CN1490329A

CN1490329A - Imidazoline substituted phenoxyacetyl oligopeptide compounds, their synthesis and medical uses

Info

Publication number: CN1490329A
Application number: CNA021349940A
Authority: CN
Inventors: 彭师奇; 赵明; 王超; 刘峻岭; 杨坚
Original assignee: YITAI MEDICINE RESEARCH (SHENZHEN) Co Ltd
Current assignee: Pepharm R & D Ltd
Priority date: 2002-10-18
Filing date: 2002-10-18
Publication date: 2004-04-21
Anticipated expiration: 2022-10-18
Also published as: CN1231495C

Abstract

An imidazoline substituted phenoxyacetyl oligopeptide, its preparing process and its application in preparing composite medicine are disclosed.

Description

Imidazoline substituted phenoxyacetyl oligopeptide derivatives, their synthesis and medical application

Technical Field

The invention relates to imidazoline substituted phenoxyacetyl modified oligopeptide derivatives, synthesis thereof and application thereof as medicines.

Technical Field

Sudden death due to cerebral ischemia has become a common phenomenon in the current medical field. Research shows that during cerebral ischemia, a great amount of glutamic acid is released in brain to excite N-methyl-D-aspartate receptor, which results in great expression and activity increase of eNOS and nNOS, so that the concentration of NO in the ischemic focal region reaches micromolar level within 20 minutes, and iNOS is also expressed in great amount within 6 to 12 hours to generate great amount of NO, so that neurons die. See Dawson YL, Dawson TM, London ED, Bredt DS, Nitric oxide media primer primers, Pro.Natl.Acad Sci, USA, 1991, 88, 6368. At present, no systemic treatment method for cerebral ischemic injury exists clinically. Inhibiting the generation of toxic substances in neurons, eliminating toxic substances, blocking the transfer of toxic substances and improving the biochemical change of the pathological neurons after ischemia are main targets of treating cerebral ischemia injury by medicaments. The measures that can be taken include, among others, improving brain energy metabolism, preventing calcium overload and sustained activation of EAA receptors, scavenging oxygen free radicals and inhibiting NO growth.

Many clinical practices prove that after cerebral blood flow is blocked due to cerebral artery obstruction or severe stenosis, irreversible damage can be formed on the central part of the cerebral tissue within minutes. Although the peripheral portion of brain tissue can maintain a low required amount of blood flow through collateral circulation, if this unstable blood circulation is not improved within 3 to 4 hours, brain tissue metabolic failure may occur. Therefore, it is important to re-establish blood circulation in time to supply oxygen and nutrients to ischemic brain tissue (Zhu nationality, thrombolytic therapy of cerebral infarction, division of foreign medicine-neurology neurosurgery, 1995, 22, 51-57).

Further brain damage can occur during ischemia reperfusion of brain tissue. Cerebral tissue ischemia reperfusion injury is an important cause of delayed cerebral ischemia injury, and is related to the generation of nerve cell metabolic toxic substances such as oxygen free radicals and NO free radicals. Timely clearance of the neurotoxic substances is an important aspect of the treatment of acute cerebral infarction (FisherM, therapeutic neuro protection for cerebral ischemia, Stroke, 1994, 25, 25, 1074).

Thus, improving blood flow in ischemic brain tissue in time to prevent re-infarction and avoiding further improvement of toxic metabolites in marginal brain tissue become two basic principles for treating ischemic stroke.

The inventor has recognized after combining the above background that the therapeutic principle of ischemic stroke can be more fully embodied if the free radical scavenging, especially NO scavenging, can be combined with thrombolysis, i.e. the free radical scavenging is carried out at the same time of thrombolysis. In view of the above, the inventors combined the drugs having the above two functions, and tried to invent a new drug that can simultaneously realize both functions.

In chinese patent 95106340.5 issued to the inventor of the present invention, which was granted to the university of beijing medical science, it was disclosed that the polypeptide compound of the general formula (I) has the effects of dilating blood vessels and dissolving thrombus.

The inventor carefully researches and analyzes the relationship between the structure of the substituted imidazoline and the NO scavenging effect and finds that the imidazoline substituted phenoxyacetic acid not only has excellent NO scavenging effect, but also can tolerate various reaction conditions in polypeptide synthesis, so that the inventor organically combines the two compounds to design a new compound, thereby providing the invention.

Summary of The Invention

It is an object of the present invention to provide a compound of the general formula (I),

4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazoline-2-yl) phenoxyacetyl-

X-Arg-Pro-Ala-Lys-Y (I)

Wherein, X is a natural amino acid fragment,

y is hydroxy, a group wherein a hydrogen on the hydroxy group is substituted with a metal ion or an ammonium ion, amino, substituted amino, lower alkoxy, lower alkenyloxy, lower alkynyloxy, aryl lower alkoxy, lower aryl lower alkoxy substituted with a substituent, aryl lower alkenyloxy substituted with a substituent, aryl lower alkynyloxy substituted with a substituent selected from the group consisting of halogen, cyano, nitro, carboxy, hydroxy, aldehyde, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl.

Another object of the present invention is to provide a pharmaceutical composition comprising a compound of formula (I), comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier.

The invention also aims to provide the application of the compound shown in the general formula (I) in preparing medicaments for treating cerebral apoplexy, in particular to preparing medicaments for scavenging oxygen, hydroxyl and nitrogen oxide free radicals and dissolving thrombus.

Detailed Description

In the compound of the general formula (1) of the present invention,

4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazoline-2-yl) phenoxyacetyl-

X-Arg-Pro-Ala-Lys-Y (I)

X is a natural amino acid fragment,

"Natural amino acids" in the present invention refer to 20 kinds of amino acids occurring in nature. The amino acid fragment refers to a part of amino acids in which hydrogen at the amino terminal and hydroxyl at the carboxyl terminal are substituted, and the abbreviations of natural amino acids in the present invention all refer to the corresponding amino acid fragments. In the present invention, the natural amino acid is preferably Gly, Gln, Ala.

The terms "alkyl", "alkenyl" and "alkynyl" generally refer to straight or branched chain groups containing from 1 to 20 carbons. The term "lower" is intended to mean having 1 to 8 carbon atoms.

"aryl" refers to a group having 6 to 40 carbon atoms and having an aromatic ring structure, such as a benzene ring and a naphthalene ring. The term "substituent" means halogen, cyano, nitro, carboxy, hydroxy, aldehyde, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl. "substituted aryl" means that a hydrogen on an aromatic ring is replaced by a substituent. In the present invention, the number of substituents on the aromatic ring is preferably 1 to 3.

In the present invention, Y is preferably hydroxy, lower alkoxy, aryl lower alkoxy, lower alkenyloxy, aryl lower alkenyloxy, lower alkynyloxy, aryl lower alkynyloxy, amino substituted by a substituent selected from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl.

The therapeutically effective amount described in the present invention is generally 0.1 to 99.9%, preferably 1 to 90% by weight of the composition of the compound of formula (I). In the composition of the present invention, the compound of the general formula (I) may be a single compound or two or more compounds may be present in the composition. The person skilled in the art can specifically determine the condition of the patient and the disease to be treated. The "carrier" is any of those commonly used in the pharmaceutical arts, such as solid carriers, e.g., corn flour, calcium carbonate, liquid carriers, e.g., ethanol, water, and gaseous carriers, depending on the form in which the composition is to be administered.

In the present invention, imidazoline substituted phenoxyacetic acids follow the route shown below.

In Br₂And NaOH to convert 2-nitropropane to 2, 3-dimethyl-2, 3-dinitrobutane. The latter being Zn/NH₄Reducing in Cl system to generate 2, 3-bis (hydroxyamino) butane. 2, 3-dimethyl-2, 3-bis (hydroxyamino) butane is condensed with p-hydroxybenzaldehyde to produce 4- (4, 4, 5, 5-tetramethyl-1, 3-dihydroxyimidazolin-2-yl) phenol (4). In PbO₂In the presence of 4, 4 is converted to 4- (4, 4, 5, 5-tetramethyl-1, 3-dioxoimidazolin-2-yl) phenol (5). In NaC₂H₅In the presence of BrCH₂COOC₂H₅And carrying out oxyalkylation reaction with 5 to obtain 4- (4, 4, 5, 5-tetramethyl-1, 3-dioxyimidazoline-2-yl) phenoxyacetic acid (7) after saponification of a product. Compound 7 is sufficiently stable to the conditions used in the peptide synthesis reaction for saponification, Boc removal, and side chain protecting group removal (e.g., HCl, NaOH, HF, or trifluoromethanesulfonic acid).

The present invention relates to the preparation of protected polypeptide intermediates X-Arg (tos) -Pro-Ala-Lys (Czl) OBzl (8-10, wherein X is Gly, Gln and Ala, respectively) according to literature methods (Ming ZHao Shiqi Pen St beans on hybrid of fragments from fibrin, J.Prakt Chem, 1999, 341, 668-676), involving coupling 4- (4, 4, 5, 5-tetramethyl-1, 3-dioxyimidazoline-2-yl) phenol acetic acid with polypeptide intermediates 8-10, respectively, and HF deprotection of the resulting products 11-13 to obtain the desired N- (4, 4, 5, 5-tetramethyl-1, 3-dioxyimidazoline-2-yl) phenoxyacetyl modified oligopeptides 14-16.

The above scheme shows the synthesis of N-4- (4, 4, 5, 5-tetramethyl-1, 3-dioxoimidazolin-2-yl) phenoxyacetyl oligopeptides, where X is Gly, Gln, Ala.

To further illustrate the invention, corresponding examples are given below. It should be noted that these examples are merely illustrative of the present invention and are not intended to limit the invention in any way.

Preparation example 1

Preparation of 2, 3-dimethyl-2, 3-dinitrobutane

3.45 g (0.039mol) of 2-nitropropane were added to 6.5ml of a 6mol/l aqueous NaOH solution, stirred in an ice bath for 5 minutes and then treated dropwise with 1ml (0.019mol) of Br₂For 1 hour. 12ml of ethanol was added to the mixture, and the mixture was refluxed in a 90-water bath for 3 hours, and a plate-like precipitate appeared in the reaction mixture. The hot reaction mixture was poured into a 40ml ice bath and after the ice had dissolved, the flaky crystals were collected by suction filtration under reduced pressure. After drying, 2.80g (81%) of the title compound were obtained, mp 110-.

Preparation example 2

Preparation of 2, 3-dimethyl-2, 3-dihydroxyaminobutane

75g (1mmol)2, 3-dimethyl-2, 3-dinitropropane and 1.00g NH₄Cl was suspended in 20ml of 50% aqueous ethanol. The suspension was stirred in an ice bath and 4.00g of zinc dust were added thereto over a period of 3 hours. The reaction mixture was then stirred at room temperature for 3 hours. Filtering the reaction mixture under reduced pressureThe cake was repeatedly washed with 50% ethanol aqueous solution. The washing solution and the filtrate were combined, adjusted to pH 2 with concentrated hydrochloric acid, and concentrated under reduced pressure to a slurry state. The resulting slurry is mixed with a suitable amount of K₂CO₃Mixing to pH 10, and placing in Soxhlet extractor with CHCl₃The extraction was carried out for 6 hours. The extract was concentrated under reduced pressure, and the residue was crystallized from petroleum ether to give 0.48g (48%) of the title compound as white flaky crystals at mp157-159 deg.C

Preparation example 3

Preparation of 3-dihydroxy-2- (4-hydroxyphenyl) -4, 4, 5, 5-tetramethylimidazole

122mg (1mmol) of p-hydroxybenzaldehyde, 148mg (1mmol) of 2, 3-dimethyl-2, 3-bishydroxybutane and 3ml of methanol were stirred at room temperature for 6 hours, TLC showed disappearance of the starting material, and the reaction mixture was suction-filtered under reduced pressure to give 125mg (47%) of the title compound as white crystals, mp198-200 deg.C

Preparation example 4

Preparation of 3-dioxo-2- (4-hydroxyphenyl) -4, 4, 5, 5-tetramethyl-2-imidazoline

To a solution of 176mg (0.5mmol)1, 3-dihydroxy-2- (4-hydroxyphenyl) -4, 4, 5, 5-tetramethylimidazolidine and 5ml methanol was added 200mg PbO₂. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was chromatographed on silica gel (petroleum ether/ethyl acetate, 20: 1). The collected fractions containing the product were concentrated under reduced pressure to yield 50mg (29%) of the title compound as a blue solid. Mp 142 deg.C, EI/MS (M/e) ═ 249[ M [ ]⁺]，217[M-32]⁺。

Preparation example 5

Preparation of 3-dioxo-2- (4-ethoxycarbonylmethoxy) -4, 4, 5, 5-tetramethyl-2-imidazoline

A solution of 250mg (1mmol) of 1, 3-dioxo-2- (4-hydroxyphenyl) -4, 4, 5, 5-tetramethyl-2-imidazoline, 0.32ml of ethyl bromoacetate, 100mg of sodium ethoxide, and 5ml of anhydrous tetrahydrofuran was stirred at 60 for 5 hours, TLC showed disappearance of the starting material spot. The reaction mixture was concentrated under reduced pressure and the residue was washed with silica gelColumn chromatography separation, CHCl₃And (4) eluting. The fractions containing the desired fractions were concentrated to dryness under reduced pressure to give 300mg (90%) of the title compound, mp107-109 ℃, EI/MS (M/e) ═ 336[ M ]]⁺。

Preparation example 6

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenylacetic acid

33mg (0.1mmol) of 1, 3-dioxo-2- (4-ethoxycarbonylmethoxy) -4, 4, 5, 5-tetramethyl-2-imidazoline are dissolved in 3ml of methanol. To the resulting solution was added 7 drops of 2mol/NaOH aqueous solution, followed by stirring at room temperature for 30 minutes, and TLC showed disappearance of the starting material spot. The reaction mixture was concentrated under reduced pressure, and the residue was mixed with 2ml of saturated saline and adjusted to pH 5 with 2mol/l hydrochloric acid. The resulting weakly acidic solution was treated with CHCl₃(3 ml. times.3) extraction.

Preparation example 7

Preparation of Boc-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

38mg (0.2mmol) of Boc-Ala-OH was dissolved in 4ml of anhydrous tetrahydrofuran, and 30mg (0.22mmol) of HOBT and 48mg (0.22mmol) of DCC were added to the solution under ice-bath, followed by stirring for 30 minutes under ice-bath. 170mg (0.20mmol) prepared as described in the literature (Ming Zhao Shiqi Pen Studies on hybrid of fragments from fibrous, J.Prakt Chem, 1999, 341, 668-one 676)

HCl Arg (Tos) -Pro-Ala-Lys (Czl) OBzl was dissolved in 5ml of anhydrous tetrahydrofuran, and N-methylmorpholine was added dropwise to adjust pH9 under ice bath. The two solutions were mixed in an ice bath, stirred at 0 ℃ for 2 hours and at room temperature for 12 hours, and TLC showed the disappearance of the starting material. The reaction mixture was filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was sequentially saturated NaHCO₃Aqueous solution washing, saturated NaCl aqueous solution washing, and 5% KHSO₄And (4) washing with an aqueous solution. Separating ethyl acetate layer, anhydrous Na₂SO₄Drying, filtration and concentration of the filtrate under reduced pressure to dryness gave 169mg (83%) of the title compound, mp 86-89 deg.C,

[α]²⁰ _D＝-13(c＝0.2，CHCl₃)，FAB/MS(m/e)＝1042[M+Na]⁺。

preparation example 8

Preparation of Boc-Gly-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

The same procedure as in preparation 7 was followed, using 35mg (0.2mmol) of Boc-Gly-OH in place of Boc-Ala-OH, to give 179mg (89%) of the title compound, mp 79-81 ℃, [ alpha ]]²⁰ _D＝+13(c＝0.2，CHCl₃)，

FAB/MS(m/e)＝1028[M+Na]⁺。

Preparation example 9

Preparation of Boc-Gln-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

The same procedure as in preparation 7 was carried out, using 49mg (0.2mmol) of Boc-Gln-OH in place of Boc-Ala-OH, to give 187mg (87%) of the title compound, mp, 84-86, [ alpha ]]²⁰ _D＝-9(c＝0.3，CHCl₃)，

FAB/MS(m/e)＝1099[M+Na]+。

Example 1

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Ala-Arg-Pro-Ala-Lys-OH

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

196mg (0.2mmol) Boc-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl was dissolved in 6ml of 4mol/l anhydrous hydrogen chloride/ethyl acetate solution, the resulting solution was stirred at room temperature for 1 hour, and TLC showed disappearance of the starting material spot. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate and concentrated under reduced pressure. The treatment is repeated for 3 times until the free hydrogen chloride is removed. The residue was triturated with anhydrous ether to give 183mg (100%) of HCl. Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl as a white powder.

61mg (0.2mmol) of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetic acid was dissolved in 3ml of anhydrous tetrahydrofuran. To the resulting solution was added 30mg (0.22mmol) of HOBT, 48mg (0.22mmol) of DCC under ice bath, and 183mg (0.2mmol) of the resulting solution was added at 0 deg.C

HCl Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl in solution with 5ml tetrahydrofuran and adjusted pH9 with N-methylmorpholine. After mixing, the reaction mixture was reacted at 0 ℃ for 2 hours,

the reaction was allowed to proceed at room temperature for 12 hours, and TLC showed the disappearance of the starting material. The reaction mixture was filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was dissolved in ethyl acetate. The resulting solution was sequentially saturated NaHCO₃Washing with aqueous solution, washing with saturated NaCl aqueous solution, and washing with 5% KHSO₄And (4) washing with an aqueous solution. The ethyl acetate layer was separated and washed with anhydrous Na₂SO₄And (5) drying. Filtration and concentration of the filtrate to dryness under reduced pressure gave 215mg (92%) of the title compound, mp, 92-94 ℃, [ alpha ]]²⁰ _D＝+24(c＝1，CH₃OH)

FAB/MS(m/e)＝1210[M+H]⁺。

136mg (0.1mmol) of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl, 1ml of anisole and 2ml of anhydrous HF, were reacted at 6 ℃ for 1 hour, HF was removed under reduced pressure, and the residue was solidified with anhydrous ether to give a white solid. It turned blue on standing in anhydrous ether. Desalting the blue solid with sephadex G10, eluting with distilled water, collecting fractions containing blue band, and freeze drying to obtain 70mg (84%) of the title compound as blue lyophilizate mp, 170 deg.C (decomposition), [ alpha ], []²⁰ _D＝+35(c＝1，H₂O)，ESI/MS(m/e)＝831[M]⁺。

Example 2

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gly-Arg-Pro-Ala-LysOH

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gly-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

The procedure is as in example 1, replacing Boc-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl with 186mg of Boc-Gly-Arg (tos) -Pro-Ala-Lys (Czl) OBzl. 210mg (90%) of the title compound are obtained, mp, 90-92 ℃, [ alpha ]]²⁰ _D＝+13(c＝2，CHCl₃)，FAB/MS(m/e)＝1196[M+H]⁺. Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gly-Arg-Pro-Ala-LysOH

The procedure is as in example 1, using 130mg of 4, 5, 4- (1, 3-dioxo-4, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gly-Arg (tos) -Pro-Ala-Lys (Czl) OBzl instead of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-

Ala-Arg (tos) -Pro-Ala-Lys (Czl) Obzl to yield 60mg (75%) of the title compound, mp, 174 ℃ (cleaved), [ alpha ], (75%),]²⁰ _D＝+21(c＝1，H₂O)，ESI/MS(m/e)＝817[M]⁺。

example 3

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gln-Arg-Pro-Ala-Lys-OH

Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gln-Arg (tos) -Pro-Ala-Lys (Czl) OBzl

The procedure of example 1 was followed, using 220mg of Boc-Gln-Arg (tos) -Pro-Ala-Lys (Czl) OBzl instead of Boc-Ala-Arg (tos) -Pro-Ala-Lys (Czl) OBzl. 220mg (90%) of the title compound are obtained, mp, 96-98 ℃, [ alpha ]]²⁰ _D＝+29(c＝2，CHCl₃)，FAB/MS(m/e)＝1267[M+H]⁺. Preparation of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gln-Arg-Pro-Ala-Lys-OH

The procedure is as in example 1, using 136mg of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Gln-Arg (tos) -Pro-Ala-Lys (Czl) OBzl instead of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-Ala-Arg (tos) -Pro-Ala-Lys (Czl) Obzl, 70mg (80%) of the title compound, mp, 178 (decomposition), [ alpha ], (ii) of]²⁰ _D＝-27(c＝1，H₂O)，ESI/MS(m/e)＝889[M+2H]⁺。

Example 4

Pharmaceutical compositions containing the compounds of the invention

Example 1 Compound 0.1 g

Ethanol 100ml

Example 5

Pharmaceutical compositions containing the compounds of the invention

EXAMPLE 2 Compound 1 g

Physiological saline 200ml

Test example 1

Determination of NO scavenger Activity of Compounds of the invention

Male Wistar rats (fasting for 12 hours before operation and free drinking water) with the weight of 250-. The vessels were cut into 3-5mm long arterial rings after stripping of the attached connective tissue. The arterial loop was placed in a 15ml perfusion bath containing 15ml Krebs Henseleit (KH) solution, thermostatted at 37 deg.C, and aerated with a mixture of 95% O2 and 5% CO 2. The hook that secures the aortic annulus is connected to the tension transducer. The aortic diastolic curve was plotted on a two-pass recorder at a paper speed of 1 mm/min. The aortic strip was pre-stressed by adjusting the tension to 1.0g and adding norepinephrine to the bath after 30 minutes of equilibration to a final concentration of 10-9 mol/l. Cleaning noradrenaline, balancing for 30 minutes, adding noradrenaline into a bath tank to ensure that the terminal concentration is 10-9mol/l, adding 15ml of ethanol (used as a blank control) or the compound of the invention with different concentrations (10-4mol/l, 10-5mol/l and 10-6mol/l) after the aortic bar tension is stabilized at a platform level, and adding 1.5ul of acetylcholine (the terminal concentration is 10-6mol/l) after the two instruments are stabilized. The NO scavenging activity of the compounds of the present invention is expressed as a percentage of inhibition of acetylcholine relaxation of the aortic strip and the results are shown in Table 1. The compounds of the invention all show definite NO scavenging activity, which indicates that the NO scavenging activity is maintained after 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazoline-2-yl) phenoxyacetic acid is introduced into the N end of the thrombolytic oligopeptide.

TABLE 1 inhibition of acetylcholine-induced vasodilation of vascular strips by compounds of the invention

Compound (I)	Diastolic inhibition rate%
Compound (I)	Diastolic inhibition rate%	7	99.2±1.4
20	88.9±1.4	7	99.2±1.4
20	88.9±1.4	21	91.2±0.5
22	88.9±5.3	21	91.2±0.5

N is 6, the final concentration is 100umol/l

Test example 2

Determination of thrombolytic Activity of Compounds of the invention

Male Wistar rats (250-300g) were anesthetized by intraperitoneal injection of sodium pentobarbital and the right common carotid artery and the left external jugular vein were isolated. 0.5ml of blood is taken from the right common carotid artery to prepare thrombus, and a fixing bolt is used as a thrombus bracket. After 15 minutes the thrombus fixed on the bolt was removed and weighed and placed into a bypass cannula constructed from polyethylene tubing. The tube was filled with a heparin saline solution from the tip of the tube with a syringe. One end of the tube was then inserted into the left external jugular vein and the syringe was used to push the heparin in saline. The other end of the tube was inserted into the right carotid artery, the proximal end of the left external jugular vein on the thrombus principle was maintained, and physiological saline (blank control), a physiological saline solution of urokinase (positive control), and a physiological saline solution of the compound of the present invention were inserted from the middle section of the polyethylene tube into the proximal vein away from the thrombus-fixing bolt with a scalp needle, and the contents were slowly injected into the blood for 6 minutes. And timing after the injection, taking out the thrombus fixing bolt after 1 hour and weighing. The amount of thrombolysis was determined by the weight of each sample and the results are shown in Table 3. The data in table 2 show that only QRPAK retains thrombolytic activity after introduction of 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl group into the N-terminus of the 5-peptide thrombolytic oligopeptide, and that a dose-effect dependence is shown between the three doses.

TABLE 2 thrombolytic effect of Compounds of the invention

Compound (I)	Dosage form	Loss of weight of thrombus (mg)
Compound (I)	Dosage form	Loss of weight of thrombus (mg)	NS	3ml/g	15.1±3.7
UK	20000IU/kg	25.4±2.5^***	NS	3ml/g	15.1±3.7
UK	20000IU/kg	25.4±2.5^***	7	10umol/kg	15.9±2.5
20	10umol/kg	19.9±7.9	7	10umol/kg	15.9±2.5
20	10umol/kg	19.9±7.9	21	10umol/kg	15.9±4.4
22	10umol/kg	28.8±3.8^***，△	21	10umol/kg	15.9±4.4
22	10umol/kg	28.8±3.8^***，△	22	5umol/kg	20.4±0.8^**
22	1umol/kg	16.4±3.4	22	5umol/kg	20.4±0.8^**

N-10, in ratio to NS,^**P＜0.01，^***p is less than 0.001, the ratio of delta to UK is less than 0.01

Claims

1. A compound of the general formula (I),

4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl

-X-Arg-Pro-Ala-Lys-Y (I)

Wherein

X is a natural amino acid fragment,

y is hydroxy, a group wherein the hydrogen of the hydroxy group is substituted with a metal ion or an ammonium ion, amino, substituted amino, lower alkoxy, lower alkenyloxy, lower alkynyloxy, aryl lower alkoxy, lower aryl lower alkoxy substituted with a substituent, aryl lower alkenyloxy substituted with a substituent, aryl lower alkynyloxy substituted with a substituent,

the substituents include halogen, cyano, nitro, carboxyl, hydroxyl, aldehyde, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl.

2. The compound according to claim 1, wherein X is selected from Gly, Gln, Ala.

3. Compounds according to claim 1 or 2, wherein Y is hydroxy, lower alkoxy, aryl lower alkoxy, lower alkenyloxy, aryl lower alkenyloxy, lower alkynyloxy, aryl lower alkynyloxy, amino substituted with a substituent selected from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl.

4. A compound according to claim 3 wherein Y is hydroxy, amino.

5. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), 4- (1, 3-dioxo-4, 4, 5, 5-tetramethyl-2-imidazolin-2-yl) phenoxyacetyl-

X-Arg-Pro-Ala-Lys-Y (I)

Wherein,

x is a natural amino acid fragment,

the substituents include halogen, cyano, nitro, carboxyl, hydroxyl, aldehyde, lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl,

and a pharmaceutically acceptable carrier.

6. The compound according to claim 5, wherein X is selected from Gly, Gln, Ala.

7. The compound according to claim 5 or 6, wherein Y is hydroxy, lower alkoxy, aryl lower alkoxy, lower alkenyloxy, aryl lower alkenyloxy, lower alkynyloxy, aryl lower alkynyloxy, amino substituted with a substituent selected from the group consisting of lower alkyl, lower alkenyl, lower alkynyl, lower alkoxy, lower alkenyloxy, lower alkynyloxy, lower alkylcarbonyl, lower alkenylcarbonyl, lower alkynylcarbonyl, lower alkoxycarbonyl, lower alkenyloxycarbonyl, lower alkynyloxycarbonyl.

8. The compound according to claim 7, wherein Y is hydroxy, amino.

9. Use of a compound according to claims 1-4 for the preparation of a medicament for scavenging oxygen, hydroxyl and nitric oxide radicals and for thrombolysis.