CN108815148A - Niclosamidum and its structural modification object Cardioprotective, anti-pulmonary hypertension and it is antitumor in application - Google Patents
Niclosamidum and its structural modification object Cardioprotective, anti-pulmonary hypertension and it is antitumor in application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
Abstract
The invention discloses niclosamidum and its structural modification object Cardioprotective, anti-pulmonary hypertension and it is antitumor in application.The structural modification object is to obtain niclosamidum using the amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl to be grafted 2- hydroxypropyhnethacrylamide copolymer (N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer-Niclosamide conjugates, HPMA-Nic).The present invention increases cardiac muscle cell ATP by research discovery niclosamidum (Niclosamide) and generates, fight the myocardial cell injury of Doxorubicin induction, inhibit fibroblastic proliferation and collagen secretion, inhibits Proliferation of Pulmonary Artery Smooth Muscle Cells and migration.HPMA-Nic significantly improves the water solubility of niclosamidum, and the mouse cardiac muscle that intraperitoneal injection can inhibit pressure load induction is plump, inhibits nude mice tumor growth in vivo.It is proposed of the invention is myocardial preservation, anti-cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and antineoplaston provide new effective technological means, is had broad application prospects.
Description
Technical field
The present invention relates to niclosamidums (niclosamide) in myocardial preservation, anti-cardiac fibrosis, resisting cardiac hypertrophy, resists
Heart failure, anti-pulmonary hypertension and resist application in other fibrosis diseases；Further relate to N- (2- hydroxypropyl) Methacrylamide
It modifies the niclosamidum that niclosamidum obtains and is grafted 2- hydroxypropyhnethacrylamide copolymer (N- (2-hydroxypropyl)
Methacrylamide (HPMA) copolymer-Niclosamide conjugates, HPMA-Nic) and its myocardial preservation,
Anti- cardiac fibrosis, anti-heart failure, anti-pulmonary hypertension, resists other fibrosis diseases and anti-tumor aspect at resisting cardiac hypertrophy
Application.The invention belongs to biomedicine fields.
Background technique
Heart failure abbreviation heart failure refers to that obstacle occurs for contractile function and (or) diastolic function due to heart, cannot incite somebody to action
Heart is sufficiently discharged in venous return, leads to venous system sludging, arterial system hemoperfusion is insufficient, so as to cause the heart
Dirty dyshaemia and life threatening.A variety of cardiovascular diseases can lead to heart failure, and process is the diseases such as myocardial infarction, hypertension
The pathologic that inducing heart occurs characterized by cardiac muscle cell loses with cardiac fibrosis is plump, and plumpness progresses to the decompensation stage
Enter heart failure.Energostim object mainly has (one) renin-angiotensin system depressant at present；(2) aldosterone antagonist
Medicine；(3) diuretics (four) beta receptor blocking agent；(5) cardiac glycosides；(6) other classes, beta receptor excitomotor and phosphodiesterase
Non- glycoside inotropic agent such as depressant etc..With the aggravation of aging of population, heart failure is one increasingly serious public
Hygienic issues need new types of therapeutic agents.Heart failure treatment should inhibit the heart to protect cell, enhancing cardiac muscle cell's mitochondrial function
Dirty fiber turns to basic strategy, and with the novel anti-heart failure medicine of this strategy Development.
Pulmonary hypertension refers to the raised pathological state of pulmonary artery pressure, can lead to right heart failure.Pulmonary hypertension is a kind of
Common disease, frequently-occurring disease, and disability rate and case fatality rate are high.Pulmonary hypertension mainly comes due to arteria pulmonalis smooth muscle cells hyper-proliferative
And migration, cause artery wall thickening, pulmonary artery chamber reduces and causes pulmonary hypertension.The therapeutic agent of pulmonary hypertension is urgently opened
Hair.
Tumour is the serious disease of harm, and new antitumoral medicine is also urgently developed.
Summary of the invention
An object of the present invention is to provide niclosamidum (niclosamide) in myocardial preservation, anti-cardiac fibrosis, anti-
Application in myocardial hypertrophy, anti-heart failure and anti-pulmonary hypertension.The structural formula of niclosamidum is shown in formula I：
The second object of the present invention is to provide a kind of structural modification drug and preparation method thereof based on niclosamidum,
The drug is remarkably improved the water solubility of niclosamidum, can internal injection application；
The third object of the present invention be to provide the structural modification drug based on niclosamidum myocardial preservation,
The application of anti-cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and anti-tumor aspect.
Present invention employs following technological means in order to reach the goals above：
The present invention handles the Neonatal Rat Primary Cardiomyocytes of culture by using niclosamidum (Niclosamide), finds it
The protection signal STAT3 for activating cardiac muscle cell, moreover, niclosamidum can fight myocardial cell injury caused by Doxorubicin.
Further study show that the ATP that niclosamidum can increase Neonatal Rat Primary Cardiomyocytes is generated；Niclosamidum can inhibit
The proliferation of cardiac fibroblast and the secretion of collagen.
Further study show that niclosamidum inhibits the proliferation and migration of human pulmonary artery smooth muscle cells.
Further, we are prepared for solubility, and the amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl obtains
Niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer (N- (2-hydroxypropyl) methacrylamide
(HPMA) copolymer-Niclosamide conjugates) (Formula II and formula III), carry out internal injection administration.Chlorine nitre willow
Amine is water-insoluble drug, it is difficult to internal systemic medication.The niclosamidum that we prepare is grafted 2- hydroxypropyl methyl acryloyl
Amine copolymer object significantly improves the water solubility of niclosamidum, can have significant advantage completely with drug administration by injection.
Further, we establish the pressure load type myocardial hypertrophy model of mouse aorta bow ligation induction, intraperitoneal injection
Niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer, it is found that it significantly inhibits the myocardial hypertrophy of pressure load induction.
Further, we establish lotus knurl in nude mouse (CT26 cell) model, and discovery intraperitoneal injection niclosamidum is grafted 2-
Hydroxypropyhnethacrylamide copolymer, which has nude mice tumor growth in vivo, significantly inhibits effect.
Therefore on the basis of the studies above, the invention proposes the following contents：
Firstly, the invention proposes niclosamidums (niclosamide) to have myocardial preservation, anti-cardiac fibers in preparation
Change, resisting cardiac hypertrophy, anti-heart failure or function of anti-pulmonary hypertension drug in application, wherein the knot of the niclosamidum
Structure formula is shown in formula I：
Secondly, the structural modification drug that the invention also provides a kind of based on niclosamidum, described with chlorine nitre willow
Structural modification drug based on amine is that the niclosamidum that the amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl obtains connects
Branch 2- hydroxypropyhnethacrylamide copolymer (N- (2-hydroxypropyl) methacrylamide
(HPMA) copolymer-Niclosamide conjugates, HPMA-Nic), structural formula such as Formula II or formula
Shown in III：
Wherein, m 30-2000, n 1-200.
Again, the present invention, which changes, proposes a kind of side for preparing the structural modification drug based on niclosamidum
Method includes the following steps：
(1) preparation of HPMA (N- (2- hydroxypropyl) Methacrylamide)：Add into the organic solvent containing isopropanolamine
Entering methacrylic chloride, 1-4h is stirred under the conditions of -20 DEG C -40 DEG C, stands 10h-24h, complete precipitation to be crystallized is filtered, washing,
Up to HPMA monomer；
(2) preparation of the niclosamidum monomer with carbon-carbon double bond：Chlorine is added in methacrylic chloride or acryloyl chloride
1-24h is stirred at room temperature under the conditions of existing for the basic catalyst in the organic solution of nitre willow amine, filters, distillation under pressure, deionization
Water washing crude product is recrystallized to give double bond containing niclosamidum monomer；
(3) preparation of niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymerization (HPMA-Nic)：By step (1) and
(2) monomer in is dissolved in organic solvent, and the catalyst of 0.1w/w%-5w/w% is added, degassing, and 40 DEG C -80 DEG C, isothermal reaction
6-48h is precipitated in organic solvent, and is washed 2-5 times repeatedly.
Wherein, it is preferred that the organic solvent in step (1) is methylene chloride, acetone, acetonitrile or DMF (N, N dimethyl first
Amide) one of or more than one organic solvent in certain ratio mixture.
Wherein, it is preferred that the crystallization temperature in step (1) is 0 DEG C or less.
Wherein, it is preferred that organic solution is tetrahydrofuran, DMF (n,N dimethylformamide), dichloro in the step (2)
The mixture of one of methane, hexamethylene or cyclohexanone or more than one organic solvent in certain ratio.
Wherein, it is preferred that basic catalyst described in step (2) includes natrium carbonicum calcinatum, Anhydrous potassium carbonate etc..
Wherein, it is preferred that the temperature recrystallized in step (2) is 0 DEG C or less.
Wherein, it is preferred that stating organic solvent in step (3) is petroleum ether, n-hexane, acetone, acetonitrile, tetrahydrofuran, uncle
The mixture of one of butyl ether or ether or more than one organic solvent in certain ratio；It is added in step (3)
The mass ratio of the monomer of step (1) and (2) is 0.5-3:7-10.
Wherein, it is preferred that catalyst described in step (3) is radical polymerization initiator, including azo-initiator
And peroxide initiator, the azo-initiator include that azodiisobutyronitrile, azobisisoheptonitrile and azo two are different
Butyric acid dimethyl ester initiator；The peroxide initiator includes hydrogen peroxide, ammonium persulfate, potassium peroxydisulfate, benzoyl peroxide
Formyl, benzoyl peroxide and methyl ethyl ketone peroxide.
Finally, the structural modification drug that the invention also provides described based on niclosamidum has cardiac muscle in preparation
Application in protection, anti-cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and anti-tumor drug.
Compared to the prior art, the beneficial effects of the invention are as follows：
1, the present invention provides a kind of novel anti-cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and
Resist other fibrosis diseases, anti-tumor drug, be cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and
Other fibrosis diseases, antitumor treatment are resisted to provide new effective technology means.
2, the present invention obtains niclosamidum by using the amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl and connects
Branch 2- hydroxypropyhnethacrylamide copolymer (N- (2-hydroxypropyl) methacrylamide (HPMA)
Copolymer-Niclosamide conjugates) (Formula II and formula III), the water solubility of niclosamidum is significantly improved, it is complete
There can be significant advantage compared to niclosamidum with drug administration by injection entirely.
Detailed description of the invention
Fig. 1 is the protection signal STAT3 that niclosamidum (Niclosamide) activates cardiac muscle cell；
Fig. 2 is that niclosamidum (Niclosamide) fights myocardial cell injury caused by Doxorubicin；
Fig. 3 is the ATP generation that niclosamidum (Niclosamide) increases Neonatal Rat Primary Cardiomyocytes；
Fig. 4 is that niclosamidum can inhibit the proliferation of cardiac fibroblast and the secretion of collagen；
Fig. 5 is human pulmonary artery smooth muscle cells (HPASMC) proliferation that niclosamidum inhibits PDGF-BB induction；
Fig. 6 is human pulmonary artery smooth muscle cells (HPASMC) migration that niclosamidum inhibits PDGF-BB induction；
Fig. 7 is that niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer infrared spectroscopy；
Fig. 8 is that niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer nuclear-magnetism spectrum；
Fig. 9 is that niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer raising niclosamidum water solubility photo knot
Fruit；
Figure 10 is that intraperitoneal injection niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer significantly inhibits pressure load
The myocardial hypertrophy of induction；
Figure 11 is that intraperitoneal injection niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer inhibition nude mice in-vivo tumour
Growth.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
1 niclosamidum of embodiment (Niclosamide) activates the protection signal STAT3 and confrontation Doxorubicin of cardiac muscle cell
Caused myocardial cell injury
1. cell and experiment reagent：
Cultivate Primary rat neonatal rat myocardial cell.Niclosamidum is purchased from Sigma company.
2. experimental method：
2.1. Primary rat neonatal rat myocardial cell is cultivated：
Suckling mouse (1-3 days) heart is taken, " mixing clostridiopetidase A+pancreatin " 37 degrees Celsius of 2500r/min centrifugations are removed supernatant, blown and beaten
Cell is collected in culture bottle, and differential velocity adherent removes fibroblast, continues to cultivate.
2.2. the expression and activity of STAT3 are detected
After cell length to Suitable Density, it is added niclosamidum (0.05 μm of ol/L), respectively at 15 minutes, 30 minutes, 1 is small
When, it samples within 2 hours, extracts total protein.Protein concentration is measured using BCA protein detection kit.With 10%SDS-PAGE points
From on protein and trace to nitrocellulose filter.It according to the difference of destination protein, is incubated for corresponding primary antibody, at 4 DEG C
Shaking table shakes overnight slowly.At room temperature with 1:10000 ratio is protected from light incubation fluorescence secondary antibody 1 hour.With Odyssey it is infrared at
As system (Odyssey CLx) and LI-COR Image Studio Software carry out imaging and interpretation of result.
2.3. myocardial cell injury effect caused by detection niclosamidum confrontation Doxorubicin
After cell length to Suitable Density, after Doxorubicin DOX (10 μm of ol/L) is added, niclosamidum (0.05 μ is added
Mol/L), cultivate 12 hours, using MTT kit measurement cell viability, using lactate dehydrogenase L DH kit detection LDH's
Release, using cell death-survival staining kit (Cell Viability Assays) detection cell
Death condition.
3, result
3.1. the protection signal STAT3 of niclosamidum (Niclosamide) activation cardiac muscle cell
By research, present invention discover that niclosamidum (0.05 μm of ol/L) can increase cultured myocardial P-STAT3
(Y705) protein level shows as activation cardiac muscle cell STAT3 signal, the result is shown in Figure 1 (* P<0.05,**P<0.01vs
Control.Nic,niclosamide)。
3.2. myocardial cell injury caused by niclosamidum (Niclosamide) confrontation Doxorubicin
By research, present invention discover that Doxorubicin DOX (10 μm of ol/L) can cause myocardial cell injury, cardiac muscle is shown as
Cell viability declines (Fig. 2A), cardiac muscle cell LDH release increases (Fig. 2 B) and cardiomyocyte cell death increases (Fig. 2 C), and chlorine nitre willow
Amine (0.05 μm of ol/L) can significantly fight the above damaging action that Doxorubicin is induced, and as a result see Fig. 2 (* * P<0.01vs
Control；#P<0.05,##P<0.01, vs.DOX Doxorubicin .Nic, niclosamide).
2 niclosamidum of embodiment (Niclosamide) increases cardiac muscle cell ATP and generates
1. cell and experiment reagent：
Ibid
2. experimental method：
2.1. Primary rat neonatal rat myocardial cell is cultivated：Ibid
2.2. cardiac muscle cell ATP assay
It after cell length to Suitable Density, is added niclosamidum (0.05 μm of ol/L), kit measurement is used after 12 hours
Cardiac muscle cell's ATP content.
3. result
3.1 niclosamidums (Niclosamide) increase cardiac muscle cell ATP content
By research, present invention discover that niclosamidum (0.05 μm of ol/L) is handled primary cardiomyocytes 12 hours, it is significant to increase
Add cardiac muscle cell's ATP content.As a result see Fig. 3 (* P<0.05,**P<0.01vs Control.Nic,niclosamide).
3 niclosamidum of embodiment (Niclosamide) inhibits the proliferation of cardiac fibroblast and the secretion of collagen
1. experimental method：
1.1. Primary rat suckling mouse cardiac fibroblast is cultivated
Suckling mouse (1-3 days) heart is taken, " mixing clostridiopetidase A+pancreatin " 37 degrees Celsius of 2500r/min centrifugations are removed supernatant, blown and beaten
Cell is collected in culture bottle, and differential attachment method obtains cardiac fibroblast.
1.2. the secretion measurement of the proliferation of cardiac fibroblast and collagen
Collagen measurement：It extracts primary SD Neonatal myocardial fibroblast and uses complete medium after cytotostatic is adherent
Starved cells 12h.Agent-feeding treatment for 24 hours, draws 32 μ L of supernatant culture solution later, and 5 × protein loading buffer is added
8 μ L, 100 DEG C are boiled sample 5min, carry out western blot experiment using 7.5%SDS-PAGE glue.
Cell Proliferation detection：This experiment is detected thin using Brdu Cell Proliferation ELISA Kit (Abcam)
Born of the same parents' proliferation.By cell with 2*105The density bed board of a/mL is inoculated in 96 orifice plates, and every hole is added 100ul and control wells are arranged.To
After cytotostatic is adherent, 100 μ L drugs (twice final concentration) are added into every hole and continue to cultivate, 2-24 is small before experiment terminates
When Brdu is added.It discards culture solution afterwards for 24 hours, the incubation at room temperature of 200 μ L denaturing solns is added to every hole, it is molten to discard denaturation after 30min
Liquid is washed three times with 1 × dcq buffer liquid, is blotted with filter paper.Backward every hole 100 μ L Anti-Brdu monoclonal antibodies are added,
It is incubated at room temperature 1h.100 hole μ L/ 1 × peroxidase sheep anti-mouse igg conjugated bodies, incubation at room temperature is added with pipettor after board-washing
30min.100 μ L TMB peroxidase substrates are added to every hole after board-washing, room temperature, which is protected from light, is incubated for 30min.Last every hole is added
100ul terminate liquid terminates reaction and reads at 450nm.
2. result
Heart is dramatically increased into fiber present invention discover that IL-6 (50ng/ml) handles cardiac fibroblast by research
The proliferation of cell and the secretion of collagen, niclosamidum (0.5 μm of ol/L) can significantly fight the heart of IL-6 induction into fiber finer
The proliferation of born of the same parents and the secretion of collagen.As a result see Fig. 4 (* P<0.05,vs Control；##P<0.01vs IL-6.Nic,
niclosamide)。
4 niclosamidum of embodiment inhibits human pulmonary artery smooth muscle cells (HPASMC) proliferation and migration of PDGF-BB induction
1. cell and reagent
Human pulmonary artery smooth muscle cells (HPASMC) is purchased from Sciencell company；Recombinant murine PDGF-BB is purchased from the U.S.
PeproTech company.
2. experimental method
2.1.DNA synthesis detection (detection proliferation)
HPASMCs cell is inoculated in 96 orifice plates with the even density in 5000/hole.Niclosamidum is incubated for by prescribed concentration
Cell is for 24 hours.BrdU reagent is added in 12h before drug incubation terminates, and continues to be incubated in 37 DEG C of incubators.After being incubated for, discard
Culture solution is separately added into the fixer of 200 μ L in every hole, is incubated at room temperature 30min.Fixer is discarded later, and dcq buffer liquid washes 3
Secondary, the anti-BrdU monoclonal detection antibody of 100 μ L is added in every hole, is incubated at room temperature 1h by each 3min.After being incubated for, punching
Wash buffer is washed 3 times, each 3min, and the peroxidase sheep anti-mouse igg conjugated body of 100 μ L is added, and is incubated at room temperature 30min.Again
It is secondary to rinse 3 times, each 3min, finally rinsed 1 time with deionized water.100 μ L peroxidase substrate TMB, room temperature is added in every hole
It is incubated for 30min, pays attention to being protected from light, blue is presented in positive hole at this time.Then 100 μ L terminate liquids are added and terminate reaction, at this time positive hole
Color is turned yellow by indigo plant, and measures absorbance at 450nm wavelength immediately.Wherein, not plus BrdU reagent processing hole be set as blank well.
2.2. cell scratch experiment (detection migration)
90% fusion, the good HPASMCs cell of growth conditions are chosen under inverted microscope.By cell with 50000/hole
Inoculation overleaf indicates in horizontal six orifice plate.Reach 90% to cell fusion degree, with specification be 200 μ l pipette tip along
Ruler makees vertical scratch in uniform cellular layer, intersects with the horizontal line of six hole backs and is accurately positioned.Use ordinary optical
Microscope photographs to record same position, finally measures not celliferous scratch using ImagePro Plus image analysis software
Area, the ratio of scored area area indicates mobility when the area from cell to scored area and 0h that are migrated with.
3. result
Platelet derived growth factor (PDGF-BB) is the important pathological factors for inducing pulmonary hypertension, we use PDGF-BB
The model drug of human pulmonary artery smooth muscle cells (HPASMC) proliferation and migration as induction, discovery PDGF-BB significantly induce people
Arteria pulmonalis smooth muscle cells (HPASMC) proliferation and migration, and niclosamidum can significantly inhibit people's pulmonary artery of PDGF-BB induction
Smooth muscle cell (HPASMC) proliferation and migration, illustrate that it has the function of anti-pulmonary hypertension.
Niclosamidum inhibits the result of human pulmonary artery smooth muscle cells (HPASMC) proliferation of PDGF-BB induction to see Fig. 5 (n
=9, * P<0.05vs.control, #P<0.05vs.PDGF-BB.Nic indicates niclosamidum).
Niclosamidum inhibits the result of human pulmonary artery smooth muscle cells (HPASMC) migration of PDGF-BB induction to see Fig. 6 (n
=12, * P<0.05vs.control, #P<0.05vs.PDGF-BB.Nic indicates niclosamidum).
The synthesis of 5 niclosamidum of embodiment grafting 2- hydroxypropyhnethacrylamide copolymer
Formula II (wherein, m 30-2000, n 1-200.)
Step：
(1) preparation of HPMA (N- (2- hydroxypropyl) Methacrylamide)：To the dichloromethane solution containing isopropanolamine
Middle addition methacrylic chloride stirs 4h under the conditions of -20 DEG C, and 0 DEG C or less stands for 24 hours, and complete precipitation to be crystallized is filtered, washing,
Up to HPMA monomer；
(2) preparation of the niclosamidum monomer with carbon-carbon double bond：It is molten that the DMF containing niclosamidum is added in acryloyl chloride
In liquid, under the conditions of existing for the natrium carbonicum calcinatum, it is stirred at room temperature for 24 hours, filtering, distillation under pressure, deionized water washs crude product, and 0
DEG C or less be recrystallized to give double bond containing niclosamidum monomer；
(3) preparation of pHPMA-Nic copolymer：By the monomer in (1) and (2) according to 1:8 mass ratios are dissolved in tetrahydro furan
Mutter in solution, the catalyst azodiisobutyronitrile of 3w/w% be added, deaerate, 60 DEG C, isothermal reaction for 24 hours, in tetrahydrofuran solution
Middle precipitating, and wash 2-5 times repeatedly.
(4) structural formula of niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer is shown in Formula II.
The synthesis of 6 niclosamidum of embodiment grafting 2- hydroxypropyhnethacrylamide copolymer
Formula III (wherein, m 30-2000, n 1-200.)
Step：
(1) preparation of HPMA (N- (2- hydroxypropyl) Methacrylamide)：It is added into the DMF solution containing isopropanolamine
Methacrylic chloride, stirs 43h under the conditions of 0 DEG C, 0 DEG C or less standing 12h, complete precipitation to be crystallized, filtering, washing to get
HPMA monomer；
(2) preparation of the niclosamidum monomer with carbon-carbon double bond：Methacrylic chloride addition is contained into niclosamidum
In tetrahydrofuran solution, under the conditions of existing for the Anhydrous potassium carbonate, 22h is stirred at room temperature, filters, distillation under pressure, deionization washing
Crude product is washed, 0 DEG C or less is recrystallized to give double bond containing niclosamidum monomer；
(3) preparation of pHPMA-Nic copolymer：By the monomer in (1) and (2) according to 3:10 mass ratios are dissolved in n-hexane
In solution, the catalyst potassium peroxydisulfate of 4w/w% is added, deaerates, 50 DEG C, isothermal reaction 36h is precipitated in four hexane solutions,
And it washs 2-5 times repeatedly.
(4) structural formula of niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer is shown in formula III.
Fig. 7 and 8 are shown in infrared spectroscopy and nuclear-magnetism spectrum identification.
In order to compare the water solubility of niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer and niclosamidum, this hair
It is bright to have carried out solubility test, i.e., the niclosamidum of phase homogenous quantities (15mg) is grafted 2- hydroxypropyhnethacrylamide copolymer
It is dissolved separately in the water of same volume with niclosamidum, observation dissolution situation, as a result as shown in figure 9, result explanation passes through
The amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl is remarkably improved the water solubility of niclosamidum.
Embodiment 7 is injected intraperitoneally niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic) and significantly presses down
The myocardial hypertrophy of pressing pressure load induction
1. experimental material：Bull Kun Ming mice (22-24 grams of weight)
2. experimental method：Mouse anesthesia, dorsal position are fixed, and ventilator is connected.Preceding midsection skin, tractor pulls open vertical
Diaphragm separates the arch of aorta, silk thread ligation.Close thoracic cavity, suture.Ventilator is withdrawn from after mouse restores autonomous respiration, revival is just
Often raising.All postoperative mouse are randomly divided into model group and model administration group, and model administration group gives intraperitoneal injection niclosamidum
It is grafted 2- hydroxypropyhnethacrylamide copolymer (suitable niclosamidum 100mg/kg), model group gives same volume physiology salt
Water.Animal is weighed after 4 weeks, puts to death animal, and coring is dirty, weighs cardiac weight, calculates the heart again than (HW/BW).
3. result
By research, present invention discover that intraperitoneal injection niclosamidum is grafted 2- hydroxypropyhnethacrylamide copolymer (phase
When to significantly inhibit the mouse cardiac muscle that pressure load induced plump by niclosamidum 100mg/kg), the result is shown in Figure 10 (sham, TAC,
The animal number of cases of TAC+drug group is respectively 5,7,5；**P<0.01vs Control；#P<0.05vs TAC.Drug is chlorine nitre willow
Amine is grafted 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic)).
It is antitumor that niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic) is injected intraperitoneally in embodiment 8
Effect
1. animal：
BALB/c-nu male nude mouse (strain：BALB/c-nu, 18~20g of weight, system in Beijing Jing tie up tonneau China experimental animal skill
Art Co., Ltd purchase)
2. experimental method：
(1) CT26 cell injuring model, condition of culture are：In RPMI1640 culture medium plus 37 DEG C of 10% fetal calf serum, 5%
CO2Culture.The CT26 cell in logarithmic phase growth is taken, cell is resuspended with PBS in digestion centrifugation.It will contain about 5 × 106A CT26 is thin
The cell suspension of born of the same parents is inoculated in the right back of 6~8 weeks BALB/c-nu.
(2) mouse model is established：After inoculation 5 days, BALB/c-nu is randomly divided into 2 groups, chlorine nitre willow is injected intraperitoneally in administration group
Amine is grafted 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic) (containing niclosamidum 100mg/kg), and model control group is to phase
The physiological saline of same volume, successive administration 15 days.
(3) mouse weight is measured daily.After administration 15 days, nude mice is put to death, tumor tissues is taken to weigh.
3, result
Niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic) is injected intraperitoneally, and to lotus knurl, (CT26 is thin
Born of the same parents) nude mice tumor growth in vivo have significantly inhibit effect.As a result it is compared as shown in figure 11 with model group, gives chlorine nitre willow
It is significant that amine is grafted 2- hydroxypropyhnethacrylamide copolymer (HPMA-Nic) (containing niclosamidum 100mg/kg) group tumor weight
Reduce (every group of n=18, * * P<0.01).
Claims (10)
1. niclosamidum (niclosamide) has myocardial preservation, anti-cardiac fibrosis, resisting cardiac hypertrophy, anti-heart failure in preparation
Or the application in the drug of function of anti-pulmonary hypertension, wherein the structural formula of the niclosamidum is shown in formula I：
2. the structural modification drug based on niclosamidum, which is characterized in that the structure based on niclosamidum
Modified medicaments are that the niclosamidum that the amine-modified niclosamidum of N- (2- hydroxypropyl) methacryl obtains is grafted 2- hydroxypropyl methyl
Acrylamide copolymer (N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer-Niclosamide
Conjugates, HPMA-Nic), structural formula is as shown in Formula II or formula III：
Wherein, m 30-2000, n 1-200.
3. a kind of method for preparing the structural modification drug as claimed in claim 2 based on niclosamidum, including following step
Suddenly：
(1) preparation of HPMA (N- (2- hydroxypropyl) Methacrylamide)：First is added into the organic solvent containing isopropanolamine
Base acryloyl chloride stirs 1-4h under the conditions of -20 DEG C -40 DEG C, stands 10h-24h, complete precipitation to be crystallized, filtering, washing to get
HPMA monomer；
(2) preparation of the niclosamidum monomer with carbon-carbon double bond：Chlorine nitre willow is added in methacrylic chloride or acryloyl chloride
1-24h is stirred at room temperature under the conditions of existing for the basic catalyst in the organic solution of amine, filters, distillation under pressure, deionization washing
Crude product is washed, double bond containing niclosamidum monomer is recrystallized to give；
(3) preparation of niclosamidum grafting 2- hydroxypropyhnethacrylamide copolymerization (HPMA-Nic)：It will be in step (1) and (2)
Monomer be dissolved in organic solvent, the catalyst of 0.1w/w%-5w/w% is added, deaerate, 40 DEG C -80 DEG C, isothermal reaction 6-
48h is precipitated in organic solvent, and is washed 2-5 times repeatedly.
4. according to the method described in claim 3, it is characterized in that the organic solvent in the step (1) is methylene chloride, third
The mixture of one of ketone, acetonitrile or DMF (N, N-dimethylformamide) or more than one organic solvent in certain ratio.
5. according to the method described in claim 3, it is characterized in that the crystallization temperature in the step (1) is 0 DEG C or less.
6. according to the method described in claim 3, it is characterized in that organic solution is tetrahydrofuran, DMF in the step (2)
One of (N, N-dimethylformamide), methylene chloride, hexamethylene or cyclohexanone or more than one organic solvent are by certain ratio
The mixture of example；Basic catalyst described in the step (2) includes natrium carbonicum calcinatum, Anhydrous potassium carbonate.
7. according to the method described in claim 3, it is characterized in that the temperature recrystallized in the step (2) is 0 DEG C or less.
8. according to the method described in claim 3, it is characterized in that in the step (3) organic solvent be petroleum ether, n-hexane,
The mixing of one of acetone, acetonitrile, tetrahydrofuran, tertbutyl ether or ether or more than one organic solvent in certain ratio
Object；The mass ratio of the monomer of the step of be added in step (3) (1) and (2) is 0.5-3:7-10.
9. according to the method described in claim 3, it is characterized in that catalyst described in the step (3) is free radical polymerization
Initiator, including azo-initiator and peroxide initiator, the azo-initiator include azodiisobutyronitrile,
Azobisisoheptonitrile and azo-bis-iso-dimethyl initiator；The peroxide initiator includes hydrogen peroxide, over cure
Sour ammonium, potassium peroxydisulfate, benzoyl peroxide, benzoyl peroxide and methyl ethyl ketone peroxide.
10. the structural modification drug as claimed in claim 2 based on niclosamidum has myocardial preservation, anti-heart in preparation
Application in fibrosis, resisting cardiac hypertrophy, anti-heart failure, anti-pulmonary hypertension and anti-tumor drug.
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| CN111233695A (en) * | 2020-03-13 | 2020-06-05 | 中国科学院成都有机化学有限公司 | Niclosamide cyclopropyl derivative, preparation method and application thereof |
| CN113143919A (en) * | 2021-03-22 | 2021-07-23 | 广州医科大学 | Novel application of bisindole maleimide compound |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233695A (en) * | 2020-03-13 | 2020-06-05 | 中国科学院成都有机化学有限公司 | Niclosamide cyclopropyl derivative, preparation method and application thereof |
| CN111233695B (en) * | 2020-03-13 | 2024-01-26 | 中国科学院成都有机化学有限公司 | Chlor Liu Anhuan propyl derivative, preparation method and application thereof |
| CN113143919A (en) * | 2021-03-22 | 2021-07-23 | 广州医科大学 | Novel application of bisindole maleimide compound |
| CN113143919B (en) * | 2021-03-22 | 2022-12-06 | 广州医科大学 | Novel application of bisindole maleimide compound |
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