Secondary amine local anesthetics and their use

Abstract

The application belongs to the field of medicine, and particularly relates to a secondary amine local anesthetic and application thereof. The secondary amine local anesthetic SPPX.HCl of the application can realize differential block of sensation and movement, and compared with PPX.HCl, the block duration of SPPX is significantly prolonged, the onset concentration is significantly reduced, and a high clinical conversion potential is shown.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a secondary amine local anesthetic and application thereof. BACKGROUND

[0002] Local anesthetics, as a non-opioid analgesic, play a very important role in the management of acute and chronic pain, which can be used alone for local anesthesia or combined with general anesthesia. Most of the local anesthetics used in clinic are amphipathic molecules, which are usually composed of a lipophilic aromatic ring, a connecting chain and a hydrophilic amine, and can reversibly block the propagation of nerve fiber action potential by blocking voltage-gated sodium ion channels in cells. The main purpose of the application of local anesthetics is to block the propagation of sensory nerve action potential to prevent pain, but the traditional local anesthetics will inevitably block the motor nerves, and the patients will often have a numb feeling, and even cause the paralysis of the body. Motor block will have an adverse effect in many clinical situations that require precise control of muscle or movement, such as motor block in labor analgesia of pregnant women threatening the life of the newborn, motor block affecting the rehabilitation training of patients, etc. Therefore, the development of local anesthetics that can achieve sensory selective peripheral nerve block will be beneficial to patients.

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

[0004] The present patent, through the systematic analysis of the main endogenous metabolites of traditional local anesthetics, we noticed that the dealkylation product of ropivacaine, 2',6'-piperidyl hydrochloride (PPX·HCl), can selectively block peripheral nerves. Here, we use the bioisosteric principle to replace the carbonyl oxygen of the amide bond in PPX·HCl with a sulfur atom, generating N-(2,6-dimethylphenyl)piperidin-2-thiocarboxamide hydrochloride (SPPX·HCl). The present invention shows that SPPX·HCl can achieve sensory selective peripheral nerve block at a lower dose, and the duration of block is significantly prolonged. Given that PPX·HCl has been proven to exist widely in patients using local anesthetics and SPPX·HCl is derived from PPX·HCl, the SPPX·HCl of the present patent invention has very high practical value and great clinical conversion prospects.

[0005] The technical scheme adopted by the present invention to solve its technical problems 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 bioisosteric bodies, which is modified by replacing the sulfur oxygen of the endogenous metabolite of local anesthetics, PPX·HCl. Compared with PPX·HCl, SPPX·HCl has a significantly prolonged duration of block, a significantly reduced onset concentration, and shows high clinical conversion potential. In addition, unlike traditional local anesthetics (commonly used local anesthetics in clinical practice are usually tertiary amine compounds), SPPX·HCl is a secondary amine compound that produces sensory selective block of peripheral nerves. SPPX·HCl is a multifunctional local anesthetic that is not only suitable for local infiltration anesthesia, nerve block anesthesia, treatment and prevention of rapid ventricular arrhythmias, ophthalmic surgery anesthesia, postoperative analgesia, obstetric anesthesia, etc., 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 comfortable experience, while showing significant advantages in reducing postoperative pain and promoting rapid recovery. Its application range is wide, from routine surgery to special treatment, and it can provide safe and reliable anesthetic effect.

[0009] In a perfect rodent sciatic nerve block model, rats were anesthetized with isoflurane in oxygen, and a 23-gauge needle was advanced into the posteromedial aspect of the greater trochanter, pointing in the anteromedial direction. After contacting the bone, 0.3 mL of SPPX·HCl solution with different concentrations was injected, and the injection was deposited on the sciatic nerve. Then every 5 min, the modified hot plate method and the extensor postures thrust test were used to evaluate the sensory and motor block of SPPX·HCl, respectively. The study showed that within a certain concentration range, SPPX·HCl could achieve differential block of sensation and movement.

[0010] In a second aspect, the present application also provides a preparation method of the aforementioned compound, which 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, stirred at 90±5℃ for 1±0.5h, after the reaction is completed, the organic solvent is removed under reduced pressure, the residue is suspended in hydrochloric acid ethyl ether solution, stirred for 0.5±0.2h, then the suspension is filtered under reduced pressure, the filter cake is washed, and the filter cake is collected.

[0011] Of course, the present application is not limited to the above preparation method, and all methods for preparing SPPX·HCl by using the prior art are within the protection scope of the present application.

[0012] In a third aspect, the present application also protects the use of the aforementioned compound SPPX·HCl in the preparation of anesthetics.

[0013] In a specific embodiment, the anesthetics are local anesthetics.

[0014] In a more specific embodiment, the local anesthetics are long-acting local anesthetics or selective local anesthetics.

[0015] In a more specific embodiment, the selective local anesthetics are sensory selective local anesthetics.

[0016] In a fourth aspect, the present application protects a pharmaceutical composition comprising the aforementioned compound.

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

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

[0019] In a specific embodiment, the pharmaceutical composition comprises oral preparations, injection preparations, transdermal administration preparations, or mucosal administration preparations.

[0020] Advantages

[0021] (1) Compared with PPX·HCl, the duration of SPPX·HCl block is significantly prolonged, the onset concentration is significantly reduced, and it shows higher clinical conversion potential;

[0022] (2) SPPX·HCl can achieve differential block of sensation and movement;

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

[0024] Figure 1 The dose-effect curve of SPPX·HCl.

[0025] Figure 2 Cell toxicity study of SPPX·HCl on C2C12.

[0026] Figure 3 Cell toxicity study of SPPX·HCl on PC12.

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

[0028] The application will be further described in detail below in conjunction with examples. If the reagents or instruments used are not specified by the manufacturer, they are all considered to be 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)-tert-butyl 2-((2,6-dimethylphenyl)carbamoyl)piperidine-1-carboxylate

[0032]

[0033] (S)-tert-butyl 2-((2,6-dimethylphenyl)carbamoyl)piperidine-l-carboxylate (1, 10 g, 43.61 mmol) was dissolved in dry dichloromethane (DCM) (90 mL) and the solution was added triethylamine (TEA) (9.1 ml, 65.42 mmol). The solution was cooled to 4°C with an ice bath and isobutyl chloroformate (6.22 mL, 47.97 mmol) was added dropwise. A white precipitate was observed and the mixture was allowed to react for 1 h at 4°C. Then 2,6-dimethylaniline (2, 5.9 mL, 47.97 mmol) was 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%) of 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)-tert-butyl 2-((2,6-dimethylphenyl)carbamoyl)piperidine-l-carboxylate (10.29 g) was dissolved in dry 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 then concentrated under vacuum. The resulting mixture was diluted with deionized water and its pH was adjusted to 2 with a 1 M NaOH solution. The aqueous layer was extracted with DCM (100 mL) three times and then the combined organic layers were washed with saturated NaHCO3(100 mL) twice and finally the organic phase was 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 give the product as a white solid (4.86 g, 68% yield).

[0037] Target product: SPPX-HCl

[0038]

[0039] (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 dry 1,4-dioxane (14 mL) under a nitrogen atmosphere 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 a hydrochloric acid ethyl ether solution and stirred at room temperature for 30 min. The suspension was filtered under reduced pressure and the filter cake was washed with a cold acetonitrile solution to give the product SPPX-HCl as a white solid (558.7 mg, 1.96 mM).1 HNMR (300 MHz, D20) δ 7.27 - 7.10 (m, 3H), 4.30 (dd, J = 12.0, 3.1 Hz, 1H), 3.54 - 3.44 (m, 1H), 3.11 (td, J = 12.8, 3.5 Hz, 1H), 2.38 (d, J = 14.2 Hz, 1H), 2.09 (d, J = 4.6 Hz, 6H), 1.92 (dt, J = 25.3, 11.7 Hz, 3H), 1.79 - 1.61 (m, 2H). 13 C NMR (300 MHz, D20) δ 200.5, 172.16, 169.86, 135.31, 129.93, 52.00, 22.91.C 12 H 13 Calculated for N04S2 248.1347, found 248.1342.

[0040] 2. In vitro cytotoxicity assessment

[0041] (1) Experimental Section

[0042] C2C12 mouse myoblast cells (American Type Culture Collection (ATCC), Manassas, VA, USA) and PC12 rat pheochromocytoma cells (ATCC, Manassas, VA, USA) were used to assess the toxicity of SPPX-HCl on muscle and nerve, respectively. 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 assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) colorimetric assay. C2C12 cells and PC12 cells were treated with different concentrations of SPPX-HCl, with bupivacaine, a commonly used local anesthetic in the clinic, as a positive control. After 24 hours of incubation, 40 μΐ^of MTS solution was added to each well, and the cells were incubated for an additional 4 hours, after which the absorbance was measured at 490 nm. Synergy TMAbsorbance (490 nm) was measured on a Mx microplate reader (BioTek Inc., VT, USA). Survival is expressed as a percentage of the results of untreated cells.

[0044] (2) Experimental results and discussion

[0045] The experimental results show that the toxicity of bupivacaine and SPPX-HCI to PC12 and C2C12 cells is concentration-dependent, and the survival rate of cells gradually decreases with the increase of the concentration of the compounds. The IC50 values of bupivacaine in PC12 and C2C12 cells are 1.12 mM and 0.72 mM, respectively. In contrast, the IC50 values of SPPX-HCI are significantly increased, and the IC50 values in PC12 and C2C12 cells are 4.51 mM and 1.69 mM, respectively. In vitro data show that SPPX-HCI has lower toxicity to nerve cells and muscle cells compared with bupivacaine, and it is also likely to have less damage to nerves and muscles.

[0046] 3. Evaluation of SPPX-HCI on peripheral nerve block

[0047] (1) Experimental section

[0048] The experimenter was blinded to the treatment the animals received. A modified hot plate test was used to determine the presence and extent of sciatic sensory nerve block. The experimenter was blinded to the treatment the animals received, placed the animals above the hot plate, and placed the hind paws of the animals one at a time on the 56°C hot plate in triplicate. The time for the animals to withdraw their hind paws, i.e., the thermal latency, was measured, and if the animals did not withdraw after 12 seconds, the experimenter removed the hind paws from the hot plate to avoid causing injury to the hind paws of the animals. The assessment was performed every 5 min after sciatic nerve injection until the nerve block subsided. The extensor postural thrust (EPT) test was used to assess motor block. The experimenter hung the hind paws of the animals on a digital balance in order (left first, then right). The maximum weight that the animals could bear was measured in triplicate.

[0049] (2) Experimental results and discussion

[0050] To determine the concentration dependence and concentration range of SPPX-HCl to produce sensory selective peripheral block, we plotted the concentration (dose)-effect curve of SPPX-HCl. As the concentration increased from 10 mM to 15 mM, the duration of sensory block by SPPX-HCl increased from 4.667 ± 4.163 min to 17.5 ± 6.5 min without producing any motor block as defined here, and the animal behavior was normal. At 20 mM, SPPX-HCl produced sensory and motor block of 23.9 ± 12.03 min and 13 ± 12.77 min, respectively. At 50 mM, SPPX-HCl produced sensory and motor block of 50.33 ± 7.638 min and 46.67 ± 9.609 min, respectively, and there was no difference in the duration of sensory and motor block by SPPX-HCl at higher concentrations. These results indicate that the block of sensory and motor by SPPX-HCl is concentration dependent. SPPX-HCl produced block of sensory only in the concentration range of 10 mM to 20 mM; further increase in the drug concentration, SPPX-HCl started producing block of motor and the duration of sensory and motor block by SPPX-HCl gradually increased, and SPPX-HCl was not selective for sensory and motor block.

[0051] 4. Tissue compatibility assessment of SPPX-HCl

[0052] (1) Experimental section

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

[0054] Muscle samples were fixed in 10% neutral buffered formalin solution and muscle tissue was embedded in paraffin. After sectioning, the muscle was stained with hematoxylin and eosin using standard techniques. Each section was examined by light microscopy in a blinded fashion. For each rat muscle tissue section evaluated, 4 independent sections were evaluated and graded according to the area of most severe damage. As previously described, inflammation (0-4) and muscle toxicity (0-6) of muscle samples were scored. The inflammation score reflects a subjective assessment of the severity of inflammation, with 0 = none, 1 = minimal, 2 = mild, 3 = moderate inflammation, and 4 = severe inflammation. The muscle toxicity score takes into account two independent but related markers of SPPX-HCI muscle toxicity, specifically nuclear internalization and regeneration. Nuclear internalization is characterized by normal muscle cell size and cytoplasmic color intensity, but nuclei displaced from their normal peripheral location. Regenerating muscle cells are characterized by a shrunken appearance and basophilic cytoplasm. The scores are as follows: 0 = normal; 1 = perifascicular internalization; 2 = deep internalization (> 5 cell layers); 3 = perifascicular regeneration; 4 = deep regeneration; 5 = half bundle regeneration; 6 = full bundle regeneration.

[0055] Rat sciatic nerves were fixed in Karnovsky's KII solution, processed accordingly, embedded in resin, and stained with toluidine blue. To ensure objectivity of the results, the experimenter evaluated the samples in a blinded fashion by light microscopy.

[0056] (2) Experimental Results and Discussion

[0057] Muscle and nerve toxicity of 15 mM SPPX-HCI, which produces a sensory-selective block. Four days after injection, rats exhibited inflammation with lymphocytes and macrophages and a few scattered neutrophils. Rats had mild inflammation (median 1.0 (1.0, 2.0) out of a maximum of 4). Inflammation was localized to the area surrounding the fascia adjacent to the nerve, i.e., the injection site. No residual inflammation was observed 14 days after injection of SPPX-HCI ( Figure 3 ). All animals had mild muscle damage four days after injection of SPPX-HCI, including edematous muscle cells, muscle cells with nuclear condensation, from edematous muscle cells (nuclear condensation) to atrophic muscle fibers (basophilic cytoplasm, i.e., regenerating muscle cells). However, regenerating muscle cells were limited to the area surrounding the fascia and there was no muscle toxicity in the deep muscle tissue (median 1.0 (1.0, 3.0) out of a maximum of 6). Since H&E staining is relatively insensitive to nerve damage, the sciatic nerve was harvested four days after injection of SPPX-HCI, embedded in Epon, and stained with toluidine blue, and no nerve damage was observed ( Figure 4 ).

[0058] The protection scope of the present application is not limited to the above-mentioned embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application and are protected by the appended 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 includes the following steps: Under inert gas protection, N-(2,6-dimethylphenyl)piperidine-2-carboxamide and Lawson's reagent were suspended in anhydrous 1,4-dioxane and stirred at 90±5℃ for 1±0.5h. After the reaction was completed, the organic solvent was removed under reduced pressure, and the residue was suspended in diethyl hydrochloride ether solution and stirred for 0.5±0.5h. Then, the suspension was filtered under reduced pressure, the filter cake was washed, and the filter cake was collected.

3. The use of the compound of claim 1 in the preparation of anesthetic drugs.

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

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

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

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

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient.

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 formulations, injectable formulations, transdermal formulations, or mucosal formulations.

Images