CN107573404B - Active salt of dipeptide compound of ornithine and aspartate and application thereof - Google Patents
Active salt of dipeptide compound of ornithine and aspartate and application thereof Download PDFInfo
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Abstract
The invention provides an active salt of ornithine and aspartate dipeptide compounds, a preparation method thereof, a pharmaceutical composition containing the active salt, and application of the dipeptide compounds or the active salt thereof in preparing a medicament for preventing or treating hyperammonemia or liver diseases, particularly hepatic encephalopathy. The test result clearly shows that the ornithine and aspartate dipeptide compound and the active salt thereof can obviously reduce the blood ammonia concentration after administration, and can obviously improve the memory disorder secondary to TAA-induced liver injury of rats, which indicates that the dipeptide compound and the active salt thereof have certain treatment effect on hyperammonemia or liver diseases, particularly hepatic encephalopathy.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an active salt of an ornithine and aspartate dipeptide compound, a preparation method thereof, a pharmaceutical composition containing the active salt, and application of the dipeptide compound or the active salt thereof in preparing a medicine for preventing or treating hyperammonemia or liver diseases, particularly hepatic encephalopathy.
Background
Hepatic Encephalopathy (HE) is a complex neuropsychiatric disorder that occurs in a variety of clinical conditions such as acute or chronic liver disease and spontaneous portal vein bypass. In the early stages of hepatic encephalopathy, minor mental changes such as disorientation, confusion, and disorientation occur. In severe cases, hepatic encephalopathy can lead to stupor, coma, brain swelling (encephaledema) and death. The accumulation of ammonia is thought to play an important role in the progression of hepatic encephalopathy and multiple organ failure (respiratory failure, cardiovascular failure, renal failure).
Typical therapies for patients with hepatic encephalopathy include methods of reducing ammonia concentration. These methods include limiting the intake of dietary proteins, administering lactulose, neomycin, L-ornithine L-aspartate (LOLA) or sodium benzoate, and cleansing enemas.
LOLA belongs to an amino acid composite salt of L-ornithine and L-aspartic acid, is a medicine for treating hyperammonemia on the market, can reduce the blood ammonia by stimulating the synthesis of glutamine, and is a vein medicine which is proved to be more effective at reducing the blood ammonia at present. However, although LOLA is effective in reducing ammonia in patients with cirrhosis, it is not good in patients with acute liver failure, and it may be that ornithine cycle disorder exists in patients with acute liver failure, and the reduced blood ammonia is only ammonia combined with glutamic acid to generate glutamine, and glutamine can be catalyzed by glutamine enzyme to be decomposed again to generate ammonia in kidney and intestinal tract, so that blood ammonia rises. Furthermore, the literature (A clinical analysis of students assessing L-organization-L-aberration (LOLA) in pharmaceutical engineering treatment. so < rez PC, et al. Arq.gastroenterol, 46 (3)), 241-; the American Liver disease research Association and European Liver research Association 2014Practice guidelines (2014Practice guidelines by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver) also do not recommend LOLA for the treatment of HE.
It has been shown in the literature that L-Ornithine Phenylacetate (OP) has a very good therapeutic effect in the treatment of HE, particularly in the reduction of blood ammonia and cerebral edema. Currently, the secondary clinical study of OPs for the treatment of HE has ended and is ongoing.
In recent years, research on active small molecule peptides shows that compared with an amino acid transportation system, the small molecule peptides have the characteristics of quick absorption, high bioavailability, low energy consumption, low possibility of saturation and the like.
The prior art does not disclose active salts of ornithine and aspartate dipeptide compounds nor their use in the preparation of a medicament for the prevention or treatment of hyperammonemia or liver disease, especially hepatic encephalopathy.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide active salts of ornithine and aspartate dipeptide compounds having activity for preventing and treating hyperammonemia or liver diseases, especially hepatic encephalopathy, preparation methods thereof, medical uses thereof and pharmaceutical compositions comprising the same, in view of the above-mentioned current state of the art.
In a first aspect of the present invention, there is provided an active salt of an ornithine and an aspartate dipeptide compound, said ornithine being L-ornithine and said aspartate being L-aspartate, said active salt being an acid addition salt, said dipeptide compound preferably being a compound of formula (I):
examples of the acid moiety of said acid addition salts are: inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid, and organic acids such as benzoic acid, phenylacetic acid, phenylbutyric acid, trifluoroacetic acid, maleic acid, methanesulfonic acid, and p-toluenesulfonic acid.
Further, the acid addition salt is preferably hydrochloride and phenylacetate, preferably having the structures shown in the following formulas (Ia) and (Ib):
the active salts of the present invention may be administered to humans orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously). The active salts may be administered alone or in combination with other pharmaceutically acceptable compounds.
Another aspect of the present invention is to provide a process for the preparation of said compounds of formula (Ia) or (Ib).
The preparation method of the compound of the formula (Ia) is as follows:
the method specifically comprises the following steps:
(1) stirring N-delta-Boc-L-ornithine or L-ornithine hydrochloride and Boc anhydride in the presence of an acid-binding agent to react to obtain an intermediate 1;
(2) stirring a mixed solution containing the intermediate 1, N-hydroxysuccinimide NHS and a solvent until the temperature is reduced to about 0 ℃, adding Dicyclohexylcarbodiimide (DCC), stirring for reaction, performing suction filtration after the reaction is finished, and reserving a filtrate for later use; in the presence of alkali, the L-aspartic acid and the filtrate are stirred and reacted at room temperature to obtain an intermediate 2;
(3) the intermediate 2 is reacted with a saturated HCl gas in ethyl acetate at room temperature with stirring to give the compound (Ia).
Wherein, the acid-binding agent in the step (1) can be selected from sodium hydroxide, potassium carbonate, sodium bicarbonate or potassium bicarbonate, and sodium bicarbonate is preferred; the reaction temperature is 0 ℃ to room temperature; the reaction solvent is selected from tetrahydrofuran, tetrahydrofuran/water, toluene and N, N-dimethylformamide; the dosage molar ratio of the N-delta-Boc-L-ornithine, the Boc acid anhydride and the acid-binding agent is 1 (1.1-1.5) to 2-4; the molar ratio of the L-ornithine hydrochloride to the Boc anhydride is 1 (2.5-4) to (4-6).
The alkali in step (2) can be selected from sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, preferably sodium bicarbonate; the reaction temperature is 0 ℃ to room temperature; the reaction solvent is selected from tetrahydrofuran, tetrahydrofuran/water, toluene and N, N-dimethylformamide; the molar ratio of the intermediate 1 to the NHS and DCC is 1 (1-1.5) to 1-1.5; the molar ratio of the intermediate 1 to the L-aspartic acid and the alkali is 1 (1-1.5) to 2-5.
In the step (3), the dosage ratio of the intermediate 2 to the ethyl acetate solution of saturated HCl gas is 1 g: (10-15) mL.
The preparation method of the compound of the formula (Ib) is as follows:
the method specifically comprises the following steps:
(1) treating the compound shown in the formula (Ia) by using cation exchange resin to obtain a compound shown in the formula (I);
(2) heating the compound of the formula (I) and phenylacetic acid in a solvent for reaction to obtain a compound of a formula (Ib).
Wherein, the ion exchange resin treatment in the step (1) comprises the following specific operations: dissolving the compound of formula (Ia) in a small amount of water, adding the solution to a treated ion exchange column, washing with distilled water until the eluent is not colored by silver nitrate test solution, and then changing to ammonia water: and (3) eluting with distilled water at a ratio of 1:10, starting to collect eluent when the pH of the eluent is monitored to be alkaline, stopping collecting when no product is monitored, concentrating the collected eluent under reduced pressure until the eluent is dried to obtain a crude product, and further recrystallizing to obtain a pure compound of the formula (I).
In the step (2), the dosage molar ratio of the compound shown as the formula (I) to the phenylacetic acid is 1:1, and the reaction temperature is 45-55 ℃; the reaction solvent is selected from anhydrous methanol, dichloromethane or N, N-dimethylformamide.
Another aspect of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of an active salt of the above dipeptide compound, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents. Further, the therapeutically effective amount is 20-40 g.
The active salt may be prepared as an oral formulation in the form of tablets, capsules, microtablets, pills, pellets, granules, powders or syrups, optionally enteric-or film-coated, capsules, which may be soft or hard gelatin capsules; can also be prepared into lyophilized powder for injection, injection such as injection solution, etc., or suppository.
These formulations can be manufactured by known methods with the following additives: excipients (e.g., sugar derivatives such as lactose, white sugar, glucose, mannitol, and sorbitol, starch derivatives such as corn starch, potato starch, alpha-starch, and dextrin, cellulose derivatives such as methylcellulose, organic excipients such as gum arabic, dextran, and pullulan, silicate derivatives such as light silicic anhydride, synthetic aluminum silicate, calcium silicate, and magnesium aluminum silicate, phosphates such as calcium hydrogen phosphate, carbonates such as calcium carbonate, and inorganic excipients such as sulfates such as calcium sulfate), lubricants (e.g., metal stearates such as stearic acid, calcium stearate, and magnesium stearate, waxes such as talc, beeswax, and spermaceti, boric acid, adipic acid, sulfates such as sodium sulfate, hexanediol, fumaric acid, sodium benzoate, DL-leucine, lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate, silicic anhydride, silicic acid, dextrin, and dextrin), lubricants (e.g., sodium stearate, sodium lauryl sulfate, magnesium stearate, etc.), lubricants (e.g., sodium stearate, calcium stearate, etc.), lubricants, etc, Silicic acid such as silicic acid hydrate; and the above starch derivatives), binders (such as: hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyethylene glycol, and the same compounds as the above excipients), a disintegrant (e.g.: low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, and cellulose derivatives of croscarmellose sodium and cellulose; chemically modified starches/celluloses such as carboxymethyl starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and the like; the above starch derivatives), emulsifiers (such as: colloidal clay such as bentonite and V-shaped glue; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid ester, and the like), stabilizers (for example: parabens such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenethyl alcohol; phenols such as benzalkonium chloride, phenol, cresol and the like; sulfur and mercury are scattered; dehydroacetic acid; and sorbic acid), flavors (e.g.: sweeteners, souring agents, flavors), diluents, and the like, which are generally used.
Still another aspect of the present invention is to provide a medical use of the ornithine and aspartate dipeptide compound or an active salt thereof, particularly a use in preparing a medicament for preventing or treating hyperammonemia or liver disease, particularly hepatic encephalopathy.
In certain embodiments of the invention, the pharmacological effects of ornithine and an active salt of an aspartate dipeptide compound of the invention are evaluated in a Thioacetamide (TAA) induced rat acute hepatic encephalopathy and chronic liver injury model and compared to both its free base and LOLA. The test result clearly shows that the ornithine and aspartate dipeptide compound and the active salt thereof can obviously reduce the blood ammonia concentration after administration, can obviously improve the memory disorder secondary to TAA-induced liver injury of rats, and show that the dipeptide and the active salt thereof have certain treatment effect on hyperammonemia or liver diseases, particularly hepatic encephalopathy.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
EXAMPLE 1 preparation of intermediate 1
A100 ml reaction flask was charged with 0.258g (6.45mmol, 3eq) of sodium hydroxide, 10ml of purified water and 0.5g (2.15mmol, 1eq) of N-delta-Boc-L-ornithine, and a solution of Boc anhydride 0.563g (2.58mmol, 1.2eq)/10ml of THF was added at 0 to 10 ℃ and after completion of dropwise addition, stirred at room temperature. And (3) monitoring the reaction by TLC (thin layer chromatography) until the reaction is completed, cooling to below 10 ℃, dropwise adding dilute hydrochloric acid until the pH value is 4-5, extracting a water phase by using ethyl acetate (15ml multiplied by 2), combining ethyl acetate layers, washing by using purified water once, drying an organic phase by using anhydrous sodium sulfate, filtering, and concentrating under reduced pressure until the organic phase is dry to obtain 0.81g of a bubble solid crude product, namely the intermediate 1, wherein the yield is basically quantitative.
EXAMPLE 2 preparation of intermediate 1
11.8g (296.5mmol, 5eq) of sodium hydroxide and 200ml of purified water are added into a 500ml three-necked bottle, the temperature is reduced to 0 ℃, 10g (59.3mmol, 1eq) of L-ornithine hydrochloride is added, the mixture is stirred to be clear, a solution of Boc-anhydride (38.8g, 177.9mmol, 3eq) in tetrahydrofuran (200ml) is added dropwise at about 0 ℃, and after the dropwise addition is finished, the reaction is carried out at room temperature. After TLC monitoring is carried out until the reaction is finished, tetrahydrofuran is concentrated under reduced pressure at 45 ℃, the water phase is adjusted to pH 4-5 by 1N hydrochloric acid, ethyl acetate is extracted (100ml multiplied by 2), the ethyl acetate layer is combined, purified water is washed twice, the organic phase is dried by anhydrous sodium sulfate, and after filtration, the organic phase is concentrated under reduced pressure at 45 ℃ until the organic phase is dried, 20g of white bubble solid, namely the intermediate 1 is obtained, and the yield is basically quantitative.
EXAMPLE 3 preparation of intermediate 2
Adding 18.0g (54.2mmol, 1eq) of the intermediate compound 1 into a 500ml round-bottom flask, adding 180ml tetrahydrofuran, stirring to dissolve, adding 6.86g (59.6mmol, 1.1eq) of N-hydroxysuccinimide, stirring until the internal temperature is reduced to about 0 ℃, slowly adding dropwise a tetrahydrofuran (100ml) solution of DCC (12.3g, 59.6mmol, 1.1eq), stirring at room temperature after adding dropwise, slowly precipitating solids at room temperature, monitoring by TLC until the reaction is complete, performing suction filtration, and keeping the filtrate for use.
Adding 7.9g (59.35mmol, 1.1eq) of L-aspartic acid, 200ml of purified water and 18.2g (21.66mmol, 4eq) of sodium bicarbonate into a 1000ml three-necked bottle, stirring until the solid is clear, dropwise adding the remaining filtrate (namely the prepared activated ester solution) obtained in one step at room temperature, stirring overnight at room temperature after dropwise adding, monitoring by TLC until the reaction is complete, concentrating tetrahydrofuran under reduced pressure at 40 ℃, adding 20ml of purified water, washing by ethyl acetate (15ml multiplied by 2), adjusting the pH of an aqueous phase to 7 by using 2N hydrochloric acid, concentrating under reduced pressure at 60 ℃ to the remaining 30ml, cooling to below 10 ℃, adjusting the pH to about 4 by using 1N hydrochloric acid, extracting by ethyl acetate (100ml multiplied by 2), combining organic phases, drying by anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to dryness to obtain 24g of a foamed solid, and performing rapid column chromatography to obtain 17.5g of a white solid with the yield of 72.2%.
EXAMPLE 4 preparation of Compound of formula (Ia)
Adding 6.0g (13.4mmol) of the intermediate 2 compound into a 100ml round-bottom flask, slowly adding 60ml of HCl/ethyl acetate mixed solution (self-made) at room temperature till the solid is clear, stirring at room temperature, slowly separating out the solid, monitoring by TLC (thin layer chromatography) until the reaction is finished, standing, pouring off the ethyl acetate, adding 20ml of ethyl acetate, stirring, standing, pouring off the ethyl acetate, repeating the steps again, adding 20ml of ethyl acetate, concentrating at 50 ℃ under reduced pressure till the solid is dried to obtain a white bubble solid, further drying at 60 ℃ under vacuum to obtain 3.1g of the solid which is hydrochloride, wherein the yield is 81.5 percent, and the HPLC content is 98.5 percent.
The preparation process of the HCl/ethyl acetate solution comprises the following steps: 50g of sodium chloride and 500ml of concentrated hydrochloric acid are added into a 1L three-neck flask, concentrated sulfuric acid is dropwise added, generated HCl gas is dried, and ethyl acetate is introduced until the HCl gas is saturated, so that the sodium chloride is obtained.
ESI-MS m/z:248[M+H]+.
1H-NMR(300MHz,DMSO-d6)δppm:12.59(s,2H),9.01(dd,J＝41.9,7.8Hz,1H),8.37(s,3H),8.11(s,3H),4.57(dd,J＝13.2,6.2Hz,1H),3.89(s,1H),2.81(s,2H),2.71(dd,J＝10.3,6.6Hz,2H),1.90～1.70(m,4H).
EXAMPLE 5 preparation of the Compound of formula (I)
According to 732 cationic resin: preparing a cation exchange resin in an amount of 25-30: 1 (weight ratio) of the compound of formula (Ia), and pretreating a cation exchange resin column as follows: soaking analytically pure 732 cation exchange resin in deionized water for 4-8h to swell the resin, rinsing the resin for several times with purified water until the effluent is clear and not turbid, soaking the resin in 4% HCl solution with the volume 4 times that of the resin for 2-4h, and then washing the resin with purified water to be neutral; soaking the resin in 4% NaOH solution in 4 times of the volume of the resin for 2-4h, washing the resin with purified water to be neutral, and repeating the acid soaking step and the alkali soaking step once; treatment with 4% HCl solution gave 732 hydrogen cation exchange resin, which was then washed with purified water to pH 7.
The compound of formula (Ia) (5.0g, 17.6mmol) to be desalted is dissolved with as little water as possible. The solution was slowly added to the already treated ion exchange column. Washing with distilled water until the eluent is not colored by silver nitrate test solution, and then changing with ammonia water: distilled water 1:10 elution. The eluent collection was started when the pH of the eluent was monitored to change from 7 to the basic range. Collection was stopped when no product was monitored. The collected eluate was concentrated to dryness at 50 ℃ under reduced pressure to give a residual oily liquid. 100ml of absolute ethyl alcohol is added into the mixture, the mixture is stirred in a water bath at 50 ℃, and white viscous solid is separated out at the bottom of a bottle. Cooling to 0 deg.C, and removing solvent to obtain crude product (white sticky solid, slightly yellow local). Dispersing the crude product in 60ml of methanol/THF (1:1) mixed solution, heating to 50 deg.C for half an hour under stirring, slowly cooling to room temperature, cooling to 0 deg.C, stirring for half an hour, and vacuum filtering. The filter cake was washed with a small amount of THF and then dried under vacuum at 60 deg.C to obtain the refined compound of formula (I) as a white powder 3.7g with a yield of 85.0%.
EXAMPLE 6 preparation of Compound of formula (Ib)
3.5g (14.2mmol) of the compound of the formula (I) are weighed out, dissolved in 100ml of anhydrous methanol and stirred with heating at 50 ℃. 2.0g (14.7mmol, 1.04eq) of phenylacetic acid was weighed, dissolved in as small an amount of anhydrous methanol as possible, and then added dropwise to the reaction flask. When dripping, white floccule is separated out, stirring for half an hour after dripping, then stopping stirring, standing and cooling. Suction filtration, washing the filter cake with a small amount of anhydrous methanol, and then vacuum drying the filter cake at 60 ℃ to obtain 4.6g of white powder, wherein the yield is 84.8 percent, and the HPLC content is 98.0 percent.
ESI-MS m/z:248[M+H]+.
1H-NMR(500MHz,CDCl3)δppm:9.24(s,3H),7.41–7.20(m,6H),4.80(t,J＝7.7Hz,1H),4.01(s,2H),3.48(t,J＝7.6Hz,1H),3.21(s,1H),3.17(s,1H),2.92(dd,J＝12.5,7.7Hz,1H),2.79(t,2H),2.32(dd,J＝12.5,7.7Hz,1H),2.07(m,2H),1.81(m,2H).
Meanwhile, the precipitate contains a phenylacetic acid peak through HPLC analysis, and the compound of the formula (I) has no stable single peak in HPLC through comparison detection. Furthermore, the compound of formula (I) was determined to be substantially insoluble in water by dissolution test, whereas the white powder obtained above was highly soluble in water and was thus determined to be a compound of formula (Ib).
Example 7 Activity Studies on a Thioacetamide (TAA) -induced acute hepatic encephalopathy rat model
1. Solution preparation:
(1) preparation of TAA solution at an administration dose of 300 mg/kg: weighing TAA powder 6.0g in a volume of 0.5ml per rat 100g, dissolving in physiological saline as solvent, and diluting to 100ml (0.5ml per 100g weight);
(2) preparation of LOLA solution at 2g/kg dose: weighing LOLA powder 20g in a volume of 0.5ml per rat 100g, and diluting to 50ml with physiological saline as solvent (0.5ml per 100g body weight);
(3) preparation of a solution of the compound of formula (I), formula (Ia) or formula (Ib) at an administration dose of 2 g/kg: the preparation was carried out in the same manner as the LOLA solution at the above administration dose of 2 g/kg.
2. Grouping and administration:
36 healthy male SD rats (purchased from Beijing Wittingle laboratory animal technology Co., Ltd., certification number: 11400700171427, body weight range 230- "300 g) were randomly divided into 6 groups of 6 rats each. A blank control group, a model group, a LOLA administration group, a formula (I) administration group, a formula (Ia) administration group and a formula (Ib) administration group. The blank control group had normal drinking and ingestion and was given 2 consecutive days with 5ml/kg of saline i.p. administered at 9 am. TAA (dose 300mg/kg) was administered to the model group and each of the corresponding administration groups, starting at 9 am every day for 2 consecutive days; wherein animals in the LOLA administration group, the formula (I) administration group, the formula (Ia) administration group and the formula (Ib) administration group are administered with the corresponding drugs at a dose of 2g/kg immediately after 1 hour of TAA modeling administration on the next day, and are administered with the same drugs at the same doses again at time points of 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 27 hours and 30 hours after the initial administration of the corresponding drugs. The administration mode is intraperitoneal injection administration. Body weight and dietary water intake were recorded once daily. During the feeding process, the animals were examined daily for food intake and water intake.
3. Collecting blood and blood plasma:
SD Male rats before TAA administration, all groups of rats were anesthetized with ether, bled using capillary eye (0.5ml) and placed in a 1.5ml EP tube to which heparin sodium (30. mu.l) had been added. 30min after the second administration of TAA, all groups of rats collected plasma using eye bleeds; after the respective drugs were administered 6h, 12h, 24h, and 30h after the initial administration in the LOLA administration group, the formula (I) administration group, the formula (Ia) administration group, and the formula (Ib) administration group, all groups of rats collected plasma by eyeball bleeding. Animals were sacrificed 24h after the last dose.
4. Measurement of blood ammonia:
taking out a plasma sample stored at the temperature of minus 20 ℃, putting 50 mu l of the plasma sample into a 1.5ml EP tube, sequentially adding 250 mu l of a reagent I and a reagent II (from an A086 blood ammonia determination kit, Nanjing as a built organism), fully mixing, centrifuging at 3500 rpm for 10min, and collecting a supernatant. Taking 250 mu l of supernatant, placing the supernatant into a 1.5ml EP tube, sequentially adding 250 mu l of reagent III and reagent IV (from an A086 blood ammonia determination kit, Nanjing established creature) into the EP tube, fully mixing the mixture, carrying out water bath at 37 ℃ for 20min, observing blue liquid, taking out 250 mu l of the mixture to a 96-well plate, and measuring the light absorption value at 630nm by using a microplate reader.
5. Statistical analysis:
data results are expressed as mean ± standard deviation, and analysis of differences between groups was performed using One-Way ANOVA in combination with Post-Hoc (Tukey method). Statistical difference significance is indicated by P < 0.05.
6. The experimental results are as follows:
6.1 weight data
The weight measurement result shows that the weight of the rats in the normal control group is gradually increased; the body weight of the rats in the model group basically shows a descending trend and slightly rises again on the 4 th day; the LOLA, formula (I), formula (Ia) and formula (Ib) compounds are always in the descending trend, which shows that TAA can damage organs such as liver in rat body and has obvious toxic effect. The results are shown in table 1 below.
TABLE 1 Effect of test substances on animal body weight (mean. + -. standard deviation) (unit: mg)
6.2 blood Ammonia concentration determination value
The results of the blood ammonia concentration measurement showed that the blood ammonia concentration of the model group rats was significantly increased after 6 hours of administration after 2 consecutive days of TAA administration, compared to the normal control group. Compared with the model group, the blood ammonia concentration of the LOLA group was not significantly reduced after 6h administration, while the blood ammonia concentration was significantly reduced after 12h administration, suggesting that the blood ammonia-reducing effect may be a slow rather than instantaneous process. In addition, the LOLA, formula (I), formula (Ia) and formula (Ib) compounds all showed some sustained blood ammonia lowering effect compared to the model group, and the formula (Ib) compound showed faster onset and more pronounced blood ammonia lowering amplitude than LOLA. The results are shown in table 2 below.
TABLE 2 Effect of test samples on blood Ammonia concentration
Note: c-normal control group, M-model group;
raw ammonia refers to the measured value of ammonia obtained from blood taken prior to initial administration of TAA; except for the normal control group, the blood ammonia after the model building of other groups refers to the blood ammonia measured value obtained by blood sampling 30min after TAA is given for the second time;
compared with the blank control group, # P <0.05, # P < 0.01; p <0.05, P < 0.01 compared to model group.
Example 8 Effect on learning Capacity of TAA-induced rats after Chronic liver injury
1. The method comprises the following steps: healthy male SD rats were randomly divided into a blank control group, a model group, a LOLA administration group, a formula (I) administration group, a formula (Ia) administration group, and a formula (Ib) administration group, and 6 animals were administered per group. Animals in each group except the blank control group were freely drunk with water containing 0.1% TAA for 50 consecutive days, and were administered by gavage at a dose of 2g/kg starting at 30 days of TAA water administration, 1 time a day for 15 consecutive days. Each rat was allowed to learn to find the location of the survival island within the water maze on day 12 of dosing for 3 consecutive days. The test was performed 1h after the last dose and the time for rats to find a survival island in the water maze and the number of times to mount the island within 2min were observed.
2. As a result: chronic TAA poisoning can lead to subsequent dysmnesia and a decline in learning ability in rats with dry injury. This is improved by orally administering LOLA, a compound of formula (I), a compound of formula (Ia) and a compound of formula (Ib) to rats for 2 weeks. In addition, the compounds of formula (I), (Ia) and (Ib) can significantly improve the memory disorder secondary to TAA-induced liver injury in rats, and the degree of improvement of the compounds of formula (Ia) and (Ib) is statistically significant compared with the model group.
TABLE 3 influence on learning ability of TAA-induced liver injury rats (mean. + -. standard deviation)
Note: compared with the blank control group, # P <0.05, # P < 0.01;
p <0.05, P < 0.01 compared to model group.
Claims (1)
1. A process for the preparation of a compound of formula (Ib), characterized in that the reaction is:
the method specifically comprises the following steps:
(1) stirring N-delta-Boc-L-ornithine or L-ornithine hydrochloride and Boc anhydride at room temperature in the presence of an acid-binding agent to react to obtain an intermediate 1;
(2) stirring a mixed solution containing the intermediate 1, N-hydroxysuccinimide NHS and a solvent until the temperature is reduced to about 0 ℃, adding Dicyclohexylcarbodiimide (DCC), stirring for reaction, performing suction filtration after the reaction is finished, and reserving a filtrate for later use; in the presence of alkali, the L-aspartic acid and the filtrate are stirred and reacted at room temperature to obtain an intermediate 2;
(3) the intermediate 2 and an ethyl acetate solution of saturated HCl gas are stirred and reacted at room temperature to obtain a compound (Ia);
(4) treating the compound shown in the formula (Ia) by using cation exchange resin to obtain a compound shown in the formula (I);
(5) heating a compound shown in a formula (I) and phenylacetic acid in a solvent to react to obtain a compound shown in a formula (Ib);
wherein, the acid-binding agent in the step (1) is selected from sodium hydroxide; the dosage molar ratio of the N-delta-Boc-L-ornithine, the Boc acid anhydride and the acid-binding agent is 1 (1.1-1.5) to 2-4; the molar ratio of the L-ornithine hydrochloride to the Boc anhydride is 1 (2.5-4) to (4-6);
the alkali in the step (2) is selected from sodium bicarbonate; the molar ratio of the intermediate 1 to the NHS and DCC is 1 (1-1.5) to 1-1.5; the molar ratio of the intermediate 1 to the L-aspartic acid and the alkali is 1 (1-1.5) to 2-5;
the dosage ratio of the intermediate 2 in the step (3) to the ethyl acetate solution of saturated HCl gas is 1 g: (10-15) mL;
the specific operation of the ion exchange resin treatment in the step (4) is as follows: dissolving the compound of formula (Ia) in a small amount of water, adding the solution to a treated ion exchange column, washing with distilled water until the eluent is not colored by silver nitrate test solution, and then changing to ammonia water: eluting with 1:10 distilled water, starting to collect eluent when the pH of the eluent is monitored to be alkaline, stopping collecting when no product is monitored, concentrating the collected eluent under reduced pressure until the eluent is dried to obtain a crude product, and further recrystallizing to obtain a pure product of the compound shown in the formula (I);
in the step (5), the dosage molar ratio of the compound of the formula (I) to the phenylacetic acid is 1:1, and the reaction temperature is 45-55 ℃.
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| JP2817157B2 (en) * | 1989-01-13 | 1998-10-27 | 味の素株式会社 | Production method of L-amino acid by fermentation method |
| GB9222772D0 (en) * | 1992-10-30 | 1992-12-09 | Unilever Plc | Cosmetic composition |
| CN101844995A (en) * | 2010-05-13 | 2010-09-29 | 上海李氏化学科技有限公司 | Preparation method of L-ornithine-L-aspartate |
| CN102093236B (en) * | 2010-12-02 | 2012-08-01 | 海南美兰史克制药有限公司 | Ornithine aspartate compound and new preparation method thereof |
| CN102274166A (en) * | 2011-07-13 | 2011-12-14 | 辽宁科泰生物基因制药股份有限公司 | Medicinal composition containing ornithine aspartate |
| CN103655533A (en) * | 2012-09-26 | 2014-03-26 | 天津市嵩锐医药科技有限公司 | Ornithine aspartate pharmaceutical composition for injection |
| CN102993037B (en) * | 2012-11-20 | 2015-03-04 | 南京工业大学 | Preparation method of L-ornithine phenylacetate |
| CN103239434A (en) * | 2012-12-26 | 2013-08-14 | 辽宁亿灵科创生物医药科技有限公司 | Ornithine aspartate composition |
| CN106117311B (en) * | 2016-06-24 | 2018-04-13 | 沈阳兴齐眼药股份有限公司 | A kind of cyclic peptide compound and its preparation method and application |
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