CN115504927A - 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid and preparation method and application thereof - Google Patents
4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid and preparation method and application thereof Download PDFInfo
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- CN115504927A CN115504927A CN202210976338.5A CN202210976338A CN115504927A CN 115504927 A CN115504927 A CN 115504927A CN 202210976338 A CN202210976338 A CN 202210976338A CN 115504927 A CN115504927 A CN 115504927A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/72—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D211/78—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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Abstract
The invention discloses 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid and a preparation method and application thereof, and a preparation method of the 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid comprises the following steps: toluene and formic acid are catalyzed by dimethyl formamide and phosphorus oxychloride to obtain a product I; the first product reacts with nitric oxide vapor under the catalytic action of hydrogen cyanide and ammonia salt to obtain a second product; reacting the product II with n-butyric acid under the catalytic action of thionyl chloride, diazomethane and silver chloride to obtain a product III; and reacting the product III in vacuum and alkaline environment to obtain the target compound. The compound prepared by the invention has the effects of inducing autophagy of cells, delaying aging of adult stem cells, inhibiting the growth of various tumor cells, resisting osteoporosis, inhibiting senile fatty liver and resisting senile renal fibrosis.
Description
Technical Field
The invention relates to the field of biomedicine. More particularly, the invention relates to 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid and a preparation method and application thereof.
Background
Aging is a process of progressive functional decline in combination of various organs, for example, osteoporosis occurs in bones, atrophy occurs in muscles, fatty liver is easily formed in liver, fibrosis easily occurs in kidney, tumor easily occurs and the like, and is a key factor causing combination of various age-related diseases.
Revuelta M, matheu a. Autophagy in stem cell imaging. Aging cell.2017oct;16 912-915, the aging of organ tissue specific stem cells is a key factor causing organ failure, and the decline of autophagy function is a sign of cell aging.
Studies have been made (1) Kaushik S, tasset I, arias E, pampliega O, wong E, martinez-Viscente M, cuervo AM. Autophagy and the hallmarks of aging. Ageing Res Rev.2021Dec;72, (2) Leidal AM, levine B, debnath J.Autophase and the Cell biology of age-related disease. Nat Cell biol.2018Dec;20 (12): 1338-1348, it was demonstrated that enhancing autophagy can delay symptoms of aging. The current research on healthy aging drugs is still in the clinical research phase, with the best known and widely used drugs being the genus metformin and rapamycin.
Metformin originally serves as a hypoglycemic agent for treating diabetes patients, and is applied as an anti-aging drug to enter into anti-aging clinical Trials (TAME). However, in a mouse experiment, metformin is found to be not effective in improving the aging symptoms of mice. Rapamycin is currently used as an immunosuppressant, often in combination with cancer therapy and organ transplantation surgery. These two drugs, although already used clinically, have side effects. Besides the common side effects of headache, nausea, diarrhea and the like, the metformin and rapamycin can also bring about severe side effects of megaloblastic anemia, liver and kidney dysfunction, lactic acidosis, low potassium, hypomagnesemia and the like. Since the side effects of both of the above-mentioned drugs are severe, the administration of the drugs cannot be continued, and the effect of both of the above-mentioned drugs in inhibiting the growth of tumor cells has not been found.
Disclosure of Invention
An object of the present invention is to provide a compound of 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, which can enhance autophagy, can resist aging of stem cells, is useful for preventing and treating the above-mentioned diseases related to aging, and can promote healthy aging of the elderly.
Another object of the present invention is to provide the use of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, which has the effects of inducing autophagy, delaying aging of adult stem cells, inhibiting cell growth of various tumors (liver cancer, lung cancer, acute monocytic leukemia, colon cancer, prostate cancer), resisting osteoporosis, inhibiting senile fatty liver, and resisting senile renal fibrosis.
To achieve these objects and other advantages in accordance with the present invention, there is provided 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid having a chemical structure represented by formula (I):
a process for the preparation of 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid comprising the steps of:
s1, obtaining a product I by toluene and formic acid under the catalytic action of dimethylformamide and phosphorus oxychloride;
s2, reacting the product I with nitric oxide steam under the catalytic action of hydrogen cyanide and ammonium salt to obtain a product II;
s3, reacting the product II with n-butyric acid under the catalytic action of thionyl chloride, diazomethane and silver chloride to obtain a product III;
and S4, reacting the product III in a vacuum and alkaline environment to obtain the 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid.
Preferably, in the method for preparing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, the reaction conditions in S1 are 130 to 170 ℃ and 45% humidity.
Preferably, in the method for preparing 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, the reaction conditions in S2 are 55 ℃ and a dry environment.
Preferably, in the method for preparing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, the reaction conditions in S3 are 40 ℃ and 10% humidity.
Preferably, in the preparation method of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, S4 is reacted in ammonium chloride, sodium hydroxide and vacuum environment.
Preferably, in the preparation method of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, the ammonia salt is one or more of ammonium bromide, ammonium iodide and ammonium chloride.
Preferably, in the method for preparing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, the ammonium salt is ammonium chloride.
Application of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid in preparing medicine for inducing autophagy and delaying adult stem cell senility.
Application of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid in preparing medicines for treating liver cancer, lung cancer, acute monocytic leukemia, colon cancer, prostatic cancer, osteoporosis, senile fatty liver and senile renal fibrosis.
The invention at least comprises the following beneficial effects:
the compound prepared by the invention has the effects of inducing autophagy of cells, delaying aging of adult stem cells, inhibiting cell growth of various tumors (liver cancer, lung cancer, acute monocytic leukemia, colon cancer and prostatic cancer), resisting osteoporosis, inhibiting senile fatty liver and resisting senile renal fibrosis.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is an NMR chart of a target compound of the present invention;
FIG. 2 is a graph showing the test of the OD activity of various cancer cells;
FIG. 3 is a staining pattern for senescent cells;
FIG. 4 is a diagram showing the identification of molecular markers for senescent cells;
FIG. 5 is a graph showing detection of autophagy;
FIG. 6 is a graph showing the ability of differentiating osteogenic-adipogenic into adipose tissues after aging of MSCs;
FIG. 7 is a graph of P1NP content in mouse serum;
FIG. 8 is a graph showing the content of CTX-1 in mouse serum;
FIG. 9 is a graph of bone density in mice;
FIG. 10 is a graph of mouse bone mass, bone volume, trabecular bone, bone mineral quality;
FIG. 11 is a mouse liver fat detection map;
FIG. 12 is a mouse kidney fiber detection map;
FIG. 13 is a target compound of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Example 1
4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, the chemical structural formula is shown in formula (I):
The preparation method of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid comprises the following steps:
s1.92g toluene and 46g formic acid react under the catalysis of dimethyl formamide (DMF, 145 g) and phosphorus oxychloride (300 g) as catalysts, and then the temperature is reduced to-40 ℃ and the product I is obtained after cooling;
s2.120g of the first product reacts with 112g of nitric oxide steam under the catalytic action of 30g of hydrogen cyanide and 60g of ammonium chloride to obtain a second product;
s3.170g of the product II and 60g of n-butyric acid react under the catalysis of thionyl chloride (60 g), diazomethane (50 g) and silver chloride (70 g) to obtain a product III;
and (4) reacting the product III 4.264g in ammonium chloride (80 g) and alkaline (sodium hydroxide) in a vacuum environment to obtain the target compound 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid.
NMR chart of 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, as shown in FIG. 1.
The molecular formula is as follows: c 14 H 17 NO 3
Molecular mass: 247.29
The ratio of the nucleus to the nucleus: 247.12
Elemental analysis: c-68%, H-6.93%, N-5.66%, O-19.41%
In the preparation method of the 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid, the reaction condition in S1 is 130-170 ℃ and the humidity is 45%.
In the preparation method of the 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid, the reaction condition in S2 is 55 ℃ and a dry and ventilated environment.
In the preparation method of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid, the reaction conditions in S3 are 40 ℃ and the humidity is 10%.
Application of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid in preparing a medicament for inducing autophagy and delaying aging of adult stem cells.
Application of 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid in preparing medicine for treating liver cancer, lung cancer, acute monocytic leukemia, osteoporosis, senile fatty liver and senile renal fibrosis.
Experiment of
1. In vitro antitumor Activity test
In vitro cytotoxicity assays were performed using the MTT method.
Respectively inoculating Hep3B cell, HCCLM3 cell, HCT116 cell, A549 cell, THP-1 cell, and DU145 cell to RPMI-1640 culture solution containing 10% fetal calf serum and 1% penicillin-streptomycin antibiotic, placing at 37 deg.C and 5% CO 2 And culturing in a culture dish with saturated humidity, changing the liquid every 3 days, and after the culture dish is full of cells, digesting with trypsin with the mass fraction of 0.25% for subculture.
Taking cells growing to logarithmic phase in the culture solution, preparing the cells with the concentration of 2 multiplied by 10 percent by mass with the culture solution containing 10 percent by mass of embryo bovine serum and 1 percent by mass of double antibody 4 Permol of single cell suspension, 4000 cells per well, seeded in 96-well cell culture plates, at 37 ℃ and 5% CO 2 And culturing under saturated humidity24h; the compound of example 1 was then added to the cell suspension as experimental group; wherein 6 concentration gradients were set for the addition of the anti-tumor acting compound in the experimental group. And simultaneously setting a blank control group and a negative control group, wherein the blank control group only contains culture solution but no tumor cells, and the negative control group only contains 4000 cells and is added with DMSO with the same volume in the experimental group.
The plates were incubated at 37 ℃ and 5% CO 2 And after culturing for 12h under the condition of saturated humidity, adding 10 mu LCCK-8 solution into each hole, continuously culturing for 2h, placing on a shaking table, shaking for 5min at a low speed, and detecting the OD value of each hole by using an enzyme-labeling instrument (the light source is 450nm in wavelength), wherein the higher the OD value is, the more the cells survive, and the lower the OD value is, the less the cells survive. The measured results are shown in fig. 2, and the graph at the upper left corner in fig. 2 shows the cell activities measured at different drug concentrations (0, 5, 10, 20, 30, 50 μ M) for 8h, 16h and 24h, so that the hepatoma cells show obvious activity reduction after being treated by the compound for 8 h. Therefore, when the activity of other cells is tested, the cell activity of the compound after 8 hours of treatment can be measured to evaluate the regulation effect of the compound on the cell activity. Finally, the cell activity of each cell at 8h of compound treatment was scaled to obtain the drug IC50 concentration, and the results are shown in table 1.
TABLE 1 drug IC50 concentration values corresponding to each cell
| Cell line | IC50(μM) |
| Hep3B | 92.5 |
| HCCLM3 | 120 |
| HCT116 | 37.5 |
| A549 | 105 |
| THP-1 | 22.7 |
| |
40 |
As can be seen from fig. 1 and table 1, CXM102, a compound of the present invention, at a concentration of 20 μ M, can significantly inhibit the activity of liver cancer and highly metastatic liver cancer, colon cancer, lung cancer, monocytic leukemia and prostate cancer cells, and has the potential to treat these cancers.
2. In vitro anti-aging experiments of Mesenchymal Stem Cells (MSCs)
Anti-aging identification is carried out by adopting cell aging specific beta-galactosidase (SA-beta-Gal) staining, aging specific protein marker p16INK4a staining and osteogenic-adipogenic differentiation induction experiments.
Inoculating human bone marrow Mesenchymal Stem Cells (MSCs) in special stem cell culture solution containing 10% fetal calf serum and 1% penicillin-streptomycin (double antibody) at 37 deg.C, and 5% CO 2 And culturing in a culture dish with saturated humidity, changing the liquid every 3 days, digesting with trypsin with the mass fraction of 0.25% after the cells grow over the culture dish, and carrying out subculture.
Taking the cells growing to logarithmic phase in the culture medium, preparing the culture medium containing 10% of embryo bovine serum and 1% of double antibody into the culture medium with the concentration of 1 × 10 5 40000 cells per well of a single cell suspension per well inoculated in a 12-well cell culture plate, at 37 ℃ C. And 5% 2 And culturing for 24h under the condition of saturated humidity. After the cells adhered to the wall, the compound of example 1 and hydrogen peroxide at a concentration of 600 μ M were added to the experimental group as an experimental group; whereinFor the addition of the anti-cell-senescence compound in the experimental group, 3 concentration gradients (5 μ M, 10 μ M, 20 μ M) were set. A negative control group was also prepared, to which only 600. Mu.M hydrogen peroxide was added and to which the compound of example 1 was not added. Wherein, the time for treating the cells by the hydrogen peroxide is 2 hours, and then half of the fresh culture medium is replaced for continuously culturing for 12 hours.
(1) For the staining of senescent cells, the SA-. Beta. -Gal staining method was used for identification. The plates were incubated at 37 ℃ and 5% CO 2 And continuously culturing for 12h under the condition of saturation humidity, adding 500 mu L of SA-beta-Gal staining working solution into each well, incubating for 2h at 37 ℃, taking out, staining the cell nucleus for 10min by using cell nucleus specific staining solution (DAPI), cleaning, and taking a picture under a microscope, wherein as shown in figure 3, the SA-beta-Gal staining solution in the upper row of staining only stains senescent cells into blue-green, the more blue-green in the picture indicates that the senescent cells are more, the control indicates the cells without compound treatment, and the 5 mu M, 10 mu M and 20 mu M respectively indicate the cells treated by the concentration. It follows that cellular senescence decreases with increasing compound concentration. In the lower row of staining, DAPI staining solution only stained nuclei, indicating that the total cell amount remained consistent in each experiment.
(2) For the identification of the molecular marker of the senescent cells, the specific antibody is used for carrying out immunofluorescence staining identification on the intracellular p16INK4a molecules. After the medium was aspirated from the well plate, the cells were washed once with PBS buffer, treated with paraformaldehyde (500 μ L per well) with a mass fraction of 4% for 20min, then treated with Triton solution (100 μ L) with a mass fraction of 0.1% for 10min, then incubated with p16INK4a antibody diluted with BSA (50 μ L) with a mass fraction of 1% (1) at 4 ℃ for 12h, washed with PBS buffer and incubated with fluorescent antibody at room temperature for 1h, and finally stained with DAPI stain for 10min, and then washed and photographed under a microscope, as shown in fig. 4, the upper control indicates cells without compound treatment, the lower CXM102 indicates cells treated with 20 μ M compound, the p16INK4a indicates the senescence-specific protein molecule p16INK4a expressed by senescent cells, the more green indicates senescent cells, DAPI indicates cell localization, one blue indicates one cell, merge indicates the synthetic map of the molecule expressing p16INK4a and the localization of senescent-specific protein molecule p16 a, and facilitates observation of the distribution of senescent cells in-cell distribution in-cell. It follows that the compounds of the invention are resistant to aging by MSCs.
3. Experiment for promoting autophagy of Mesenchymal Stem Cells (MSCs) in vitro
Taking cells growing to logarithmic phase in the culture solution, preparing the cells with the concentration of 1 multiplied by 10 by using the culture solution containing 10 mass percent of embryo bovine serum and 1 mass percent of double antibody 5 Perml of single cell suspension, 20000 cells per well were seeded in 24-well cell slides at 37 deg.C, 5% CO 2 And culturing for 24h under the condition of saturated humidity. After cell attachment, 20 μ M of the compound of example 1 and 600 μ M of hydrogen peroxide (3.5 μ L) were added to the experimental group as an experimental group; wherein, for the addition of the anti-cell senescence compound to the experimental group, the cells were treated at 20. Mu.M. A negative control group was also set, which was supplemented with only 600. Mu.M hydrogen peroxide and no compound, but with an equal volume of dimethyl sulfoxide (DMSO). In addition, since Rapamycin (Rapamycin) has been recognized as a standard drug for inducing autophagy in cells, in order to compare the autophagy-inducing ability of the compound of the present invention and Rapamycin to cells, rapamycin was also introduced as a positive control in this experiment.
(1) For detecting the autophagy of the cells, an immunofluorescence staining method is used for identifying the marker molecule LC3II of the autophagy. Placing the culture plate at 37 deg.C, 5% CO 2 And continuously culturing for 12h under the condition of saturated humidity, fixing by using paraformaldehyde with the mass fraction of 4%, permeabilizing a cell membrane for 20min by using penetrating fluid (100 mu L) with the mass fraction of 0.1%, treating for 30min by using sealing fluid with the mass fraction of 2%, immediately incubating for 2h at room temperature by using an LC3II protein antibody, staining for 45min by using a green fluorescence-labeled antibody, taking out, staining cell nuclei for 10min by using DAPI staining solution, and then washing and taking a picture under a microscope, wherein as shown in figure 5, the stronger the green color in the figure shows that the LC3II protein is more, namely the stronger the autophagy of the cell is, and as can be seen from figure 5, compared with a negative control group, both Rapamycin and CXM102 can obviously induce the autophagy of the cell, but CXM102 has stronger capacity than that Rapamycin induces the autophagy of the cell.
4. Experiment for promoting osteogenic differentiation and inhibiting fat differentiation of bone marrow Mesenchymal Stem Cells (MSCs) in vitro
After the MSCs are aged, the osteogenic differentiation capacity of the MSCs is weakened, the fat differentiation capacity of the MSCs is enhanced, and the MSCs are consistent with the symptoms of reduction of bone synthesis and increase of bone marrow fat after clinical human body aging.
For the identification of osteogenic-adipogenic differentiation capacity after the aging of MSCs, the embodiment uses an osteogenic differentiation specific dye liquid alizarin red staining solution and an adipogenic differentiation specific dye liquid oil red O staining solution for identification. MSCs were treated with osteogenic differentiation (Osteogenesis) induction medium and adipogenic differentiation (Adipogenesis) induction medium, respectively, to which 20. Mu.M of the compound of example 1 was added; control groups were supplemented with only 2. Mu.L DMSO and no compound from example 1. After 7 days, fixing with paraformaldehyde with a mass fraction of 4%, staining the osteoblastic differentiation-inducing MSCs with alizarin red staining solution for 15min, staining the adipogenic differentiation-inducing MSCs with oil red O staining solution for 15min, and then washing and taking a picture under a microscope, as shown in fig. 6. The upper row in the figure is alizarin red staining identification of osteoblast differentiation experiments of MSCs, and more red indicates that MSCs differentiate into osteoblasts; the lower row in the figure is the identification of oil red O staining of adipogenic differentiation experiments of MSCs, more red indicating more MSCs differentiated into adipocytes. From this, CXM102 was found to be able to significantly promote osteogenic differentiation of MSCs, while inhibiting differentiation of MSCs into adipocytes, exhibiting potential against osteoporosis.
5. In vivo anti-senile osteoporosis experiment
Mice entered the aging stage at 16 months of age, thus 16 months of age mice were used as the starting point for the experiment. The experimental setup was DMSO injection control and dosing experimental. Administration experimental groups 16-month-old C57BL6 mice were administered by intramuscular injection at a dose of 10mg/kg, twice weekly until the end of 18 months. Among these, control mice were injected intramuscularly with DMSO in an equal volume (10. Mu.L) of the drug.
(1) For the detection of the osteoporosis evaluation index, the content of type I procollagen amino-terminal propeptide (P1 NP) and type I collagen carboxyl-terminal propeptide (CTX-1) in serum is detected by an enzyme-linked immunosorbent assay (ELISA) method. P1NP and CTX-1 are clinically accepted indexes for evaluating in vivo bone synthesis and bone resorption activity respectively. The comparison shows that the serum of the mouse injected with the CXM102 has obviously higher content of P1NP than the serum of the mouse of a control group (see figure 7), which indicates that the CXM102 can promote the anabolism of bones in a mouse body and increase the bone formation. By detecting CTX-1, CXM102 can obviously reduce the content of CTX-1 in the serum of mice, and the medicament is shown to inhibit the bone resorption activity in the bodies of the mice (see figure 8). Fig. 7 and fig. 8 show that CXM102 can promote bone formation and inhibit bone resorption in mice, and thus is a potential anti-senile osteoporosis drug.
(2) And detecting the bone density index and the bone volume index in the osteoporosis by adopting a micro CT scanning method. Mice were anesthetized at 16 months of age, then femurs were scanned for whole length with microCT, and then continued to be bred to 18 months of age. During the period, a micct scan was performed every two weeks, and then the femoral bone density, bone volume, and bone mineral content of the mice were evaluated. When analyzing mouse femur, the femur was divided into distal, middle and proximal ends, and a bone density map as shown in fig. 9 was obtained. Fig. 9 is a graph of bone density from left to right for the proximal, middle and distal femur in that order. In fig. 9, the lower line represents the trend line for femoral density in the control mice and the upper line represents the trend line for femoral density in mice injected with CXM102, indicating that CXM102 can delay bone density loss in older mice. Through analysis of osteogenic mass, bone volume, trabecular bone and bone mineral content in the same part of a mouse, the CXM102 was found to be capable of promoting bone mass formation in vivo (BV/TV, upper left of fig. 10), increasing trabecular bone thickness (tb.th, upper right of fig. 10) and number (tb.n, lower left of fig. 10) and bone mineral content (BMC, lower right of fig. 10).
6. Experiment for inhibiting senile fatty liver in vivo
To detect liver adipogenesis, liver tissue sections were stained with oil red O staining solution.
The experimental setup was DMSO injection control and dosing experimental. Administration experimental groups 16-month-old C57BL6 mice were administered by intramuscular injection at a dose of 10mg/kg, twice a week. The control group was injected intramuscularly with an equal volume (10. Mu.L) of drug. After 3 months, the mice are sacrificed to take livers, the livers are soaked in paraformaldehyde with the mass fraction of 4% for 48 hours, then, the livers are soaked in cane sugar with the mass fraction of 30% until the liver tissue blocks sink from the suspension state, and then, the frozen sections are carried out. During staining, the section was soaked in paraformaldehyde with a mass fraction of 4% for 20min, washed with PBS, stained with an oil red O-isopropanol solution with a mass fraction of 1.5%, washed with PBS after 10min, and photographed under a microscope, as shown in FIG. 11, the more red particles, the more fat particles formed in the liver tissue. Therefore, the compound can inhibit the occurrence of senile fatty liver.
7. In vivo renal fibrosis inhibition experiment
To detect renal fibrosis, the kidney tissue sections were stained with massson stain.
The experimental setup was DMSO injection control and dosing experimental. Administration experimental groups 16-month-old C57BL6 mice were administered by intramuscular injection at a dose of 10mg/kg, twice a week. The control group was injected intramuscularly with an equal volume (10. Mu.L) of drug. After 3 months, the mice are sacrificed, the kidneys are taken out, the mice are soaked in paraformaldehyde with the mass fraction of 4% for 48 hours, then the mice are soaked in cane sugar with the mass fraction of 30% until the kidney tissue blocks sink from the suspension state, and then the mice are frozen and sliced. During staining, the section is soaked in paraformaldehyde with the mass fraction of 4% for 20min, washed with PBS and stained with massson stain, washed with PBS after 10min, and photographed under a microscope, as shown in figure 12, the more blue in the figure indicates that the renal fibrosis is more severe, and the more red in the figure indicates that the renal tissue and muscle fiber structure is more complete. Therefore, the compound can inhibit the occurrence of renal fibrosis.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details shown and described herein without departing from the generic concept as defined by the claims and their equivalents.
Claims (10)
- 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-carboxylic acid, characterized by the chemical structural formula as shown in formula (I):
- a process for producing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid, which comprises the steps of:s1, obtaining a product I by toluene and formic acid under the catalytic action of dimethylformamide and phosphorus oxychloride;s2, reacting the product I with nitric oxide steam under the catalytic action of hydrogen cyanide and ammonia salt to obtain a product II;s3, reacting the product II with n-butyric acid under the catalytic action of thionyl chloride, diazomethane and silver chloride to obtain a product III;s4, reacting the product III in an ammonium chloride, alkaline and vacuum environment to obtain the 4-methyl-2-p-tolyl-6-carbonyl pyridine-3-formic acid.
- 3. The process for producing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 2, wherein the reaction condition in S1 is 130 to 170 ℃ and a humidity of 45%.
- 4. The process for producing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 2, wherein the reaction condition in S2 is 55 ℃ and dry atmosphere.
- 5. The process for producing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 2, wherein the reaction conditions in S3 are 40 ℃ and 10% humidity.
- 6. The process for preparing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 2, wherein the product III of S4 is reacted in the presence of ammonium chloride, sodium hydroxide and vacuum.
- 7. The method of claim 2, wherein the ammonium salt is one or more of ammonium bromide, ammonium iodide, and ammonium chloride.
- 8. The process for producing 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 7, wherein the ammonium salt is ammonium chloride.
- 9. The use of 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 1 for preparing a medicament for inducing autophagy and delaying senescence of adult stem cells.
- 10. The use of 4-methyl-2-p-tolyl-6-carbonylpyridine-3-carboxylic acid according to claim 1, in the preparation of a medicament for treating liver cancer, lung cancer, acute monocytic leukemia, colon cancer, prostate cancer, osteoporosis, senile fatty liver, and senile renal fibrosis.
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| 陈绘如等: ""4-哌啶酮类化合物的合成及其在医药领域中的应用"", 《化工中间体》, no. 8, pages 1 - 4 * |
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