Process for the preparation of isodihydrokounidine and its use in anti-inflammatory, immunosuppressive and analgesic medicaments

Abstract

The application discloses a preparation method of isodihydrokayasonide and application of the isodihydrokayasonide in anti-inflammatory immune and analgesic drugs, and belongs to the field of medicinal chemistry. The isodihydrokayasonide is obtained by catalytic hydrogenation of kayasonide and has a chemical structural formula as shown in formula (I). Cell experiments show that the isodihydrokayasonide can significantly inhibit the release of NO and the generation of proinflammatory factors IL-6 and TNF-α in an LPS-induced RAW264.7 macrophage inflammatory model, and the isodihydrokayasonide has a better anti-inflammatory immune effect than the kayasonide. Pharmacokinetic experiments show that the in-vivo exposure of the compound after oral administration is much greater than that of the kayasonide, indicating that the compound has a higher bioavailability and good safety. The prevention effect of the compound on acute peritonitis of mice is stronger than that of the kayasonide, and the compound has a significant analgesic effect on acute joint pain of rats with rheumatoid arthritis, and can be used for preparing anti-inflammatory immune drugs and analgesic drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the preparation method of isodihydrogellecithin and its application in the preparation of anti-inflammatory and immunomodulatory drugs and analgesic drugs. Background Technology

[0002] Gelsemium elegans Benth., a perennial evergreen climbing vine belonging to the genus Gelsemium in the family Loganiaceae, is a medicinal plant with a long history in my country. It can be used to treat carbuncles, scrofula, tumors, traumatic injuries, boils, scabies, eczema, and other ailments. The main active ingredient of Gelsemium elegans is indole alkaloids, with koumine being the primary alkaloid and exhibiting relatively low toxicity. Its complex structure endows it with unique biological activity. Several authorized patents have been published concerning the use of koumine in treating various diseases. For example, US Patent 9,078,890 B2 and Chinese Patent CN102293768B disclose the use of koumine in the preparation of drugs for treating rheumatoid arthritis; Chinese Patent CN101322705B discloses the anti-chronic pain effect of koumine and its use in the preparation of drugs for treating chronic pain; Chinese Patent CN109953991B discloses the use of koumine in treating amyloid deposition and / or tau lesion-related diseases or conditions (such as Alzheimer's disease); and so on. All these patents indicate that koumine possesses significant value in the creation of novel drugs.

[0003] Despite the promising efficacy of gelsemium elegans, its pharmacokinetic properties present challenges. Previous research by our group revealed that in SD rats, a single oral and intravenous injection of 3 mg / kg gelsemium elegans hydrochloride resulted in [a specific effect / effect] through AUC [a specific mechanism / method]. 0-t The calculated absolute bioavailability was 9.47%; when Beagle dogs received a single oral or intravenous injection of Gelsemium elegans hydrochloride at 1 mg / kg, the bioavailability was [data missing - likely related to bioavailability]. 0-t The calculated absolute bioavailability is 10.3%, indicating that the in vivo exposure of gelsemium elegans after oral administration is low and its bioavailability is not high. Therefore, structural modification of gelsemium elegans to impart superior pharmacokinetic characteristics is of great significance.

[0004] Structural modification using natural products as skeletal molecules is an important approach to new drug discovery. Patent application CN1159 91712A discloses a method for preparing dihydrokoumine, an alkaloid from *Gelsemium elegans* with dual target activities against opioid and muscarinic receptors, and its use in treating pain and other neurological disorders. This dihydrokoumine is obtained by reacting *Gelsemium elegans* with the selective reducing agent LiAlH4. Structurally, it involves the hydrogenation reduction of the double bond on the N1 of the indole ring of *Gelsemium elegans*. Pharmacokinetic experiments show that this compound has good oral bioavailability and high blood-brain barrier permeability. Currently, there are no reports on methods for preparing isodihydrokoumine by reducing the C-18 and C-19 double bonds from *Gelsemium elegans*, and no reports on the activity of isodihydrokoumine have been found. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing isodihydrogelelinin and its application in the preparation of anti-inflammatory and analgesic drugs. Specifically, the compound provided by this invention is isodihydrogelelinin, which is obtained by catalytic hydrogenation of gelelinin as a raw material. The reaction requires only one step, and the product yield and purity are high, with good controllability and reproducibility. Cell experiments show that this compound has superior anti-inflammatory and immunomodulatory effects compared to gelelinin. Pharmacokinetic experiments show that the in vivo exposure of this compound after oral administration is much greater than that of gelelinin, indicating that it has higher bioavailability and good safety. Animal experiments show that this compound has a stronger preventive effect against acute peritonitis in mice than gelelinin and has a significant analgesic effect on acute joint pain in rats with rheumatoid arthritis. The above provides a foundation for its subsequent drug development.

[0006] The isodihydrokoumine described in this invention has the following structural formula:

[0007]

[0008] Furthermore, the isodihydrogespermon described in this invention has the molecular formula C2. 20 H 24 N₂O, white powder, m / z 309.1961 [M+H] as determined by high-resolution mass spectrometry. + .

[0009] The present invention also provides a method for preparing the above-mentioned isodihydrogellanin, comprising the following steps: catalytic hydrogenation of gellanin in an organic solvent under high pressure in the presence of a catalyst to obtain gellanin derivatives.

[0010] Furthermore, in the above technical solution, the organic solvent is selected from tetrahydrofuran, dichloromethane, dimethyl sulfoxide, or N,N-dimethylformamide; tetrahydrofuran is preferred.

[0011] Furthermore, in the above technical solution, the catalyst is selected from platinum on carbon or palladium on carbon; the catalyst content is selected from 1% to 30%, preferably 10%;

[0012] Furthermore, in the above technical solution, the reaction pressure is 1 to 10 atmospheres; preferably 5 atmospheres.

[0013] Furthermore, in the above technical solution, a typical preparation method is as follows: 0.1–5 g of gelsemium elegans is dissolved in 1–100 mL of reaction solvent in a high-pressure reactor, a catalyst is added and hydrogen is introduced, and a catalytic hydrogenation reaction is carried out under heating conditions. After the reaction is complete, the mixture is filtered to remove excess palladium on carbon, the organic phase is washed with saturated brine, and dried over anhydrous sodium sulfate to obtain the crude target product. The purified target product is then obtained by column chromatography.

[0014] The present invention also provides the use of the said isodihydrogespermine and its pharmaceutically acceptable salts in the preparation of anti-inflammatory and analgesic drugs.

[0015] Furthermore, in the above technical solution, the pharmaceutically acceptable salt is selected from hydrochloride, sulfate, hydrobromide, hydroiodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, citrate, acetate, oxalate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pyrazocyanate.

[0016] Further applications include the fact that this compound can significantly inhibit LPS-induced inflammatory responses in RAW264.7 macrophages, has a preventive effect against LPS-induced acute peritonitis in mice, and has a significant analgesic effect on acute joint pain in rats with rheumatoid arthritis. It can be used to prepare anti-inflammatory and immunomodulatory drugs and analgesics.

[0017] The beneficial effects of this invention are:

[0018] This invention provides isodihydrogelelinin, obtained by catalytic hydrogenation of gelelinin as a raw material. The reaction requires only one step, resulting in high product yield and purity, and good controllability and reproducibility. In cell and animal experiments, this compound exhibits superior anti-inflammatory and immunomodulatory effects compared to gelelinin. Pharmacokinetic results show that its in vivo exposure after oral administration is approximately 10 times that of gelelinin, suggesting potentially higher bioavailability and good safety, making it more advantageous for drug development. This invention provides isodihydrogelelinin with significant anti-inflammatory, immunomodulatory, and analgesic effects, offering a candidate molecule for the development of anti-inflammatory and immunomodulatory drugs and analgesics. Attached Figure Description

[0019] Figure 1 The high-performance liquid chromatogram of isodihydrogespermite in Example 1;

[0020] Figure 2 The HRESIMS spectrum of isodihydrogesnezidin in Example 2;

[0021] Figure 3 The isodihydrogellansinin in Example 2 1 H-NMR spectrum (600MHz, DMSO-d6);

[0022] Figure 4 The isodihydrogellansinin in Example 2 13 C-NMR spectrum (400MHz, DMSO-d6);

[0023] Figure 5 This study describes the effects of isodihydrogellansin (KMD) and gellansin (KM) on the levels of (A) IL-6, (B) TNF-α, and (C) NO in LPS-induced RAW264.7 inflammatory cells in Example 3. All data are expressed as Mean ± SEM (n = 4); the positive control was dexamethasone (DXMS). ### p<0.001 vs normal control group; *p<0.05,**p<0.01,***p<0.001 vs model group; ※ p<0.01 Comparison of the same dose groups;

[0024] Figure 6 The drug-time curves (Mean±SEM, n=6) of rats administered 15 mg / kg isodihydrogellecithin (KMD) and gellecithin (KM) by gavage in Example 4 are shown.

[0025] Figure 7 This study investigated the effects of isodihydrogellansin (KMD) and gellansin (KM) on the inflammatory response of LPS-induced acute peritonitis in mice, as described in Example 5. The levels of IL-6 (A), TNF-α (B), and IL-1β (C) in the peritoneal lavage fluid of peritonitis-affected mice, as well as the serum IL-6 (D) level, were determined using ELISA. Dexamethasone (DXMS) was used as the positive control. All data are expressed as Mean ± SEM (n = 6). # p<0.05, ### p<0.001 vs normal control group; *p<0.05, **p<0.01, ***p<0.001 vs model group; ns indicates no significant difference;

[0026] Figure 8This is the effect of isodihydrogellansin (KMD) on the mechanical pain threshold in rats with acute rheumatoid arthritis in Example 6. (A) Schematic diagram of the experimental protocol; (B) Effect of KMD on the mechanical pain threshold of immunized rats. Data for each group are expressed as Mean±SEM (n=6~10); *p<0.05, ***p<0.001 vs model group. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. The following shows and describes the basic principles and main features of the present invention and its advantages. Those skilled in the art should understand that the present invention is not limited to the following embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the present invention as claimed.

[0028] Example 1: Preparation of target gelseminine derivatives

[0029] Gelsemium elegans was dissolved in a reaction solvent in a high-pressure reactor, a catalyst was added, and hydrogen gas was introduced. A catalytic hydrogenation reaction was then carried out under heating conditions to generate the target gelsemium elegans derivative. When the mass-to-volume ratio of gelsemium elegans to the reaction solvent was 15:1, the reaction conditions were screened based on the yield of the target product. The screened conditions included the reaction solvent (tetrahydrofuran, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide), the catalyst (10% palladium on carbon and 10% platinum on carbon), and the pressure (2 atm, 3 atm, 4 atm, 5 atm, and 6 atm). Experimental results showed that under the above reaction conditions, the yield of the target product ranged from 19% to 92%. The highest yield, reaching 92%, was achieved when tetrahydrofuran was used as the reaction solvent, 10% palladium on carbon was used as the catalyst, and the pressure was 5 atm.

[0030] Under the optimized conditions described above, *Gelsemium elegans* seeds were hydrogenated and reduced to obtain the target *Gelsemium elegans* derivative. *Gelsemium elegans* seeds (306 mg, 1 mmol, supplied by the research group) were weighed and dissolved in 20.0 mL of tetrahydrofuran. After complete dissolution, the solution was transferred to a high-pressure reactor equipped with a magnetic stirrer. 50.0 mg of 10% (w / w) palladium on carbon was added to the reactor, and hydrogen was purged three times. Then, hydrogen gas at 5 atmospheres was introduced into the high-pressure reactor, and the reactor was heated to 40 °C for 16 h. After the reaction, the reaction was monitored using thin-layer chromatography (CH3Cl2:CH3OH = 10:1). Once the reactants had completely reacted, excess palladium on carbon was removed by filtration. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solution was mixed with silica gel and purified by column chromatography to obtain 270 mg of the target *Gelsemium elegans* derivative as a white powder, with a yield of 88% and a purity greater than 99.0%. (See [link to relevant documentation]). Figure 1 .

[0031] Example 2: Structural identification of the target gelsemin derivative

[0032] The high-resolution mass spectrum of the target gelsemin derivative is shown below. Figure 2 (G6520B liquid chromatography-quadrupole time-of-flight tandem mass spectrometry, Agilent Technologies, USA), high-resolution mass spectrometry shows a quasi-molecular ion peak at m / z 309.1961 [M+H]. + The molecular formula of the compound is presumed to be C. 20 H 24 N2O; see the carbon and hydrogen NMR spectra. Figures 3-4 (Bruker IMPACT-II spectrometer, Bruker GmbH, Germany) The peak assignments of the 1H and 1C NMR spectra of the target gelsemin derivative are shown in Table 1. The results confirm that the C-18 and C-19 double bonds of the reactant gelsemin are hydrogenated, and the target gelsemin derivative is isodihydrogelsemin.

[0033] Table 1. Peak assignments of the proton (600 MHz, DMSO-d6) and carbon (400 MHz, DMSO-d6) NMR spectra of heterodihydrogellansin.

[0034]

[0035] Example 3: Comparison of the anti-inflammatory and immunomodulatory effects of isodihydrogelsemonin and gelsemonin

[0036] The experimental groups were as follows: control group, LPS group, positive control drug group, and low, medium, and high dosage groups. RAW264.7 macrophages in the logarithmic growth phase were used at a concentration of 1.0 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. Using 100 μM dexamethasone as a positive control, the treatment groups received isodihydrogellansin or gellansin at doses of 50, 100, and 200 μM. After 1 h of drug pretreatment, LPS (1 μg / mL) was added to the LPS group, positive control group, and treatment groups, and cultured for another 24 h. NO content in the cell supernatant was measured using the Griess method, and TNF-α and IL-1β levels in the cell supernatant were detected using an ELISA kit. Results are shown in [Figure number missing]. Figure 5The results showed that pretreatment with 50, 100, and 200 μM isodihydrogelelin significantly reduced the release levels of NO, IL-6, and TNF-α from RAW264.7 macrophages (p<0.01), and this effect was dose-dependent. Gelsemium elegans also inhibited the release of NO, IL-6, and TNF-α in a dose-dependent manner, with significantly lower inhibition of IL-6 release at 50 μM compared to isodihydrogelelin (p<0.01), and significantly lower inhibition of NO release at 200 μM compared to isodihydrogelelin (p<0.01). These results suggest that isodihydrogelelin exhibits superior anti-inflammatory and immunomodulatory effects on LPS-induced RAW264.7 inflammatory cells compared to gelelin.

[0037] Example 4: Comparison of pharmacokinetic characteristics of isodihydrogelsemonin and gelsemonin administered by gavage to rats

[0038] The content of isodihydrogellansin or gellansin in rat plasma was determined by UPLC-MS / MS. The chromatographic conditions used for UPLC-MS / MS were as follows: ZOBXA XDB-C18 column (4.6 × 50 mm, 3.5 μm), flow rate 0.4 mL / min, elution time 4 min, injection volume 5 μL, column temperature 30℃, mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, and the elution method was 28%. Phase B isocratic elution; Mass spectrometry conditions: detection mode positive ion mode, ionization mode electrospray ionization (ESI) source, ion source temperature (TEM) 550℃, spray voltage (IS) 5500V, nebulizing gas (Gas1) 55psi, heating gas (Gas2) 55psi, mass scan range m / z 50-1000, declustering voltage (DP) 226.00V (isodihydrogellansinolide), 197.28V (Gelsemium elegans), 184.83V (internal standard, gelseminine A), collision energy (CE) is 60.84V (isodihydrogelseminine elegans), 62.08V (gelseminine elegans), 34.01V (internal standard, gelseminine A), MRM ion pairs are 309.1→180.0 (isodihydrogelseminine elegans), 307.2→180.1 (gelseminine elegans), 323.1→236.1 (internal standard, gelseminine A); residence time: 500ms.

[0039] Male SD rats were fasted for 12 hours but allowed free access to water. They were then randomly divided into two groups of six rats each. Each group was given a single oral gavage dose of 15 mg / kg of isodihydrogellansin or gellansin. Blood samples were collected from the inner canthus of the rats at 3 min, 6 min, 10 min, 30 min, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h after administration. The blood samples were placed in heparin sodium-treated centrifuge tubes and centrifuged at 3500 rpm at 4 °C for 10 min. The supernatant plasma was collected and stored at -80 °C for analysis. Take 50 μL of thawed plasma sample and place it in a 1.5 mL centrifuge tube. Add 50 μL of serial working solution or 80% acetonitrile blank solution, then add 150 μL of gelseminalis A (internal standard) working solution. Vortex for 3 min, then centrifuge (18000 rpm, 4℃) for 10 min. Take 50 μL of the supernatant and determine it by LC-MS / MS. Calculate the blood drug concentration of rats at each time point using a standard curve and plot the drug-time curve. Figure 6 The pharmacokinetic parameters of isodihydrogelelin and gelelin were fitted using DAS 3.2.8 software to investigate the in vivo pharmacokinetic characteristics of isodihydrogelelin and gelelin after a single oral administration (Table 2). The results showed that isodihydrogelelin and gelelin exhibited similar dynamic trends in vivo, and the in vivo exposure of isodihydrogelelin was more than 10 times higher than that of gelelin, indicating that its bioavailability was significantly higher. Furthermore, no abnormalities were observed in the rats treated with 15 mg / kg isodihydrogelelin in the pharmacokinetic experiment, indicating that it remained safe even with an in vivo exposure 10 times higher than that of gelelin.

[0040] Table 2. Pharmacokinetic parameters of rats administered isodihydrogellanin and gellanin via gavage.

[0041]

[0042] Example 5: Effects of isodihydrogellecithin and gellecithin on LPS-induced inflammatory response in mice with acute peritonitis

[0043] An acute peritonitis model was established in mice. After weighing, the mice were randomly divided into 5 groups of 6 mice each. The groups included a control group, an LPS (1.0 mg / kg) model group, an isodihydrogellansin (15 mg / kg) group, a gellansin seed administration group (15 mg / kg), and a positive control drug dexamethasone (DXMS, 1.0 mg / kg) group. The treatment group and positive control group were administered LPS intraperitoneally, while the control group and LPS model group were administered an equal volume of sterile saline intraperitoneally. One hour later, the LPS model group, isodihydrogelsnezin, gelsnezin treatment group, and positive control group were given 1.0 mg / kg LPS, while the control group was administered an equal volume of sterile saline intraperitoneally. Six hours later, blood was collected from the eyes of mice. The blood was allowed to stand at room temperature for 30 minutes, and serum was collected at 3000 rpm for 5 minutes. IL-6 levels were detected by ELISA. Mice were sacrificed in a CO2 chamber, and the peritoneum was irrigated with 5 mL of PBS. The abdomen was gently massaged for 1 minute to allow the irrigating fluid to permeate the entire peritoneal cavity. A small incision was made in the abdomen with surgical scissors, and the irrigating fluid was drawn into a centrifuge tube. The supernatant was collected at 3000 rpm, 4℃, for 5 minutes. IL-6, IL-1β, and TNF-α levels were detected by ELISA. Figure 7 The results showed that after pretreatment with isodihydrogellusrin, the release levels of IL-6 in mouse serum and IL-6, IL-1β, and TNF-α in peritoneal lavage fluid were decreased to varying degrees, and the differences were statistically significant compared with the LPS model group (p<0.001), suggesting that isodihydrogellusrin has a preventive effect against LPS-induced acute peritonitis in mice. The release levels of IL-1β and TNF-α in peritoneal lavage fluid of mice in the isodihydrogellusrin group were significantly lower than those in the gellusrin group (p<0.05), suggesting that isodihydrogellusrin has a stronger preventive effect against LPS-induced acute peritonitis in mice than gellusrin alone.

[0044] Example 6: Analgesic effect of isodihydrogellansin on joint pain in the acute phase of rheumatoid arthritis in rats.

[0045] A rat model of rheumatoid arthritis (AIA) induced by complete Freund's adjuvant was established. The skin of the right hind paw of each rat was disinfected with 75% alcohol, and 100 μL of 10 mg / mL complete Freund's adjuvant was injected intradermally to induce the AIA model. Using an Electronic Von Frey Aesthesiometer (IITC LifeScience), three different sites were taken on the soles of each hind paw of each rat with a semi-flexible stimulation needle to measure mean mechanical pain (MWT). Rats were kept still during measurements, with 5-minute intervals between each measurement. The mean value of all measurements was used. The baseline mechanical pain threshold was measured one day before modeling. Animals with abnormal MWT (MWT < 35 g) were excluded. Rats with a qualified baseline MWT were randomly assigned to three groups: a control group, a model group, and a group treated with 15 mg / kg isodihydrogellansine.

[0046] Using MWT as a pain assessment index, changes in the mechanical pain threshold of the hind paw in rats were measured during the experiment. The room temperature was maintained at 22-26℃, and the measurement time was consistent. On day 1 (D1) after AIA modeling in rats, a single dose of 15 mg / kg of isodihydrogellecithin was administered. The MWT of the injected paw in each group was measured before administration (0 h) and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 9, 15, and 21 h after administration. The rats were kept quiet during the measurement. Each animal was measured 3 times, with each measurement 5 min apart. Data are expressed as mean ± standard error (Mean ± SEM). A time-effect curve of isodihydrogellecithin was plotted. Figure 8 The results showed that at 0 h, the MWT in the injected paw of the model mice was significantly lower than that in the control group (p<0.001), indicating that the model mice were still in the acute phase and had persistent joint pain symptoms in the injected paw, consistent with the characteristics of the AIA model. 0.5 h after administration, the MWT in the injected paw of the isodihydrogelelinin treatment group was significantly higher than that of the model group (p<0.05), suggesting that the drug could alleviate the pain and sensitivity symptoms of arthritis in the injected paw of the model. Compared with the model group, the analgesic effect of isodihydrogelelinin reached its peak 1 h after administration, then gradually weakened, and the analgesic effect persisted for 9 h (p<0.05). There was no statistically significant difference in analgesic effect after 15 h. These results suggest that isodihydrogelelinin has a significant analgesic effect on joint pain in the acute phase of rheumatoid arthritis in rats, with a clear time-effect relationship and a duration of action of approximately 9–15 h.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. The use of isodihydrogellecithin and its pharmaceutically acceptable salts in the preparation of anti-inflammatory, immunomodulatory, or analgesic drugs, wherein the chemical structure of the isodihydrogellecithin is as follows: 。 2. The use of isodihydrogellansin and its pharmaceutically acceptable salts according to claim 1 in the preparation of anti-inflammatory, immunomodulatory, or analgesic drugs, characterized in that: The pharmaceutically acceptable salts are selected from hydrochloride, sulfate, hydrobromide, hydroiodide, nitrate, hydrogen sulfate, phosphate, acid phosphate, citrate, acetate, oxalate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronide, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pyrazocyanate.

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