CN102552235B - Application of eonurine-cysteine conjugate in preparing medicine used for treating ischaemic cardiomyopathy - Google Patents
Application of eonurine-cysteine conjugate in preparing medicine used for treating ischaemic cardiomyopathy Download PDFInfo
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Abstract
The invention belongs to the field of pharmacy, and relates to an application of eonurine-cysteine conjugate in preparing medicines used for treating ischaemic cardiomyopathy, and especially medicines used for treating hypoxia-ischemic myocardial damages. According to the invention, in-vitro cardiac muscle cell cultivation experiments show that, the eonurine-cysteine conjugate assists in improving cell survival rate, regulating H2S content, reducing cardiac muscle cell LDH leakage rate, improving SOD and CAT activities, reducing lipid peroxidation (MDA) and ROS levels, and reducing cardiac muscle cell apoptosis. The provided eonurine-cysteine conjugate can be adopted as a treatment medicine, and can be applied for treating myocardial damages, and especially hypoxia-ischemic myocardial damages.
Description
Technical field
The invention belongs to pharmaceutical field, relate to the purposes of leonurine-cysteine conjugate in preparation treatment target-organ protection medicine, especially the purposes in preparation treatment ischemia myocardial damage medicine.
Background technology
Ischemic cardiomyopathy is cardiac muscle ischemia sharply, temporary transient and the hypoxia syndrome causing due to coronary insufficiency, and its typical clinical manifestation is squeeze type pain radiating to left upper extremity after paroxysmal breastbone.Its main pathophysiological basis is the imbalance of supply and demand of cardiac muscular tissue's oxygen.The medicine that is used for the treatment of clinically ischemic cardiomyopathy mainly contains nitrate esters, beta-adrenoceptor blocking drug and calcium channel blocker etc.Three class medicines all have certain therapeutical effect to ischemic cardiomyopathy.Noval chemical compound leonurine-cysteine conjugate of the present invention is brand-new compound, the adjusting H of its uniqueness
2the effect of S content, clinical medicine used does not possess at present.
Summary of the invention
The object of this invention is to provide the purposes of leonurine-cysteine conjugate in preparation treatment myocardial damage medicine.Leonurine-cysteine conjugate is the brand-new compound that leonurine and cysteine analogs are connected to form by ester bond.So far there are no about leonurine-cysteine conjugate and the report to hypoxic-ischemic injury of myocardium cytoprotection thereof.
The structural formula of leonurine-cysteine conjugate of the present invention:
The preparation of noval chemical compound, taking syringic acid as initiation material, is passed through carbonylation successively, the reactions such as the hydrolysis of esterification and ester, the synthetic conjugate that obtains leonurine and cysteine analogs.
specific embodiment is as follows
1. 3; the synthetic route of 5-dimethoxy-4 '-(2-amino-3-(2-alkynes-rosickyite alkane)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester (1a) and 3,5-dimethoxy-4 '-(2-amino-3-(2-alkene-rosickyite alkane)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester (1b)
Syringic acid is carbonylation first; by its hydroxyl protection; then with N; N '-tertbutyloxycarbonyl-N ' '-(4-hydroxyl-butyryl)-guanidine reaction preparation 3; 5-dimethoxy-4 '-acetyl group-benzoic acid 4-(N; N '-tertbutyloxycarbonyl-guanidine radicals)-butyryl ester, the latter after deacetylation with 2-tertiary butyloxycarbonyl amide groups-3-
s-propargylic acid (or 2-tertiary butyloxycarbonyl amide groups-3-
s-alkene-propanoic acid) condensation obtains intermediate 3; 5-dimethoxy-4 '-(2-tertiary butyloxycarbonyl amide groups-3-(2-alkynes-rosickyite alkane)-propiono)-benzoic acid 4-(N; N '-tertbutyloxycarbonyl-guanidine radicals)-butyryl ester (or 3; 5-dimethoxy-4 '-(2-tertiary butyloxycarbonyl amide groups-3-(2-alkene-rosickyite alkane)-propiono)-benzoic acid 4-(N; N '-tertbutyloxycarbonyl-guanidine radicals)-butyryl ester); (3,5-dimethoxy-4 '-(2-amino-3-(2-alkynes-rosickyite alkane)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester will be obtained after the latter's Deprotection
1a) (or 3,5-dimethoxy-4 '-(2-amino-3-(2-alkene-rosickyite alkane)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester (
1b).
the synthetic route of dimethoxy-4 '-(3-(2-carboxyl-ethylene dithiol base)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester (2)
Syringic acid is carbonylation first, its hydroxyl is protected, then with N, N '-tertbutyloxycarbonyl-N ' '-(4-hydroxyl-butyryl)-guanidine reaction preparation 3, 5-dimethoxy-4 '-acetyl group-benzoic acid 4-(N, N '-tertbutyloxycarbonyl-guanidine radicals)-butyryl ester, the latter is chloroformyl with 3-(2-after deacetylation-second dimercapto) and-propionyl chloride condensation obtains intermediate 3, 5-dimethoxy-4 '-(3-(2-carboxyl-ethylene dithiol base)-propiono)-benzoic acid 4-(N, N '-tertbutyloxycarbonyl-guanidine radicals)-butyryl ester, the latter's Deprotection obtains 3, 5-dimethoxy-4 '-(3-(2-carboxyl-ethylene dithiol base)-propiono)-benzoic acid 4-guanidine radicals-butyryl ester (
2).
This method separation process is few, easy and simple to handle, reaction temperature and, be easy to control and implement, product yield is high, purity is high.This preparation method is applied for a patent, patent name: leonurine derivative and preparation method thereof application number: 201010100153.5.
The present invention, by external myocardial cell culture experiment, shows that leonurine-cysteine conjugate has raising cell survival rate; Reduce the rate that spills of anoxia-induced apoptosis myocardial cell LDH; Regulate H
2the content of S; Improve the activity of SOD and CAT; Suppress the generation of lipid peroxide (MDA) and ROS; Reduce the effect of apoptosis of cardiac muscle, show that leonurine-cysteine conjugate can be used as medicine and is applied to treatment ischemic cardiomyopathy.
Brief description of the drawings
Figure 1A is the impact of the survival rate of leonurine-cysteine conjugate on Anoxia 5 h injury of myocardium cells.Figure 1B is leonurine-cysteine conjugate spills rate impact on Anoxia 5 h injury of myocardium cell LDH.Fig. 1 C is the H of leonurine-cysteine conjugate to Anoxia 5h injury of myocardium cell
2the impact of S burst size.Anoxia 5h after the pre-administration 12h of leonurine-cysteine conjugate.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *: the each concentration processed group of leonurine-cysteine conjugate compared with model group,
p<0.05; $: with
1arelatively,
p<0.05.
Fig. 2 is that fluorescein(e) dye Hoechst 33258 measures apoptosis of cardiac muscle experiment.Anoxia 5h after leonurine-cysteine conjugate and administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 3 is
1ato cell survival rate, LDH spills rate, to H with its precursor compound
2the comparison of the impact of S content.Each administration group is Anoxia 5h after pre-administration 12h all.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *: each processed group compared with model group,
p<0.05, $: each administration group with
1acompare,
p<0.05.
Fig. 4 is
1aon the impact of the total SOD of Anoxia 5h injury of myocardium cell and MDA content.
1aanoxia 5h after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 5 is
1aon the impact of Anoxia 5h injury of myocardium cell CAT and ROS content.
1aanoxia 5h after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 6 is
1aon the impact of Anoxia 5 h injury of myocardium cell bcl-2, bax, gene expression of caspase-3.
1aanoxia 5h again after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 7 is
1aon the impact of Anoxia 5 h injury of myocardium apoptosis gene Bcl-2, Bax, Caspase-3 protein expression.
1aanoxia 5h again after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 8 is
1aon the apoptotic impact of Anoxia 5 h injury of myocardium.
1aanoxia 5h after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Fig. 9 is
1aon the impact of Anoxia 5 h injury of myocardium cell ultrastructures.
1aanoxia 5h after pre-administration 12h.Wherein, Control: Normal group (C); Model: model group (M); #: model group compared with Normal group,
p<0.05; *:
1aprocessed group compared with model group,
p<0.05.
Detailed description of the invention
Former culture 1-3 age in days SD neonatal rat, cores under aseptic condition and dirtyly in PBS, cleans, and shreds, and moves in the conical flask that contains 0.125% pancreatin 37 DEG C of digestion 10 minutes, continues to digest altogether 8 times under magnetic agitation.After each digestion, collect supernatant, serum is ended digestion, centrifugal 5 minutes of 2000rpm, and it is 10 that collecting cell precipitation is adjusted cell density
6/ ml, cultivates containing in the DMEM of 10% calf serum, and the 3rd day for experiment.Be divided into Normal group (not awarding pharmaceutical intervention, the high sugar of normal oxygen), model group (do not award pharmaceutical intervention, Anoxia 6 h), leonurine-cysteine conjugate group (pre-medicine 10 of giving respectively
-5mol/L, 10
-6mol/L, 10
-7mol/L, 10
-8mol/L, 10
-9after mol/L 12h, carry out Anoxia 5h).Mtt assay detects cell survival rate, and as shown in Figure 1A, model group survival rate is starkly lower than Normal group to result, and leonurine-cysteine conjugate group is 10
-7mol/L can obviously improve cell survival rate (
p<0.05).
Every group of (n=6) each sample gets 10
6individual cell, fully cracking, acetone acid system detects the content of LDH in cell, and in Normal group cell, LDH is 100%, and in each medication group and model group cell, the difference of LDH and Normal group is that LDH spills rate.As shown in Figure 1B, the LDH of each medication group spills rate all obviously to be reduced compared with model group result.
Use colorimetric determination H
2the content of S.As shown in Figure 1 C, model group compared with normal matched group is compared H
2s concentration obviously decline (
p<0.05), and
1awith
1ball can obviously improve H
2s concentration (
p<0.05).
embodiment 4. 1a and its precursor compound are to cell survival rate, and LDH spills rate, H
2
the impact of S content and the comparison of morphological change
Specific implementation method is measured apoptosis of cardiac muscle experiment with embodiment 1-3. fluorescein(e) dye Hoechst 33258: each processed group cell 4% paraformaldehyde room temperature is fixed 20 ~ 30 min, washes 3 times with PBS.Add fluorescein(e) dye Hoechst 33258(20 μ g/ml), 37 ° of C are hatched 20 min, and PBS washes three times, each 10 min.10% buffering glycerol mounting, fluorescence microscopy Microscopic observation, shooting.As shown in Figure 2, each medication group all can significantly reduce the apoptosis of myocardial cell to result.Based on the above results, as shown in Figures 2 and 3,
1adosage be respectively leonurine 1/10 and SPRC 1/100, show noval chemical compound
1abe better than its precursor compound, and the mechanism of action of 1a is furtherd investigate.
embodiment 5.1a improves SOD activity experiment and suppresses lipid peroxidation (MDA) experiment
Hydroxylamine assay detects SOD, every milligram of histone when in 1ml reactant liquor, SOD suppression ratio reaches 50% corresponding SOD amount be a SOD unit of activity (U).Result shows total SOD as shown in Figure 4 A,
1amedication group all can obviously improve the vigor of total SOD, compared with model group significantly raise (
p<0.05).Oxygen-derived free radicals is attacked the polyunsaturated fatty acid in biomembrane, causes lipid peroxidation, and therefore produces malondialdehyde (MDA).MDA can with thiobarbituricacidα-(TBA) condensation, form red product, have maximum absorption band at 532 nm places.As shown in Figure 4 B, model group MDA content significantly increase (
p<0.05 is compared with Normal group).
1aprocessed group obviously reduces compared with model group, confirms
1acan suppress the lipid peroxidation of active oxygen induction.
embodiment 6.1a improves CAT activity experiment and suppresses ROS experiment
At hydrogen peroxide (H
2o
2) in relatively sufficient situation, catalase can catalysis H
2o
2produce H
2o and O
2.Remaining H
2o
2under the catalysis of peroxidase, can be oxidized chromogenic substrate, produce red product (N-(4-antipyryl)-3-chloro-5-sulfonate-pbenzoquinonemonoimine), maximum absorption wavelength is 520 nm.Use H
2o
2standard substance, production standard curve, so just can calculate CAT in sample in unit interval unit volume catalysis the H of how many amounts
2o
2change H into
2o and O
2thereby, can calculate the enzyme activity of CAT in sample.1 enzyme activity unit (1 U) is at 25 DEG C, under the condition of pH7.0, and can catalytic decomposition 1 μ mol H in 1 min
2o
2.Structure as shown in Figure 5A,
1amedication group all can obviously improve the vigor of total CAT, compared with model group significantly raise (
p<0.05).Dihydroethidium this as blue-fluorescence, after dehydrogenation and RNA or DNA in conjunction with producing red fluorescence.In the time that intracellular superoxide anion level is higher, redness should be stronger, otherwise.Result as shown in Fig. 5 B and C, model group ROS content significantly increase (
p<0.05 is compared with Normal group).
1aprocessed group obviously reduces compared with model group, confirms
1acan reduce the generation of active oxygen.
embodiment 7.1a can improve bcl-2 anti-apoptotic genes expression and reduce bax and the expression of the short apoptogene of caspase3
1acan improve the expression of apoptosis-related genes bcl-2, (*
p<0.05, * *
p<0.01 is compared with Normal group) expression of short apoptogene bax, caspase3 of reduction simultaneously (
# p<0.05,
## p<0.01 is compared with model group).As shown in Figure 6.
embodiment 8.1a can improve Bcl-2 and reduce Bax, the expression of Caspase-3 protein level
Model group Bcl-2 protein expression level significantly reduces, Bax protein level significantly increase (
p<0.05 is compared with Normal group).,
1aprocessed group can obviously increase Bcl-2 protein expression level compared with model group and reduce expression and cleaved-caspase-3/pro-caspase-3 albumen ratio of Bax protein level.
1areduced Apoptosis-Related Factors Bax at protein level, improve anti-apoptosis because of and sub-Bcl-2(Fig. 7).
the experiment of embodiment 9.1a protecting myocardial cell ultrastructure
collect each group of myocardial cell, the centrifugal 15min of 1500 rpm, abandons supernatant, more than 4 ° of fixing 2h of C pre-cooling 2.5% glutaraldehyde; At PBS rinsing 3 times for 4 ° of C, each 10 min; Starve acid at 4 ° of C with 2% and fix 2 h, then use PBS rinsing 3 times; With 50%, 70%, 90%, 100% acetone gradient dehydration l time, each 10 min; Embedding in bulk is also made into ultrathin section, after plumbi nitras double staining, at Philip transmission electron microscope observation.Result as shown in FIG. 8 and 9.
1aprocessed group, apoptosis obviously reduces, wherein mitochondrial crista fracture or the minimizing of collapsing, chromatin margination is not obvious, and myofilament form is substantially complete.
Claims (3)
1. the purposes of the conjugate of leonurine-cysteine analogs in preparation treatment ischemic myocardial medicine, the conjugate of described leonurine-cysteine analogs is selected from 1a, and 1b or 2 has respectively following molecular formula:
2. by purposes claimed in claim 1, it is characterized in that the conjugate of described leonurine-cysteine analogs is by improving cell survival rate, regulate the content of the content of H2S, the rate that spills that reduces myocardial cell LDH, raising SOD, CAT activity, reduction malonaldehyde, ROS, reduce apoptosis of cardiac muscle effect treatment myocardial damage cardiomyopathy.
3. by purposes claimed in claim 2, it is characterized in that described myocardial damage cardiomyopathy is ischemic cardiomyopathy.
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| CN106146573A (en) * | 2015-04-16 | 2016-11-23 | 复旦大学 | Leonurine metabolite and preparation method thereof |
| WO2021010344A1 (en) * | 2019-07-12 | 2021-01-21 | 国立大学法人九州大学 | Development of therapy for improving myocardial contraction and method for inhibiting cardiomyocyte death |
| CN112552211B (en) * | 2020-12-30 | 2023-04-14 | 青岛海合生物科技有限公司 | Leonurine derivative and application thereof in preparation of medicines for preventing or treating ischemic cerebrovascular diseases |
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Effective date of registration: 20170125 Address after: 519020 Guangdong city of Zhuhai province Hengqin Hengqin town hongqicun Baoxing Road No. 119, room 103 Patentee after: Zhuhai Hengqin in CHINT medical management company Address before: 200433 Handan Road, Shanghai, No. 220, No. Patentee before: Fudan University |