CN112047972A - ROS-responsive capsaicin prodrug, preparation method and application - Google Patents
ROS-responsive capsaicin prodrug, preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/025—Boronic and borinic acid compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pain & Pain Management (AREA)
- Pharmacology & Pharmacy (AREA)
- Rheumatology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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Abstract
The invention discloses a ROS-responsive capsaicin prodrug, a preparation method and application. The structural formula of the prodrug is as follows:
the prodrug can improve physicochemical properties of capsaicin, reduce irritation of capsaicin, and treat pain in inflammation.
Description
Technical Field
The invention relates to the field of chemical medicine, in particular to a ROS-responsive capsaicin prodrug, a preparation method and application.
Background
Capsaicin is a major active ingredient of capsicum, has various beneficial effects in the human body including the treatment of pain, atherosclerosis, hyperlipidemia, inflammation, rheumatoid arthritis, vasomotor rhinitis and the like, and has been proved to be an effective anticancer agent. Most capsaicin-sensitive afferents contain Calcitonin gene-related peptide (CGRP) and/or substance P, and various studies have shown that capsaicin can alter macrophage function, and that capsaicin preincubated rat peritoneal macrophages inhibit arachidonic acid binding to membrane lipids. The consumption of sensory neuropeptides is caused by repeated use of large doses of capsaicin, and there is increasing evidence that CGRP is a potent anti-inflammatory agent, capsaicin also inhibits macrophage secretion of hydrolytic enzymes (collagenase, elastase and hyaluronidase) in rat abdominal cavity, and these in vitro studies indicate that capsaicin can control the release of inflammatory mediators and the secretion of lysosomal enzymes from macrophages, thereby acting to reduce inflammation.
Topical capsaicin acts in the skin to reduce the processes of skin irritation and pain, best described as "defunctionalized" nociceptor fibers. Defunctionalization is due to a range of effects including temporary loss of membrane potential, inability to transport neurotrophic factors resulting in phenotypic changes, and reversible contraction of the terminals of the epidermal and dermal nerve fibers. Therefore, capsaicin can exert analgesic effect. Although capsaicin has a good analgesic effect, it is difficult to be absorbed orally and is highly irritant due to its poor water solubility, limiting its use. Further in vivo studies have shown that capsaicin is not sufficiently active in vivo, poorly absorbed, metabolized and poorly bioavailable, limiting its use.
In addition inflammation is associated with a number of pathological conditions, including infection, cancer, atherosclerosis, neurodegenerative diseases and arthritis. The inflammatory response of the joint is largely dependent on activated macrophages, which produce Reactive Oxygen Species (ROS) within the cell. Therefore, the high active oxygen environment of the inflammation part can be utilized to design a prodrug which can be activated by active oxygen, so that the medicine with improved property and safety is obtained. Among them boronic acids and their esters are the most common ROS-triggering groups, which can be substituted by H2O2Selective oxidation, release at inflammatory sites, effect of pain delay, and H2O2Is the ROS with the most abundant content and the highest stability in the inflammation process.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a ROS-responsive capsaicin prodrug, improve the physicochemical property of capsaicin and reduce the stimulation effect of the capsaicin.
It is another object of the present invention to provide a method for preparing the ROS-responsive capsaicin prodrug.
It is a final object of the invention to provide the use of ROS-responsive capsaicin prodrugs.
The technical scheme is as follows: the invention provides a ROS-responsive capsaicin prodrug, which has the following structural formula:
wherein R is1Represents:
the preparation method of the ROS-responsive capsaicin prodrug comprises the following steps:
(1)
dissolving 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-diazaborane-2-yl) benzyl bromide in an organic solvent, and mixing with capsaicin for reaction;
(2)
dissolving the product in (1) in an organic solvent, and adding NaIO4And NH4And (5) mixing and reacting the OAc.
The method for measuring the content of the ROS-responsive capsaicin prodrug is to combine the prodrug with H2O2And (3) incubating in PBS (phosphate buffer solution) containing DMSO (dimethyl sulfoxide), and detecting the concentration change and in-vitro release process of the prodrug by adopting Shimadzu liquid chromatograph-mass spectrometer LC-MS (liquid chromatography-mass spectrometer) at a wavelength of 254nm from reaction liquid incubated for different times. Each test needs to be repeated three more times.
Further, the measurement was repeated three times or more.
Use of the ROS-responsive capsaicin prodrug in the treatment of pain in inflammation.
Further, the ROS-responsive capsaicin prodrug has the effect of reducing the toxicity of capsaicin to cells.
Further, the cells were L02 cells.
Has the advantages that: the prodrug of the invention can improve the physicochemical property of capsaicin and reduce the stimulation of the capsaicin. Can be used for treating pain caused by inflammation.
Drawings
FIG. 1 shows the prodrug LK-1, LK-2(1mM) is substituted by H2O2(5mM, 5equiv) activated content change profile;
FIG. 2 shows the prodrug LK-1, LK-2(1mM) being treated with different concentrations of H2O2Content change graph after 2h of activation;
FIG. 3 shows the stability of the prodrugs LK-1, LK-2(1mM) at different pH (5-9);
FIG. 4 shows the stability of the prodrugs LK-1, LK-2(1mM) in mouse plasma and human plasma conditions;
FIG. 5 is a graph showing the results of the anti-proliferative activity of capsaicin and the prodrugs LK-1, LK-2 on L02 cells.
FIG. 6 shows the analgesic effect of capsaicin and the prodrug LK-1, LK-2.
Detailed Description
The following partial compounds are preferred according to the invention:
the capsaicin prodrug can be prepared by the following method:
(E) -8-methyl-N- (3-methoxy-4- ((4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) -6-nonenamide (compound LK-1)
The specific synthesis steps are as follows:
reaction reagents and conditions: (a) potassium carbonate, acetone, 60 ℃, 2h, 90.5%.
(E) -8-methyl-N- (3-methoxy-4- ((4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) -6-nonenamide (compound LK-1)
4- (4, 4, 5, 5-tetramethyl-1.3.2-diazaboran-2-yl) benzyl bromide (1g, 3.367mmol) was dissolved in a quantity of acetone, capsaicin (1.03g, 3.367mmol) and potassium carbonate (1.4g, 10.11mmol) were added, the reaction was warmed to 60 ℃ for 2 hours and the potassium carbonate solid was removed by suction filtration, the solvent was removed under reduced pressure and the crude product was subjected to column chromatography to give about 1.59g of compound LK-1 in 90.5% yield.1H NMR(300MHz，MeOD-d4)：7.73(d，J＝8.0Hz，2H)，7.42(d，J＝8.0Hz，2H)，6.94-6.85(m，2H)，6.80-6.70(m，1H)，5.42-5.27(m，2H)，5.10(s，2H)，4.28(d，J＝3.6Hz，2H)，3.84(d，J＝3.2Hz，3H)，2.21(t，J＝7.4Hz，3H)，1.98(dd，J＝12.5，7.3Hz，2H)，1.68-1.54(m，2H)，1.40-1.30(m，14H)，0.95(d，J＝6.7Hz，6H).HRMS(ESI)：m/z，calcd for C31H45NO5[M+H]+，522.3385；found：522.3421。
(E) -8-methyl-N- (3-methoxy-4-phenylboronic acid) benzyl) -6-nonenamide (Compound LK-2)
The specific synthesis steps are as follows:
reaction reagents and conditions: (a) NaIO4，NH4OAc, acetone, rt, 12h, 73.5%.
(E) -8-methyl-N- (3-methoxy-4-phenylboronic acid) benzyl) -6-nonenamide (Compound LK-2)
Dissolving compound LK-1(1g, 1.918mmol) in a certain amount of acetone, adding NaIO4(1.23g, 5.754mmol) and NH4OAc (0.33g, 4.219mmol), the reaction solution was stirred overnight at room temperature, the reaction solvent was removed under reduced pressure, and the crude product was subjected to column chromatography to give the compound LK-2 in an amount of about 0.62g with a yield of 73.5%.1H NMR(300MHz，MeOD-d4)：7.74(d，J＝7.4Hz，1H)，7.61(d，J＝6.9Hz，1H)，7.40(t，J＝9.0Hz，2H)，6.90(d，J＝7.8Hz，2H)，6.80-6.72(m，1H)，5.43-5.27(m，2H)，5.08(s，2H)，4.28(s，2H)，3.83(s，3H)，2.27-2.14(m，3H)，1.98(dd，J＝12.5，7.2Hz，2H)，1.69-1.54(m，2H)，1.41-1.27(m，5H)，0.95(d，J＝6.7Hz，6H).13C NMR(75MHz，DMSO-d6)：172.48，149.53，147.05，139.47，137.80，134.66，134.03，133.10，127.05，119.64，114.03，111.89，70.43，55.88，35.71，32.20，30.88，29.18，25.41，22.99.HRMS(ESI)：m/z，calcd for C25H35BNO5[M+H]+，440.2603；found：440.2572。
Reactive oxygen species (ROS, e.g. H) in physiological environment2O2) Plays an important role in the human immune system; in pathological conditions, H in diseased tissue due to elevated levels of oxidative stress2O2The concentration can be up to about 1mM, which is more than 100 times higher than that of normal tissue. Therefore, the graph of the change of the content of the compounds LK-1, LK-2 activated by hydrogen peroxide was tested next.
Firstly, whether the mono-substituted borate prodrug LK-1, LK-2 can be tested by ROS (H) in vitro or not is verified2O2) Activated to release capsaicin. The release process of the prodrug was monitored using liquid chromatography mass spectrometer (LC-MS). Prodrug LK-1, LK-2 with concentration of 1mM and H respectively2O2(5mM, 5equiv) was incubated in 10% DMSO in PBS (pH 7.4) at 37 ℃ and the reaction solutions incubated for various periods were analyzed by LC-MS (FIG. 1). Significant differences in the release rates of the compounds were found. LK-1 is rapidly transformed into LK-2 to release capsaicin. The compound LK-2 may be in oneThe capsaicin is completely released within half an hour. At the same time, the prodrugs LK-1, LK-2 were found to react with different concentrations of H2O2Upon incubation, it released the drug in a concentration-dependent manner (fig. 2).
The stability of the monosubstituted boronate ester prodrug LK-1, LK-2 was next examined under different relevant physiological conditions, including chemical stability at various pH values and plasma stability in humans and mice.
The acid-base stability of the mono-substituted boronate ester prodrug LK-1, LK-2 in buffers of different pH (FIG. 3) and plasma stability in humans and mice (FIG. 4) were tested by means of High Performance Liquid Chromatography (HPLC). PBS buffers were prepared at various pH values (pH 5-9) using 0.1M HCl and 0.1M NaOH solutions. In a 1.5mL microcentrifuge tube, 50. mu.L of prodrug (1mM) was mixed with 450. mu.L of PBS buffer at various pH values (pH 5-9), placed in a homomixer Thermomixer C (1.5mL, 1000rpm) and incubation commenced at 37 ℃. The results show that the compound LK-2 shows good chemical stability. In addition, prodrug LK-2 with concentration of 1mM is incubated with human plasma and mouse plasma at 37 deg.C, reaction solution incubated for different time is taken for HPLC analysis, and the prodrug content is not reduced obviously after prodrug LK-2 is incubated with human plasma and mouse plasma for 12 hours. Taken together, the results show that the mono-substituted boronic acid ester prodrug LK-2 is relatively stable under physiological conditions and can be used for further in vivo research.
Compounds were next tested for toxicity by MTT assay using L02 cells. L02 cells in log phase were seeded into 96-well plates at a density of 70-80% confluence per well at 37 ℃ and 5% CO2Under the conditions of (1) overnight. Use/non-use 0.1mM H2O2After treatment of the drug or prodrug, 20 μ L of 5mg/mLMTT was added and the cells were incubated for 4 hours at 37 ℃. The supernatant was discarded, and 150 μ L DMSO was added to each well. The mixture was shaken on a mini-shaker at room temperature for 5 minutes and the spectrophotometric absorbance was measured at 570nm by a Multiskan Spectrum Microplate Reader (Thermo, USA). Three replicates were performed for each concentration point in parallel and the results are expressed as mean ± SEM. By passing throughThe percent survival of the treated cells was calculated by the following formula:
the survival rate (%) (OD of experimental group OD-blank OD)/(OD of control group OD-blank OD × 100%.
Measure H2O2Antiproliferative activity of prodrugs LK-1 as well as LK-2 on normal hepatocyte L02 in the presence and absence. The results are shown in FIG. 5, where capsaicin itself has some inhibitory activity, IC, on L02 cells50115.7 mu mol, the prodrug LK-1 has lower toxicity to cells and IC50168.4. mu. mol, when H is added2O2Releases capsaicin, so the inhibitory activity to L02 is increased to 127.4 mu mol, and the prodrug LK-2 has lower toxicity to cells and IC50197.7. mu. mol, but when H was added2O2Capsaicin is released. Thus, the inhibitory activity against L02 was also increased to 129.9. mu. mol. Therefore, the prodrugs LK-1 and LK-2 have lower toxicity and can release capsaicin, and can be used for further animal experiments.
The analgesic effect of LK-1, LK-2 on a rat model of pain after plantar incision was subsequently evaluated.
Firstly, an animal model is made: after the rats are adaptively raised for 3-5 days, all rats are induced to be anesthetized by 3.5-4% isoflurane oxygen, and then are maintained to be anesthetized by 1.5-2% isoflurane (using a small animal anesthesia machine). Shortly before incision (1-2min), the infiltration drug (150 μ L) was injected plantar. Under sterile conditions, each rat was placed 0.5cm posterior to the foot and a longitudinal 1cm incision was made anteriorly using a 11-gauge surgical blade, the fascia and muscle were isolated, the flexor was raised, and a longitudinal incision was made through blunt dissection to leave the muscle source and insertion intact. After hemostasis by gentle pressure, the drug was injected into the flexor muscle (30 μ L) before closing the wound, and two needles were sutured discontinuously at the incision site with 5 gauge nylon thread, and the skin was sutured.
The specific administration mode is as follows: 1-2min before incision, 150 μ L of drug was injected into sole, and the remaining 30 μ L of drug was injected into flexor muscle before wound closure. And then detecting the analgesic effect of the thermal pain by using a hot plate method, namely placing the mouse on a hot plate with a certain temperature, thermally stimulating the foot of the mouse to generate a pain response, and judging whether the compound has the analgesic effect, wherein the detection indexes are as follows: the time from heat stimulation to paw withdrawal is s, and the time points are 1h, 4h, 8h, 12h, 16h and 20h after operation.
The experimental result is shown in figure 6, and the result shows that the compound LK-1 has a certain analgesic effect, but the effect is not as obvious as that of LK-2, and compared with a blank group, the compound LK-2 can obviously increase the time from thermal stimulation to paw withdrawal of a mouse, has a good analgesic effect, and has the advantages of quick response and long duration of analgesic effect, and the analgesic effect is at the same level as that of capsaicin. Therefore, LK-2 may be used for treating pain effect in inflammation.
Claims (7)
1. A ROS-responsive capsaicin prodrug having the structural formula:
wherein R is1Represents:
2. the method of making a ROS-responsive capsaicin prodrug of claim 1, wherein: the method comprises the following steps:
(1)
dissolving 4- (4, 4, 5, 5-tetramethyl-1, 3, 2-diazaborane-2-yl) benzyl bromide in an organic solvent, and mixing with capsaicin for reaction;
(2)
dissolving the product in (1) in an organic solvent, and adding NaIO4And NH4And (5) mixing and reacting the OAc.
3. The method of measuring the amount of a ROS-responsive capsaicin prodrug of claim 1, wherein: reacting a prodrug with H2O2And (3) incubating in PBS (phosphate buffer solution) containing DMSO (dimethyl sulfoxide), and detecting the concentration change and in-vitro release process of the prodrug by adopting Shimadzu liquid chromatograph-mass spectrometer LC-MS (liquid chromatography-mass spectrometer) at a wavelength of 254nm from reaction liquid incubated for different times. Each test needs to be repeated three more times.
4. The method of measuring a ROS-responsive capsaicin prodrug according to claim 3, wherein: the measurement was carried out at a wavelength of 254nm and was repeated three times or more.
5. The use of the ROS-responsive capsaicin prodrug of claim 1, to treat pain in inflammation.
6. The ROS-responsive capsaicin prodrug according to claim 1, wherein: reducing the toxicity of capsaicin to cells.
7. The ROS-responsive capsaicin prodrug according to claim 6, wherein: the cells were L02 cells.
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Application publication date: 20201208 Assignee: CHANGZHOU SIYAO PHARMACY Co.,Ltd. Assignor: CHINA PHARMACEUTICAL University Contract record no.: X2023320000090 Denomination of invention: ROS responsive capsaicin prodrug, preparation method and use Granted publication date: 20220412 License type: Common License Record date: 20230208 Application publication date: 20201208 Assignee: Nanjing Baize Pharmaceutical Technology Co.,Ltd. Assignor: CHINA PHARMACEUTICAL University Contract record no.: X2023320000091 Denomination of invention: ROS responsive capsaicin prodrug, preparation method and use Granted publication date: 20220412 License type: Common License Record date: 20230208 |