WO2018068696A1 - 一种青藤碱及其衍生物的peg修饰物、其制备方法和用途 - Google Patents

一种青藤碱及其衍生物的peg修饰物、其制备方法和用途 Download PDF

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WO2018068696A1
WO2018068696A1 PCT/CN2017/105515 CN2017105515W WO2018068696A1 WO 2018068696 A1 WO2018068696 A1 WO 2018068696A1 CN 2017105515 W CN2017105515 W CN 2017105515W WO 2018068696 A1 WO2018068696 A1 WO 2018068696A1
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sinomenine
derivative
peg
pharmaceutically acceptable
acceptable salt
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PCT/CN2017/105515
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English (en)
French (fr)
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李思成
张祖兵
张青松
杨强
仲伟铭
李盎
包旭
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成都一平医药科技发展有限公司
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Publication of WO2018068696A1 publication Critical patent/WO2018068696A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/22—Bridged ring systems
    • C07D221/28—Morphinans
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • 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
    • C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
    • C08G65/48—Polymers modified by chemical after-treatment

Definitions

  • the invention relates to the field of medicine, in particular to a PEG modification of sinomenine and its derivatives, a pharmaceutical composition thereof, a preparation method thereof and medical use in anti-inflammatory, analgesic, immunosuppressive and the like.
  • Qingfeng vine is the dry rhizome of the anti-family plant vine and vine, and the qingfeng vine has the functions of collaterals, pain relief, rheumatism and so on.
  • Qingfeng vine is used as a Chinese herbal medicine for internal and external use. It has been used for the treatment of diseases and symptoms such as rheumatism and rheumatoid arthritis, joint swelling, numbness and pain for thousands of years.
  • Sinomenine is a kind of alkaloid monomer obtained from the genus Qingfeng vine by modern Chinese medicine extraction and separation technology.
  • the structural formula is as follows:
  • sinomenine hydrochloride preparations have been used in clinical treatment.
  • sinomenine hydrochloride preparations also expose many disadvantages in clinical applications, such as short half-life and low oral bioavailability, so long-term oral administration is generally required in clinical applications. Taking clothes, and taking a large dose, can easily cause more serious gastrointestinal side effects, and also have different degrees of toxicity to liver, kidney and heart.
  • Sinomenine is one of the very strong histamine-releasing agents extracted from plants. It has been found that sinomenine can make tissues such as guinea pig aorta, trachea, diaphragm, heart, uterus, skin and stomach. Release of histamine.
  • sinomenine derivative is a chemically modified derivative on the nucleus of sinomenine. Because its mother nucleus structure is the same as sinomenine, its medicinal activity is similar to that of sinomenine, but it has different physical and chemical properties. Also suitable for preparation into different drugs, sinomenine derivatives which have been found and have been prepared have the following chemical structures,
  • R represents a plurality of groups.
  • drugs such as protein drugs, peptide drugs, and chemical drugs
  • polyethylene glycol modification technology can reduce the side effects of drugs, increase the water solubility of drugs, prolong the half-life of drugs, and reduce The number of doses can reduce enzymatic hydrolysis and increase bioavailability.
  • sinomenine monomer can be effectively evaded by immunogenic reaction, avoiding the release of histamine from mast cells, reducing severe toxic side effects such as allergies, and prolonging the half-life.
  • the present invention provides a PEG modification of sinomenine or a derivative thereof of the formula I or a pharmaceutically acceptable salt thereof:
  • PEG represents polyethylene glycol having a molecular weight of 200 to 60,000 Daltons or a modifier thereof
  • A represents an amino acid
  • i is an integer from 0 to 20, representing the number of amino acids
  • j is an integer from 0 to 20, representing the number of amino acids
  • n is an integer from 0-20;
  • n is an integer from 0-20;
  • r is an integer from 0-20;
  • t is an integer from 0 to 20, representing the number of connecting arms when PEG is connected to Q;
  • h is 0 or 1;
  • Q represents sinomenine (sinomenine having the structural formula shown in formula V) or a derivative thereof;
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 50,000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 40,000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 30000 Daltons or a modifier thereof.
  • the PEG represents polyethylene glycol having a molecular weight of from 200 to 20000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 10,000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 8000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 200 to 5000 Daltons or a modifier thereof.
  • the PEG represents a polyethylene glycol having a molecular weight of from 300 to 5000 Daltons or a modifier thereof.
  • the PEG is selected from the group consisting of PEG 20K -(NH 2 ) 2 , mPEG 5K -SC (ie ), mPEG 340 and mPEG 5K -CH 2 CH 2 COOH.
  • the amino acid is a natural amino acid or a synthetic unnatural amino acid.
  • the amino acid is lysine.
  • the amino acid is glycine.
  • i is zero.
  • i 1 or 2.
  • i is one.
  • j is 0 or 1.
  • j is zero.
  • m is 0 or 1.
  • n is zero.
  • n is zero.
  • n 1 or 2.
  • n is one.
  • r is 1-3.
  • r is 2.
  • t is from 2 to 5.
  • t is 3-5.
  • t is one.
  • t is 2.
  • t is 4.
  • h is zero.
  • x is one.
  • x is 3-5.
  • x is 4.
  • the amino acid is glycine or lysine
  • i is 0, 1 or 2
  • j is 0 or 1
  • B is 0 or 1
  • n is 0, 1 or 2
  • r 1-3 t is 1-5 (for example, 3-5)
  • D is 0 or 1
  • x is 1-10 (for example, 3-5)
  • Q represents sinomenine.
  • the amino acid is glycine or lysine, i is 1, j is 0, B is, m is 0, n is 1 or 2, r is 2, and t is 2 or 4, D Where h is 0 or 1, x is 4, and Q represents sinomenine.
  • i is 0 or 1
  • j is 0 or 1
  • n is 0 or 2
  • r is 2
  • t is 1 or 2
  • x is 1 or 4 and Q represents sinomenine.
  • B is, m is 0, n is 0, r is 2, t is 1, D is, h is 0, x is 1, and Q is Sinomenine.
  • i 1, j is 0, B is 0, n is 0 or 2, r is 2, t is 1 or 2, in D, h is 0, x is 1 or 4, Q stands for sinomenine.
  • sinomenine described herein has the structure shown in Formula V:
  • sinomenine derivative described herein refers to a series of compounds having the same or similar biological activity as sinomenine obtained by chemically modifying the sinomenine core.
  • the sinomenine derivative is selected from the group consisting of compounds of formula IV:
  • R 1 -R 17 are selected from the group consisting of:
  • the 7-position to the 8-position carbon in the structure of the sinomenine or its derivative is a single bond in which a double bond is reduced.
  • the PEG comprises a one-armed PEG blocked at one end and a modifier thereof; an active double-armed PEG at both ends and a modifier thereof; and a multi-arm PEG and a modifier thereof.
  • the PEG modifying agent refers to a structure obtained by the one-arm PEG blocked at one end, the active double-armed PEG or the multi-arm PEG at both ends, optionally modified by one or more modifying groups.
  • the modifying group is selected from a C 1-6 alkoxy group (eg, C 1-4 alkoxy or C 1-2 alkoxy), —(CH 2 ) k —OH, —(CH 2 ) k — NH 2 , -(CH 2 ) k -COOH, Where k is an integer from 0 to 6 (eg, 0-4 or 0-2).
  • the PEG is optionally modified with one or more modifying groups, wherein the modifying group is selected from the group consisting of methoxy, hydroxy, amino, carboxy, And -(CH 2 ) 2 COOH.
  • the hydroxyl modification site in the structure of the sinomenine or derivative thereof is linked to the linking arm of the PEG by a chemical bond.
  • the hydroxyl modification site in the structure of the sinomenine or derivative thereof comprises a hydroxyl group at the 4 or 6, 7, 8 position.
  • the chemical bond comprises: an ester bond, an amide bond, and an ether bond.
  • the PEG comprises a one-armed PEG blocked at one end and a modifier thereof; a double-armed PEG with a living wave at both ends and a modifier thereof; and a multi-arm PEG and a modifier thereof; wherein sinomenine or The hydroxyl modification sites in the structure of the derivative include hydroxyl groups at the 4 or 6, 7, and 8 positions.
  • the hydroxyl modification site in the structure of sinomenine or a derivative thereof is linked to the linking arm of PEG by a chemical bond including: an ester bond, an amide bond, and an ether bond.
  • the PEG, B, A, D, Q in the general formula (I) are linked by a chemical bond, wherein the chemical bond includes an ester bond, an amide bond, and an ether bond.
  • the PEG modification of sinomenine or a derivative thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of:
  • the PEG modification described herein may comprise a four-part structure as described in the structure of Formula I: PEG, tether, D, and sinomenine or a derivative thereof, wherein the tether is B m -A i - B n -A j , which may be attached to the multi-arm PEG at one end and to D or sinomenine or a derivative thereof at one end, the number of which depends on the number of arms of the PEG.
  • Formula I is When PEG is a double-armed PEG or a modifier thereof, it extends two arms A 1 -B 2 (ie, one A is connected to two Bs) and is connected to four Qs, that is, two Qs are connected to each arm. .
  • pharmaceutically acceptable salt means that (1) an acidic functional group (for example, -COOH, -OH, -SO 3 H, etc.) present in the compound of the present invention is formed with a suitable inorganic or organic cation (base).
  • an acidic functional group for example, -COOH, -OH, -SO 3 H, etc.
  • a salt for example a salt of a compound of the invention with an alkali metal or alkaline earth metal, an ammonium salt of a compound of the invention, and a salt of a compound of the invention with a nitrogen-containing organic base; and (2) a basic functional group present in the compound of the invention (for example, -NH 2 , etc.) a salt formed with a suitable inorganic or organic anion (acid), for example a salt of a compound of the invention with an inorganic or organic carboxylic acid.
  • a suitable inorganic or organic anion for example a salt of a compound of the invention with an inorganic or organic carboxylic acid.
  • salts of the compounds of the invention include, but are not limited to, alkali metal salts such as sodium, potassium, lithium, and the like; alkaline earth metal salts such as calcium, magnesium, and the like; other metal salts, Such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc.; inorganic alkali salt, such as ammonium salt; organic alkali salt, such as t-octylamine salt, dibenzylamine salt, morpholine salt, Portuguese Glycosylamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'- Dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperaz
  • the pharmaceutically acceptable salt of the PEG modification of sinomenine or a derivative thereof is its oxalate salt.
  • the present invention also provides a pharmaceutical composition comprising the PEG modification of the sinomenine or a derivative thereof or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition is in the form of a unit dose of a pharmaceutical formulation.
  • the pharmaceutical composition is an injection (including a lyophilized formulation and a small volume injection).
  • the pharmaceutical preparations can be prepared using conventional techniques of formulation.
  • some pharmaceutically acceptable carriers may be added to the pharmaceutical composition as needed.
  • the pharmaceutically acceptable carrier is selected from the group consisting of: mannitol, sorbitol, glucose, sodium chloride, disodium EDTA, calcium EDTA, sodium dihydrogen phosphate, disodium hydrogen phosphate, citric acid and sodium citrate, hydrochloric acid And sodium hydroxide and so on.
  • the pharmaceutical composition of the present invention determines the usage amount according to the condition of the patient at the time of use.
  • a unit dose of the medicament may contain 0.1 to 1000 mg of the pharmaceutically active substance of the present invention, and the balance is a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be from 0.1 to 99.9% by weight based on the total weight of the formulation.
  • the present invention provides the use of the PEG modification of the sinomenine or a derivative thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for anti-inflammatory, analgesic, and immunosuppressive.
  • the invention provides a method of anti-inflammatory, analgesic or immunosuppressive comprising administering to a subject in need thereof an effective amount of a PEG modification of sinomenine or a derivative thereof according to the invention Or a step of its pharmaceutically acceptable salt.
  • the invention provides a PEG modification of sinomenine or a derivative thereof, or a pharmaceutically acceptable salt thereof, for use in anti-inflammatory, analgesic or immunosuppressive.
  • the present invention provides a process for the preparation of a PEG modification of the sinomenine or a derivative thereof, comprising the steps of:
  • sinomenine or a derivative thereof is prepared into an ester compound such as succinate or glycinate, and then with polyethylene glycol or a derivative thereof such as bislysyl-polyethylene glycol or methoxypolyethylene.
  • the alcohol succinimide carbonate is reacted to obtain a PEG modification of the sinomenine or a derivative thereof;
  • the PEG having a carboxyl group at the terminal group is esterified with the hydroxyl group of sinomenine or a derivative thereof to obtain a corresponding PEG modification of sinomenine or a derivative thereof.
  • the ester can be directly condensed with a hydroxyl group on sinomenine via a carboxy group having a carboxyl group.
  • the present invention provides a PEG modification of sinomenine and derivatives thereof of formula (I),
  • PEG represents polyethylene glycol having a molecular weight of from 100 to 60,000 Daltons
  • A represents an amino acid, and i and j are integers from 0 to 20, representing the number of amino acids;
  • n is an integer from 0 to 20
  • r is an integer from 0 to 20
  • h 0 or 1.
  • Q represents sinomenine and its derivatives, x is an integer from 0 to 20,
  • t is an integer from 0 to 20, representing the number of connecting arms.
  • the amino acid is lysine, i is 1 or 2, j is 0 or 1, in B, m is 0 or 1, n is 1 or 2, and r is 1-3, t For 3-5, D, h is 0 or 1, x is 3-5, and Q represents sinomenine.
  • the amino acid is lysine, i is 1, j is 0, B is, m is 0, n is 1, r is 2, t is 4, D is, h is 0 or 1, x is 4, Q represents sinomenine.
  • the 7-position to the 8-position carbon in the structure of sinomenine and its derivatives is a single bond in which a double bond is reduced.
  • the PEG comprises a one-armed PEG blocked at one end and a modifying group thereof; a double-armed PEG having a living wave at both ends and a modifying group thereof; and a multi-arm PEG and a modifying group thereof, wherein sinomenine and The hydroxyl group of the modification site in the structure of the derivative includes a hydroxyl group at the 4-position or the 6,7,8-position.
  • the hydroxyl group of the modification site in the structure of sinomenine and its derivatives is linked to the linking arm of PEG by a chemical bond including an ester bond, an amide bond or an ether bond.
  • the PEG, B, A, D, Q are linked by a chemical bond, the chemical bond comprising: an ester bond, an amide bond, or an ether bond.
  • the PEG modification of the sinomenine or derivative thereof is selected from
  • sinomenine modified with PEG Anti-inflammatory, analgesic, immunosuppressive and mast cell release of histamine and ileal contraction were performed on sinomenine modified with PEG to observe the changes in the pharmacological effects of the compounds obtained after modification.
  • the control drug was sinomenine hydrochloride, which was called the sinomenine group in the experiment.
  • Inhibition rate% (1 - number of writhings before administration/number of writhing in negative group) ⁇ 100%
  • Kunming female mice were placed in an aluminum pan in a water bath of 55 ⁇ 0.5°C. The mice were treated with hind paws and hindered feet and returned to the head as pain response indicators. The time of pain response (pain threshold) was recorded and measured twice. Mice with an average value of less than 30 seconds and no jump were selected for the experiment. 60 eligible mice were randomly divided into 6 groups according to their body weight. The rats in the negative control group were injected with normal saline 0.4 ml/20 g body weight, and the aspirin group per mouse.
  • mice Sixty Kunming mice were randomly divided into 6 groups according to their gender, male and female.
  • the rats in the negative control group were injected with 0.4ml/20g body weight of normal saline in the tail vein, 0.4ml/20g body weight per mouse in the aspirin group, sinomenine group, PEG20K-green group, PEG5K-green group and mPEG-6-green group.
  • the rats were injected with 0.4ml/20g body weight of the corresponding drugs in the tail vein respectively, and the concentration of each group was 5mg/ml.
  • One hour after the administration each mouse was exposed to inflammation with xylene 0.05 ml/mouse, and the left ear was used as a control.
  • mice were sacrificed from the cervical vertebra 30 minutes after the inflammation, and the left and right ears of the ear were removed with a 7 mm diameter puncher.
  • the tablets were weighed with an electronic balance, and the degree of swelling was expressed by the difference in weight between the right and left ears, and a significant comparison between the groups was performed.
  • Inhibition rate% (1 - number of writhings before administration/number of writhing in negative group) ⁇ 100%
  • mice were sacrificed by cervical dislocation. Disinfect the chest and abdomen skin, expose the peritoneum, inject 5ml/pre-cooled RPMI-1640 medium into the abdominal cavity, massage the abdomen, return the abdominal cavity cleaning solution, and centrifuge the cells at low speed to make a cell suspension. After counting, 5 ⁇ 10 4 / The wells were inoculated on the culture plate, and after incubation for 2 hours at 5% CO 2 at 37 ° C, macrophages were obtained after washing off the non-adherent cells.
  • each test drug was configured to have two concentrations of 10 ⁇ M and 50 ⁇ M.
  • TNF- ⁇ was detected by the cytotoxicity method of L929 cell line, and the result was expressed by OD570 value.
  • IL-1 was detected by the L929 cell proliferation method, and the results were also expressed by the OD570 value.
  • Table 4 shows inhibition of TNFa and IL-1 secretion
  • the sterile fresh synovial membrane obtained by the operation was washed twice with physiological saline in a petri dish, and the ophthalmic scissors were cut into small pieces of 1 ⁇ 1 (mm 3 ).
  • a small amount (type dip) and an equal volume of DMEM medium at a concentration of 0.8 mg/mL were digested for 3 h at 37 ° C in a 5% CO 2 incubator. Conventional centrifugation, discard the supernatant, add 0.2 mL of 0.25% trypsin, mix and digest in a CO 2 incubator for 30 min. After filtration through a 120 mesh copper mesh screen, the digest was collected and placed in a 37 ° C, 5% CO 2 incubator overnight. Change the liquid once a day.
  • Synovial cells were stained with trypan blue and Giemsa, and their viability and purity were ⁇ 95%. Thereafter, the liquid is changed once every 2 to 3 days, and once every 5 to 7 days. After 3 passages, a synovial cell suspension (1 ⁇ 10 6 /mL) was prepared by using DMEM containing 5 ⁇ g/mL of LPS, and added to a 6-well cell culture plate, 3 mL per well, in duplicate. The cells were cultured in a CO 2 incubator for 48 hours, and the nuclear protein and total RNA were extracted by the corresponding methods.
  • the total cellular RNA was extracted by conventional methods for reverse transcription-PCR. Scanning with a coagulation pulse image analyzer, the ratio of the target band gray scale to the internal reference ⁇ -actin standard band was obtained by LabImage 2.6 gel image analysis software.
  • the electrophoretic mobility shift assay was used to detect the activity of NF- ⁇ B in synovial cells.
  • the electrophoresis hysteresis band was scanned with a gel image analyzer for density scanning and analyzed by LabImage 2.6 gel image analysis software.
  • guinea pig peritoneal mast cells The in vitro hatching test of guinea pig peritoneal mast cells (PMC) was slightly modified by the method reported by Nemeth. Animals were decapitated and sacrificed. 15 ml of pre-cooled mast cell culture solution was intraperitoneally injected. The abdomen was opened by gently agitating the abdomen for 2 minutes. Abdominal lavage solution was aspirated, centrifuged at 4 ° C, 1500 rmin for 10 min, and the cell suspension was adjusted to about 0.5 ml. In 7 ml of P-F10, the cells were separated by centrifugation at 2500 rpm for 15 min at 4 °C.
  • the isolated and purified mast cell suspension was taken, 0.9 ml per tube, and 0.1 ml of different drugs or CP48/80 were added respectively, and the blank control group was replaced with physiological saline. Incubate in a 37 ° C incubator for 15 min. Immediately after removal, put in an ice water bath for 5 min to terminate the reaction. Centrifuge each tube at 4 ° C, 400 ⁇ g for 10 min, add the supernatant to 25% trichloroacetic acid, mix, centrifuge, and take.
  • Histamine release rate supernatant histamine / (intracellular histamine + supernatant histamine) x 100%.
  • Determination of contractile tension of guinea pig isolated ileum smooth muscle guinea pig fasting for 12h, drinking water is not limited.
  • guinea pig fasting for 12h open the abdominal cavity, cut the ileum 2 cm away from the cecum, remove the ileum for about 15 cm, place it in the pre-cooled Krebs solution, cut off the fat tissue around the ileum, rinse the intestinal contents with Krebs solution, cut Specimens growing about 1.5 to 2.0 cm.
  • the experiment was started after the load of 1 g was adjusted for 30 minutes, and the change of the ileus tension was recorded by the Powerlab biological signal acquisition and analysis system.
  • the experiment was divided into 5 groups, namely blank control group, sinomenine group, PEG20K-cyan group, PEG5K-cyan group and mPEG-6-cyan group.
  • the blank control group was given the same volume of physiological saline instead of the drug.
  • Ten specimens from each group were taken from different guinea pig ileum.
  • the experiment was divided into two parts. One was to observe the effect of each group on the autonomic rhythm contraction of normal ileum: firstly, the normal intestinal smooth muscle contraction curve was traced for 10 min at the beginning of the experiment, and then 0.1 ml of different concentrations of the test drug were added to the bath for 5 min. The 10 min ileal contraction curve was traced. The second is to observe the effect of each group on histamine-induced ileal contraction: after a normal ileal contraction curve is traced, 0.1 ml of histamine phosphate solution is given to maximize the contraction tension of intestinal smooth muscle, and then the Krebs solution is used to restore the smooth muscle contraction of the intestine.
  • Eight Japanese white rabbits were divided into four groups according to body weight, namely sinomenine group, PEG20K-cyan group, PEG5K-cyan group and mPEG-6-cyan group, with 2 groups in each group.
  • Each group was slowly injected with 2ml/kg of drug in the left ear vein at a concentration of 5mg/ml.
  • the right ear vein was slowly injected with physiological saline 2ml/kg once a day for three consecutive days, starting from the first injection. Every day, the injection site was observed to have edema and erythema. 24 hours after the last injection, the ear was cut from the ear and fixed in 10% formaldehyde solution, and then examined by pathological examination. Local pain is an indication of whether the rabbit is struggling when administered.
  • the preparation of the synthetic drug and the dosage form of the present invention can be carried out in a conventional pharmaceutical laboratory under the operation of a pharmacy professional.
  • diamine polyethylene glycol is also commercially available as a self-made product, dimethylaminopyridine (DMAP), N-tert-butoxycarbonylglycine, N,N-diisopropylethylamine (DIEA), two rings.
  • DMAP dimethylaminopyridine
  • DIEA N,N-diisopropylethylamine
  • DCC Hexyl carbodiimide
  • Monomethoxy polyethylene glycol succinimide carbonate (5KD) (mPEG-SC), sinomenine, 1-bromo-3,6,9,12,15,18,21-heptaoxene Dioxane is a commercially available raw material, and the solvent is a laboratory purely pure solvent.
  • Example 1 PEG20k-[lysine-(succinic acid-4- sinomenine) 2 ] 2 was called PEG20K-cyan in the experiment.
  • Example 2 mPEG5k-CO-glycine-4- sinomenine is called PEG5K-cyan in the experiment.
  • Monomethoxypolyethylene glycol succinimide carbonate (5KD) (mPEG-SC) (8.5g, 1.7mmol) and B1 (0.7g, 1.9mmol) were sequentially added to a 250ml single-mouth bottle, and 100ml of dichlorochloride was added. The methane was dissolved, and DCC (1 g, 4.8 mmol) was added at room temperature, and the reaction was stirred overnight. The reaction was monitored by thin layer chromatography. After the reaction was completed, the filtrate was concentrated, and the filtrate was concentrated to give 8.4 g of white powdery solid.
  • 5KD Monomethoxypolyethylene glycol succinimide carbonate
  • B1 0.7g, 1.9mmol
  • Example 3 mPEG340-6- sinomenine oxalate (6-(3,6,9,12,15,18,21-heptaxodocosyl) sinomenine ether oxalate experiment MPEG-6-cyan
  • the sinomenine (10 g, 30 mmol) was added to a 500 ml round bottom flask, 300 ml of dichloromethane was added, then DIPEA (23.5 ml, 18.4 g, 180 mmol) was added and stirred, and MEMCl (13.7 ml, 14.9 g, 120 mmol) was added to the ice water bath. After the completion of the dropwise addition for 30 minutes, the mixture was warmed to room temperature and then reacted overnight. The reaction mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate for 1 hour, filtered, and concentrated to give 12.
  • 4-MEM- sinomenine (12.01 g, 28.8 mmol) was added to a 500 ml round bottom flask, then 120 ml of a mixed solution of acetonitrile and 120 ml of acetic acid was added, and the temperature was lowered to -15 ° C to add tetramethylammonium triacetoxyborohydride ( 11.37g, 43.2mmol), stirred for 20min, warmed to room temperature, stirred for 2 hours, acetonitrile was added to dryness under reduced pressure, the pH was made basic, extracted with DCM, dried over anhydrous sodium sulfate, filtered and concentrated to give 4-MEM-6 - OH- sinomenine product 12.14 g.
  • Example 4 mPEG5k-(CH 2 ) 2 CO-4- sinomenine is called PEG5K-cyan 2 in the experiment.

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Abstract

公开了一种青藤碱及其衍生物的PEG修饰物、其制备方法和用途，所述青藤碱或其衍生物的PEG修饰物可显著降低免疫原性反应，过敏反应；可延长青藤碱半衰期；口服苦味降低；注射时血管刺激性降低；和/或恶心和呕吐副作用降低。

Description

一种青藤碱及其衍生物的PEG修饰物、其制备方法和用途 技术领域
本发明涉及医药领域，具体涉及一种青藤碱及其衍生物的PEG修饰物、其药物组合物、制备方法和在消炎、镇痛、免疫抑制等方面的医药用途。
技术背景
青风藤是防己科植物青藤和毛青藤的干燥根茎，青风藤具有通络、止痛、祛风湿等功效。在中国传统中医学中青风藤作为内服外用的中草药，用于治疗风湿和类风湿性关节炎、关节肿大、麻木和疼痛等疾病或症状已经有上千年的应用历史。
青藤碱是通过现代中药提取分离技术从青风藤中分理得到一种生物碱单体，结构式如下：
Figure PCTCN2017105515-appb-000001
化学名称:(9alpha,13alpha,14alpha)-7,8-二脱氢-4-羟基-3,7-二甲氧基-17-甲基吗啡喃-6-酮；
英文名称:Sinomenine；
性质:针状结晶(由苯中结晶)。熔点161℃，熔化后熔点又升至182℃。旋光度-71(c＝2.1，乙醇)。溶于乙醇、丙酮、氯仿和稀碱，微溶于水、乙醚和苯。其盐酸盐，结晶(水或乙醇)，278℃分解。其氢碘酸盐，针晶(由水中结晶)，272℃分解。其苦味酸盐，黄色针晶，176℃分解。
CAS:115-53-7；
分子式：C19H23NO4；
分子量:329.38；
现代药理学研究表明青藤碱是青风藤中一种主要的生物活性碱，具有免疫抑制、祛风镇痛、消炎、降压、抗心律失常、抗神经退行性疾病等作用。在我国和日本已经有青藤碱盐酸盐制剂应用于临床治疗当中。但是青藤碱盐酸盐制剂在临床应用中，也暴露出很多缺点，例如：半衰期较短，口服生物利用率低，因此在临床应用中一般需要长期口 服，且服用剂量大，容易引起较严重的胃肠道不良反应，同时对肝肾和心脏也有不同程度的毒性。青藤碱是目前所知的从植物中提取的非常强的组胺释放剂之一，试验发现青藤碱对豚鼠大动脉、气管、横膈膜、心脏、子宫、皮肤、胃等组织均能使之释放组胺。
青藤碱衍生物是在青藤碱结构母核上进行化学修饰后的衍生物，因其母核结构和青藤碱相同，因此其药物活性和青藤碱相似，但具有不同的理化性质，也适合制备成不同的药物，已经发现并已经制备的青藤碱衍生物具有以下化学结构，
Figure PCTCN2017105515-appb-000002
其中的R代表多种基团。
许多药物如蛋白药物，多肽药物，化学药物等经过聚乙二醇修饰技术修饰后在体内能够缓慢的释放出药物产生疗效，能够降低药物毒副作用，增加药物的水溶性，延长药物的半衰期，降低用药次数，能够降低酶解作用从而提高生物利用度。但对于青藤碱类结构复杂的药物，目前尚无用聚乙二醇修饰的报道。本发明人通过研究发现青藤碱单体经聚乙二醇修饰后，能够有效的屏避免疫原性反应，避免肥大细胞组胺释放，降低过敏等严重的毒副反应，延长半衰期。
发明内容
本发明提供通式Ⅰ的青藤碱或其衍生物的PEG修饰物或其可药用盐：
Figure PCTCN2017105515-appb-000003
其中，
PEG代表分子量为200-60000道尔顿的聚乙二醇或其修饰剂；
A代表氨基酸；
i为0-20的整数，代表氨基酸的数量；
j为0-20的整数，代表氨基酸的数量；
B代表通式(Ⅱ)
Figure PCTCN2017105515-appb-000004
m为0-20的整数；
n为0-20的整数；
r为0-20的整数；
t为0-20的整数，代表PEG与Q连接时连接臂的数量；
D代表通式(Ⅲ)
Figure PCTCN2017105515-appb-000005
h为0或者1；
Q代表青藤碱(sinomenine,其结构式如式Ⅴ所示)或其衍生物；
x为0-20的整数。在一些优选的实施方案中，所述PEG代表分子量为200-50000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-40000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-30000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-20000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-10000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-8000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为200-5000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG代表分子量为300-5000道尔顿的聚乙二醇或其修饰剂。
在一些优选的实施方案中，所述PEG选自PEG20K-(NH2)2、mPEG5K-SC(即
Figure PCTCN2017105515-appb-000006
)、mPEG340和mPEG5K-CH2CH2COOH。
在一些优选的实施方案中，所述氨基酸为天然氨基酸或合成的非天然氨基酸。
在一些优选的实施方案中，所述氨基酸为赖氨酸。
在一些优选的实施方案中，所述氨基酸为甘氨酸。
在一些优选的实施方案中，i为0。
在一些优选的实施方案中，i为1或2。
在一些优选的实施方案中，i为1。
在一些优选的实施方案中，j为0或1。
在一些优选的实施方案中，j为0。
在一些优选的实施方案中，m为0或1。
在一些优选的实施方案中，m为0。
在一些优选的实施方案中，n为0。
在一些优选的实施方案中，n为1或2。
在一些优选的实施方案中，n为1。
在一些优选的实施方案中，r为1-3。
在一些优选的实施方案中，r为2。
在一些优选的实施方案中，t为2-5。
在一些优选的实施方案中，t为3-5。
在一些优选的实施方案中，t为1。
在一些优选的实施方案中，t为2。
在一些优选的实施方案中，t为4。
在一些优选的实施方案中，h为0。
在一些优选的实施方案中，x为1。
在一些优选的实施方案中，x为3-5。
在一些优选的实施方案中，x为4。
在一些优选的实施方案中，所述氨基酸为甘氨酸或赖氨酸，i为0、1或2，j为0或1，B中，m为0或1，n为0、1或2，r为1-3，t为1-5(例如3-5)，D中，h为0或者1，x为1-10(例如3-5)，Q代表青藤碱。
在一些优选的实施方案中，所述氨基酸为甘氨酸或赖氨酸，i为1，j为0，B中，m为0，n为1或2，r为2，t为2或4，D中，h为0或者1，x为4，Q代表青藤碱。
在一些优选的实施方案中，i为0或1,j为0或1,B中,m为0,n为0或2,r为2,t为1或2,D中,h为0,x为1或4,Q代表青藤碱。
在一些优选的实施方案中，i为0,j为0或1,B中,m为0,n为0,r为2,t为1,D中,h为0,x为1,Q代表青藤碱。
在一些优选的实施方案中，i为1,j为0,B中,m为0,n为0或2,r为2,t为1或2,D中,h为0,x为1或4,Q代表青藤碱。
本文中所述青藤碱具有式V所示结构：
Figure PCTCN2017105515-appb-000007
本文中所述青藤碱衍生物是指对青藤碱母核进行化学修饰后得到的一系列具有与青藤碱相同或相似生物学活性的化合物。
在一些优选的实施方案中，所述青藤碱衍生物选自式IV所示化合物：
Figure PCTCN2017105515-appb-000008
其包括R1-R17选自下述取代基时的一系列化合物：
Figure PCTCN2017105515-appb-000009
Figure PCTCN2017105515-appb-000010
其余取代基以及未修饰的部位同青藤碱。
在一些优选的实施方案中，所述青藤碱或其衍生物结构中的7位与8位碳之间为一双键被还原的单键。
在一些优选的实施方案中，所述PEG包含一端封闭的单臂PEG及其修饰剂；两端活泼的双臂PEG及其修饰剂；以及多臂PEG及其修饰剂。
在一些优选的实施方案中，所述PEG修饰剂是指所述一端封闭的单臂PEG、两端活泼的双臂PEG或多臂PEG任选地被一个或多个修饰基修饰后得到的结构，其中所述修饰基 选自C1-6烷氧基(例如C1-4烷氧基或C1-2烷氧基)、-(CH2)k-OH、-(CH2)k-NH2、-(CH2)k-COOH、
Figure PCTCN2017105515-appb-000011
其中，k为0-6的整数(例如0-4或0-2)。
在一些优选的实施方案中，所述PEG任选地被一个或多个修饰基修饰，其中所述修饰基选自甲氧基、羟基、氨基、羧基、
Figure PCTCN2017105515-appb-000012
和-(CH2)2COOH。
在一些优选的实施方案中，所述青藤碱或其衍生物结构中的羟基修饰位点通过化学键与PEG的连接臂连接。
在一些优选的实施方案中，所述青藤碱或其衍生物结构中的羟基修饰位点包括4位或者6,7,8位的羟基。
在一些优选的实施方案中，所述化学键包括：酯键、酰胺键和醚键。
在一些优选的实施方案中，所述PEG包含一端封闭的单臂PEG及其修饰剂；两端活波的双臂PEG及其修饰剂；以及多臂PEG及其修饰剂；其中青藤碱或其衍生物结构中的羟基修饰位点包括4位或者6,7,8位的羟基。
在一些优选的实施方案中，所述青藤碱或其衍生物结构中的羟基修饰位点通过化学键与PEG的连接臂连接，所述化学键包括：酯键、酰胺键和醚键。
在一些优选的实施方案中，所述通式(I)中PEG、B、A、D、Q之间通过化学键连接，其中所述化学键包括：酯键、酰胺键和醚键。
在一些优选的实施方案中，所述青藤碱或其衍生物的PEG修饰物或其可药用盐选自：
PEG20k-[赖氨酸-(琥珀酸-4-青藤碱)2]2；
mPEG5k-CO-甘氨酸-4-青藤碱；
mPEG340-6-青藤碱；和
mPEG5k-(CH2)2CO-4-青藤碱。
本文中所述PEG修饰物可包含通式I结构中所述的四部分结构：PEG、连接臂、D和青藤碱或其衍生物，其中，所述连接臂是指Bm-Ai-Bn-Aj，它可以一端与多臂PEG连接，一端与D或青藤碱或其衍生物连接，该连接臂的数量取决于PEG的臂数。例如，当通式I为
Figure PCTCN2017105515-appb-000013
时，代表PEG为双臂PEG或其修饰剂，其伸出两条臂A1-B2(即一个A与2个B相连)与四个Q连接，即，每条臂上连接两个Q。
本文中所用术语“可药用盐”是指，(1)本发明化合物中存在的酸性官能团(例如-COOH、-OH、-SO3H等)与适当的无机或者有机阳离子(碱)形成的盐，例如本发明化合物与碱金属或碱土金属形成的盐、本发明化合物的铵盐，和本发明化合物与含氮有 机碱形成的盐；以及(2)本发明化合物中存在的碱性官能团(例如-NH2等)与适当的无机或者有机阴离子(酸)形成的盐，例如本发明化合物与无机酸或有机羧酸形成的盐。
因此，本发明化合物的“药学上可接受的盐”包括但不限于，碱金属盐，如钠盐、钾盐、锂盐等；碱土金属盐，如钙盐、镁盐等；其他金属盐，如铝盐、铁盐、锌盐、铜盐、镍盐、钴盐等；无机碱盐，如铵盐；有机碱盐，如叔辛基胺盐、二苄基胺盐、吗啉盐、葡糖胺盐、苯基甘氨酸烷基酯盐、乙二胺盐、N-甲基葡糖胺盐、胍盐、二乙胺盐、三乙胺盐、二环己基胺盐、N,N’-二苄基乙二胺盐、氯普鲁卡因盐、普鲁卡因盐、二乙醇胺盐、N-苄基-苯乙基胺盐、哌嗪盐、四甲基胺盐、三(羟甲基)氨基甲烷盐；氢卤酸盐，如氢氟酸盐、盐酸盐、氢溴酸盐、氢碘酸盐等；无机酸盐，如硝酸盐、高氯酸盐、硫酸盐、磷酸盐等；低级烷磺酸盐，如甲磺酸盐、三氟甲磺酸盐、乙磺酸盐等；芳基磺酸盐，如苯磺酸盐、对苯磺酸盐等；有机酸盐，如醋酸盐、苹果酸盐、富马酸盐、琥珀酸盐、柠檬酸盐、酒石酸盐、草酸盐、马来酸盐等；氨基酸盐，如甘氨酸盐、三甲基甘氨酸盐、精氨酸盐、鸟氨酸盐、谷氨酸盐、天冬氨酸盐等。
在一些优选的实施方案中，所述青藤碱或其衍生物的PEG修饰物的可药用盐为其草酸盐。
在另一个方面，本发明还提供含有所述青藤碱或其衍生物的PEG修饰物或其可药用盐的药物组合物。
在一些优选的实施方案中，所述药物组合物为单位剂量的药物制剂形式。在一些优选的实施方案中，所述药物组合物为注射剂(包括冻干制剂和小容量注射剂)。可以采用制剂学常规技术制备所述药物制剂。
在一些优选的实施方案中，所述药物组合物中，根据需要可以加入一些药物可接受的载体。所述药物可接受的载体选自：甘露醇、山梨醇、葡萄糖、氯化钠、EDTA二钠、EDTA钙钠，磷酸二氢钠、磷酸氢二钠、枸橼酸及枸橼酸钠、盐酸和氢氧化钠等。
本发明的药物组合物在使用时根据病人的情况确定用法用量。
本发明的药物组合物，在制成药剂时，单位剂量的药剂可含有本发明的药物活性物质0.1-1000mg，其余为药学上可接受的载体。药学上可接受的载体以重量计可以是制剂总重量的0.1-99.9％。
在另一个方面，本发明还提供所述青藤碱或其衍生物的PEG修饰物或其可药用盐在制备消炎、镇痛、免疫抑制的药物中的用途。
在另一个方面，本发明提供一种消炎、镇痛或免疫抑制的方法，其包括向有此需要的受试者施用有效量的本发明所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐的步骤。
在另一个方面，本发明提供青藤碱或其衍生物的PEG修饰物、或其可药用盐，其用于消炎、镇痛或免疫抑制。
在另一个方面，本发明提供所述青藤碱或其衍生物的PEG修饰物的制备方法，其包括以下步骤：
先将青藤碱或其衍生物制备成酯类化合物如琥珀酸酯或甘氨酸酯等，再和聚乙二醇或其衍生物如双赖氨酰-聚乙二醇或甲氧基聚乙二醇琥珀酰亚胺碳酸酯进行反应得到所述青藤碱或其衍生物的PEG修饰物；
或者，端基带有羧基的PEG与青藤碱或其衍生物的羟基发生酯化反应得到相应的青藤碱或其衍生物的PEG修饰物。
在一些优选的实施方案中，可以通过端基为羧基的PEG直接与青藤碱上的羟基缩合成酯。
在另一个方面，本发明提供一种通式(Ⅰ)的青藤碱及其衍生物的PEG修饰物，
Figure PCTCN2017105515-appb-000014
其中，
PEG代表分子量为100-60000道尔顿的聚乙二醇；
A代表氨基酸，i和j为0-20的整数，代表氨基酸的数量；
B代表通式(Ⅱ)，
Figure PCTCN2017105515-appb-000015
m为0-20的整数，n为0-20的整数，r为0-20的整数，
D代表通式(Ⅲ)，
Figure PCTCN2017105515-appb-000016
h为0或者1。
Q代表的青藤碱及其衍生物，x为0-20的整数，
t为0-20的整数，代表连接臂的数量。
在一些优选的实施方案中，所述氨基酸为赖氨酸，i为1或2，j为0或1，B中，m为0或1，n为1或2，r为1-3，t为3-5，D中，h为0或者1，x为3-5，Q代表的青藤碱。
在一些优选的实施方案中，所述氨基酸为赖氨酸，i为1，j为0，B中，m为0，n为1，r为2，t为4，D中，h为0或者1，x为4，Q代表的青藤碱。
在一些优选的实施方案中，所述青藤碱及其衍生物结构中的7位与8位碳之间为一双键被还原的单键。
在一些优选的实施方案中，所述PEG包含一端封闭的单臂PEG及其修饰基；两端活波的双臂PEG及其修饰基；以及多臂PEG及其修饰基，其中青藤碱及其衍生物结构中的修饰位点的羟基，包括4位或者6,7,8位的羟基。
在一些优选的实施方案中，所述青藤碱及其衍生物结构中的修饰位点的羟基，通过化学键与PEG的连接臂连接，所述化学键包括：酯键、酰胺键或者醚键。
在一些优选的实施方案中，所述PEG、B、A、D、Q之间通过化学键连接，所述化学键包括：酯键、酰胺键或者醚键。
在一些优选的实施方案中，所述青藤碱或其衍生物的PEG修饰物选自
PEG20k-[-Lys-(-CO-CH2CH2-CO-O-青藤碱)2]2；
mPEG5k-CO-NHCH2-CO-O-青藤碱；
mPEG-6-青藤碱醚草酸盐。
以下通过实验数据进一步说明本发明的有益效果。
药效试验
对采用PEG修饰后的青藤碱进行了抗炎、镇痛、免疫抑制及肥大细胞释放组胺和离体回肠收缩等方面的试验，以观察经过修饰后获得的化合物药理作用的相关变化情况。对照药物为盐酸青藤碱，实验中称为青藤碱组。
实验动物信息：
昆明种小鼠，SPF级，由四川省医学科学院实验动物研究所养殖研究室提供，许可证号：SCXK(川)2013-15。
豚鼠，普通级，四川省实验动物专委会养殖场提供，许可症号：SCXK(川)2013-14。
1、对小鼠扭体试验的影响：
实验方法
取健康昆明种小鼠60只，依性别体重随机分为6组，阴性对照组各鼠尾静脉注射生理盐水0.4ml/20g体重，阿司匹林组每鼠灌服0.4ml/20g体重，青藤碱组、PEG20K-青组、PEG5K-青组及mPEG-6-青组各鼠分别尾静脉注射相应药物0.4ml/20g体重，其中各组药物浓度均为5mg/ml。给药后30min，各组各鼠腹腔注射0.6％醋酸0.2ml/鼠，观察记录各鼠在腹腔注射醋酸后的15min内扭体次数，进行组间比较其显著差异性。
抑制率％＝(1-给药前扭体次数/阴性组扭体次数)×100％
表1对小鼠扭体试验的影响(n＝10)
组别 剂量/mg.kg-1 扭体数(15min内) 抑制率(％) ID50/mg.kg-1
生理盐水组 -- 41.2±5.2 -- --
青藤碱组 100 14.1±2.5** 65.8 26.7
PEG20K-青 100 13.7±2.7** 66.7 25.4
PEG5K-青 100 13.2±2.1** 68.0 25.1
mPEG-6-青 100 13.3±2.4** 67.7 25.3
阿斯匹林 100 29.5±4.7* 28.4  
注：与生理盐水组比较，*P<0.05,**P<0.01
2、对小鼠热板试验的影响：
实验方法
取昆明种雌性小鼠放置入55±0.5℃水浴中的铝盘上，以小鼠舔后足及抬后足并回头为痛反应指标，记录痛反应出现的时间(痛阈值)，测定两次平均值小于30秒且不跳跃的小鼠选供实验用，将合格的小鼠60只按体重随机分成6组，阴性对照组各鼠尾静脉注射生理盐水0.4ml/20g体重，阿司匹林组每鼠灌服0.4ml/20g体重，青藤碱组、PEG20K- 青组、PEG5K-青组及mPEG-6-青组各鼠分别尾静脉注射相应药物0.4ml/20g体重，其中各组药物浓度均为5mg/ml。以上各组在给药后的30′、60′、90′、180′以上述同样的方法测定各小鼠的痛阈值，痛阈值若超过60秒则以60秒计，并以下列公式计算各组小鼠痛阈提高率(％)，并进行组间比较其显著差异性。
表2对小鼠热板试验的影响(n＝10)
Figure PCTCN2017105515-appb-000017
与生理盐水组比较，*P<0.05
3、对小鼠耳肿胀试验的影响：
实验方法
取昆明种小鼠60只，按性别体重随机分6组，雌雄各半。阴性对照组各鼠尾静脉注射生理盐水0.4ml/20g体重，阿司匹林组每鼠灌服0.4ml/20g体重，青藤碱组、PEG20K-青组、PEG5K-青组及mPEG-6-青组各鼠分别尾静脉注射相应药物0.4ml/20g体重，其中各组药物浓度均为5mg/ml。给药后1小时，用二甲苯0.05ml/鼠涂抹每鼠右耳致炎，左耳作对照，致炎后30min脱颈椎处死小鼠，以7mm直径打孔器取下耳缘左、右耳片，分别用电子天平称重，以右、左耳片重量差表示肿胀度，进行组间显著比较。
抑制率％＝(1-给药前扭体次数/阴性组扭体次数)×100％
表3对二甲苯致小鼠耳肿胀试验(
Figure PCTCN2017105515-appb-000018
n＝10)
组别 剂量/mg.kg-1 肿胀度/mg 抑制率(％)
生理盐水组 -- 21.5±4.7 --
青藤碱组 100 15.7±2.6** 27.0
PEG20K-青 100 14.9±1.9** 30.7
PEG5K-青 100 15.0±2.1** 30.2
mPEG-6-青 100 14.7±2.5** 31.6
阿斯匹林 100 16.1±3.3** 25.1
注：与生理盐水比较，**P<0.01
4、对TNFa和IL-1分泌的抑制
实验方法
小鼠腹腔巨噬细胞的制备
小鼠颈椎脱臼处死。消毒胸腹部皮肤,暴露腹膜,腹腔内注入预冷RPMI-1640培养基5ml/只,按摩腹部,回抽腹腔清洗液,低速离心沉淀细胞,制成细胞悬液,计数后,5×104/孔接种于培养板,5％CO2 37℃孵育2小时后,洗掉非贴壁细胞后获得巨噬细胞。
诱生TNF-α、IL-1
加入RPMI-1640完全培养液及受试药物各0.5ml,5％CO2 37℃下继续孵育。于16小时、48小时吸取上清,-80℃保存,分别供检测TNF-α、IL-1用。其中，各受试药物均配置成10μM和50μM两个浓度。
采用L929细胞株细胞毒法,检测TNF-α，结果以OD570值表示。采用L929细胞增殖法,检测IL-1，结果亦以OD570值表示。
表4对TNFa及IL－1分泌抑制试验
Figure PCTCN2017105515-appb-000019
5、不同结构青藤碱对AA大鼠滑膜细胞因子表达TNF-αmRNA、IL-1βmRNA及IL-10mRNA的影响：
实验方法
动物模型的建立及滑膜细胞的培养：Wistar大鼠30只,雄性,体重(180±10)g,随机分组:正常对照组,AA组,AA+青藤碱组,AA+PEG20K-青组，AA+PEG5K-青组及AA+mPEG-6-青组,每组5只。AA大鼠模型的建立:在大鼠右后足垫皮内注射完全弗氏佐剂每只0.1mL(含卡介苗2g·L-1)。于致炎后18d时在清醒状态下腋下放血处死大鼠,无菌取出正常大鼠及 佐剂性关节炎(AA)大鼠双侧膝关节滑膜组织。将手术取到的无菌新鲜滑膜在培养皿内用生理盐水冲洗2次,眼科剪剪碎成1×1(mm3)的小块。以浓度为0.8mg/mL的Ⅱ型胶原酶少量(浸及为度)及等体积DMEM培养液,在37℃、5％CO2孵箱中消化3h。常规离心,弃上清后加0.25％胰酶1mL,混匀,在CO2孵箱内消化30min。120目铜网筛过滤后,收集消化液,放至37℃、5％CO2孵箱中过夜。次日换液1次。滑膜细胞经台盼蓝及Giemsa染色,其活力与纯度均≥95％。此后每2～3d换1次液,5～7d传代1次。3代后用含5μg/mL的LPS的DMEM培养液配制成滑膜细胞悬液(1×106/mL),加入6孔细胞培养板内,每孔3mL,一式两孔。于CO2孵箱培养48h,用相应的方法提取细胞核蛋白及总RNA。
TNF-αmRNA、IL-1βmRNA及IL-10mRNA的表达
常规方法提取细胞总RNA,作反转录-PCR。用凝脉图像分析仪扫描,用LabImage 2.6凝胶图像分析软件求出目的条带灰度与内参照β-actin标准条带的比值。
核蛋白抽提及电泳迁移率改变分析法(EMSA)检测滑膜细胞NF-κB的活性方法参照文献,电泳滞后带用凝胶图像分析仪进行密度扫描,LabImage 2.6凝胶图像分析软件分析。
表5大鼠滑膜细胞因子TNF-αmRNA IL-1βmRNA及IL-10mRNA的表达及NF-κB活性(x±s)
组别 TNF-α/βactin IL-1β/βactin IL-10/βactin NF-κB活性
正常对照组 0.41±0.08* 0.61±0.13* 0.47±0.16* 15±3*
AA 0.73±0.13 0.73±0.09 0.78±0.14 32±4
AA+青藤碱组 0.35±0.09 0.43±0.05* 0.36±0.13* 21±7*
AA+PEG20K-青 0.31±0.07 0.41±0.03* 0.37±0.12* 20±6*
AA+PEG5K-青 0.30±0.03 0.46±0.19* 0.29±0.16* 19±4*
AA+mPEG-6-青 0.29±0.05 0.42±0.16* 0.26±01.9* 19±7*
6、不同结构青藤碱对豚鼠腹腔肥大细胞组胺释放的影响
实验方法
豚鼠腹腔肥大细胞(PMC)离体孵化试验采用Nemeth报道的方法略加修改。取动物断头放血处死，用预冷的肥大细胞培养液15ml腹腔注射,轻轻按摩腹部2min后打开腹腔,吸取腹腔冲洗液,4℃、1500rmin离心10min，调节细胞悬液约0.5ml,缓慢加入7ml P-F10中,在4℃、2500rpm离心15min分离细胞。用培养液洗涤2次(1000rpm离心10min), 收集界面细胞,镜下计数,调细胞密度至106个细胞/L,锥虫蓝染色检查细胞活力大于90％,甲苯胺蓝染色细胞纯度90％以上。
取分离纯化的肥大细胞悬液,每管0.9ml,分别加入0.1ml不同药物或CP48/80,空白对照组以生理盐水取代药物。置入37℃温箱孵育15min,取出后立即置入冰水浴中5min终止反应,将各管以4℃,400×g离心10min,取上清加入25％三氯乙酸,混合后离心,取上清1ml加入0.5ml NaOH,立即加入0.1％邻苯二甲醛0.1ml,混匀,25℃反应10min,加入0.5mlHCl终止反应。在荧光分光光度计上比色(激发波长λex 360nm,发射波长λem460nm)。细胞用1mlMC培养液重悬,加热煮沸1min,测定细胞裂解液的组胺含量。
组胺释放率＝上清液组胺/(细胞内组胺+上清液组胺)×100％。
表6不同结构青藤碱对豚鼠腹腔肥大细胞组胺释放的影响(n＝10)
组别 浓度 组胺释放率(％)
空白对照 -- 5.21±1.10
青藤碱组 100μmol/L 28.11±4.34*
PEG20K-青 100μmol/L 6.71±2.15
PEG5K-青 100μmol/L 5.33±1.13
mPEG-6-青 100μmol/L 5.80±1.78
CP48/80 10μmol/L 66.25±9.84**
与空白对照组比较*P<0.05,**P<0.01
7、不同结构青藤碱对豚鼠腹腔离体回肠收缩张力的影响
实验方法
豚鼠离体回肠平滑肌收缩张力的测定：豚鼠禁食12h,饮水不限。击头处死后,立即打开腹腔,在离盲肠2cm处剪断回肠,取出回肠一段长约15cm,置于预冷的Krebs液中,剪除回肠周围脂肪组织,用Krebs液将肠内容物冲洗干净,剪成长约1.5～2.0cm的标本。将标本浸入含30ml Krebs液的恒温浴槽中,持续通入95％O2和5％CO2混合气体,pH7.4,T＝37±0.5℃。标本的一端固定于标本板钩上,另一端连接于张力换能器,调标本负荷1g平衡30min后开始实验,用Po werlab生物信号采集分析系统记录回肠张力变化。实验分5组,即空白对照组、青藤碱组、PEG20K-青组、PEG5K-青组及mPEG-6-青组。空白对照组给予同容量生理盐水取代药物。每组10条标本,取自不同的豚鼠回肠。
实验分两部分,一是观察各组对正常回肠自动节律收缩的影响:实验开始时先描记10min正常肠平滑肌收缩曲线,然后在浴槽中加入不同浓度的受试药物0.1ml,作用5min后 再描记10min回肠收缩曲线。二是观察各组对组胺所致回肠收缩的影响:描记一段正常回肠收缩曲线后,给予磷酸组胺溶液0.1ml,使肠管平滑肌收缩张力达最大,随后用Krebs液冲洗使肠管使平滑肌收缩恢复到基线水平,然后加入不同浓度的受试药物溶液0.1ml,作用5min后,再给予磷酸组胺溶液0.1ml,以回肠收缩曲线张力的峰值为指标,观察药物对组胺致肠管最大收缩效应的影响。
表7不同结构青藤碱对豚鼠腹腔离体回肠收缩张力的影响(
Figure PCTCN2017105515-appb-000020
n＝10)
Figure PCTCN2017105515-appb-000021
与空白对照组比较，**P<0.01
8、注射剂在应用中的影响
实验方法[1]
取日本大耳白兔8只，按体重分为4组，即青藤碱组、PEG20K-青组、PEG5K-青组及mPEG-6-青组，各组2只。各组均于左耳缘静脉缓慢注射药物2ml/kg，给药浓度为5mg/ml，右耳缘静脉缓慢注射生理盐2ml/kg，每天一次，连续注射三天，于注射第一次开始，每天观察注射部位有无水肿、红斑，末次注射后24小时，从耳根剪下耳朵置10％甲醛溶液中固定，然后作病理切片检查。局部疼痛以给药时兔是否挣扎为指标。
考察现有技术和本发明药物制备成冻干注射剂后的各项指标
Figure PCTCN2017105515-appb-000022
Figure PCTCN2017105515-appb-000023
本发明青藤碱或其衍生物的PEG修饰物，其至少能够实现下述一种技术效果：
(1)相较于青藤碱，可显著降低免疫原性反应，过敏反应；
(2)可延长青藤碱半衰期；
(3)口服苦味降低；
(4)注射时血管刺激性降低；以及
(5)恶心和呕吐副作用降低。
具体实施方式
以下通过实施例进一步说明本发明。
本发明合成药物以及剂型的制备都可以在常规的药学实验室，在药学专业技术人员操作下完成，以下为实施举例，但本发明不仅限于如下实施例：
实验中，二胺聚乙二醇为自制产品也可商购获得，二甲氨基吡啶(DMAP)，N-叔丁氧羰基甘氨酸，N,N-二异丙基乙胺(DIEA)，二环己基碳二亚胺(DCC)，双-叔丁氧碳基-L-赖氨酸均为国产分析纯试剂。单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(5KD)(mPEG-SC)、青藤碱、1-溴-3,6,9,12,15,18,21-七氧代二十二烷为市购原料，溶剂为实验室常规分析纯溶剂。
实施例1：PEG20k-[赖氨酸-(琥珀酸-4-青藤碱)2]2实验中称为PEG20K-青
Figure PCTCN2017105515-appb-000024
(1)青藤碱-4-琥珀酸单酯(A1)合成
将青藤碱(3.3g,10mmol)、丁二酸酐(2g,20mmol)、4-二甲氨基吡啶(DMAP，300mg，2.5mmol)、二环己基碳二亚胺(DCC，2.1g10mmol)加入100ml单口瓶中，氮气保护下加入60ml二氯甲烷，升温到50℃搅拌反应48h，薄层色谱检测反应。将反应液浓缩，残液搅拌下加入冰水混合物中，搅拌30min，0℃结晶过夜，过滤，滤饼用冰水洗涤，滤饼干燥，的淡黄色固体4.9g。
(2)双赖氨酰-聚乙二醇(A2)的合成
在单口瓶中依次加入，双氨基聚乙二醇(20KD)(30g，1.5mmol)和双-叔丁氧碳基-L-赖氨酸(1.38g,4mmol)溶于100ml二氯甲烷中，冰水浴冷却搅拌下，加入二环己基碳二亚胺(DCC，1.03g5mmol)，加完料后，自然回温至室温，搅拌反应过夜，薄层色谱监测反应。反应完全，过滤，滤液依次用饱和碳酸氢钠溶液和饱和食盐水溶液洗涤，无水硫酸钠干燥。过滤，减压蒸出二氯甲烷，加入乙醚得到白色固体粉末，29.3g。
将上述白色固体粉末20g，溶于100ml二氯甲烷中，加入35ml三氟乙酸和7ml三异丙基硅烷，室温搅拌2小时，减压浓缩，加入无水乙醚，得到白色固体16g。
(3)PEG20k-[赖氨酸-(琥珀酸-4-青藤碱)2]2合成
将2.2g(5mmol)中间体A1和24.8g(1.24mmol)A2依次加入250ml单口瓶中，加入100ml二氯甲烷溶解，冰浴冷却下，加入DCC(1g，4.8mmol)，然后加入1ml DIEA，自然回温到室温，搅拌反应过夜，薄层色谱监测反应，反应完全后，过滤，滤液浓缩，异丙醇重结晶得到白色粉末状固体24.5g。H1-NMR(CDCl3):6.74(d，1H),6.40(d，1H),5.80(s，1H),4.90(m，2H)，4.23(s，3H),3.63(m，4H)，3.51(m，1809H),3.37(m，4H),3.21(m，4H),3.10(d，2H),2.95(t，2H),2.70(q，1H),2.58(m，8H),2.48(m，8H),2.39(t，2H),2.23(s，3H),2.00(m，1H),1.82(t，2H),1.71(m，4H),1.55(m，4H)，1.28(m，4H)
实施例2：mPEG5k-CO-甘氨酸-4-青藤碱实验中称为PEG5K-青
Figure PCTCN2017105515-appb-000025
(1)青藤碱-4-甘氨酸单酯(B1)合成
将青藤碱(3.3g,10mmol)、N-叔丁氧羰基甘氨酸(8.7g,50mmol)、4-二甲氨基吡啶(DMAP，300mg，2.5mmol)、二环己基碳二亚胺(DCC，3.1g15mmol)加入250ml单口瓶中，氮气保护下加入100ml二氯甲烷，室温搅拌反应48h，薄层色谱检测反应。过滤，将反应液浓缩，加入100ml乙醚，搅拌30min，过滤，真空干燥，得白色青藤碱-4-(N-叔丁氧羰基)甘氨酸单酯固体11g。
将上一步青藤碱-4-(N-叔丁氧羰基)甘氨酸单酯10g加入250ml单口瓶中，加入100ml二氯甲烷溶解，全溶后加入30ml三氟乙酸，室温搅拌3小时，薄层色谱监测。反应完全后，减压浓缩，加入500ml乙醚，搅拌30min。过滤，滤饼真空干燥，得到青藤碱-4-甘氨酸单酯7.5g。
(2)mPEG5k-CO-甘氨酸-4-青藤碱合成
将单甲氧基聚乙二醇琥珀酰亚胺碳酸酯(5KD)(mPEG-SC)(8.5g,1.7mmol)和B1(0.7g,1.9mmol)依次加入250ml单口瓶中，加入100ml二氯甲烷溶解，室温下，加入DCC(1g，4.8mmol)，搅拌反应过夜，薄层色谱监测反应，反应完全后，过滤，滤液浓缩，异丙醇重结晶得到白色粉末状固体8.4g。H1-NMR(CDCl3):6.73(d，1H),6.41(d，1H),5.76(s，1H),4.23(s，3H),3.60(m，3H),3.48(m，116H)，3.15(d，2H),2.95(t，2H),2.70(q，1H),2.46(t，2H),2.23(s，3H),2.00(m，1H),1.80(t，2H)。
实施例3：mPEG340-6-青藤碱草酸盐(6-(3,6,9,12,15,18,21-七氧代二十二烷基)青藤碱醚草酸盐实验中称为mPEG-6-青
Figure PCTCN2017105515-appb-000026
(1)4-MEM-6-OH-青藤碱合成
将青藤碱(10g，30mmol)加入到500ml圆底烧瓶中，加入300ml二氯甲烷，然后加入DIPEA(23.5ml，18.4g，180mmol)搅拌，冰水浴下加入MEMCl(13.7ml，14.9g，120mmol)30min滴加完毕后，自然升温到室温，反应过夜，反应液用饱和氯化钠溶液洗涤，无水硫酸钠干燥1小时，过滤，浓缩得到4-MEM-青藤碱产物12.01g。
将4-MEM-青藤碱，(12.01g，28.8mmol)加入到500ml圆底烧瓶中，然后加入120ml乙腈和120ml乙酸混合溶液，降温至-15℃加入四甲基三乙酰氧硼氢化铵(11.37g,43.2mmol)搅拌20min，升至室温，搅拌2小时，减压旋干乙腈，调节pH值成碱性，用DCM萃取，无水硫酸钠干燥，过滤，浓缩后得4-MEM-6-OH-青藤碱产物12.14g。
(2)mPEG340-6-青藤碱合成
将上述4-MEM-6-OH-青藤碱，(6g,14.3mmol)和100ml DMF加入到500ml圆底烧瓶中，冰水浴下加入氢化钠(1.71g,42.9mmol)，升温到回流；将mPEG-Br(1-溴-3,6,9,12,15,18,21-七氧代二十二烷)(17.3g,42.9mmol)的DMF(100ml)溶液滴入反应液中，回流1小时，冷却到室温，除去溶剂。加入150ml DCM完全溶解后，加入对甲苯磺酸(4.95g，28.6mmol)，室温搅拌3小时，反应液用0.5mol/L的盐酸(50mlx3)萃取，合并水相，加入碳酸钠调节pH＝10，用DCM萃取，合并有机相用无水硫酸钠干燥，过滤，旋干后过硅胶柱得产品5.76g。
(3)mPEG340-6-青藤碱草酸盐的制备
将上述mPEG340-6-青藤碱(5.76g,8.8mmol)溶于乙醇：甲基叔丁醚＝1:4的50ml混合溶液中；将草酸(0.75ml,1.23g，9.8mmol)溶解于50ml甲基叔丁醚中，缓慢加入到混合液中，搅拌过夜。过滤，滤饼用50ml甲基叔丁醚洗涤，滤饼真空干燥，得白色固体5.1g。H1-NMR(CDCl3):7.2(m，1H),6.70(d，1H),6.42(d，1H),5.67(s，1H),4.23(s，3H),3.63(m，3H),3.48(m，28H)3.36(m，1H)3.24(S，3H),3.15(d，2H),2.95(t，2H),2.70(q，1H),2.46(t，2H),2.23(s，3H),2.00(m，1H),1.80(t，2H)
实施例4：mPEG5k-(CH2)2CO-4-青藤碱实验中称为PEG5K-青2
Figure PCTCN2017105515-appb-000027
将青藤碱(1.32g,4mmol)、mPEG丙酸-5000(2g,0.4mmol)、4-二甲氨基吡啶(DMAP，0.025g，0.2mmol)、二环己基碳二亚胺(DCC，0.82g 4mmol)加入250ml单口瓶中，氮气保护下加入100ml二氯甲烷，室温搅拌反应48h，薄层色谱检测反应。过滤，将反应液浓缩，加入100ml乙醚，搅拌30min，过滤，真空干燥，异丙醇重结晶，得白色mPEG5k-(CH2)2CO-4-青藤碱固体1.8g。
实施例5：
处方：1000支
Figure PCTCN2017105515-appb-000028
配制工艺
加入配制总量70％的注射用水置配液罐中，加入依地酸二钠2g，加热搅拌充分溶解后，放凉至10～20℃，再加入PEG20K-青、甘露醇搅拌至完全溶解，加入配制量0.1％(W/V)的药用炭，搅拌15min，脱碳过滤；补充10～20℃的注射用水至配制量的足量，充氮(氮气压力0.3～0.5MPa)，用0.22μm滤芯的过滤器过滤；分装于西林瓶中，半盖胶塞，冻干。
实施例6：
Figure PCTCN2017105515-appb-000029
配制工艺
加入配制总量80％的注射用水置配液罐中，加入依地酸二钠2g，加热搅拌充分溶解后，放凉至10～20℃，再加入磷酸二氢钠、甘露醇搅拌至完全溶解，再加入氢氧化钠调节PH至4.0，再加入PEG5K-青溶解完全，加入配制量0.1％(W/V)的药用炭，搅拌15min，脱碳过滤；补充10～20℃的注射用水至配制量的足量，充氮(氮气压力0.3～0.5MPa)，用0.22μm滤芯的过滤器过滤；分装于西林瓶中，半盖胶塞，冻干。
实施例7
Figure PCTCN2017105515-appb-000030
配制工艺
加入配制总量70％的注射用水置配液罐中，加入依地酸二钠2g，加热搅拌充分溶解后，放凉至10～20℃，再加入mPEG-6-青、甘露醇搅拌至完全溶解，加入配制量0.1％(W/V)的药用炭，搅拌15min，脱碳过滤；补充10～20℃的注射用水至配制量的足量，充氮(氮气压力0.3～0.5MPa)，用0.22μm滤芯的过滤器过滤；分装于西林瓶中，半盖胶塞，冻干。

Claims (14)

  1. 通式(Ⅰ)所示青藤碱或其衍生物的PEG修饰物、或其可药用盐，
    Figure PCTCN2017105515-appb-100001
    其中，
    PEG代表分子量为100-60000道尔顿的聚乙二醇及其修饰剂；
    A代表氨基酸，i和j为0-20的整数，代表氨基酸的数量；
    B代表通式(Ⅱ)，
    Figure PCTCN2017105515-appb-100002
    m为0-20的整数，n为0-20的整数，r为0-20的整数；
    D代表通式(Ⅲ)，
    Figure PCTCN2017105515-appb-100003
    h为0或者1；
    Q代表的青藤碱及其衍生物，x为0-20的整数；
    t为0-20的整数，代表连接臂的数量。
  2. 根据权利要求1所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述氨基酸为甘氨酸或赖氨酸，i为0、1或2，j为0或1，B中，m为0或1，n为0、1或2，r为1-3，t为1-5(例如3-5)，D中，h为0或者1，x为1-10(例如3-5)，Q代表青藤碱。
  3. 根据权利要求1所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述氨基酸为甘氨酸或赖氨酸，i为1，j为0，B中，m为0，n为1或2，r为2，t为2或4，D中，h为0或者1，x为4，Q代表青藤碱。
  4. 根据权利要求1所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，i为0或1,j为0或1,B中,m为0,n为0或2,r为2,t为1或2,D中,h为0,x为1或4,Q代表青藤碱。
  5. 根据权利要求1-4任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，青藤碱或其衍生物结构中的7位与8位碳之间为一双键被还原的单键。
  6. 根据权利要求1-5任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述PEG包含一端封闭的单臂PEG及其修饰剂；两端活泼的双臂PEG及其修饰剂；以及多臂PEG及其修饰剂；其中青藤碱或其衍生物结构中的羟基修饰位点，包括4位或者6,7,8位的羟基。
  7. 根据权利要求1-6任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述青藤碱或其衍生物结构中的羟基修饰位点，通过化学键与PEG的连接臂连接，所述化学键包括：酯键、酰胺键和醚键。
  8. 根据权利要求1-7任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述通式(I)中PEG、B、A、D、Q之间通过化学键连接，所述化学键包括：酯键、酰胺键和醚键。
  9. 根据权利要求1所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其中，所述PEG修饰物选自
    PEG20k-[赖氨酸-(琥珀酸-4-青藤碱)2]2；
    mPEG5k-CO-甘氨酸-4-青藤碱；
    mPEG340-6-青藤碱；和
    mPEG5k-(CH2)2CO-4-青藤碱。
  10. 权利要求1-9任一项所述的青藤碱或其衍生物的PEG修饰物的制备方法，其包括以下步骤：
    先将青藤碱或其衍生物制备成酯类化合物如琥珀酸酯或甘氨酸酯，再和聚乙二醇或其衍生物如双赖氨酰-聚乙二醇或甲氧基聚乙二醇琥珀酰亚胺碳酸酯进行反应得到所述青藤碱或其衍生物的PEG修饰物；
    或者，端基带有羧基的PEG与青藤碱或其衍生物上的羟基发生酯化反应得到相应的青藤碱或其衍生物的PEG修饰物。
  11. 药物组合物，其含有权利要求1-9任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐。
  12. 权利要求1-9任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐在制备消炎、镇痛或免疫抑制药物中的用途。
  13. 一种消炎、镇痛或免疫抑制的方法，其包括向有此需要的受试者施用有效量的权利要求1-9任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐的步骤。
  14. 权利要求1-9任一项所述的青藤碱或其衍生物的PEG修饰物、或其可药用盐，其用于消炎、镇痛或免疫抑制。
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CN102579340A (zh) * 2011-01-07 2012-07-18 北京因科瑞斯医药科技有限公司 青藤碱囊泡及其制剂及制备方法
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