Secondary amine local anesthetic and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a secondary amine local anesthetic and application thereof. The secondary amine local anesthetic SPPX.HCl can achieve distinctive retardation of feeling and movement, compared with PPX.HCl, the retardation duration of SPPX is remarkably prolonged, the onset concentration is remarkably reduced, and the secondary amine local anesthetic SPPX.HCl shows high clinical transformation potential.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a secondary amine local anesthetic and application thereof. Background Art

[0002] As a non-opioid analgesic, local anesthetics play an important role in the management of acute and chronic pain. They can be used alone for local anesthesia or in combination with general anesthesia. Most of the local anesthetics used clinically are amphiphilic molecules, usually composed of a lipophilic aromatic ring, an intermediate connecting chain, and a hydrophilic amine. They reversibly block the propagation of action potentials in nerve fibers by blocking voltage-gated sodium ion channels in the cell. The main purpose of the application of local anesthetics is to block the propagation of action potentials in sensory nerves to prevent pain, but traditional local anesthetics will inevitably block motor nerves, and patients often feel numbness or even paralysis of the body. Motor blockade can have adverse effects in many clinical situations that require precise control of muscles or movements, such as motor blockade in labor analgesia for pregnant women, which can threaten the life of newborns and affect patients' rehabilitation training. Therefore, the development of local anesthetics that can achieve sensory selective peripheral nerve blockade will be beneficial to patients.

[0003] Traditional local anesthetics, such as ropivacaine, bupivacaine, etc., block both sensation and movement, and the duration of sensory and motor blockade is almost the same. Inevitably, motor blockade brings great inconvenience to patients. Therefore, in order to achieve sensory selective peripheral nerve blockade, various local anesthetic combination strategies have been widely explored and have achieved a certain degree of success. For example, capsaicin, lidocaine and surfactants are used in combination with QX-314. However, these combination strategies still have some challenges in clinical practice, such as the initial irritation induced by capsaicin and the adverse tissue toxicity caused by surfactants. In addition, QX-314 alone does not produce sensory selective blockade, and it will have certain toxicity to muscle tissue. Therefore, there is still a need to develop safer strategies to achieve sensory selective blockade of peripheral nerves, which is undoubtedly beneficial to patients. Summary of the invention

[0004] Through a systematic analysis of the main endogenous metabolites of traditional local anesthetics, this patent noticed that the dealkylation product of ropivacaine, 2′,6′-piperidine hydrochloride (PPX·HCl), can selectively block peripheral nerves. Here, we use the principle of bioisostericity to replace the carbonyl oxygen of the amide bond of PPX·HCl with a sulfur atom to generate N-(2,6-dimethylphenyl)piperidine-2-thiocarboxamide hydrochloride (SPPX·HCl). The research of the present invention shows that SPPX·HCl can achieve sensory selective peripheral nerve block at a lower dose, and the duration of blockade is significantly prolonged. Given that PPX·HCl has been shown to be widely present in the bodies of patients using local anesthetics and SPPX·HCl is derived from PPX·HCl, the SPPX·HCl invented in this patent has extremely high practical value and great prospects for clinical transformation.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

[0006] In a first aspect, the present invention provides a compound SPPX·HCl, which has the following structure:

[0007]

[0008] SPPX·HCl is a new compound based on the principle of bioisosteres, which is formed by replacing the endogenous metabolite PPX·HCl of local anesthetics with sulfur and oxygen. Compared with PPX·HCl, the duration of blockade of SPPX·HCl is significantly prolonged and the effective concentration is significantly reduced, showing a high potential for clinical transformation. In addition, unlike traditional local anesthetics (local anesthetics commonly used in clinical practice are usually tertiary amine compounds), SPPX·HCl is a secondary amine compound that produces sensory selective blockade of peripheral nerves. SPPX·HCl is a multifunctional local anesthetic. It is not only suitable for local infiltration anesthesia, nerve block anesthesia, treatment and prevention of rapid ventricular arrhythmias, ophthalmic surgery anesthesia, postoperative analgesia and obstetric anesthesia, but also can achieve the effects of epidermal anesthesia and local desensitization. This new anesthetic can provide patients with more comprehensive and effective pain control and comfort experience, while showing significant advantages in reducing postoperative pain and promoting rapid recovery. It has a wide range of applications, from routine surgery to special treatments, and can provide safe and reliable anesthesia effects.

[0009] In a well-established rodent sciatic nerve block model, after anesthetizing rats with oxygen containing isoflurane, a 23-gauge needle was advanced to the posteromedial aspect of the greater trochanter, pointing in an anteromedial direction. After contact with the bone, 0.3 mL of SPPX·HCl solution of varying concentrations was injected, depositing the injection onto the sciatic nerve. Then, every 5 minutes, the sensory and motor blockade of SPPX·HCl was evaluated by a modified hot plate method and an extensor postural thrust test, respectively. The study showed that within a certain range of concentrations, SPPX·HCl could achieve differential sensory and motor blockade.

[0010] In a second aspect, the present invention also provides a method for preparing the aforementioned compound, which comprises the following steps: under the protection of an inert gas, suspending N-(2,6-dimethylphenyl)piperidine-2-carboxamide and Lawesson's reagent in anhydrous 1,4-dioxane, stirring at 90±5°C for 1±0.5h, after the reaction is completed, removing the organic solvent under reduced pressure, suspending the residue in a hydrochloric acid ether solution, stirring for 0.5±0.2h, and then filtering the suspension under reduced pressure, washing the filter cake, and collecting the filter cake.

[0011] Of course, the present invention is not limited to the above-mentioned preparation method, and all methods for preparing the SPPX·HCl of the present invention using the existing technology are within the protection scope of the present invention.

[0012] In a third aspect, the present invention also protects the use of the compound SPPX·HCl described above in the preparation of anesthetic drugs.

[0013] In a specific embodiment, the anesthetic drug is a local anesthetic drug.

[0014] In more specific embodiments, the local anesthetic is a long-acting local anesthetic or a selective local anesthetic.

[0015] In a more specific embodiment, said selective local anesthetic drug is a sensory selective local anesthetic drug.

[0016] In a fourth aspect, the present invention protects a pharmaceutical composition comprising the compound described above.

[0017] In a specific embodiment, the pharmaceutical composition further contains a pharmaceutically acceptable carrier or excipient.

[0018] In a more specific embodiment, the pharmaceutically acceptable carrier or excipient is selected from sodium bicarbonate and physiological saline or water for injection.

[0019] In a specific embodiment, the pharmaceutical composition includes an oral preparation, an injection preparation, a transdermal preparation or a mucosal preparation.

[0020] Beneficial Effects

[0021] (1) Compared with PPX·HCl, SPPX·HCl has a significantly longer blocking duration and a significantly lower effective concentration, showing a high potential for clinical translation;

[0022] (2) SPPX·HCl can achieve differential sensory and motor blockade;

[0023] (3) Unlike traditional local anesthetics (local anesthetics commonly used in clinical practice are usually tertiary amine compounds), SPPX·HCl is a secondary amine compound that produces sensory selective blockade of peripheral nerves. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the dose-effect curve of SPPX·HCl.

[0025] Figure 2 .Study on the cytotoxicity of SPPX·HCl to C2C12 cells.

[0026] Figure 3 .Study on the cytotoxicity of SPPX·HCl on PC12 cells.

[0027] Figure 4 .Toxicity and tissue compatibility evaluation of SPPX·HCl. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0029] Example 1 Synthesis of SPPX·HCl and Evaluation of Its Biological Activity

[0030] 1. Synthesis of related intermediates

[0031] Compound intermediate 1: (S)-2-((2,6-dimethylphenyl)carbamoyl)piperidine-1-carboxylic acid tert-butyl ester

[0032]

[0033] Tert-butyl (S)-2-((2,6-dimethylphenyl)carbamoyl)piperidine-1-carboxylate (1,10 g, 43.61 mmol) was dissolved in anhydrous dichloromethane (DCM) (90 mL) and triethylamine (TEA) (9.1 ml, 65.42 mmol) was added to the solution. The solution was cooled to 4°C with an ice bath and isobutyl chlorocarbonate (6.22 mL, 47.97 mmol) was added dropwise. A white precipitate was observed and the mixture was reacted at 4°C for 1 h. 2,6-Dimethylaniline (2,5.9 mL, 47.97 mmol) was then added dropwise and the mixture was stirred at room temperature for 24 h. The resulting milky mixture was washed with brine (3 x 100 mL) and Na2CO3 (3 x 100 mL). The combined organic phases were dried over anhydrous Na2SO4. After concentration under reduced pressure, the resulting 10.29 g (71%) yellow residue was used directly in the next reaction step without further purification.

[0034] Compound intermediate 2: (S)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide

[0035]

[0036] (S)-2-((2,6-dimethylphenyl)carbamoyl)piperidine-1-carboxylic acid tert-butyl ester (10.29 g) was dissolved in anhydrous dichloromethane (100 mL), and trifluoroacetic acid (16.2 mL, 218.05 mmol) was added dropwise. The mixture was stirred at room temperature for 24 h and concentrated under vacuum. The resulting mixture was diluted with deionized water and its pH was adjusted to 2 with 1 M NaOH solution. The aqueous layer was extracted 3 times with DCM (100 mL), and the combined organic layer was washed twice with saturated NaHCO3 (100 mL), and the organic phase was finally dried over anhydrous Na2SO4. After concentration under reduced pressure, the residue was purified by column chromatography on silica gel (n-hexane / ethyl acetate, 1:1) to obtain a white solid product (4.86 g, yield 68%).

[0037] Target product: SPPX·HCl

[0038]

[0039] Under nitrogen atmosphere, (S)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide (1 g, 4.3 mM) and Lawesson's reagent (0.96 g, 2.37 mM) were dissolved in anhydrous 1,4-dioxane (14 mL), and the mixture was stirred at 90 ° C for 1.5 h. After the reaction was completed, the volatiles were removed under reduced pressure. The residue was suspended in hydrochloric acid ether solution and stirred at room temperature for 30 minutes. The suspension was filtered under reduced pressure, and the filter cake was washed with cold acetonitrile solution to obtain a white solid product SPPX·HCl (558.7 mg, 1.96 mM).1 HNMR(300MHz,D2O)δ7.27–7.10(m,3H),4.30(dd,J=12.0,3.1Hz,1H),3.54–3.44(m,1H),3.11(td,J=12.8,3 .5Hz,1H),2.38(d,J=14.2Hz,1H),2.09(d,J=4.6Hz,6H),1.92(dt,J=25.3,11.7Hz,3H),1.79–1.61(m,2H). 13 C NMR(300MHz,D2O)δ200.5,172.16,169.86,135.31,129.93,52.00,22.91.C 12 H 13 The calculated value of NO4S2 is 248.1347, and the actual value is 248.1342.

[0040] 2. In vitro Cytotoxicity Assessment

[0041] (1) Experimental part

[0042] The toxicity of SPPX·HCl to muscle and nerve was evaluated using C2C12 mouse myoblasts (American Type Culture Collection (ATCC), Manassas, VA, USA) and PC12 rat adrenal pheochromocytoma cells (ATCC, Manassas, VA, USA). Briefly, C2C12 cells were seeded at a density of 1,000 cells per well in DMEM containing 20% ​​FBS and 1% penicillin-streptomycin and then incubated in DMEM containing 2% horse serum and 1% penicillin-streptomycin for 10-14 days to differentiate into myotubes. PC12 cells were seeded at a density of 4,000 cells per well in DMEM containing 2.5% FBS, 12.5% ​​horse serum, and 1% penicillin-streptomycin and then incubated in DMEM containing 1% horse serum, 50 ng / mL nerve growth factor, and 1% penicillin-streptomycin for 7 days.

[0043] The cytotoxicity of SPPX·HCl was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolyl) (MTS) colorimetric assay. C2C12 cells and PC12 cells were treated with different concentrations of SPPX·HCl, and bupivacaine (a local anesthetic commonly used in clinical practice) was used as a positive control drug. After incubation for 24 h, 40 μL of MTS solution was added to each well, and the cells were then incubated for another 4 h. Synergy TMThe absorbance (490 nm) was measured on an Mx microplate reader (BioTek Inc., VT, USA). The viability was expressed as a percentage of the result of untreated cells.

[0044] (2) Experimental results and discussion

[0045] The experimental results showed that the toxicity of bupivacaine and SPPX·HCl to PC12 and C2C12 cells was concentration-dependent, and the survival rate of cells gradually decreased with the increase of compound concentration. The IC50 values ​​of bupivacaine in PC12 and C2C12 cells were 1.12mM and 0.72mM, respectively. In contrast, the IC50 values ​​of SPPX·HCl were significantly increased, and the IC50 values ​​in PC12 and C2C12 cells were 4.51mM and 1.69mM, respectively. In vitro data showed that compared with bupivacaine, SPPX·HCl was less toxic to nerve cells and muscle cells, and its damage to nerves and muscles may also be less.

[0046] 3. Evaluation of SPPX·HCl for peripheral nerve block

[0047] (1) Experimental part

[0048] The experimenter of neurobehavioral testing was blinded to the treatment the animals had received. A modified hot plate test was used to determine the presence and extent of sciatic sensory nerve block. The experimenter, blinded to the treatment the animals had received, placed the animals over a hot plate and placed the animals' hind paws on the 56°C hot plate in triplicate. The time it took for the animals to withdraw their hind paws, i.e., the thermal latency, was measured, and if the animal did not withdraw after 12 seconds, the experimenter removed the hind paw from the hot plate to avoid injury to the animal's hind paw. After the sciatic nerve injection, assessments were performed every 5 minutes until the nerve block subsided. The extensor postural thrust (EPT) test was used to assess motor block. The experimenter suspended the animals' hind paws in sequence (first left and then right) on a digital balance. The maximum weight that the animals could withstand was measured in triplicate.

[0049] (2) Experimental results and discussion

[0050] To determine the concentration dependence of SPPX·HCl and the concentration range that produces sensory selective peripheral blockade, we plotted the concentration (dose)-effect curve of SPPX·HCl. As the concentration increased from 10mM to 15mM, the duration of SPPX·HCl sensory block increased from 4.667±4.163 minutes to 17.5±6.5 minutes, without producing any motor block as defined herein, and the animal behavior was normal. At 20mM, SPPX·HCl produced 23.9±12.03 minutes and 13±12.77 minutes of sensory and motor blockade. At 50mM, SPPX·HCl produced 50.33±7.638 minutes and 46.67±9.609 minutes of sensory and motor blockade, and at higher concentrations, there was no difference in the duration of SPPX·HCl blockade of sensory and motor. These results indicate that the blockade of sensory and motor activity by SPPX·HCl is concentration-dependent. SPPX·HCl only blocks sensation in the concentration range of 10mM to 20mM; further increasing the drug concentration, SPPX·HCl begins to block movement and the duration of sensory and motor blockade gradually prolongs. SPPX·HCl has no selectivity for sensory and motor blockade.

[0051] 4. Histocompatibility assessment of SPPX·HCl

[0052] (1) Experimental part

[0053] Four and fourteen days after SPPX·HCl injection, animals were euthanized by carbon dioxide inhalation to assess inflammation (acute and chronic, respectively) and toxicity of SPPX·HCl. The sciatic nerve and adjacent connective tissue proximal to the site of nerve block injection were obtained using sterile surgical instruments.

[0054] Muscle samples were fixed in 10% neutral buffered formalin solution and paraffin embedded. After sectioning, the muscle was stained with hematoxylin and eosin using standard techniques. Each section was examined in a blinded manner by light microscopy. For each rat evaluated, 4 separate sections of muscle tissue were evaluated and graded according to the most severely damaged area. Muscle samples were scored for inflammation (0-4) and myotoxicity (0-6) as described previously. The inflammation score reflects the subjective assessment of the severity of inflammation, with 0 = none, 1 = minimal, 2 = mild, 3 = moderate inflammation, and 4 = severe inflammation. The myotoxicity score takes into account two independent but related hallmarks of SPPX·HCl myotoxicity, specifically nuclear internalization and regeneration. Nuclear internalization is characterized by normal myocyte size and cytoplasmic color, but the nucleus is far away from its normal peripheral position. Regenerating myocytes are characterized by a shrunken appearance and basophilic cytoplasm. Scoring was as follows: 0 = normal; 1 = perifascicular internalization; 2 = deep internalization (>5 cell layers); 3 = perifascicular regeneration; 4 = deep regeneration; 5 = half-fascicular regeneration; 6 = full-fascicular regeneration.

[0055] The rat sciatic nerve was fixed in Karnovsky's KII solution, treated accordingly, embedded in resin, and stained with toluidine blue. In order to ensure the objectivity of the experimental results, the experimenter evaluated them blindly using an optical microscope.

[0056] (2) Experimental results and discussion

[0057] 15mM SPPX·HCl, which produces sensory selective blockade, is toxic to muscles and nerves. Four days after injection, inflammation was found in rats with lymphocytes and macrophages and a small number of scattered neutrophils. Inflammation in rats was mild (median score was 1.0 (1.0, 2.0) (full score of 4 points). Inflammation was confined to the area around the fascia adjacent to the nerve, i.e., the injection site. No residual inflammation was observed on day 14 after SPPX·HCl injection ( Figure 3 ). All animals showed mild muscle damage on the 4th day after SPPX·HCl injection, including edematous muscle cells, muscle cells with concentrated nuclei, and atrophic muscle fibers (basophilic cytoplasm, i.e., regenerating muscle cells). However, the regenerated muscle cells were limited to the area around the fascia, and there was no myotoxicity in the deep layers of the muscle tissue. The median score was 1.0 (1.0, 3.0) (out of 6 points). Since H&E staining has a relatively low sensitivity for nerve injury, the sciatic nerve was harvested 4 days after SPPX·HCl injection, embedded in Epon, and stained with toluidine blue, and no nerve injury was observed ( Figure 4 ).

[0058] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A compound having the following structure:

2. The method for preparing the compound according to claim 1, characterized in that: The method comprises the following steps: Under the protection of inert gas, N-(2,6-dimethylphenyl)piperidine-2-carboxamide and Lawesson's reagent are suspended in anhydrous 1,4-dioxane and stirred at 90±5°C for 1±0.5h. After the reaction is completed, the organic solvent is removed under reduced pressure, and the residue is suspended in a hydrochloric acid ether solution and stirred for 0.5±0.5h. The suspension is then filtered under reduced pressure, the filter cake is washed, and the filter cake is collected.

3. Use of the compound according to claim 1 in the preparation of anesthetic drugs.

4. The use according to claim 3, characterized in that: The anesthetic drug is a local anesthetic drug.

5. The use according to claim 4, characterized in that: The local anesthetic is a long-acting local anesthetic or a selective local anesthetic.

6. The use according to claim 5, characterized in that: The selective local anesthetic is a sensory selective local anesthetic.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable carriers or excipients.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable carrier or excipient is selected from sodium bicarbonate and physiological saline or water for injection.

10. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition includes oral preparations, injection preparations, transdermal preparations or mucosal preparations.

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