CN1159006C

CN1159006C - Application of bilirubin and its derivative as preparation for resisting influenza virus A1 type

Info

Publication number: CN1159006C
Application number: CNB991006666A
Authority: CN
Inventors: 马金石; 王鹏; 阎芳
Original assignee: Institute of Chemistry CAS
Current assignee: Institute of Chemistry CAS
Priority date: 1999-02-12
Filing date: 1999-02-12
Publication date: 2004-07-28
Anticipated expiration: 2019-02-12
Also published as: CN1262928A

Abstract

The present invention belongs to the application of bilirubin and a derivative thereof in the field of medicine, particularly to the application of the bilirubin and the derivative thereof as Influenza A1 virus resisting agents. The present invention is characterized in that the bilirubin and the derivative of the bilirubin with the following structuresare used for preparing the Influenza A1 virus resisting agents.

Description

The purposes that bilirubin and derivant thereof are used to prepare prevention and treat the grippal medicine that is caused by Influenza A1 virus

The invention belongs to the application in field of medicaments of bilirubin and derivant thereof, particularly relate to bilirubin and derivant thereof purposes as preparation for resisting influenza virus A 1 type.

Bilirubin is the metabolic product of haemachrome in the mammalian body, and haemachrome forms biliverdin IX α under the effect of heme oxidase, and it is reduced to bilirubin IX α (BilirubinIX α is commonly called as bilirubin) through the biliverdin reductase effect.Bilirubin is in the intravital effect of people, and now clear and definite, it has antioxidation.Bilirubin is present in the bile, through blood circulation, metabolism, can excrete.When metabolism is undesired, can form cholelithiasis together with other components.Calculus Bovis is called Calculus Bovis; Calculus Bovis has the history in more than 2000 year in China as Chinese medicine ingredients; kind surplus having 200 with the Chinese patent medicine of Calculus Bovis (comprising the artificial Calculus Bovis), as Bezoar Sedative Pills, cow-bezoar bolus for resurrection, Xi Huan Wan, LIUSHEN WAN, Pien Tze Huang, SUXIAO GANMAO JIAONANG, GANMAOTONG etc.These medicines are sick medicines commonly used such as treatment apoplexy, encephalitis, tonsillitis, flu.Bilirubinic content in the Calculus Bovis is more than 50%; and only contain bilirubin 0.7% among the artificial Calculus Bovis of China's preparation; be on the one hand because bilirubinic too expensive; be owing to suspect bilirubinic pharmacological action (Zhang Nengrong, " bilirubin ", Chinese Medicine science and technology publishing house on the other hand; Beijing; 1994), thus not with bilirubin as effective ingredient, and be used to separately to make up a prescription.Calculus Bovis has heat-clearing and toxic substances removing, the eliminating phlegm of having one's ideas straightened out, calm the nerves the arresting convulsion effect.Bilirubinic pharmacological action also once had some researchs in the past, as promoting erythrocyte new life, strengthened the contraction of blood vessel; Refrigeration function; Anti-encephalitis b virus; Anticancer; Antiinflammation etc. (Zhang Nengrong, " bilirubin " Chinese Medicine science and technology publishing house, Beijing, 1994).External lot of documents has in recent years been reported the antioxidation (A.F.McDonagh of bile pigments, clinical enclosing produced (Clinics in perinatology), 17 (2) 359-3691990), anti-HIV (people's immunity lacks virus) effect (H.Mari et.al., Japan's cancer research (Jpn.J.Cancer Res.), 82,755-757,1991), anti-HHV-6 (herpes virus hominis-6) effect (T.Nakagami et.al., microbial immunology (Microbiol.Immunal.), 36,381-390,1992).Also there is the patent report bile pigments to can be used as anticomplement (Japan, openly specially permit communique (A), the spy opens flat 6-183969) and antimalaric (Japan openly speciallys permit communique (A), Te Kaiping 6-157308), but not useful as yet so far bilirubin and derivant thereof as preparation for resisting influenza virus A 1 type.

Human body can produce, drain the bilirubin of about 300mg every day, when using in the liver function experiment with for the treatment of ameliorate osteoarthritis, reaches 20mg/kg for adult and neonate intravenous injection dosage, injurious effects do not occur.Bilirubin has potential toxicity, also only in that metabolism is abnormal in particular cases just can take place (Zhang Nengrong, " bilirubin " Chinese Medicine science and technology publishing house, Beijing, 1994).So, be in the ascendant at present to bilirubinic medicinal research.

Purpose of the present invention is intended to show that bilirubin and derivant mesobilirubin XIII α thereof can suppress Influenza A1 virus, and can be used to prepare prevention and treat the grippal medicine that is caused by Influenza A1 virus.

Used bilirubin and derivant structure thereof are in the technical scheme of the present invention:

R ¹-R ⁸Be alkyl, vinyl, fatty acid substituent group.

R ¹ R ² R ³ R ⁴ R ⁵ R ⁶ R ⁷ R ⁸

Bilirubin (bilirubin IX α) M V M P P M M V

Mesobilirubin XIII α M E M P P M E M

M＝CH ₃，E＝CH ₂CH ₃，V＝CH＝CH ₂，P＝CH ₂CH ₂COOH

R ¹, R ², R ³, R ⁶, R ⁷, R ⁸Can also be other alkyl, thiazolinyl, alkyl-carbonyl, R ⁴, R ⁵Can also be fatty acid-based, the salt of fatty acid, the amide of fatty acid, the ester of fatty acid.

Bilirubin and derivant physicochemical properties thereof:

Bilirubin (bilirubin IX α): molecular weight 584.7, the red solid powder dissolves in chloroform, dimethyl sulfoxide, be soluble in the sodium hydrate aqueous solution of 0.1M, stable in water insoluble, methanol, petroleum ether, the normal hexane equal solvent, solid-state and alkaline solution, easy oxidation under the acid condition in the solution.

Mesobilirubin XIII α: molecular weight 588.7, the glassy yellow pressed powder, its dissolubility, stable identical with bilirubin, 310 ℃ begin to decompose.

The preparation of bilirubin and derivant thereof:

1. bilirubin (bilirubin IX α)

Bilirubin is bought from market, because of purity is too low, and must be through purification, recrystallization.Method of purification list of references (A.F.McDonagh and F.Assisi, journal of biological chemistry (Biochem.J.), 129797-800,1972) is also done improvement.Concrete grammar is as follows: the bilirubin 500mg that market is buied adds 500mL and sloughs in the chloroform of stabilizing agent, reflux half an hour under nitrogen current, is cooled to room temperature then.Filtering solution is used 0.1M NaHCO ₃Wash chloroformic solution (3 * 100mL).Then by double-deck filter paper filtering, to remove water in the chloroform.Concentrate chloroformic solution at 30 ℃ of following rotary evaporation in vacuo.In spissated chloroformic solution, add methanol then,, put into refrigerator,, filter to crystallization occurring to becoming turbid.Wash with the methanol that contains minimum of chloroform, dry under vacuum then.According to the raw material sources difference, the response rate is 50-90%, and purity is (is 65000 in molar extinction coefficient) more than 98%, and whole operation will in the dark be carried out.The logical in advance argon of all solvents half an hour.

2. bilirubin derivative mesobilirubin XIII α's is synthetic

The oxime of ethyl acetoacetate and pentanedione are condensed into 4-7 acyl group-3 under acetic acid-zinc powder reduction; 5-dimethyl-1 hydrogen pyrroles-2-carboxylic acid, ethyl ester; further through reduction; hydrolysis; decarboxylation; oxidation; bromination obtains 5-bromine methene base-4-ethyl-3-methyl-2-oxo-2; the 5-pyrrolin; this pyrroles and 2; 4-dimethyl-5-carboxyl-1 hydrogen pyrroles-3-propanoic acid acid catalysis in methanol refluxes and is condensed into the Xanthobilirubic acid methyl ester; again through DDQ (2; 3-two chloro-5; 6-dicyano-1; the 4-benzoquinone) oxidative condensation becomes mesobiliverdin XIII α dimethyl ester, after sodium borohydride reduction; be hydrolyzed into end product, purity is more than 99%.List of references (D.A.lightner, J.S.Ma et.al., heterocyclic chemistry magazine (J.Heterocyclic Chem.), 21 139-144,1984.R.T.Trull, R.W.Franklin and D.A.Lightner, heterocyclic chemistry magazine (J.Heterocyclic Chem.), 24 1573-1579 1987).

Structural parameters: IR (liquid film): v 3420,3260,2966,1704,1689,1629cm ^-1 ¹HNMR (deuterochloroform): δ 1.12 (t, 6H, J=7Hz), 1.85 (s, 6H, 2,18 CH ₃), 1,85 (s, 6H, 7,13 CH ₃), 2.48 (q, 4H, J=7Hz), 2.78 (m, 8H ,-CH ₂CH ₂COOH), 4.06 (s, 2H, 10-CH2-), 6.04 (s, 2H, 5,10=CH), 9.15 (s, 2H, pyrroles NH), 10.52 (s, 2H, lactams NH), 13.31 (s, 2H, COOH) ppm; MS:M ⁺587.7.UV-Vis (chloroform): λ max431nm, ε, 60,000. ¹³CNMR (deuterochloroform): 8.54 (q), 9.62 (q), 15.29 (q), 17.64 (t), 19.74 (t), 23.96 (t), 34.81 (t), 98.19 (d), 119.72 (s), 122.43 (s), 123.00 (s), 123.39 (s), 128.28 (s), 130.81 (s), 147.67 (s), 172.42 (s), 174.46 (s).

The preparation of bilirubin and derivant resisting influenza virus A 1 type reagent thereof:

The preparation of bilirubin aqueous solution: accurately take by weighing above-mentioned pure 2.790mg bilirubin IX α in the volumetric flask of a 25mL, add the analytical pure dimethyl sulfoxide (DMSO) of 250 μ L with microsyringe, then accurately pipette the potassium dihydrogen phosphate (KH of 5mL 0.2M with pipet ₂PO ₄) standard water solution and adding in the volumetric flask, vibration is fully dissolved bilirubin; Accurately pipette sodium hydroxide (NaOH) the standard water solution of 4.28mL 0.2M again, dropwise add in the volumetric flask with dropper, reuse deionization washing dropper and transfer container add in the volumetric flask in the lump, use the deionized water standardize solution at last.Ultimate density: bilirubin is that 111.6 μ g/mL are equivalent to 190.87 μ M, and DMSO is 1% (v/v).Buffer system (KH ₂PO ₄-NaOH) pH value 7.6, concentration 0.05M.

The preparation of mesobilirubin XIII α aqueous solution: sample size is the aqueous solution (125.8 μ g/mL are equivalent to 214 μ M) that 3.146mg is configured to 25mL, and its operation is the same, and DMSO is 0.5% (v/v), and buffer system concentration and pH value are the same.

The configuration of bilirubin and derivant aqueous solution thereof also has following several method:

1. introduce water solublity group, in the direct water-soluble solution;

A introduces two taurine sodium salts on bilirubinic two carboxyls, make two taurine sodium bilirubinate (DTBR):

B introduces quaternary ammonium salt cationic (as: tetraethyl ammonium) on bilirubinic two carboxyls, make bilirubinic quaternary ammonium salt.

2. adopt pharmaceutical carrier to increase its water solublity

The a serum albumin;

B beta-schardinger dextrin-inclusion.

The preparation of bilirubin of the present invention and derivant mesobilirubin XIII α thereof is significantly to the inhibitory action of Influenza A1 virus, therefore can be used as the drug use that suppresses Influenza A1 virus.Bilirubin and derivant mesobilirubin XIII α's is synthetic simple, and cost is lower.This class medicine can suppress Influenza A1 virus., it then is to observe through secular clinical medicine to have proved to organism and the low toxic and side effects of human body.In sum, bilirubin and derivant thereof have greatly the value of further exploitation, research as a class resisting influenza virus A 1 type medicine.

Description of drawings:

Fig. 1. the logarithm of bilirubin micro-molar concentration is to the average suppression ratio mapping of Influenza A1 virus;

Fig. 2. the logarithm of derivant micro-molar concentration is to the average suppression ratio mapping of Influenza A1 virus.

Below in conjunction with embodiment technical scheme of the present invention is further described.

Bilirubin and derivant extracorporeal antivirus effect experimental technique and experimental result:

Experiment material

1. Embryo Gallus domesticus: 10-12 day instar chicken embryo (available from China Agricultural University)

2. virazole: (available from Xinyi pharmaceutical factory, lot number: 960330)

3. bacterial strain: Influenza A1 virus strain (China prevention academy of science Ins of Virology provides)

(1) bilirubin

. the experiment reagent preparation:

The preparation of bilirubin aqueous solution: the bilirubin IX α that accurately takes by weighing purity and be the 2.790mg more than 98% is in a 25mL volumetric flask, add the analytical pure dimethyl sulfoxide (DMSO) of 250 μ L with microsyringe, then accurately pipette the potassium dihydrogen phosphate (KH of 5mL 0.2M with pipet ₂PO ₄) standard water solution and adding in the volumetric flask, vibration is fully dissolved bilirubin; Accurately pipette sodium hydroxide (NaOH) the standard water solution of 4.28mL 0.2M again, dropwise add in the volumetric flask with dropper, reuse deionization washing dropper and transfer container add in the volumetric flask in the lump, use the deionized water standardize solution at last.Ultimate density: bilirubin is that 111.6 μ g/mL are equivalent to 190.87 μ M, and DMSO is 1% (v/v).Buffer system (KH ₂PO ₄-NaOH) pH value 7.6, concentration 0.05M.

Experimental technique;

Is 1: 2,1.4,1: 8, the solution of-----1: 64 with physiological saline solution with bilirubin stock solution dilution, gets different dilution medicinal liquid 1mL and 100EID respectively ₅₀Viral liquid 1mL with in vitro mix room temperature effect 1 hour; Virazole is pressed 5mg/mL concentration and viral liquid mixed in equal amounts.Get said mixture 0.2mL inoculated into chick embryo allantoic cavity respectively, normal saline matched group inoculation normal saline 0.2mL, virus control winding kind 100EID ₅₀Virus liquid 0.1mL.Inoculate the hole with sealing with wax, put 37 ℃ of ovum and educate case cultivation 48 hours.The results chick embryo allantoic liquid is done the blood clotting experiment.

Experimental result:

Bilirubin is to Influenza A1 virus breeding inhibitory action result

Group virus experiment number

1 2 3 4

Stock solution first 1------

First 1 in 1: 2---+

First 1 in 1: 4---++-

1: 8 first 1 of bilirubin ++ ++---

1: 16 first 1+++ ++-

1: 32 first 1+++ ++ +++

First 1 in 1: 64 +++++++++++ ++

NS matched group------

Virus control group first 1 ++ ++ ++ ++ ++ ++ ++ ++

Virazole group first 1------

Annotate :+representative has virus breeding;-represent virus-free seedlings NS to represent normal physiological saline.

(2) bilirubin derivative mesobilirubin XIII α

The preparation of experiment reagent:

The preparation of mesobilirubin XIII α aqueous solution: the mesobilirubin XIII α that accurately takes by weighing purity and be 99% above 3.146mg is in the volumetric flask of a 25mL, with the dimethyl sulfoxide (DMSO) of microsyringe adding analytical pure 125 μ L, then accurately pipette the potassium dihydrogen phosphate (KH of 5mL 0.2M with pipet ₂PO ₄) standard water solution and adding in the volumetric flask, vibration is fully dissolved mesobilirubin XIII α; Accurately pipette sodium hydroxide (NaOH) the standard water solution of 4.28mL 0.2M again, dropwise add in the volumetric flask with dropper, reuse deionization washing dropper and transfer container add in the volumetric flask in the lump, use the deionized water standardize solution at last.Ultimate density: mesobilirubin XIII α is that 125.8 μ g/mL are equivalent to 214 μ M, and DMSO is 0.5% (v/v).Buffer system (KH ₂PO ₄-NaOH) pH value 7.6, concentration 0.05M.

Experimental technique

Is 1: 2,1: 4,1: 8, the solution of-----1: 64 with physiological saline solution with bilirubin derivative mesobilirubin XIII α stock solution dilution, gets different dilution medicinal liquid 1mL and 100EID respectively ₅₀Viral liquid 1mL with in vitro mix room temperature effect 1 hour; Virazole is pressed 5mg/mL concentration and viral liquid mixed in equal amounts.Get said mixture 0.2mL inoculated into chick embryo allantoic cavity respectively, normal saline matched group inoculation normal saline 0.2mL, virus control winding kind 100EID ₅₀Virus liquid 0.1mL.Inoculate the hole with sealing with wax, put 37 ℃ of ovum and educate case cultivation 48 hours.The results chick embryo allantoic liquid is done the blood clotting experiment.

Experimental result

Mesobilirubin XIII α is to the external breeding inhibitory action of influenza virus group virus experiment number as a result

1 2 3 4

Stock solution first 1------

First 1 in 1: 2---+

First 1 in 1: 4---++

1: 8 first 1 of mesobilirubin-++ ++-

1: 16 first 1 of XIII α ++++++++ ++ ++ ++

First 1 in 1: 32 ++ ++ ++ ++ ++ ++ ++ ++

First 1 in 1: 64 ++ ++ ++ ++ ++ ++ ++ ++

NS matched group------

Virus control group first 1 ++ ++ ++ ++ ++ ++ ++ ++

Virazole group first 1------

Annotate :+representative has virus breeding;-represent virus-free seedlings; NS represents normal physiological saline.

Experimental results show that bilirubin and derivant mesobilirubin XIII α thereof do not influence the normal growth growth of Embryo Gallus domesticus under experimental concentration; The mechanism that non-specific inhibition has been got rid of in experiment is coagulated in heat insulating culture laggard promoting the circulation of blood in 48 hours.Analytically show data as can be seen, stock solution has the complete inhibition effect to Influenza A1 virus; Bilirubin and mesobilirubin XIII α have Influenza A1 virus 50% inhibition concentration (IC ₅₀) be respectively 13.95 μ g/mL (23.86 μ M) and 15.73 μ g/mL (26.75 μ M), be the original liquid concentration of 1/8 dilution.Can find out also that by accompanying drawing in the experimental concentration scope, when crossing certain threshold values concentration, along with the increase of pigment concentration, pigment significantly improves the inhibitory action of virus, until suppressing fully.All these explanation has the effect of obvious suppression Influenza A1 virus in the aqueous buffer solution of bilirubin and derivant mesobilirubin XIII α above concentration about its 25 μ M thereof, and has no side effect.

Claims

1. the bilirubin of a following formula structure and derivant thereof are used to prepare the purposes of preventing and treating the grippal medicine that is caused by Influenza A1 virus, and it is characterized in that: described structure is:

R ¹ R ² R ³ R ⁴ R ⁵ R ⁶ R ⁷ R ⁸

Bilirubin IX α M V M P P M M V

Mesobilirubin X III α M E M P P M E M

M＝CH ₃，E＝CH ₂CH ₃，V＝CH＝CH ₂，P＝CH ₂CH ₂CO0H