Calcium ion channel regulation activity polysubstituted cyclopropyl carbonyl compound and application thereof

Abstract

The invention discloses a polysubstituted cyclopropyl carbonyl compound with calcium ion channel regulation activity as well as a pharmaceutical composition and application thereof, and belongs to the technical field of medicines. An alpha-chlorocyclobutanone compound is used as a raw material and reacts with aryl magnesium bromide with different substituent groups in an organic solvent, and the polysubstituted cyclopropyl carbonyl compound is obtained in one step. The polysubstituted cyclopropyl carbonyl compound is novel in structure, the preparation method has the advantages of being low in cost, simple and convenient to operate, mild in condition and the like, and the polysubstituted cyclopropyl carbonyl compound novel in structure can be rapidly obtained. The prepared cyclopropyl carbonyl compound has T-type calcium channel regulation activity and has an analgesic effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to a compound containing a multi-substituted cyclopropyl carbonyl group and its pharmaceutical composition, as well as their application in the preparation of analgesic drugs targeting calcium ion channels. Background Technology

[0002] T-type calcium channels, as an important subtype of voltage-gated calcium channels, play a crucial role in pain transmission, regulation of neural excitability, and cardiovascular function, and have become core targets for the development of analgesic, antihypertensive, anti-Parkinson's tremor, and antiepileptic drugs. However, existing clinical-stage drug candidates are mostly based on structural modifications of traditional parent nuclei, sharing the common problem of insufficient subtype selectivity, and long-term use may induce serious adverse reactions such as neurodegenerative diseases. The incomplete three-dimensional structural analysis of these channel proteins hinders structure-based rational drug design, severely restricting the development of novel, highly effective, and low-toxicity modulators. Therefore, developing T-type calcium channel modulators with novel structures, high activity, and high subtype selectivity has become a major challenge in global drug development, urgently requiring the discovery of more novel lead compounds to overcome existing technological bottlenecks.

[0003] Cyclopropanes are widely found in the structures of bioactive natural products and drug molecules. Due to their unique spatial configuration and electronic properties, they occupy an important position in the field of medicinal chemistry and are widely used in the design of small molecule drugs, thereby significantly improving the properties of small molecule drugs. Current synthetic methods for cyclopropane compounds mainly revolve around the following three systems: 1) [2+1] cycloaddition systems based on olefin substrates, involving carbene intermediates (such as transition metal catalytic systems), carbene-like reagents (such as the Simmons-Smith reaction), and thioylides (such as the Corey-Chaykovsky reaction); 2) Kulinkovich cyclopropanization reactions based on carboxylic acid derivatives; and 3) intramolecular cyclization condensation strategies. These methods generally have some drawbacks: they require noble metal catalysts (such as rhodium, ruthenium, etc.), have limited functional group tolerance and require specific directing groups, are highly hazardous in operation and have complex post-processing, and in particular, are difficult to achieve efficient construction of multi-substituted cyclopropyl carbonyl derivatives. Existing literature reports a severe lack of synthetic methods for trisubstituted and more cyclopropyl ketone compounds, which greatly restricts the in-depth development of this type of advantageous skeleton in drug design.

[0004] To date, there are no reports in the prior art regarding the structure and biological activity of polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a new polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3, a pharmaceutical composition comprising the compounds, a method for preparing the composition, and its application in the preparation of pharmaceuticals.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] The following structural formula represents a polysubstituted cyclopropyl carbonyl compound.

[0008]

[0009] R1 is selected from a bromine atom, or one of the following structures:

[0010] .

[0011] R2 is selected from fluorine atoms or methoxy groups.

[0012] The following structural formulas show the polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3.

[0013] .

[0014] The application of the aforementioned multi-substituted cyclopropyl carbonyl compounds in the preparation of T-type calcium channel modulators.

[0015] The use of the polysubstituted cyclopropyl carbonyl compounds according to claim 1 or 2 in the preparation of analgesic drugs.

[0016] A pharmaceutical composition comprising at least one of the aforementioned polysubstituted cyclopropyl carbonyl compounds and a pharmaceutically acceptable carrier.

[0017] The application of the pharmaceutical composition in the preparation of T-type calcium channel modulators.

[0018] The application of the pharmaceutical composition in the preparation of analgesic drugs.

[0019] The method for preparing the polysubstituted cyclopropyl carbonyl compounds includes the following steps:

[0020] Using α-chlorocyclobutanone compounds with R1 substituents as raw materials, aryl magnesium bromide was reacted with them in an organic solvent to obtain polysubstituted cyclopropyl carbonyl compounds as shown in Formula B.

[0021] The reaction formula is as follows:

[0022]

[0023] In formula A, R1 is selected from bromine atoms and X is selected from chlorine atoms;

[0024] In formula B, R1 has the same meaning as R1 in formula A. In formula B, R2 is selected from para-substituted methoxy groups and meta-substituted fluorine atoms.

[0025] The method for preparing the polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3 includes the following steps:

[0026] Preparation of polysubstituted cyclopropyl carbonyl compounds B1: First, olefin feedstock S1 was synthesized according to the method reported in the literature (Angew. Chem. Int. Ed. 60(23), 12859), namely: (4aS,5S,8aS)-5-(2-bromoethyl)-1,1,4a,6-tetramethyl-1,2,3,4,4a,5,8,8a-octahydronaphthalene; Raw material S1 was dissolved in anhydrous diethyl ether, zinc powder was added, and then trichloroacetyl chloride was dissolved in anhydrous diethyl ether and slowly added dropwise to the system. The addition was completed within 1 hour, and the mixture was stirred vigorously at room temperature. After TLC detection showed complete reaction, the solid residue was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain the crude reaction product. The crude reaction product was dissolved in dry tetrahydrofuran, and a commercially available samarium diiodide tetrahydrofuran solution was added at -78°C. The mixture was stirred and the reaction was completed by TLC detection. A saturated potassium carbonate aqueous solution was added, and the mixture was extracted 1-5 times with an equal volume of organic solvent. The extracts were then combined. The organic phase was concentrated under reduced pressure to obtain a crude product, which was then purified by conventional methods to obtain an oily compound α-chlorocyclobutanone compound A1. Under nitrogen protection, compound A1 was dissolved in ethylene glycol dimethyl ether or dioxane, and commercially available 4-methoxyphenyl magnesium bromide was slowly added dropwise at -20-25°C. The mixture was then stirred at -20-25°C for 1-48 hours. After the reaction was confirmed to be complete by TLC, a saturated ammonium chloride aqueous solution was added, and the mixture was extracted 1-4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was then purified by conventional methods to obtain a white solid compound B1.

[0027] Preparation of polysubstituted cyclopropyl carbonyl compound B2: Under nitrogen protection, α-chlorocyclobutanone compound A1 was dissolved in ethylene glycol dimethyl ether. Commercially available 3-fluorophenyl magnesium bromide was slowly added dropwise at -20–25 °C, and the reaction was stirred for 1–48 hours after reaching -20–25 °C. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1–4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was then purified conventionally to obtain a white solid polysubstituted cyclopropyl carbonyl compound B2.

[0028] Preparation of polysubstituted cyclopropyl carbonyl compound B3:

[0029] First, prepare α-chlorocyclobutanone compound A2: using commercially available labda-7,13E-dien-15-oic acid as a raw material, dissolve it in dichloromethane, and add EDCI. TMSEOH and DMAP were reacted at room temperature for 1-12 hours. After TLC confirmation of complete reaction, saturated sodium bicarbonate solution was added to quench the reaction, followed by extraction with organic solvent 1-4 times. The organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was then purified to obtain an oily ester. The ester was dissolved in anhydrous diethyl ether, zinc powder was added, and trichloroacetyl chloride was dissolved in anhydrous diethyl ether and slowly added dropwise over 1 hour until complete. The mixture was stirred vigorously at room temperature. After TLC confirmation of complete reaction, the solid residue was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in dry tetrahydrofuran and added to a commercially available samarium diiodide tetrahydrofuran solution at -78°C. The mixture was stirred and, after TLC confirmation of complete reaction, saturated potassium carbonate aqueous solution was added, followed by extraction with an equal volume of organic solvent 1-5 times. The extracts were then combined. The organic phase was concentrated under reduced pressure to obtain a crude product, which was then purified by conventional methods to obtain an oily compound A2. Then, the polysubstituted cyclopropyl carbonyl derivative B3 was prepared: similar to the synthesis of compound B2, α-chlorocyclobutanone compound A2 was dissolved in ethylene glycol dimethyl ether under nitrogen protection. Commercially available 3-fluorophenyl magnesium bromide was slowly added dropwise at -20-25°C, and the reaction was stirred for 1-48 hours at -20-25°C. After the reaction was confirmed to be complete by TLC, a saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was then dissolved in tetrahydrofuran. Tetrabutylammonium fluoride was added, and after the reaction was confirmed to be complete by TLC, a saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was then purified by conventional methods to obtain a white solid compound B3.

[0030] The organic solvents involved in the above preparation steps are all industrial, analytical or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0031] When used as a medicine, the compounds of the present invention can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the compounds of the present invention, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutically viable carrier for humans and animals.

[0032] The pharmaceutical carrier is one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical excipients. The pharmaceutical composition of the present invention is used in a dose per unit body weight. The drug of the present invention can be administered in various forms (liquid formulation, solid formulation, injection, topical formulation, spray, compound formulation).

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1. This invention provides a new class of polysubstituted cyclopropyl carbonyl compounds, filling a gap in the prior art.

[0035] 2. This invention provides a method for preparing novel polysubstituted cyclopropyl carbonyl compounds. This method uses readily available raw materials, is easy to operate, has a high yield, and is suitable for industrial production.

[0036] 3. This invention provides a novel pharmaceutical composition with a novel multi-substituted cyclopropyl carbonyl compound as the active ingredient, providing a new drug with good pharmaceutical effects for novel T-type calcium channel modulators and analgesics.

[0037] 4. The pharmacological test results of this invention suggest that B1-B3, as a novel 6 / 6 / 3 tricyclic skeleton compound, has unique channel regulation properties; in addition, it also shows that B1-B3 has a potent analgesic effect.

[0038] 5. Polysubstituted cyclopropyl carbonyl compounds can be used as drugs to treat related diseases. They can be used to prepare analgesics and T-type calcium channel modulators. Attached Figure Description

[0039] Figure 1 Compounds B1 and B2 provided by this invention are effective against Ca v 3.2 Effect of loss of activity kinetics. Figures A and B show that compound B1 significantly delayed the degradation of Ca2+ at concentrations of 10 μM and 30 μM. v 3.2 Channel inactivation mechanics, recording representative peak current trajectories. Figure C: Tau values ​​increased from 15.98 ms to 19.68 ms (10 μM) and further increased (30 μM), with the elution effect being irreversible (unpaired two-tailed t-test). Figures D and E: Compound B2 also significantly prolonged Tau values ​​at concentrations of 10 μM and 30 μM. Figure F: Increased from 12.85 ms to 26.87 ms (10 μM) and a more significant change (30 μM), with the inhibitory effect being irreversible at high concentrations. These results indicate that B1 and B2 regulate Ca through a unique mechanism. v 3.2 The inactivation process, whose mode of action differs from that of known active molecules, further supports the novel regulatory properties of this class of 6 / 6 / 3 tricyclic skeleton compounds.

[0040] Figure 2 Compound B3 provided by this invention is effective against Ca v 3.1-3.3, hERG and Na v 1.5 Channel Electrophysiological Study. Figure A: Ca2+ in the presence or absence of 3 μM B3.v 3.2 Representative current-voltage (I (V) relationship. Figure BC: shows the effect of different concentrations of B3 on Ca at clamping voltages (HPs) of −100mV and −75mV. v 3.2 Suppression of Peak Current. Figure D: The effect of B3 on Ca at -100mV and -75mV HPs. v 3.2 Dose-response relationship for peak current suppression. Solid curves represent the fit to the Hill equation. Figure EF: Represents the effect of B3 (3 μM) on Ca v 3.2 Effects of activation and deactivation. The left figure shows the effect of Ca in the presence or absence of B3. v 3.2 Activation or deactivation curves. Solid curves represent the fit to the Boltzmann equation. The upper right corner shows the half-activation potential (V) with or without B3. 1 / 2act ) and half-activation potential (V 1 / 2 inact The comparison of slope factors with and without B3 is shown in the lower right panel. Figure G: shows the Ca of B3 at stimulation frequencies of 0.1 and 1 Hz. v 3.2 Peak current suppression. Figure H: shows the effect of B3 (3μM) on Ca. v 3.2 Effect of B3 on inactivation recovery kinetics. Figure I: shows the effect of B3 on Ca at 3 μM. v 3.1, Ca v 3.2, Ca v Peak current suppression of 3.3. Figure JK: shows the effect of B3 on hERG and Na, respectively. v The effect of 1.5. An unpaired two-tailed t-test was used to evaluate whether the difference was statistically significant.

[0041] Figure 3 Compound B3 provided by this invention has the effect of 3 μM concentration on Ca v 3.1 Suppression of Peak Current. Under a peak current suppression condition of -100 mV, Ca was induced by applying a pulse stimulus with a depolarization period of 250 ms to -40 mV (repeated every 4 seconds). v 3.1 Representative peak current trajectory, normalized to the peak current before drug administration, showed that 3 μM B3 had an effect on Ca v 3.1 Peak current has a significant suppression effect.

[0042] Figure 4 Compound B3 provided by this invention has the effect of 3 μM concentration on Ca v 3.3 Suppression of Peak Current. Under a peak current suppression condition of -100 mV, Ca was induced by applying a pulse stimulation with a depolarization period of 500 ms to -40 mV (repeated every 4 seconds). v3.3 Representative peak current trajectories were analyzed and normalized to the peak current before drug administration. The results showed that 3 μM B3 had an effect on Ca v 3.3 The peak current suppression is relatively weak, further verifying its effect on Ca. v 3.2 Selective inhibition characteristics of subtypes.

[0043] Figure 5 The compound B3 provided by this invention affects Ca at different concentrations v 3.2 Suppression of Peak Current. Under a peak current suppression condition of -100 mV, Ca was induced by a pulse stimulation (repeated every 4 seconds) that depolarized to -40 mV over 250 ms. v 3.2 Representative peak current trajectories were obtained, and the inhibitory effect of compound B3 at specified concentrations was recorded. All current data were normalized based on the peak current before drug administration to quantify the degree of drug inhibition of channel currents.

[0044] Figure 6 The compound B3 provided by this invention affects Ca at different concentrations v 3.2 Suppression of Peak Current. Under a peak current suppression condition of -75 mV, Ca was induced by a pulse stimulation (repeated every 4 seconds) that depolarized to -40 mV for 150 ms. v 3.2 Representative peak current trajectories were obtained, and the currents were normalized based on the peak current before drug administration to evaluate the inhibitory effect of B3 on channel currents at specified concentrations.

[0045] Figure 7 Z944 is a positive control drug for Ca v 3.2 Inhibition of the channel. Figure A: Under HP of -100 mV, Ca was recorded by applying a pulse stimulation of 250 ms depolarization to -40 mV (repeated every 4 seconds). v 3.2 Representative peak current trajectories, comparing current changes in the blank control (Bath) and treatments with different concentrations of Z944. Figure B: Further analysis shows that Z944 affects Ca... v 3.2 The suppression of peak current exhibits a dose-dependent effect. The dose-response curve based on HP -100 mV was fitted by the Hill equation (solid line). The mean ± standard error (SEM) of five independent measurements shows its half-maximal inhibitory concentration (IC50). 50 The value was 0.54 μM.

[0046] Figure 8 The compound B3 provided by this invention has the effect of reacting with Na at a concentration of 3 μM. v1.5 Effect of Peak Current. Under a peak current (HP) of -110 mV, Na+ was induced by applying a 30 ms depolarization pulse stimulation to -30 mV (repeated every 5 seconds). v 1.5 Representative peak current trajectories were plotted and normalized to the peak current before drug administration. The results showed that 3 μM B3 had an effect on Na v The peak current of 1.5 showed no significant suppression effect, indicating that it had no effect on the sodium channel Na. v The 1.5 subtype has a lower risk of cardiovascular off-target effects.

[0047] Figure 9 The effect of compound B3 provided in this invention on hERG channels at a concentration of 3 μM was investigated. hERG current-voltage (IV) curves were recorded at an HP of -80 mV using the following method: a 2000 ms incremental voltage step (range: -60 to +60 mV, step size 10 mV) was applied, followed by repolarization to -50 mV and maintenance for 2000 ms to induce tail current, with an 8-second pulse interval. The experimental results showed that 3 μM B3 had no significant effect on the current-voltage curves and tail current amplitude of the hERG channels, indicating a low risk of hERG-related cardiovascular side effects.

[0048] Figure 10 The present invention illustrates the allergic effects of compound B3 in different mouse pain models. Figures AB show that compound B3 dose-dependently reduced the number of writhing responses induced in a mouse visceral pain model induced by intraperitoneal injection of 0.6% acetic acid (AA) and prolonged the initial writhing latency. Results were analyzed using one-way ANOVA combined with Tukey's multiple comparison test. There was no significant difference between the B3 (20 / 30 mg / kg) and positive control Z944 (10 mg / kg) groups (NS). Figure C shows that B3 (10-30 mg / kg) significantly alleviated mechanical hyperalgesia in a paclitaxel-induced peripheral neuropathy model. Figure D shows the area under the curve (AUC) calculated at 0-6 hours using von Frey fibers to determine the paw withdrawal threshold (PWT). The results showed that its effect was superior to the PTX group (using Brown-Forsythe and Welch ANOVA combined with Tamhane T2 multiple comparison test), but there was still no statistically significant difference between the high-dose B3 and Z944 groups (NS).

[0049] Figure 11This invention relates to the analgesic effect of compound B3 on paclitaxel (PTX)-induced mechanosensitive hyperalgesia in wild-type (WT) mice. Compound B3 significantly alleviated PTX-induced mechanosensitive hyperalgesia at doses of 10 mg / kg (Figure A), 20 mg / kg (Figure B), and 30 mg / kg (Figure C). The positive control Z944 (10 mg / kg, Figure D) also showed a similar analgesic effect. Statistical analysis using two-way ANOVA combined with the Benjamini-Krieger-Yekutieli two-stage stepwise multiple comparison test confirmed that compound B3 effectively reduced neuropathic pain-related behavioral phenotypes at different doses.

[0050] Figure 12 This is a schematic diagram of the structures of the polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3 of this invention. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1

[0053] Preparation of compound B1.

[0054] 1. Preparation of α-chlorocyclobutanone compound A1:

[0055] The method reported in the reference (Angew. Chem. Int. Ed. 60(23), 12859) can synthesize olefin raw material S1, namely (4aS,5S,8aS)-5-(2-bromoethyl)-1,1,4a,6-tetramethyl-1,2,3,4,4a,5,8,8a-octahydronaphthalene. Olefin S1 (0.01–10 g) was dissolved in anhydrous diethyl ether (0.01–100 mL), zinc powder (0.01–20 g) was added, and trichloroacetyl chloride (0.01–10 mL) was dissolved in anhydrous diethyl ether (0.01–100 mL) and slowly added dropwise to the system, completing the addition within 1 hour. The mixture was stirred vigorously at room temperature. After the reaction was confirmed to be complete by TLC, the solid residue was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the crude reaction product. The crude product was dissolved in dry tetrahydrofuran (0.01–100 mL), and a commercially available tetrahydrofuran solution of samarium diiodide (0.01–300 mL) was added at -78 °C. The mixture was stirred and reacted. After the reaction was confirmed to be complete by TLC, a saturated potassium carbonate aqueous solution was added, and the mixture was extracted 1–5 times with an equal volume of organic solvent. The organic phases were combined, concentrated under reduced pressure, and the crude product was obtained. The crude product was purified by conventional methods to obtain an oily substance, which was identified as compound A1 (yield 20%–80%) by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

[0056] The organic solvents are industrial, analytical, or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0057]

[0058] The characterization of compound A1 is as follows: [α]20 D = –34.94 (c = 0.245, methanol); 1H NMR (400 MHz, CDCl3) δ = 4.54(d, J = 2.5 Hz, 1H), 3.62–3.45 (m, 2H), 3.11–3.05 (m, 1H), 2.08–1.95 (m, 3H),1.65–1.59 (m, 1H), 1.59–1.53 (m, 1H), 1.56 (s, 3H), 1.53–1.42 (m, 3H), 1.42–1.35 (m, 1H), 1.13 (td, J = 13.3, 4.1 Hz, 1H), 1.06 (dd, J = 5.6, 4.1 Hz,1H), 0.89 (s, 3H), 0.89 (s, 3H), 0.88–0.82 (m, 1H), 0.82 (s, 3H) ppm; 13 C NMR (125 MHz, CDCl3) δ = 200.58, 71.98, 59.43, 52.30, 50.06, 41.76, 39.22, 38.98,37.38, 33.13, 32.98, 32.93, 32.49, 25.13, 21.37, 18.04, 17.74, 14.42 ppm; HRMS (ESI) m / z: [M + H] + calcd for C 18 H 29 BrClO 375.1085, found 375.1090.

[0059] 2. Preparation of polysubstituted cyclopropyl carbonyl derivative B1:

[0060] Under nitrogen protection, compound A1 (0.01–100 g) was dissolved in (0.1–100 mL) of ethylene glycol dimethyl ether or dioxane. Commercially available 4-methoxyphenyl magnesium bromide (1.0 M tetrahydrofuran solution, 0.1–100 mL) was slowly added dropwise at -20–25 °C, followed by stirring for 1–48 hours at -20–25 °C. After the reaction was confirmed to be complete by TLC, a saturated ammonium chloride aqueous solution was added, followed by extraction with an organic solvent 1–4 times. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was then purified conventionally to obtain a white solid. The solid was identified as compound B1 by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance (NMR) (yield 65%–90%).

[0061] The organic solvents are industrial, analytical, or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0062]

[0063] The characterization of compound B1 is as follows: [α]20 D = +92.4 (c = 0.249, methanol); 1 H NMR (400 MHz, CDCl3): δ = 7.90 (d,J = 8.9 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H), 3.27 (td, J = 10.0,4.2 Hz, 1H), 2.87 (td, J = 9.5, 7.2 Hz, 1H), 2.24 (d, J = 8.2 Hz, 1H), 2.12(dd, J = 14.7, 6.7 Hz, 1H), 2.07–1.96 (m, 1H), 1.93–1.81 (m, 1H), 1.81–1.70(m, 1H), 1.62–1.41 (m, 4H), 1.41–1.30 (m, 2H), 1.34 (s, 3H), 1.27–1.09 (m,2H), 0.96–0.81 (m, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H) ppm; 13 C NMR (150 MHz, CDCl3): δ = 197.72, 162.96, 132.67, 129.84, 113.58, 55.43, 49.66,45.72, 41.98, 38.02, 36.58, 35.29, 33.87, 33.17, 32.43, 29.59, 29.36, 28.95,26.35, 21.27, 18.87, 17.60, 14.77 ppm; HRMS (ESI) m / z: [M+H] + calcd forC 25 H 36 BrO2 447.1893, found 447.1899.

[0064] Example 2

[0065] Preparation of polysubstituted cyclopropyl carbonyl compound B2.

[0066] Under nitrogen protection, α-chlorocyclobutanone compound A1 (10-100 mg) was dissolved in ethylene glycol dimethyl ether (0.1-10 mL). Commercially available 3-fluorophenyl magnesium bromide (1 M tetrahydrofuran solution, 0.1-20 mL) was slowly added dropwise at -20-25°C, and the reaction was stirred for 1-48 hours after reaching -20-25°C. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain a crude product, which was purified conventionally to obtain a white solid. The solid was identified as compound B2 (yield 70%-85%) by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

[0067] The organic solvents are industrial, analytical, or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0068]

[0069] The characterization of compound B2 is as follows: [α]20 D = +53.9 (c = 0.186, methanol); 1 H NMR (400 MHz, CDCl3): δ = 7.69 (d,J = 7.7 Hz, 1H), 7.63–7.55 (m, 1H), 7.48–7.38 (m, 1H), 7.27–7.19 (m, 1H), 3.34–3.24 (m, 1H), 2.92 (q, J = 8.7 Hz, 1H), 2.27 (d, J = 8.0 Hz, 1H), 2.12–1.96 (m, 2H), 1.94–1.83 (m, 1H), 1.83–1.72 (m, 1H), 1.66–1.51 (m, 3H), 1.51–1.37 (m, 3H), 1.35 (s, 3H), 1.28–1.14 (m, 2H), 0.99–0.79 (m, 1H), 0.88 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H) ppm; 13C NMR (150 MHz, CDCl3): δ = 197.68,163.68, 162.03 (d, J = 247.7 Hz), 141.76 (d, J = 6.0 Hz), 130.08 (d, J = 7.7Hz), 123.36 (d, J = 2.9 Hz), 119.33 (d, J = 21.3 Hz), 114.41 (d, J = 22.2Hz), 49.63, 45.10, 41.96, 38.00, 36.63, 35.85, 33.89, 33.02, 32.41, 31.00,30.09, 29.30, 26.40, 21.21, 18.85, 17.58, 14.78 ppm; 19 F NMR (471 MHz, CDCl3): δ = -112.12 ppm; HRMS (ESI) m / z: [M+H] + calcd for C 24 H 33 BrFO 435.1693, found435.1691.

[0070] Example 3

[0071] Preparation of compound B3.

[0072] 1. Preparation of α-chlorocyclobutanone compound A2:

[0073] Using commercially available labda-7,13E-dien-15-oic acid as a raw material, 0.1-1 g of it was dissolved in 0.1-200 ml of dichloromethane. EDCI [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] (0.2-2 g), TMSEOH [2-(trimethylsilyl)ethanol] (0.1-2 g), and DMAP (4-dimethylaminopyridine, 0.2-2 g) were added. The reaction was carried out at room temperature for 1-12 hours. After the reaction was checked by TLC to be complete, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure, and the crude product was obtained. The crude product was then purified by conventional methods to obtain the pure oily esterified product. The esterified compound (0.01–10 g) was dissolved in anhydrous diethyl ether (0.01–100 mL), and zinc powder (0.01–20 g) was added. Trichloroacetyl chloride (0.01–10 mL) was then dissolved in anhydrous diethyl ether (0.01–100 mL) and slowly added dropwise to the system, completing the addition within 1 hour. The mixture was stirred vigorously at room temperature. After the reaction was confirmed to be complete by TLC, the solid residue was filtered through diatomaceous earth. The resulting filtrate was concentrated under reduced pressure to obtain the crude reaction product. The crude product was dissolved in dry tetrahydrofuran (0.01–100 mL), and commercially available samarium diiodide tetrahydrofuran solution (0.01–300 mL) was added at -78°C. The mixture was stirred and reacted. After the reaction was confirmed to be complete by TLC, saturated potassium carbonate aqueous solution was added, and the mixture was extracted 1–5 times with an equal volume of organic solvent. The organic phases were combined, concentrated under reduced pressure, and the crude product was obtained. The crude product was purified by conventional methods to obtain an oily substance, which was identified as compound A2 (yield 40%–80%) by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

[0074] The organic solvents are industrial, analytical, or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0075]

[0076] The characterization of compound A2 is as follows: 1H NMR (600 MHz, CDCl3): δ = 5.62 (s, 1H), 4.52 (d, J = 2.4 Hz, 1H), 4.20–4.15 (m, 2H), 3.08–3.03 (m, 1H), 2.39–2.29 (m, 2H), 2.16 (s, 1.03–0.93 (m, 4H), 0.90–0.85 (m, 1H), 0.89 (s,3H), 0.88 (s, 3H), 0.82 (s, 3H), 0.04 (s, 9H) ppm; 13 C NMR (150 MHz, CDCl3): δ= 200.98, 167.18, 160.58, 115.40, 72.14, 61.65, 59.29, 51.76, 50.11, 42.06,41.86, 39.49, 39.42, 37.85, 33.16, 33.00, 26.25, 25.26, 21.44, 18.93, 18.11,17.79, 17.45, 14.57, −1.51 ppm; HRMS (ESI) m / z: [M+Na] + calcd forC 27 H 45 ClO3SiNa 480.2827, found 503.2719.

[0077] 2. Preparation of polysubstituted cyclopropyl carbonyl derivative B3:

[0078] Similar to the synthesis of compound B2, under nitrogen protection, α-chlorocyclobutanone compound A2 (10-300 mg) was dissolved in (0.1-50 mL) ethylene glycol dimethyl ether, and commercially available 3-fluorophenyl magnesium bromide (1M tetrahydrofuran solution, 0.1-100 mL) was slowly added dropwise, and the reaction was stirred at -20-25°C for 1-48 hours. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in (0.1-50 mL) tetrahydrofuran, and tetrabutylammonium fluoride (1M tetrahydrofuran solution, 0.1-20 mL) was added. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined and concentrated under reduced pressure to obtain the crude product. The crude product was purified by conventional methods to obtain a white solid, which was identified as compound B3 (yield 65%-80%) by mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance.

[0079] The organic solvents are industrial, analytical, or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

[0080]

[0081] The characterization of compound B3 is as follows: [α]20 D = +31.74 (c = 0.218 in MeOH); 1 H NMR (400 MHz, CDCl3) δ 7.73(d, J = 7.7 Hz, 1H), 7.67–7.59 (m, 1H), 7.51–7.41 (m, 1H), 7.30–7.21 (m, 1H),5.53 (s, 1H), 2.31 (d, J = 8.0 Hz, 1H), 2.17–1.97 (m, 2H), 2.04 (s, 3H), 1.87–1.74 (m, 1H), 1.73–1.51 (m, 5H), 1.51–1.35 (m, 4H), 1.38 (s, 3H), 1.32–1.16 (m, 1H), 1.08–1.00 (m, 1H), 0.96–0.84 (m, 1H), 0.91 (s, 3H), 0.90 (s, 3H), 0.86 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 197.82, 171.17, 164.11, 161.65(d, J = 247.6 Hz), 163.06, 141.80 (d, J = 5.5 Hz), 130.04 (d, J = 7.7 Hz), 123.45 (d, J = 2.9 Hz), 119.26 (d, J = 21.6 Hz), 114.53 (d, J = 22.4 Hz), 114.42, 49.84, 46.20, 42.10, 40.98, 38.18, 36.79, 36.14, 33.93, 32.46, 31.55, 30.14, 26.35, 22.75, 21.25, 19.23, 18.93, 17.65, 14.66. 19 F NMR (471 MHz, CDCl3) δ -112.16; HRMS (ESI) m / z: [M+ Na] + calcd for C 28 H 37 FO3Na 463.2619,found 463.2633.

[0082] Example 4

[0083] Compounds B1-B3 of this invention affect Ca v 3.1-3.3, Na v 1.5 The experimental methods and results of hERG channel regulation are as follows:

[0084] 1. Cell preparation and expression

[0085] HEK 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin (100 U / ml) / streptomycin (0.1 mg / ml). HEK 293T cells were transiently transfected with the following plasmid: pCDNA3.1-human Na+ using LipoD293 in vitro DNA transfection reagent. v 1.5, human Ether-à-go-go-Related Gene (hERG), human Ca v 3.1, human Ca v 3.2, human Ca v 3.3 pEGFPN1, experiments were conducted within 48 hours after transfection.

[0086] 2. Electrophysiological experiments

[0087] All experiments were conducted at room temperature (approximately 22°C). Borosilicate glass pipettes with impedances of 4–6 MΩ were fabricated using a microelectrode puller (P-1000, Sutter Instrument) and thermopolishing (World Precision Instruments) for whole-cell current recording. The current was amplified using a Double IPA patch-clamp amplifier (Sutter Instrument), which integrates signal amplification and data acquisition. The current signal was low-pass filtered (cutoff frequency 2 kHz) and acquired at a sampling rate of 10 kHz. Data acquisition and analysis were performed using SutterPatch 2.1 software (Sutter Instrument). Data were only included in the analysis when the initial series resistance was below 25 MΩ and the resistance change during recording did not exceed 30%.

[0088] Record Ca v 3.1-3.3 The extracellular solution during current recording contained (in mM) 10⁵ CsCl, 40 TEA-Cl, 2 CaCl₂, 1 MgCl₂, 10 D-glucose, and 10 HEPES (pH = 7.4, adjusted with CsOH). The intracellular solution contained (in mM) 130 CsCH₃SO₃, 10 TEA-Cl, 10 EGTA, 10 HEPES, 5 MgCl₂, and 5 Na²⁻ATP (pH = 7.4, adjusted with CsOH). The current recording program was as follows: Ca v 3.1 and Ca v 3.2 Starting from the clamping potential (HP) of -100 mV or -75 mV, apply a -40 mV depolarization pulse every 4 seconds for 250 ms to induce peak current. v 3.3 The stimulation time was extended to 500 ms, and other parameters remained the same as above.

[0089] In order to study Ca v 3.2 Usage Dependence: The repetitive stimulation frequencies were set to 0.1 Hz and 1 Hz, respectively, to analyze the dependence of channel activity on the stimulation frequency. v 3.2 The current-voltage (IV) curves of the channel were recorded by applying a depolarization pulse lasting 250 ms with an HP of -100 mV, the voltage range being from -90 mV to +40 mV (5 mV step, 1 second pulse interval).

[0090] Ca v3.2 The voltage-dependent activation of the channel was determined as follows: A series of step depolarization pulses (5 mV step, 1 s interval) ranging from -90 mV to +40 mV, lasting 250 ms, were applied at an HP of -100 mV. The peak current at each voltage was recorded, and the voltage was then calculated using the formula G = I / (V − V). rev Calculate the corresponding conductance value (G), where V is the test voltage. rev (Reversal potential) is obtained by linear extrapolation of the peak current at depolarization potentials from +10 to +40 mV. The normalized conductance (G / Gmax) is plotted against voltage, and the activation curve is fitted using the Boltzmann equation: G / G max = 1 / (1 + exp((V 1 / 2 -V) / k)). In the formula, G max V is the maximum conductivity value. 1 / 2 is the half-activation voltage, and k is the slope factor.

[0091] Ca v 3.2 The inactivation characteristics of the channels were studied using the following method: Under a HP of -110 mV, a series of 250 ms inactivation step pulses (voltage range: -100 to -30 mV, step size 5 mV, pulse interval 4 seconds) were applied. The current amplitude P1 induced by a -40 mV (10 ms) voltage step pulse was then recorded without rapid inactivation. After the above inactivation pulse sequence, the same -40 mV pulse with a current amplitude P2 was applied again. A 1-second recovery period (-110 mV) was set after P1 to eliminate any potential inactivation. The ratio of P2 / P1 reflects the proportion of available channels. The normalized residual current was plotted against the conditional pulse voltage, and the inactivation curve was fitted using the Boltzmann equation: I / I max = 1 / (1 + exp((V 1 / 2 In the formula − V) / k)), I max For the maximum current, V 1 / 2 is the half-deactivation voltage, and k is the slope factor.

[0092] Cell inactivation recovery characteristics were determined using a dual-pulse stimulation protocol: Cells were initially stimulated at -100 mV HP. A first -40 mV (20 ms) pulse was applied, followed by a hyperpolarization pulse (duration variable) at the same HP value to induce channel recovery. Finally, a second -40 mV (50 ms) test pulse was applied. The channel recovery ratio was calculated by the ratio of the second test pulse current to the first conditional pulse current, and this ratio was plotted against recovery time. The recovery curves were obtained by fitting a biexponential function.

[0093] Record Na vAt current 1.5, the intracellular fluid composition (in mM) consisted of 135 CsF, 5 KCl, 2 MgCl2, 2 Na2-ATP, 10 HEPES, 10 EGTA, and 5 NaCl2, adjusted to pH 7.4 with CsOH. The extracellular fluid composition (in mM) consisted of 25 NaCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 122 TEA-Cl, and 10 Glucose, adjusted to pH 7.4 with NaOH. The current recording program was as follows: starting from a pH of -110 mV, a -30 mV depolarization pulse was applied every 5 seconds for 30 ms to induce a peak current.

[0094] The extracellular fluid composition for recording hERG channel currents consisted of (in mM) 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 Glucose, 10 HEPES, and 5 TEA-Cl (adjusted to pH 7.4 with NaOH). The intracellular fluid composition consisted of (in mM) 130 KCl, 1 MgCl2, 5 EGTA, 5 Mg-ATP, and 10 HEPES (adjusted to pH 7.4 with KOH). The current recording program was as follows: starting at -80 mV, a 2000 ms incremental voltage step (range: −60 to +60 mV, step size 10 mV) was applied, followed by repolarization to -50 mV and maintenance for 2000 ms to induce tail currents, with an 8-second pulse interval.

[0095] 3. Data Analysis

[0096] Data collection and statistical analysis were performed using GraphPad 8.0.1 (GraphPad Software, San Diego, CA USA). IC 50 The values ​​and Hill coefficients are obtained through the Hill equation Y = I Min + (I Max - IC Min ) / [1+10(LogEC 50 [-C)×Hillslope] is calculated using the collected data. Here, IC 50 It is the concentration when the channel current is suppressed by half, C is the concentration of the compound, and I is the concentration of the compound. Min It is the minimum inhibition rate, I Max This represents the maximum inhibition rate, and Hillslope is the Hill coefficient. All data are mean ± standard deviation.

[0097] 4. Compounds B1-B3 affect Ca v 3.1-3.3 Calcium channel inhibitory activity experiments, such as... Figure 1 As shown.

[0098] Preliminary studies of compounds B1 and B2 revealed that at a concentration of 10 mM, B1 and B2 had an effect on Ca2+. v 3.2 The peak current has a weak effect, but significantly delays its loss of kinetic energy, as evidenced by the increase in Tau value from 15.98 ms to 19.68 ms (B1) and from 12.85 ms to 26.87 ms (B2). Figure 1 (AF), and the post-elution effect is irreversible ( Figure 1 (A, B, D, E); at 30 mM, both affect Ca v 3.2 The retardation effect of deactivation of biomechanics is enhanced, with B1 at this concentration exerting a moderate inhibition on the peak current. Figure 1 (B)

[0099] Whole-cell patch-clamp experiments (using plasmids constructed by inserting human cDNA into the pCDNA3.1(+) vector) showed that 3 μM B3, in an HP range of −100 mV and a voltage step of −50 to 0 mV, was significantly better than Ca2+. v 3.1 and Ca v 3.3 Subtype, B3 against Ca v The inhibitory effect of 3.2 is more significant, and this inhibitory effect is essentially reversible after elution. Figure 2 in A and I, Figure 3 , Figure 4 (as shown in Table 1), and this effect is also voltage-dependent.

[0100] Experimental results show that when HP is −100 mV, the IC50 of B3 inhibition is [value missing]. 50 The concentration was 3.10 μM, while the IC50 of B3 inhibition was 75 mV. 50 1.10 μM ( Figure 2 Chinese BD, Figure 5 , Figure 6 (and Table 2), while the positive control Z944 showed stronger inhibitory activity (IC50). 50 = 0.54 ± 0.03 μM, Figure 7 (and Table 2). Further analysis showed that 3 μM B3 reduced Ca... v 3.2 half-conductivity potential (V 1 / 2act The value shifted left from −52.69 ± 0.86 mV to −56.98 ± 0.81 mV. Figure 2 (middle D), half-inactivation potential (V) 1 / 2 inact The value shifted left from −58.98 ± 0.36 mV to -62.63 ± 0.58 mV. Figure 2 While it showed a moderate effect on the activity level (F), it had a negligible impact on the slope factors of the activation and inactivation curves. B3 (3 μM) exhibited significant use-dependent inhibitory properties, showing a strong effect on Ca2+ at a stimulation frequency of 1 Hz.v 3.2 The peak current blocking speed and intensity are both higher than 0.1 Hz ( Figure 2 G), but it had no significant effect on its inactivation recovery curve (G), Figure 2 (H). This type of regulation pattern is similar to known Ca. v 3.2 The different regulators suggest that B1-B3, as a novel 6 / 6 / 3 tricyclic skeleton compound, has unique channel regulation properties.

[0101] 5. The effect of compound B3 on cardiovascular toxicity target ion channels.

[0102] Na, a cardiovascular off-target risk target that needs to be assessed in the early stages of drug development. v 1.5 and hERG (recommended by the US FDA and European EMA), 3 mM B3 had no significant effect on either ( Figure 2 J, K, Figure 8-9 The above results indicate that B3 can act as a target for Ca. v 3.2 Optimized prototype of a novel analgesic lead compound, and B3 for Ca v 3.2 It exhibited a different regulatory effect than PN-4 and PN-403.

[0103] Table 1 shows the effects of compound B3 and positive control Z944 provided in this invention on Ca. v 3.2 Suppression of peak current. The text mentions "compound B3..." −75 mV "and "compound B3 −100 mV "" represents the average inhibition rate of the channel current by compound B3 at clamping potentials (HP) of -75 mV and -100 mV, respectively.

[0104] Table 2 shows the effect of compound B3 provided in this invention on Ca at a concentration of 3 μM. v 3.1 and Ca v 3.3 Suppression of peak current. The average suppression rate of channel current by compound B3 under a clamping potential of -100 mV.

[0105] Table 1. Effects of compound B3 and positive control Z944 on Ca v 3.2 Suppression of Peak Current

[0106] Table 2. Effect of compound B3 on Ca at 3 μM concentration v 3.1 and Ca v 3.3 Suppression of Peak Current

[0107] Example 5

[0108] The experimental methods and results of the present invention's compound B3 in relieving acetic acid-induced visceral pain are as follows:

[0109] I. Experimental Methods:

[0110] Before the experiment, SPF-grade 8-10 week old C57BL / 6 mice were placed in an acrylic box for 1 hour to acclimatize. Then, they were injected intraperitoneally with solvent (10 ml / kg), B3 (10, 20, 30 mg / kg) or Z944 (10 mg / kg). 30 minutes later, they were injected with 0.6% acetic acid (10 ml / kg). The number of abdominal writhing movements (defined as abdominal muscle contraction accompanied by trunk extension and hind limb extension) and the time of the first writhing movement were recorded for each mouse within 30 minutes.

[0111] Experimental Groups:

[0112] 1. Solvent control group (n = 8): Mice were injected intraperitoneally with 200 μL of solvent.

[0113] 2. Test drug group (n = 8): Mice were injected intraperitoneally with 200 μL of the drug.

[0114] 3. Visceral pain model group (n = 12): Mice were injected intraperitoneally with 200 μL of 0.6% acetic acid solution.

[0115] 4. Z944 (10 mg / kg) + visceral pain model group (n = 8): 30 min before modeling, mice were injected intraperitoneally with 200 μL of Z944.

[0116] 5. Test drug + visceral pain model group (n = 8, 10): 30 min before mouse modeling, 200 μL of different doses of test drug were injected intraperitoneally.

[0117] Note: When the number of animals is even, there are half males and half females.

[0118] Drug dosage:

[0119] Z944: 10 mg / kg, intraperitoneal injection.

[0120] B3: 10, 20, or 30 mg / kg, administered intraperitoneally.

[0121] Drug solvent: Contains 20% (v / v) β-cyclodextrin and 0.4% DMSO physiological saline solution.

[0122] The methods for analyzing the significance between groups were One-way ANOVA test and Tukey MCT.

[0123] II. Data Analysis and Statistics

[0124] Data are expressed as mean ± standard error (mean ± sem). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, USA). The Anderson-Darling method or Shapiro-Wilk method was used to analyze the normality of the data. Homogeneity of variance was tested using the Brown-Forsythe test for two groups and the Bartlett test for multiple groups. Unpaired two-tailed t-tests were used for comparisons between two groups. For comparisons among multiple groups, one-way ANOVA with Tukey's multiple comparison test (MCT) was used, along with Brown-Forsythe and Welch ANOVA with Tamhane's T2 multiple comparison test. Two-way ANOVA combined with the Benjamini-Krieger-Yekutieli two-stage stepwise multiple comparisons was used. Differences with p < 0.05 were considered statistically significant, defined as **<0.05, **p<0.01, ***p<0.001, ****p < 0.0001 or #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. Differences in ns or NS were not statistically significant.

[0125] III. In the acetic acid writhing test, compound B3 at doses of 20 and 30 mg / kg showed an analgesic effect comparable to Z944 (10 mg / kg) in reducing the number of writhing episodes and prolonging the time to the first writhing episode. Figure 10 (Among the AB group), but the 10 mg / kg dose was ineffective. It is worth noting that the B3 series of molecules with the same parent nucleus have such different regulatory mechanisms, and their structural basis for regulating channels and their in vivo pharmacodynamic effects deserve further investigation.

[0126] Example 6

[0127] The experimental methods and results of the behavioral assessment of the effect of compound B3 of the present invention in relieving paclitaxel-induced neuropathic pain are as follows:

[0128] I. Experimental Methods:

[0129] C57BL / 6J mice aged 6-8 weeks and weighing 18-22 g were used in the experiment and grouped by sex (8-12 weeks old, weighing 22-26 g). They were housed in an animal room with constant temperature (22℃), constant humidity (55%), and a 12-hour light-dark cycle (maximum 5 mice per cage, cage size 30×20×15 cm), with free access to food and water. During the experiment, mice were cared for by designated personnel. Lab coats and gloves were changed for different batches of operations. Mice were housed individually after at least one week of acclimatization to avoid fighting. Mice were intraperitoneally injected (ip) with PTX (10 mg / kg) or 10 ml / kg of a solvent (physiological saline containing 0.5% CMC-Na and 2% DMSO) every other day for a total of 4 times (days 1, 3, 5, and 7), with a cumulative total PTX dose of 40 mg / kg. Baseline assessment of mechanical hyperalgesia was performed on day 9 by placing mice in plastic cages with a metal mesh bottom. Testing began after the mice ceased exploring and primary grooming behaviors. The 50% mechanical withdrawal threshold (PWT) was determined using von Frey fibers (NorthCoast Medical Inc.) via up-and-down measurements. One hour after baseline testing, a single intraperitoneal injection of solvent, B3 (10, 20, or 30 mg / kg), or Z944 (10 mg / kg) was administered, and behavioral assessments were performed at 30 minutes, 1, 2, 4, and 6 hours post-administration.

[0130] Experimental Groups:

[0131] a. Solvent control group (n = 8): Rats were injected intraperitoneally with 200 μL of solvent.

[0132] b. Model group (n = 12): PTX administered intraperitoneally for a cumulative total of 40 mg / kg.

[0133] c. Test drug + inflammatory pain model group (n = 8, 10): 1 hour after baseline testing, 200 μL of compound B3 of the test drug was injected intraperitoneally.

[0134] d. Z944 (10 mg / kg) + Inflammatory Pain Model Group (n = 10): 200 μL of Z944 was injected intraperitoneally 1 hour after baseline testing.

[0135] Note: When the number of animals is even, there are half males and half females.

[0136] Drug dosage:

[0137] a. Drug solvent: physiological saline of 0.5% CMC-Na and 2% DMSO

[0138] b. Paclitaxel (PTX): 10 mg / kg, intraperitoneal injection

[0139] c. Compound B3: 10, 20, 30 mg / kg, intraperitoneal injection

[0140] d. Z944: 10 mg / kg, intraperitoneal injection

[0141] e. The method for analyzing the significance between groups was: Brown-Forsythe and Welch analysis of variance combined with Tamhane T2 multiple comparison test.

[0142] II. Data Analysis and Statistics

[0143] Data are expressed as mean ± standard error (mean ± sem). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, USA). The Anderson-Darling method or Shapiro-Wilk method was used to analyze the normality of the data. Homogeneity of variance was tested using the Brown-Forsythe test for two groups and the Bartlett test for multiple groups. Unpaired two-tailed t-tests were used for comparisons between two groups. For comparisons among multiple groups, one-way ANOVA with Tukey's multiple comparison test (MCT) was used, along with Brown-Forsythe and Welch ANOVA with Tamhane's T2 multiple comparison test. Two-way ANOVA combined with the Benjamini-Krieger-Yekutieli two-stage stepwise multiple comparisons was used. Differences with p < 0.05 were considered statistically significant, defined as **<0.05, **p<0.01, ***p<0.001, ****p < 0.0001 or #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. Differences in ns or NS were not statistically significant.

[0144] III. In vivo pharmacodynamic studies have shown that intraperitoneal injection of vitamin B3 (10, 20, and 30 mg / kg) can significantly alleviate paclitaxel-induced peripheral neuropathic mechanodysia, manifested by a significant increase in the Paw withdrawal threshold (PWT). Particularly at doses of 20 and 30 mg / kg, it exerted a significant analgesic effect as early as 0.5 hours after administration, with a faster onset of action than the positive control Z944. Figure 10 C, Figure 11). Calculation of the area under the curve (AUC) for Z944 (0–6 hours) and B3 (0–6 hours) showed that B3 was comparable to Z944 (10 mg / kg) in relieving pain at doses of 20 and 30 mg / kg. Figure 10 The results (D) indicate that both B3 and Z944 have potent analgesic effects.

[0145] Formulation Examples

[0146] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. These examples only demonstrate the preparation of different formulations of the polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3 described in this invention, and do not specifically limit the specific reagents and operations.

[0147] 1. Dissolve any one or any combination of compounds B1, B2, and B3 in DMSO, add water for injection according to conventional methods, filter, fill and sterilize to prepare an injection solution with a concentration of 0.5-5 mg / mL.

[0148] 2. Dissolve any one or any combination of compounds B1, B2, and B3 in DMSO, then dissolve them in sterile water for injection, stir until dissolved, filter using a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then aseptically seal to obtain a powder for injection.

[0149] 3. Add any one or any combination of compounds B1, B2, and B3 to the excipient at a mass ratio of 9:1 to prepare a powder.

[0150] 4. Add any one or any combination of compounds B1, B2, and B3 to the excipient at a mass ratio of 5:1, and then granulate and compress the mixture into tablets.

[0151] 5. Prepare an oral liquid by taking any one or any combination of compounds B1, B2, and B3 according to conventional oral liquid preparation methods.

[0152] 6. Add any one or any combination of compounds B1, B2, and B3 to the excipient at a mass ratio of 5:1 to make capsules.

[0153] 7. Add any one or any combination of compounds B1, B2, and B3 to the excipient at a mass ratio of 5:1 to prepare granules.

Claims

1. A polysubstituted cyclopropyl carbonyl compound as shown in the following structural formula, ； in, R1 is selected from a bromine atom, or one of the following structures: ； R2 is selected from para-substituted methoxy groups and meta-substituted fluorine atoms.

2. The following structural formulas show polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3. 。 3. The use of the polysubstituted cyclopropyl carbonyl compounds according to claim 1 or 2 in the preparation of T-type calcium channel modulators.

4. The use of the polysubstituted cyclopropyl carbonyl compounds according to claim 1 or 2 in the preparation of analgesic drugs.

5. A pharmaceutical composition comprising at least one of the polysubstituted cyclopropyl carbonyl compounds of claim 1 or 2, and a pharmaceutically acceptable carrier.

6. The use of the pharmaceutical composition of claim 4 in the preparation of a T-type calcium channel modulator.

7. The use of the pharmaceutical composition of claim 4 in the preparation of analgesic drugs.

8. The method for preparing the polysubstituted cyclopropyl carbonyl compound according to claim 1, characterized in that, The method includes the following steps: Using α-chlorocyclobutanone compounds with R1 substituents as raw materials, aryl magnesium bromide or aryl lithium bromide are reacted in an organic solvent to obtain polysubstituted cyclopropyl carbonyl compounds as shown in Formula B. The reaction formula is as follows: ； In formula A, R1 is selected from bromine atoms and X is selected from chlorine atoms; In formula B, R1 has the same meaning as R1 in formula A. In formula B, R2 is selected from para-substituted methoxy groups and meta-substituted fluorine atoms.

9. The method for preparing the polysubstituted cyclopropyl carbonyl compounds B1, B2, and B3 according to claim 2, characterized in that, The method includes the following steps: Preparation of polysubstituted cyclopropyl carbonyl compounds B1: Refer to the method reported in the literature (Angew. Chem. Int. Ed. 60(23), 12859) The olefin raw material S1, namely (4aS,5S,8aS)-5-(2-bromoethyl)-1,1,4a,6-tetramethyl-1,2,3,4,4a,5,8,8a-octahydronaphthalene, was synthesized. S1 was dissolved in anhydrous diethyl ether, zinc powder was added, and trichloroacetyl chloride was dissolved in anhydrous diethyl ether and slowly added dropwise to the system. The addition was completed within 1 hour. The mixture was stirred vigorously at room temperature. After the reaction was confirmed to be complete by TLC, the solid residue was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in dry tetrahydrofuran and added to a commercially available tetrahydrofuran solution of samarium diiodide at -78°C. The mixture was stirred and the reaction was confirmed to be complete by TLC. Saturated potassium carbonate aqueous solution was added, and the mixture was extracted 1 to 5 times with an equal volume of organic solvent. The filtrates were combined and concentrated under reduced pressure to obtain the crude product. After conventional purification, the oily compound α-chlorocyclobutanone compound A1 was obtained. Under nitrogen protection, compound A1 was dissolved in ethylene glycol dimethyl ether or dioxane, and commercially available 4-methoxyphenyl magnesium bromide was slowly added dropwise at -20~25℃. The mixture was stirred at -20~25℃ for 1~48 hours. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added, and the mixture was extracted 1~4 times with an organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain the crude product, and then purified by conventional methods to obtain white solid compound B1. Preparation of polysubstituted cyclopropyl carbonyl compound B2: Under nitrogen protection, α-chlorocyclobutanone compound A1 was dissolved in ethylene glycol dimethyl ether, and commercially available 3-fluorophenyl magnesium bromide was slowly added dropwise at -20~25℃. The reaction was stirred at -20~25℃ for 1~48 hours. After the reaction was confirmed to be complete by TLC, saturated ammonium chloride aqueous solution was added to quench the reaction, followed by extraction with an organic solvent 1~4 times. The organic phases were combined, concentrated under reduced pressure to obtain the crude product, and compound B2 was obtained by conventional purification. Preparation of polysubstituted cyclopropyl carbonyl compound B3: First, prepare α-chlorocyclobutanone compound A2: using commercially available labda-7,13E-dien-15-oic acid as a starting material, dissolve it in dichloromethane, and then add EDCI, TMSEOH, and DMAP. The reaction was carried out at room temperature for 1-12 hours. After TLC detection showed that the reaction was complete, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was then extracted 1-4 times with an organic solvent. The organic phases were combined and concentrated under reduced pressure to obtain a crude product. After routine purification, an oily esterified product was obtained. The esterified product was dissolved in anhydrous diethyl ether, zinc powder was added, and trichloroacetyl chloride was dissolved in anhydrous diethyl ether and slowly added dropwise to the system. The addition was completed within 1 hour. The mixture was stirred vigorously at room temperature. After TLC detection showed that the reaction was complete, the solid residue was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in dry tetrahydrofuran and added to a commercially available samarium diiodide tetrahydrofuran solution at -78°C. The mixture was stirred and reacted. After TLC detection showed that the reaction was complete, saturated potassium carbonate aqueous solution was added, and the mixture was extracted 1-5 times with an equal volume of organic solvent. The organic phases were combined and concentrated under reduced pressure to obtain a crude product. After routine purification, an oily compound A2 was obtained. Then, the polysubstituted cyclopropyl carbonyl derivative B3 was prepared: similar to the synthesis of compound B2, under nitrogen protection, α-chlorocyclobutanone compound A2 was dissolved in ethylene glycol dimethyl ether, and commercially available 3-fluorophenyl magnesium bromide was slowly added dropwise at -20~25℃. After stirring at -20~25℃ for 1~48 hours, the reaction was confirmed to be complete by TLC. The reaction was then quenched by adding saturated ammonium chloride aqueous solution, and extracted 1~4 times with organic solvent. The organic phases were combined, concentrated under reduced pressure to obtain crude product, and then purified by conventional methods to obtain white solid compound B3. The organic solvents involved in the above preparation steps are all industrial, analytical or chromatographically pure ethyl acetate, chloroform, dichloromethane, and 1,2-dichloroethane.

Images