative and its preparation method and application Pending CN110358078A (en)

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