CN107619428B - Acylated derivative of ornithine and aspartate dipeptide compound and application thereof - Google Patents
Acylated derivative of ornithine and aspartate dipeptide compound and application thereof Download PDFInfo
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Abstract
The invention provides an acylated derivative of ornithine and aspartate dipeptide compound or a pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the derivative or the pharmaceutically acceptable salt thereof, and application of the derivative or the pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating hyperammonemia or liver diseases, particularly hepatic encephalopathy. Test results clearly show that the acylated derivative of the ornithine dipeptide compound and the aspartic acid dipeptide compound can obviously reduce the blood ammonia concentration after administration, has faster effect than LOLA, can obviously improve the memory disorder secondary to TAA-induced liver injury of rats, and shows that the acylated derivative of the dipeptide has 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 acylated derivative of an ornithine dipeptide compound and an aspartate dipeptide compound, a preparation method thereof, a pharmaceutical composition containing the derivative, and application of the derivative 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 pathological engineering treatment. so. rez PC, actual. 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 LiverDisases 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 acylated derivatives of ornithine and aspartate dipeptide compounds nor their use in the preparation of a medicament for the prevention or treatment of hyperammonemia or liver diseases, especially hepatic encephalopathy.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide acylated derivatives of ornithine and aspartate dipeptide compounds having activity in preventing and treating hyperammonemia or liver diseases, especially hepatic encephalopathy, preparation methods thereof, pharmaceutical uses thereof and pharmaceutical compositions comprising the same, in view of the above-mentioned state of the art.
In a first aspect of the present invention there is provided an acylated derivative of an ornithine and an aspartate dipeptide compound, the ornithine being L-ornithine and the aspartate being L-aspartate, the acylated derivative being a phenylacetated or phenylbutylated derivative, the dipeptide compound preferably being a compound of formula (I) wherein the acylation is either monoacylated or polyacylated at a primary amino group or is monoacylated at a secondary amino group:
further, the acylated derivative of ornithine and aspartate dipeptide compound is preferably a compound of the following formula (IIa) and formula (IIb):
the acylated derivatives of the present invention may be administered to humans orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously). The acylated derivatives may be administered alone or in combination with other pharmaceutically acceptable compounds.
Another aspect of the present invention provides a process for the preparation of said compounds of formula (IIa) or (IIb) according to the following reaction scheme:
the method specifically comprises the following steps:
(1) under ice bath, adding Dicyclohexylcarbodiimide (DCC) into a solution containing phenylacetic acid or phenylbutyric acid, N-hydroxysuccinimide (NHS) and a solvent, stirring for reaction after the addition is finished, quenching a reaction solution after the reaction is finished, and performing suction filtration to obtain a filtrate for later use; in the presence of alkali, N-Boc-L-ornithine reacts with the filtrate obtained before at room temperature by stirring to obtain an intermediate 1a or 1 b;
(2) under ice bath, adding Dicyclohexylcarbodiimide (DCC) into a solution containing the intermediate 1a or 1b, N-hydroxysuccinimide (NHS) and a solvent, stirring for reaction after the addition is finished, quenching a reaction solution after the reaction is finished, and performing suction filtration to obtain 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 2a or 2 b;
(3) and (3) dissolving the intermediate 2a or 2b with saturated HCl gas in ethyl acetate to obtain the compound of the formula (IIa) or the formula (IIb).
Wherein, the alkali in the step (1) can be selected from sodium hydroxide, potassium carbonate, sodium bicarbonate or potassium bicarbonate, and sodium bicarbonate is preferred; the solvent is selected from tetrahydrofuran, tetrahydrofuran/water, toluene, N-dimethylformamide; the dosage molar ratio of the phenylacetic acid or the phenylbutyric acid to the NHS and the DCC is 1 (1-1.2) to 1-1.3, and the dosage molar ratio of the phenylacetic acid or the phenylbutyric acid to the N-Boc-L-ornithine and the alkali is 1 (1-1.2) to 2-4.
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 solvent is selected from tetrahydrofuran, tetrahydrofuran/water, toluene, N-dimethylformamide; the molar ratio of the intermediate 1a or 1b to NHS and DCC is 1 (1-1.2) to 1-1.3, and the molar ratio of the intermediate 1a or 1b to L-aspartic acid and alkali is 1 (1-1.2) to 2.5-5.
The reaction temperature in the step (3) is 0 ℃ to room temperature; the preparation process of the ethyl acetate solution of the saturated HCl gas comprises the steps of drying the self-made HCl gas and then introducing ethyl acetate until the saturated HCl gas is saturated; the reaction is carried out in the absence of solvent or in the presence of one or more of ethyl acetate, methanol, ethanol and isopropanol.
Another aspect of the present invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of an acylated derivative 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 acylated derivative can be prepared into oral preparations in the form of tablets, capsules, micro-tablets, pills, micro-pills, granules, powders or syrups and the like, the oral preparations can be optionally coated by enteric coating or film coating, and the capsules can 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, 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 acylated derivative of ornithine and aspartate dipeptide compound, especially in the preparation of a medicament for the prevention or treatment of hyperammonemia or liver disease, especially hepatic encephalopathy.
In certain embodiments of the invention, the pharmacological effects of ornithine and an acylated derivative of an aspartate dipeptide compound of the invention are evaluated in a Thioacetamide (TAA) induced model of acute hepatic encephalopathy and chronic liver injury in rats and compared to LOLA. Test results clearly show that the acylated derivative of the ornithine dipeptide compound and the aspartic acid dipeptide compound can obviously reduce the blood ammonia concentration after administration, has faster effect than LOLA, can obviously improve the memory disorder secondary to TAA-induced liver injury of rats, and shows that the acylated derivative of the dipeptide has 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 1a
10.892g (0.08mol) of phenylacetic acid and 9.66g (0.084mol, 1.05eq) of NHS were dissolved in 100ml of tetrahydrofuran under ice bath. 20g of DCC (0.097mol, 1.2eq) was additionally weighed and dissolved in 100ml of tetrahydrofuran and added dropwise to the reaction flask. Stirring for reaction for 1 hour, monitoring the reaction completion by TLC, adding 10ml distilled water into the reaction solution, stirring for 20min, and quenching excess DCC; filtering to remove insoluble substances, and collecting filtrate.
19.2g (0.083mol, 1.03eq) of N-Boc-L-ornithine and 13.44g (0.160mol, 2.0eq) of sodium bicarbonate were weighed out and dispersed in 100ml of distilled water, and 50ml of THF was added thereto; dropwise adding the filtrate obtained in the previous step into the reaction solution, and stirring at room temperature for 4 hours; after TLC monitoring reaction, using 2N HCl solution to adjust pH to 7, then at 45 degrees C under reduced pressure evaporation of THF, using 2N HCl to adjust the residual liquid pH to 3 ~ 4, white solid precipitation, adding 200ml ethyl acetate extraction. The extract was filtered once before separation, the insoluble matter was filtered off, the separated liquid was separated, and the aqueous layer was extracted once with 100ml of ethyl acetate and combined with the former organic phase. The organic phase was washed with distilled water (100 ml. times.2) and once with saturated brine (100 ml). After drying with anhydrous sodium sulfate, concentrating to dryness under reduced pressure, and separating by flash column chromatography to obtain white solid 19.8g with yield of 70.6%.
EXAMPLE 2 preparation of intermediate 1b
12.697g (0.077mol) of phenylbutyric acid and 8.87g (0.077mol, 1.0eq) of NHS were weighed out and dissolved in 100ml of tetrahydrofuran and stirred in ice bath. DCC 18g (0.087mol, 1.1eq) was additionally weighed and dissolved in 100ml tetrahydrofuran and added dropwise to the reaction flask. Stirring for reaction for 1 hour, monitoring the reaction completion by TLC, adding 10ml distilled water into the reaction solution, stirring for 20min, and quenching excess DCC; filtering to remove insoluble substances, and collecting filtrate.
19.2g (0.083mol, 1.07eq) of N-Boc-L-ornithine and 13.44g (0.160mol, 2.1eq) of sodium bicarbonate were weighed out and dispersed in 100ml of distilled water, and 50ml of THF was added thereto; dropwise adding the filtrate obtained in the previous step into the reaction solution, and stirring at room temperature for 4 hours; after TLC monitoring reaction, using 2N HCl solution to adjust pH to 7, then at 45 degrees C under reduced pressure evaporation of THF, using 2N HCl to adjust the residual liquid pH to 3 ~ 4, white solid precipitation, adding 200ml ethyl acetate extraction. The extract was filtered once before separation, the insoluble matter was filtered off, the separated liquid was separated, and the aqueous layer was extracted once with 100ml of ethyl acetate and combined with the former organic phase. The organic phase was washed with distilled water (100 ml. times.2) and once with saturated brine (100 ml). After drying over anhydrous sodium sulfate, concentrating to dryness under reduced pressure, and separating by flash column chromatography to obtain 21.4g of white solid with 73.1% yield.
EXAMPLE 3 preparation of intermediate 2a
Intermediate 1a 7.36g (0.021mol) and NHS 2.53g (0.022mol, 1.05eq) were weighed out and dissolved in 100ml tetrahydrofuran and stirred under ice bath. DCC 4.97g (0.024mol, 1.15eq) was additionally weighed and dissolved in 100ml tetrahydrofuran and added dropwise to the reaction flask. Stirring and reacting for 1 hour, adding DCC (dichloro-C) when an unreacted intermediate 1a exists in a TLC (thin layer chromatography) monitoring system, adding 10ml of distilled water into the reaction solution when the monitoring reaction is complete, and stirring for 20min to quench excessive DCC; filtering to remove insoluble substances, and collecting filtrate.
Weighing 2.942g (0.022mol, 1.05eq) of L-Asp and 7.06g (0.084mol, 4.0eq) of sodium bicarbonate, dissolving with 150ml of distilled water, and generating bubbles when dissolving; dropwise adding the filtrate obtained in the previous step into the reaction solution, and stirring at room temperature overnight; after TLC monitoring reaction, using 2N HCl solution to adjust pH to 7, then rotary evaporating at 45 ℃ to remove THF, then using 2N HCl to adjust pH of residual liquid to 3-4, separating out white solid, adding 200ml ethyl acetate to extract. The mixture was filtered once before separation, the insoluble matter was filtered off, the separated liquid was separated, and the aqueous layer was extracted once with 100ml of ethyl acetate and combined with the former organic phase. The organic phase was washed with distilled water (100 ml. times.2), saturated brine (100 ml. times.1), dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and subjected to flash column chromatography to obtain 5.75g of a white solid with a yield of 58.8%.
EXAMPLE 4 preparation of intermediate 2b
10.05g (0.027mol) of intermediate 1b and 3.42g (0.030mol, 1.12eq) of NHS were weighed out and dissolved in 120ml of tetrahydrofuran and stirred under ice bath. 6.68g (0.032mol, 1.22eq) of DCC was further weighed and dissolved in 100ml of tetrahydrofuran, and added dropwise to the reaction flask, followed by stirring and reacting. When the TLC monitoring reaction is complete, adding 12ml of distilled water into the reaction solution, stirring for 20min, and quenching excessive DCC; filtering to remove insoluble substances, and collecting filtrate.
3.78g (0.028mol, 1.07eq) of L-Asp and 7.95g (0.095mol, 3.6eq) of sodium bicarbonate are weighed and dissolved by 150ml of distilled water, and bubbles are generated when the L-Asp and the sodium bicarbonate are dissolved; dropwise adding the filtrate obtained in the previous step into the reaction solution, and stirring at room temperature overnight; after TLC monitoring reaction, using 2N HCl solution to adjust pH to 7, then rotary evaporating at 45 ℃ to remove THF, then using 2N HCl to adjust pH of residual liquid to 3-4, separating out white solid, adding 250ml ethyl acetate to extract. The mixture was filtered once before separation, the insoluble matter was filtered off, the separated liquid was separated, and the aqueous layer was extracted once with 120ml of ethyl acetate and combined with the former organic phase. The organic phase was washed with distilled water (120 ml. times.2), saturated brine (120 ml. times.1), dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and subjected to flash column chromatography to obtain 7.96g of a white solid with a yield of 60.7%.
EXAMPLE 5 preparation of Compound of formula (IIa)
In a 100ml reaction bottle, dissolving the weighed intermediate 2a compound (7g, 0.015mol) in ethyl acetate 50ml, then weighing 7ml of HCl/ethyl acetate solution and diluting with 20ml of ethyl acetate, dropwise adding the diluted solution into the reaction bottle under ice bath, stirring after dropwise adding, slowly separating out solid, monitoring by TLC (thin layer chromatography) until the reaction is finished, evaporating the solvent under reduced pressure, further adding 200ml of ethyl acetate into the obtained solid after evaporation, and concentrating under reduced pressure to remove residual HCl to obtain a crude product. The crude product was separated by flash column chromatography to give 4.67g of a white solid, 85.0% yield, 98.7% HPLC content.
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:366.2[M+H]+；
1H NMR(500MHz,DMSO-d6)ppm:12.70(s,2H),8.48–8.24(m,2H),7.99(s,3H),7.33–7.26(m,4H),7.23(d,J＝6.9Hz,1H),4.52(m,J＝13.9,6.6Hz,1H),4.36(s,1H),3.53(d,J＝14.8Hz,2H),2.76(s,2H),2.69(dd,J＝16.6,6.2Hz,1H),2.59(dd,J＝16.5,6.3Hz,1H),1.77(s,1H),1.59(m,J＝7.4Hz,4H).
EXAMPLE 6 preparation of the Compound of formula (IIb)
In a 100ml reaction bottle, dissolving the weighed intermediate 2b compound (7.3g, 0.015mol) in 50ml ethyl acetate, then weighing 7ml HCl/ethyl acetate solution (same as the preparation method) and diluting with 20ml ethyl acetate, dropwise adding the diluted solution into the reaction bottle under ice bath, stirring after dropwise adding, slowly separating out solid, after TLC monitoring till the reaction is finished, evaporating the solvent under reduced pressure, further adding 200ml ethyl acetate into the obtained solid after evaporation, and concentrating under reduced pressure to remove residual HCl to obtain a crude product. The crude product was isolated by flash column chromatography to give 4.51g of a white solid, 77.5% yield, 98.8% HPLC content.
ESI-MS m/z:394.4[M+H]+；
1H-NMR(500MHz,CDCl3)ppm:7.58(d,J＝7.9Hz,2H),7.27–7.15(m,5H),5.23(s,1H),4.80(s,1H),3.03(s,1H),2.70(t,J＝8.2Hz,5H),2.53(s,2H),1.95(s,1H),1.85(s,1H),1.74(s,2H),1.63(s,2H),1.05(s,2H).
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 compounds of the formulae (IIa) and (IIb) 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:
30 healthy male SD rats (purchased from Beijing Wittingle laboratory animal technology Co., Ltd., certification number: 11400700171427, body weight range 230-. A blank control group, a model group, a LOLA administration group, a formula (IIa) administration group and a formula (IIb) administration group were each included. 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 (IIa) administration group and the formula (IIb) 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 (IIa) administration group, and the formula (IIb) administration group, blood plasma was collected from all groups of rats by eyeball blood collection. 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 (IIa) and formula (IIb) 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. Furthermore, the LOLA, formula (IIa) and formula (IIb) compounds all showed some sustained blood ammonia lowering effect compared to the model group, and the formula (IIa) and formula (IIb) compounds acted faster than LOLA and the blood ammonia lowering amplitude was more pronounced. 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, an IIa administration group and a formula (IIb) administration group, each of which was 6. 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: as shown in table 3 below, chronic TAA poisoning may lead to memory impairment and learning decline following liver damage in rats. This is improved by orally administering LOLA, a compound of formula (IIa) and a compound of formula (IIb) to rats for 2 weeks. In addition, the compounds of formula (IIa) and formula (IIb) can obviously improve the memory disorder secondary to TAA-induced liver injury of rats, and the improvement degree is statistically significant compared with that of a 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 (10)
1. An acylated derivative of ornithine and aspartate dipeptide compound or a pharmaceutically acceptable salt thereof, wherein the dipeptide compound is a compound of the following formula (I), the acylated derivative is a phenylacetated or phenylbutylated derivative, and the acylation is a monoacylation of the primary amino group near the chiral carbon atom in the ornithine structure in formula (I),
2. an acylated derivative of an ornithine and aspartate dipeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 wherein the acylated derivative of the dipeptide compound is selected from the group consisting of compounds of formula (IIa) or formula (IIb):
3. a process for the preparation of a compound of formula (IIa) or formula (IIb) according to claim 2, wherein the reaction is of the formula:
the method specifically comprises the following steps:
(1) under ice bath, adding Dicyclohexylcarbodiimide (DCC) into a solution containing phenylacetic acid or phenylbutyric acid, N-hydroxysuccinimide (NHS) and a solvent, stirring for reaction after the addition is finished, quenching a reaction solution after the reaction is finished, and performing suction filtration to obtain a filtrate for later use; in the presence of alkali, N-Boc-L-ornithine reacts with the filtrate obtained before at room temperature by stirring to obtain an intermediate 1a or 1 b;
(2) under ice bath, adding Dicyclohexylcarbodiimide (DCC) into a solution containing the intermediate 1a or 1b, N-hydroxysuccinimide (NHS) and a solvent, stirring for reaction after the addition is finished, quenching a reaction solution after the reaction is finished, and performing suction filtration to obtain 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 2a or 2 b;
(3) and (3) reacting the intermediate 2a or 2b with a saturated HCl gas ethyl acetate solution in a solvent to obtain the compound of the formula (IIa) or the formula (IIb).
4. The method according to claim 3, wherein the base in the step (1) is selected from the group consisting of sodium hydroxide, potassium carbonate, sodium hydrogencarbonate and potassium hydrogencarbonate; the dosage molar ratio of the phenylacetic acid or the phenylbutyric acid to the NHS and the DCC is 1 (1-1.2) to 1-1.3, and the dosage molar ratio of the phenylacetic acid or the phenylbutyric acid to the N-Boc-L-ornithine and the alkali is 1 (1-1.2) to 2-4.
5. The method according to claim 3, wherein the base in the step (2) is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide; the molar ratio of the intermediate 1a or 1b to NHS and DCC is 1 (1-1.2) to 1-1.3, and the molar ratio of the intermediate 1a or 1b to L-aspartic acid and alkali is 1 (1-1.2) to 2.5-5.
6. A pharmaceutical composition comprising a therapeutically effective amount of an acylated derivative of an ornithine and aspartate dipeptide compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2, and one or more pharmaceutically acceptable carriers, excipients or diluents.
7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is for oral, rectal, or parenteral administration.
8. Use of an acylated derivative of ornithine and aspartate dipeptide compounds or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the manufacture of a medicament for the prevention or treatment of hyperammonemia or liver disease.
9. The use according to claim 8, wherein the liver disease is hepatic encephalopathy.
10. The use as claimed in claim 8 or 9, wherein the medicament comprises from 20g to 40g of the acylated derivative or a pharmaceutically acceptable salt thereof.
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| WO2011083556A1 (en) * | 2010-01-08 | 2011-07-14 | キリンホールディングス株式会社 | Highly flavored ornithine-containing alcohol-free malt beverage |
| CN102688202A (en) * | 2012-06-07 | 2012-09-26 | 北京莱瑞森医药科技有限公司 | Temozolomide freeze-dried preparation |
| CN103622121A (en) * | 2013-11-13 | 2014-03-12 | 精晶药业股份有限公司 | Solid beverage containing N(2)-L-alanyl-L-glutamine |
| WO2015161448A1 (en) * | 2014-04-22 | 2015-10-29 | Wuhan Ll Science And Technology Development Co., Ltd. | Ornithine-containing or aspartate-containing compositions and the uses thereof |
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| WO2011083556A1 (en) * | 2010-01-08 | 2011-07-14 | キリンホールディングス株式会社 | Highly flavored ornithine-containing alcohol-free malt beverage |
| CN102688202A (en) * | 2012-06-07 | 2012-09-26 | 北京莱瑞森医药科技有限公司 | Temozolomide freeze-dried preparation |
| CN103622121A (en) * | 2013-11-13 | 2014-03-12 | 精晶药业股份有限公司 | Solid beverage containing N(2)-L-alanyl-L-glutamine |
| WO2015161448A1 (en) * | 2014-04-22 | 2015-10-29 | Wuhan Ll Science And Technology Development Co., Ltd. | Ornithine-containing or aspartate-containing compositions and the uses thereof |
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