Propofol-metoprolol twin drug as well as preparation method and application thereof

Abstract

The invention discloses a propofol-metoprolol twin drug as well as a preparation method and application thereof, the propofol-metoprolol twin drug is formed by connecting propofol and metoprolol, and the propofol-metoprolol twin drug is named as WSW-E01-166-P. According to the present invention, the WSW-E01-166-P can achieve the deanesthesia effect, and has good drug stability; the compound has relatively high inhibition efficiency on ICa-L, and shows an anti-arrhythmia effect in a barium chloride induced ventricular arrhythmia rat model. The novel twin drug WSW-E01-166-P disclosed by the invention has relatively good performance in the aspects of IVAs resistance, pharmacokinetics, toxicology and the like, and is greatly helpful for later promotion of entering a phase I clinical research process.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a propofol-metoprolol twin drug and a preparation method and application thereof. Background Art

[0002] Idiopathic ventricular arrhythmias (IVAs) are common clinical tachyarrhythmias that can cause chest tightness, palpitations, heart failure, and even sudden death. Treatments for IVAs currently primarily include antiarrhythmic drug therapy, percutaneous catheter radiofrequency ablation, and implantable cardioverter-defibrillator (ICD) implantation for high-risk patients with malignant arrhythmias such as ventricular tachycardia and ventricular fibrillation. Percutaneous catheter radiofrequency ablation has a success rate of over 90% for IVAs and has become the first-line treatment in large research centers. However, catheter ablation has disadvantages such as being invasive, expensive, and limited to large medical centers, making it difficult to promote and apply in primary care hospitals. Because the pathogenesis of IVAs is not fully understood, the efficacy of various antiarrhythmic drugs on IVAs varies. Currently, there is a lack of specific therapeutic drugs for IVAs.

[0003] Studies have shown that propofol has varying degrees of effects on the electrophysiological activity of the sinoatrial node, atrioventricular node, and ventricular myocardium. Multiple clinical reports have demonstrated propofol's antiarrhythmic effects, including those against supraventricular and ventricular arrhythmias. Our research group has found in clinical practice that propofol has a therapeutic effect on IVAs, particularly those originating in the RVOT. However, as a commonly used anesthetic in clinical practice, propofol's anesthetic effects limit its practical application. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide a propofol-metoprolol twin drug and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect is to provide a propofol-metoprolol twin drug, which is formed by connecting propofol and metoprolol, named WSW-E01-166-P, and its chemical structure is shown in the following formula (I):

[0007]

[0008] The second aspect is to provide a method for preparing the above-mentioned propofol-metoprolol twin drug, comprising the following steps:

[0009] Step 1: Propofol and NaOH are added to acetonitrile and stirred to dissolve, and then phthalic anhydride is slowly added to react at room temperature. After the reaction is completed, the pH is adjusted to 2-3, and post-treatment is performed to obtain compound 1-10;

[0010] Step 2: Dissolve the compound 1-10 and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in dichloromethane, then add triethylamine dropwise and transfer to an ice bath with stirring, then add a dichloromethane solution of metoprolol dropwise. After the reaction is completed, purification is performed to obtain WSW-E01-166-P.

[0011] Furthermore, the molar ratio of propofol to phthalic anhydride is 1:(1.1-3).

[0012] Furthermore, in the step 1, the reaction time is 30 to 60 minutes.

[0013] Furthermore, in the step 1, the post-treatment is specifically as follows: extraction with ethyl acetate three times, combining the organic phases, washing with saturated brine, drying the organic phase over anhydrous sodium sulfate, and distilling under reduced pressure to obtain a crude product, which is separated by silica gel column chromatography to obtain compound 1-10.

[0014] Furthermore, in the step 2, the molar ratio of compound 1-10 to metoprolol is 1:(1-5).

[0015] Furthermore, in the step 2, the purification treatment is specifically as follows: after the reaction is completed by TLC monitoring, H2O is added to the reaction solution, extracted three times with EA, the organic phases are combined, washed with brine, and the organic phases are dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product, which is separated by silica gel column chromatography to obtain a white solid 1-11, named WSW-E01-166-P.

[0016] The third aspect is to provide the use of the above-mentioned propofol-metoprolol twin drugs in the preparation of drugs for treating idiopathic ventricular arrhythmias.

[0017] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0018] The present invention successfully synthesized four propofol twin drugs (propofol connected with metoprolol: WSW-E01-159-P and WSW-E01-166-P; propofol connected with propranolol: WSW-E01-172-P and WSW-E01-173-P), achieved the deanesthesia effect, and all four propofol twin drugs showed good drug stability.

[0019] Compared with WSW-E01-172-P and WSW-E01-173-P, WSW-E01-159-P and WSW-E01-166-P have Ca-L Higher inhibition efficiency; WSW-E01-159-P and WSW-E01-166-P showed antiarrhythmic effects in the barium chloride-induced ventricular arrhythmia rat model.

[0020] Combining drug metabolite identification and pharmacokinetic data, the target twin drug WSW-E01-166-P was initially identified. This novel twin drug, WSW-E01-166-P, demonstrates promising anti-IVAs efficacy, pharmacokinetics, and toxicology, significantly assisting in advancing the drug into Phase I clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The experimental results of in vitro plasma stability of three propofol twin drugs (WSW-E01-159-P / WSW-E01-166-P / WSW-E01-173-P) are shown.

[0022] Figure 2 The experimental results of in vivo plasma stability of four propofol twin drugs (WSW-E01-159-P / WSW-E01-166-P / WSW-E01-172-P / WSW-E01-173-P) are shown.

[0023] Figure 3 The inhibitory effects of four propofol twins on L-type calcium channel Cav1.2 currents are shown.

[0024] Figure 4 Shown are representative electrocardiograms (A) and changes in myocardial electrophysiological parameters (B) of rats in the control group and propofol-drug coadministration group after barium chloride-induced arrhythmia.

[0025] Figure 5 HE staining images of myocardial tissues of rats in each group.

[0026] Figure 6 Figure 3 is the immunohistochemical staining of Cx43 in myocardial tissue of rats in each group.

[0027] Figure 7 Figure 3 is the quantitative results of Cx43 immunohistochemical expression in myocardial tissue of rats in each group.

[0028] Figure 8 This is the metabolic chromatogram of WSW-E01-159-P reference substance.

[0029] Figure 9 This is the metabolic chromatogram of WSW-E01-159-P Parent-1.

[0030] Figure 10 This is the metabolic chromatogram of WSW-E01-159-P Parent-2.

[0031] Figure 11 This is the metabolic chromatogram of WSW-E01-159-P M1.

[0032] Figure 12 This is the metabolic chromatogram of WSW-E01-159-P M2.

[0033] Figure 13 This is the metabolic chromatogram of WSW-E01-159-P M3.

[0034] Figure 14 This is the metabolic chromatogram of WSW-E01-166-P reference substance.

[0035] Figure 15 This is the metabolic chromatogram of WSW-E01-166-P Parent.

[0036] Figure 16 This is the metabolic chromatogram of WSW-E01-166-P M1.

[0037] Figure 17 This is the WSW-E01-166-P standard curve (0.5-500 ng / mL).

[0038] Figure 18 The figure shows the scatter plot of the mean concentration of WSW-E01-166-P drug in plasma samples (PO: 20 mg / kg) versus time.

[0039] Figure 19 The figure shows the scatter plot of the mean concentration of WSW-E01-166-P drug in plasma samples (IV: 10 mg / kg) versus time. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0041] Example 1

[0042] This embodiment provides a propofol-metoprolol twin drug (WSW-E01-166-P), the chemical structure of which is shown in the following formula (I):

[0043]

[0044] The preparation method is as follows:

[0045] 1) At room temperature, propofol (1.0 mmol, 1.0 eq.) and NaOH (1.1 mmol, 1.1 eq.) were weighed into a reaction flask with a stirrer. 5.0 mL of MeCN was added and stirred for 15 min.

[0046] 2) After stirring, phthalic anhydride (2.0 mmol, 2.0 eq.) was slowly added and reacted at room temperature for about 30 min. After the reaction, the pH was adjusted to 2 with 2 M HCl and extracted with ethyl acetate (3 × 15 mL);

[0047] 3) The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a crude product, which was separated by silica gel column chromatography to obtain 1-10 as a white solid;

[0048] 4) At room temperature, compound 1-10 (1.0 mmol, 1.0 eq.) and HATU (1.2 mmol, 1.2 eq.) were weighed into a reaction flask equipped with a stirrer. 5 mL of anhydrous DCM was added. Triethylamine (3.0 mmol, 3.0 eq.) was added dropwise to the reaction solution, which was transferred to an ice bath and stirred for 15 min. Subsequently, 2 mL of a DCM solution of metoprolol (1.1 mmol, 1.1 eq.) was slowly added dropwise.

[0049] 5) After the reaction was completed, 10 mL of H₂O was added to the reaction solution, and the mixture was extracted with EA (3 × 15 mL). The organic phases were combined, washed with brine (15 mL), dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography to obtain 1-11 as a white solid, designated WSW-E01-166-P.

[0050]

[0051] Example 2

[0052] This example uses a similar method to that of Example 1. Based on propofol (2,6-diisopropylphenol), a suitable naturally derived linker was selected to esterify the propofol molecule with the existing antiarrhythmic drugs metoprolol or propranolol via an -OH chemical bond. Propofol-metoprolol twin drugs (WSW-E01-159-P) and propofol-propranolol twin drugs (WSW-E01-172-P and WSW-E01-173-P) were also synthesized.

[0053]

[0054]

[0055] Furthermore, this example verifies the anesthetic effects of the four propofol twins (WSW-E01-159-P, WSW-E01-166-P, WSW-E01-172-P, and WSW-E01-173-P):

[0056] Four propofol twins (WSW-E01-159-P, WSW-E01-166-P, WSW-E01-172-P, and WSW-E01-173-P) were injected intraperitoneally into mice at a low dose of 100 mg / kg and a high dose of 500 mg / kg, respectively, and observed for at least 120 minutes. Loss of the righting reflex is a standard endpoint for general anesthetics. The mice generally remained in good condition during the experiment, and no deaths occurred. During the 120-minute observation period following injection of the four twins, the mice showed normal breathing, clear consciousness, pink skin, and the ability to move their limbs independently, with none exhibiting the standard endpoint of loss of activity (see Table 1).

[0057] Table 1

[0058]

[0059] The above results show that the four propofol twin drugs WSW-E01-159-P, WSW-E01-166-P, WSW-E01-172-P and WSW-E01-173-P have no anesthetic effect.

[0060] Example 3

[0061] This example uses liquid chromatography-mass spectrometry (HPLC-MS) to preliminarily investigate the stability of the four propofol twin drugs in mouse plasma in vitro and in vivo. In vitro and in vivo drug concentration analysis of new drugs is the basis for future personalized drug delivery.

[0062] 1. In vitro plasma stability test

[0063] 1) Preparation: Collect blood using a 5 mL EP tube and add EDTA-2K (blood:EDTA-2K = 4:1 or 4.5:0.5 is acceptable) to the tube.

[0064] 2) Weighing and Blood Collection: The experimental animals were fasted but not watered for 12 hours, weighed, and anesthetized intraperitoneally with sodium pentobarbital. Blood was collected from the apex of the heart (approximately 500-900 μL per mouse). The blood was added to a pre-rinsed EP tube and mixed gently and rapidly.

[0065] 3) The blood samples were divided into 1.5 mL EP tubes, with a total blood volume of 50 μL for each sample;

[0066] 4) The compound was diluted in equal proportions to a low concentration of 4 μM and a high concentration of 10 μM, and four time points (0 min, 30 min, 60 min, and 120 min) were set for each concentration;

[0067] 5) At least three equilibrium samples were prepared at each time point. After adding the compound to each EP tube, the tube was incubated at 37°C for the corresponding time.

[0068] 6) After incubation, quickly add 45 μl of saturated ZnSO4 + 135 μl of ACN (HPLC grade) to extract the compound and vortex vigorously using a homogenizer (saturated ZnSO4 is a high salt that can quickly denature proteins, so vortex until the blood is completely coagulated, which takes about 30 seconds to 1 minute).

[0069] 7) Centrifuge at room temperature and high speed at 12,000 g for 15 min (three layers should be visible: the lower layer is salt, the middle layer is red blood cells, etc., and the upper layer is the target product);

[0070] 8) Pipette the supernatant (about 100-120 μl) into a 1.5 mL EP tube, seal it with parafilm, and label it.

[0071] 9) LC-MS quantitative analysis.

[0072] The results are as follows Figure 1 As shown, WSW-E01-159-P and WSW-E01-173-P remained stable in whole blood samples at both low concentrations of 4 μM and high concentrations of 10 μM. WSW-E01-166-P decreased to 59.53% at a low concentration of 4 μM and to 71.73% at a high concentration of 10 μM within 2 hours in whole blood samples. WSW-E01-172-P showed a poor signal (suitable LC / MS conditions have not yet been found). These experimental results indicate that WSW-E01-159-P, WSW-E01-166-P, and WSW-E01-173-P exist as parent drugs in whole blood and degrade well in plasma, providing important theoretical basis for the further development of propofol twin drugs.

[0073] 2. In vivo plasma stability experiment

[0074] 1) Mice were randomly divided into groups and weighed, and then intraperitoneally injected with 10 mg / kg propofol.

[0075] 2) For the mouse self-control, 10 μL of blood was collected from the tail vein at different time points (0 min, 15 min, 30 min, 60 min, 2 h, 4 h, 8 h, and 24 h) and placed in 40 μL of 0.2% EDTA. The samples were placed on ice and can be frozen at -80°C before extraction.

[0076] 3) Each independent sample was extracted with 45 μL saturated ZnSO4 and 135 μL ACN, vortexed rapidly for 30 s-1 min, and centrifuged at 12000 rpm for 15 min;

[0077] 4) Aspirate the supernatant, seal with parafilm and label;

[0078] 5) LC-MS quantitative analysis.

[0079] The results are as follows Figure 2 As shown in the figure, the in vivo plasma stability study of WSW-E01-159-P and WSW-E01-166-P showed that the degradation amount of the drug in whole blood gradually increased with time; the in vivo plasma stability study of WSW-E01-172-P and WSW-E01-173-P showed that the degradation amount of the drug in whole blood remained stable within 24 hours, with almost no degradation.

[0080] In summary, the results of in vitro and in vivo plasma stability experiments in this example indicate that the four propofol twin drugs WSW-E01-159-P, WSW-E01-166-P, WSW-E01-172-P and WSW-E01-173-P have good stability.

[0081] Example 4

[0082] As an important messenger in cells, calcium ions have functions such as participating in cell contraction, secretion and signal transmission. Under normal circumstances, the amount of calcium ions entering and flowing out of cells is roughly equal, but under pathological conditions, the amount of calcium ions entering cells will be much greater than the amount flowing out, resulting in intracellular calcium overload. Calcium overload can lead to DADs, which in turn leads to the occurrence of arrhythmias such as atrial fibrillation and even ventricular fibrillation. At the same time, calcium overload can affect the activity of intracellular enzymes, causing cellular physiological dysfunction. L-type calcium channels (LTCC) are widely expressed and distributed in various types of myocardial cells and conduction cells, and are characterized by continuous opening. LTCC is crucial for maintaining myocardial automaticity and even normal heart function. It is also often used as a therapeutic target for paroxysmal supraventricular tachycardia and ventricular fibrillation. LTCC function directly affects myocardial excitation conduction and contraction function. LTCC mediates calcium influx to promote the release of Ca from the sarcoplasmic reticulum. 2+ LTCCs are involved in myocardial excitation-contraction coupling. They are often targeted by antiarrhythmic and anti-heart failure drugs. L-type calcium channel mutations (G406R) can cause Timothy syndrome, characterized by cardiomegaly and the frequent development of severe arrhythmias such as atrioventricular block, LQTS, and multifocal ventricular tachycardia, leading to sudden death.

[0083] In this example, the whole-cell patch clamp technique was used to observe the L-type calcium channel current (I Ca-L From an electrophysiological perspective, this study explored whether propofol twins could retain their inhibitory effect on calcium currents after the anesthetic effect was removed, providing a theoretical basis for subsequent in vivo drug screening and future clinical applications.

[0084] 1. Experimental Procedure

[0085] 1) Cell Recovery: Remove the CHO cell line stably overexpressing the Cav1.2 channel (gene information: CACNA1C: NM_000719, selected for resistance to 100 μg / mL Zeocin; CACNB2: NM_000724, selected for resistance to 800 μg / mL G418; CACNA2D1: NM_000722, selected for resistance to 200 μg / mL Hygromycin B) from liquid nitrogen. Check the seal of the centrifuge tube. Once the seal is confirmed, immediately place the tube in a 37°C water bath and shake to thaw. Once the tube is completely thawed and a small amount of ice crystals remain, remove the tube from the water bath and transfer it to a pre-prepared centrifuge tube containing culture medium. Centrifuge at 1000 rpm / min for 5 minutes. Carefully aspirate the supernatant and resuspend in an appropriate amount of culture medium. The cells were cultured in HAM'S / F-12 medium containing 10% fetal bovine serum and 100 μg / mL Zeocin, 800 μg / mL G418, and 200 μg / mL Hygromycin B at 37°C and 5% carbon dioxide;

[0086] 2) Cell passaging: Remove the old culture medium and wash once with PBS, then add 1 mL of 0.25%-Trypsin-EDTA solution and incubate at 37°C for about 1.5 minutes. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently blow the cell suspension with a pipette to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish with 2.5×10 cells in each cell culture dish. 5 cells (final volume: 5 mL);

[0087] 3) To maintain the electrophysiological activity of cells, the cell density must not exceed 80%;

[0088] 4) Patch clamp assay: Before the test, cells were separated with 0.25%-Trypsin-EDTA and 8×10 3 Cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). Tetracycline was added and assayed 24–72 hours later.

[0089] 5) After whole-cell seal formation, the cell membrane voltage was clamped at -80 mV. The clamping voltage was depolarized from -80 mV to +10 mV for 0.3 s (refer to the specific depolarization voltage in the pilot IV test). Data were collected every 20 s to observe the effect of the drug on the peak Cav1.2 current. The experimental data were acquired using an EPC 10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

[0090] 6) Use a microelectrode puller to pull a capillary glass tube into a recording electrode. Install the electrode filled with intracellular fluid into the electrode head, manipulate the microelectrode manipulator under an inverted microscope to immerse the electrode in the extracellular fluid and record the electrode resistance (Rpip). Contact the electrode to the cell surface and apply negative pressure to form a high-resistance seal (GΩ). At this time, perform fast capacitance compensation, and continue to apply negative pressure to break the cell membrane and form a whole-cell recording mode. Then perform slow capacitance compensation and record experimental parameters such as membrane capacitance (Cm) and series resistance (Rs) (no leakage compensation is given);

[0091] 7) When the whole-cell recorded Cav1.2 current stabilizes, drug administration is started. After each drug concentration is applied for 5 minutes (or the current stabilizes), the next concentration is detected. Three concentrations are detected for each test compound. Place the coverslip with cells in the recording bath under an inverted microscope. The blank control external solution and the working solution of the test compound are passed through the recording bath in sequence from low concentration to high concentration by gravity perfusion to act on the cells. A peristaltic pump is used for liquid exchange during recording. The current detected for each cell in the external solution without the compound serves as its own control group. At least two cells are used for each concentration and the test is repeated twice independently. All electrophysiological experiments are performed at room temperature.

[0092] 2. Experimental Results

[0093] By administering varying concentrations of four propofol-binding drugs to CHO cells stably overexpressing Cav1.2 channels, we observed the inhibitory effects of WSW-E01-159-P, WSW-E01-166-P, WSW-E01-172-P, and WSW-E01-173-P on L-type calcium channel Cav1.2 currents. In the positive control group, treatment with 1 μM nifedipine increased the Cav1.2-CHO pA from -493.81±84.10 to -5.70±8.07, achieving an inhibitory efficiency of 98.97%±1.46%. When WSW-E01-159-P was intervened at 3μM, 10μM and 30μM, respectively, Cav1.2-CHO pA changed from -1196.80±598.64 to -961.05±416.42, -267.81±152.84 and -108.39±97.81, with inhibition efficiencies reaching 18.16%±6.14%, 78.07%±1.80% and 91.98%±4.16%. When WSW-E01-166-P was intervened at 1μM, 3μM, 10μM and 30μM, respectively, Cav1.2-CHO pA changed from -1708.17±1205.57 to -931.89±786.32, -664.24±282.04, -109.41±9.95 and -61.74, with inhibition efficiencies reaching 16.30%±6.44%, 72.32%±4.18%, 95.21%±0.93% and 97.81%. When WSW-E01-172-P was intervened at 3μM, 10μM and 30μM, respectively, the Cav1.2-CHO pA increased from -848.82±459.39, -810.64±237.01, -641.11±184.34 and -261.35±102.87, and the inhibition efficiency reached 22.53%±6.01%, 38.68%±5.06% and 56.50%±1.47%. When WSW-E01-173-P was intervened at 3μM, 10μM and 30μM, respectively, Cav1.2-CHO pA changed from -526.31±326.15 to -629.91±265.68, -510.14±394.82 and -504.25±388.56, and the inhibition efficiency reached 5.33%±2.66%, 7.07%±4.76% and 7.76±5.16%.

[0094] The experimental results are shown in Table 2. Figure 3 As shown: Through equation calculation and curve fitting, we get WSW-E01-159-P IC 50 5.820μM, Hillslope 2.190; WSW-E01-166-PIC 50 2.004μM, Hillslope 2.305; WSW-E01-172-PIC50 20.14μM, Hillslope 0.6508; WSW-E01-173-P IC 50 >30μM. Previous data showed that metoprolol IC 50 The propofol IC is 25.1 μM. 50 The concentration of propofol and metoprolol in the twin drugs WSW-E01-159-P and WSW-E01-166-P was 60.9 μM, and the inhibition efficiency of L-type calcium channels was higher.

[0095] Table 2

[0096]

[0097]

[0098] In summary, the above studies show that the four propofol twins have Ca-L Both have a blocking effect in a concentration-dependent manner. Ca-L The blocking efficiency of WSW-E01-172-P and WSW-E01-173-P, two twin drugs formed by propofol and propranolol, was significantly better. We will also conduct further pharmacodynamic verification on WSW-E01-159-P and WSW-E01-166-P.

[0099] Example 5

[0100] This example verifies the antiarrhythmic effect of the propofol twin drugs (WSW-E01-159-P and WSW-E01-166-P) that were initially screened.

[0101] 1. Experimental steps:

[0102] 1) Weighing and Grouping: Thirty male Sprague-Dawley rats were randomly divided into six groups, with five rats in each group. These groups included a blank control group (Control), a barium chloride group (BaCl2), a metoprolol group (BaCl2+Metoprolol), a propofol group (BaCl2+Propofol), a WSW-E01-159-P group (BaCl2+WSW-E01-159-P), and a WSW-E01-166-P group (BaCl2+WSW-E01-166-P).

[0103] 2) The experimental animals were kept in a fasting state for 12 hours and anesthetized with isoflurane gas via mask: induction: 2-2.5%, maintenance: 1-1.5%;

[0104] 3) After anesthesia, rats in each group were fixed in the supine position on a mouse board with the ventral surface exposed and maintained at normal body temperature. Electrocardiogram (ECG) electrodes were inserted subcutaneously into both upper and lower limbs of the rats and connected to an Ensite Lab Abbott electrophysiology instrument to record lead II ECG (50 mm / s).

[0105] 4) Disinfect the iliac fossa skin with iodine, make an oblique incision, dissect the soft tissue to expose the femoral neurovascular bundle, and perform a sudden dissection.

[0106] 5) Rats in each group were given a preventive femoral intravenous injection of CMC-Na / CMC-Na / Metoprolol / Propofol / WSW-E01-159-P / WSW-E01-166-P;

[0107] 6) After the electrocardiogram stabilized (approximately 5 minutes), all rats except the normal control group were rapidly injected with 25 mg / kg barium chloride into the femoral vein within 10 seconds to induce arrhythmia.

[0108] 7) Statistical analysis: The incidence of ventricular tachycardia (VT) and mortality (Mortality) of rats in each group within 20 minutes were statistically analyzed, and the changes in PR interval, QT interval, QRS complex and heart rate (HR) were statistically analyzed.

[0109] 2. Effects of propofol and its combination on barium chloride-induced arrhythmias in rats

[0110] Each group of rats received a prophylactic injection of the corresponding drug. After electrocardiogram (ECG) stabilization (approximately 5 minutes), barium chloride was rapidly injected intravenously to induce ventricular arrhythmias. The rats were observed for at least 20 minutes, and ECG changes were recorded and analyzed. The incidence and mortality rates of malignant arrhythmias were compared between the groups. The results are shown in Table 3. In the barium chloride group, ventricular arrhythmias developed rapidly after injection, with a 60% incidence of ventricular tachycardia (VT) and a 60% mortality rate. Similar to the metoprolol and propofol groups, the WSW-E01-159-P and WSW-E01-166-P groups significantly reduced the incidence of malignant arrhythmias (0%) and mortality (0%), and improved survival, demonstrating a strong antiarrhythmic effect.

[0111] Table 3

[0112]

[0113] like Figure 4 As shown in the figure, from the representative electrocardiogram and electrocardiogram parameters, it can be seen that the RR interval, PR interval, QRS complex and QT interval of the rats in the barium chloride group were prolonged after the injection of barium chloride, rapid ventricular arrhythmia occurred, and a few rats showed ventricular fibrillation, indicating that the barium chloride model was successful.

[0114] The positive drug metoprolol (5 mg / kg) inhibited barium chloride-induced PR interval prolongation, but failed to inhibit RR interval, QRS complex, and QT interval prolongation, potentially leading to atrioventricular block and bradyarrhythmias in rats. Pretreatment with metoprolol resulted in a slower heart rate in normal rats (0 min), making it unsuitable for long-term prophylactic use.

[0115] Prophylactic intravenous administration of propofol (5 mg / kg) also resulted in prolonged RR intervals, but these prolongations returned to normal within 1 minute of barium chloride induction. In contrast to metoprolol, propofol pretreatment did not affect the normal heart rate of rats. Propofol inhibited barium chloride-induced prolongation of the PR interval and QRS complex. QT interval prolongation occurred within 1 minute of barium chloride induction but was subsequently rapidly reversed.

[0116] Prophylactic intravenous injection of WSW-E01-159-P and WSW-E01-166-P (10 mg / kg) did not affect the ECG parameters of normal rats. WSW-E01-159-P inhibited barium chloride-induced prolongation of the PR interval, QRS complex, and QT interval, with RR interval prolongation returning to normal 1 minute after barium chloride induction. WSW-E01-166-P inhibited barium chloride-induced prolongation of the RR and PR intervals. QRS interval prolongation did not fully return to normal within 20 minutes of observation, but it did improve. QT interval prolongation returned to normal 1 minute after barium chloride induction.

[0117] The above results show that the preventive intravenous injection of WSW-E01-159-P and WSW-E01-166-P can significantly improve ventricular arrhythmias caused by barium chloride without causing obvious arrhythmogenic side effects, and are suitable for use as preventive antiarrhythmic drugs.

[0118] 3. Pathophysiological effects of propofol twins on rat cardiac tissue (H&E staining, Cx43 immunohistochemistry)

[0119] The effects of WSW-E01-159-P and WSW-E01-166-P on the cardiac histology of rats with barium chloride-induced arrhythmia were evaluated by H&E staining. Figure 5As shown, the blank control group showed clear myocardial staining, neatly arranged cardiomyocytes, clearly centered nuclei, and consistent myofiber orientation. No significant inflammatory response, fibrosis, or necrosis was observed. When rats were treated with propofol, WSW-E01-159-P, or WSW-E01-166-P, no inflammatory infiltration or cell necrosis was observed in the heart, similar to the blank control group. This suggests that propofol, WSW-E01-159-P, or WSW-E01-166-P do not induce histological changes in the hearts of rats with arrhythmias, and that intravenous toxicity is low.

[0120] like Figure 6 、 Figure 7 As shown, immunohistochemistry results in rat heart tissue showed that metoprolol, propofol, and WSW-E01-159-P significantly increased Cx43 expression compared with the blank control group. The expression level of Cx43 in WSW-E01-166-P increased compared with the blank control group (Metoprolol: 7.78±0.98 vs. 5.55±1.26, P<0.01; Propofol: 7.27±1.62 vs. 5.55±1.26, P<0.05; WSW-E01-159-P: 7.86±1.85 vs. 5.55±1.26, P<0.01; WSW-E01-166-P: 6.96±0.91 vs. 5.55±1.26, P=0.11).

[0121] In summary, we were surprised to find in animal experiments that, in addition to inhibiting I Ca-L Besides the channel, the preventive intravenous injection of propofol and its twin drugs may also inhibit barium chloride-induced ventricular arrhythmias by regulating Cx43 expression, significantly improving survival rates. They are even superior to traditional antiarrhythmic drugs in preventing arrhythmias, and have minimal cardiotoxicity. Therefore, propofol and its twin drugs are ideal candidates for the development of drugs for the prevention and treatment of arrhythmias.

[0122] Example 6

[0123] Drug metabolism research runs through the entire process of drug development. Understanding the metabolic pathways and metabolite structures of compounds is very important for the design and optimization of lead compounds, the selection of clinical candidate drugs, and supporting clinical research and drug interactions and safety assessments in the development stage. Metabolite identification studies help understand the clearance mechanism, analyze potentially active or reactive metabolites, and observe high-proportion or unique metabolites in compounds to better understand the biotransformation ability of the compounds. In the process of screening lead compounds, metabolite identification can screen whether the compound will form reactive metabolites and whether it has potential safety risks. Studies based on in vitro metabolite identification can quickly identify metabolic soft spots to optimize the structure of lead compounds, thereby obtaining better drug efficacy. This example further identifies metabolites for WSW-E01-159-P and WSW-E01-166-P, two of the four propofol twin drugs.

[0124] 1. Mother drug testing

[0125] The results are shown in Tables 4-7. Plasma was collected from rats in each group 20 minutes after drug injection. The propofol concentration in the BaCl2+Propofol group was 265.45829±34.47954 ng / mL; the metoprolol concentration in the BaCl2+Metoprolol group was 634.7758±85.9471 ng / mL; neither the parent drugs (propofol, metoprolol) nor the prototype drug WSW-E01-159-P was detected in the BaCl2+WSW-E01-159-P group; the parent drug metoprolol was not detected in the BaCl2+WSW-E01-166-P group, but a large amount of the prototype drug WSW-E01-166-P and a very small amount of the parent drug propofol were detected.

[0126] Table 4 BaCl2 + Propofol group

[0127]

[0128] Table 5 BaCl2 + Metoprolol group

[0129]

[0130] Table 6 BaCl2 + WSW-E01-159-P group

[0131]

[0132]

[0133] Table 7 BaCl2 + WSW-E01-166-P group

[0134]

[0135] 2. Detection of WSW-E01-159-P plasma metabolites

[0136] MetabolitePilot software was used to process the in vitro rat plasma incubation samples of WSW-E01-159-P to obtain high-resolution mass spectra and UV chromatograms of the relevant metabolites. In addition to the parent drug, three metabolites were detected in the rat plasma incubation samples, named M1, M2, and M3. UHPLC-UV-Q Exactive Plus MS was used to identify the structures of the metabolites (e.g. Figure 8-13 ).

[0137] Parent-1([M+H] + ,m / z 526.31531)

[0138] In the rat plasma sample chromatogram, a peak was selectively detected at m / z 526.31531 and a retention time of 8.71 min, designated Parent-1. Parent-1 exhibited identical chromatographic and mass spectrometric behavior to WSW-E01-159-P, confirming it to be the unmetabolized parent drug. The relative mass spectrometric peak area accounted for 3.20%.

[0139] Parent-2([M+H] + ,m / z 526.31531)

[0140] In the rat plasma sample chromatogram, a peak at m / z 526.31531 and a retention time of 9.08 min were selectively detected, designated Parent-2. Parent-2 exhibited identical chromatographic and mass spectrometric behavior to WSW-E01-159-P, confirming it to be the unmetabolized parent drug. The relative mass spectrometric peak area accounted for 0.75%.

[0141] Parent-1 M1([M+H] + ,m / z 268.19009)

[0142] In the rat plasma sample chromatogram, m / z 268.19009 was selectively detected, and a chromatographic peak, designated M1, was detected at a retention time of 5.16 min. The relative mass spectrum peak area accounted for 3.88%.

[0143] M2([M+H] + ,m / z 366.19028)

[0144] In the rat plasma sample chromatogram, m / z 366.19028 was selectively detected, and a chromatographic peak, named M2, was detected at a retention time of 6.30 min. The relative mass spectrum peak area accounted for 74.78%.

[0145] M3([M+H] + ,m / z 366.19028)

[0146] In the rat plasma sample chromatogram, m / z 366.19028 was selectively detected, and a chromatographic peak, named M3, was detected at a retention time of 6.52 min. The relative proportion of the mass spectrometry peak area was 17.39%.

[0147] 3. Detection of WSW-E01-166-P plasma metabolites

[0148] MetabolitePilot software was used to process the in vivo rat plasma incubation samples of WSW-E01-166-P to obtain high-resolution mass spectra and UV chromatograms of the relevant metabolites. In addition to the parent drug, a metabolite was detected in the rat plasma incubation samples, named M1. The structures of the metabolites were identified using UHPLC-UV-Q Exactive Plus MS (e.g. Figure 14-16 ).

[0149] Parent([M+H] + ,m / z 576.33087)

[0150] In the rat plasma sample chromatogram, a peak was selectively detected at m / z 576.33087 at a retention time of 9.30 min, named Parent. Parent exhibited identical chromatographic and mass spectrometric behavior to WSW-E01-166-P, confirming it to be the unmetabolized parent drug. The relative mass spectrometric peak area accounted for 42.03%.

[0151] M1([M+H] + ,m / z 416.20587)

[0152] In the rat plasma sample chromatogram, m / z 416.20587 was selectively detected, and a chromatographic peak, named M1, was detected at a retention time of 6.85 min. The relative proportion of the mass spectrometry peak area was 57.89%.

[0153] In summary, the metabolites of WSW-E01-159-P and WSW-E01-166-P contain little or no propofol, which also verifies their lack of anesthetic effect. Ca-LThe inhibitory efficiency and metabolite identification of WSW-E01-166-P were further analyzed in the future.

[0154] Example 7

[0155] The above series of experiments preliminarily demonstrate that the propofol twin drug, while eliminating the anesthetic effects of propofol, still exhibits significant antiarrhythmic effects, demonstrating its promising research and development potential. As a newly synthesized compound, there are currently no reports on the pharmacokinetics and pharmacological mechanisms of WSW-E01-166-P, either domestically or internationally. This example, using the propofol twin drug WSW-E01-166-P, further investigates its pharmacokinetic profile and clarifies its in vivo disposition, laying a solid foundation for subsequent research.

[0156] 1. Experimental Procedure

[0157] 1) Anesthetize Sprague-Dawley (SD) rats with intraperitoneal injection of sodium pentobarbital;

[0158] 2) After fixed-position anesthesia, the patient lies supine on the operating table;

[0159] 3) Intubation: Prepare and disinfect the skin. Make a longitudinal incision along the midline of the neck slightly to the right. Free the subcutaneous tissue, expose the right jugular vein, clamp the freed jugular vein with a vascular clamp, insert a probe, and place the epidural catheter into the rat's jugular vein. Turn the rat over and place it on the operating table. Insert the epidural puncture needle subcutaneously to the neck incision. Under the guidance of the needle, insert the epidural catheter into the puncture needle, exit at the back of the neck, connect the connector, and secure.

[0160] 4) Continued observation after surgery.

[0161] 5) Drug administration (Table 8)

[0162] Table 8

[0163]

[0164] 6) Blood Sampling: Weigh and record the experimental animals before the experiment, and administer the drug according to the dosing schedule. Blood samples were collected from the jugular vein 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 7 hours, and 24 hours after oral administration; and 0.2 mL of blood was collected from the jugular vein 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 7 hours, and 24 hours after intravenous administration.

[0165] 7) Sample processing and storage: The collected blood samples were transferred to microcentrifuge tubes containing EDTA-K2 anticoagulant, centrifuged at 4000 g for 5 min at 4°C, and the supernatant was collected and stored in a refrigerator at -75°C ± 15°C.

[0166] 8) Sample injection analysis:

[0167] Sample pretreatment was performed according to the Full PK method, and the data obtained from the analysis were used to calculate pharmacokinetic parameters using WinNonlin software. PO pharmacokinetic parameters included T1 / 2, Tmax, Cmax, AUC, MRT, F%, etc.; IV pharmacokinetic parameters included T1 / 2, C0, AUC, Vss, CL, etc., and a sample time-drug concentration scatter plot was drawn.

[0168] 2. Experimental Results

[0169] 1) Standard curve

[0170] The standard curve of WSW-E01-166-P-HPLC absorption peak area in plasma is shown in Figure 17 Within the spike concentration range of 0.5-500 ng / mL, the peak area of ​​WSW-E01-166-P was linearly correlated with concentration. The linear regression equation was: Y = 0.00425X + 0.000361, with a correlation coefficient of r = 0.9989.

[0171] 2) Pharmacokinetic results of WSW-E01-166P solution in rats

[0172] After oral administration of 20 mg / kg of WSW-E01-166-P solution to rats, the time-average concentration of WSW-E01-166-P in plasma samples is shown in 18, and the pharmacokinetic parameters of rat plasma samples are shown in Table 9. 0.667 h after administration, the maximum blood concentration (C max ) is 12 ng / mL, and the elimination half-life (T 1 / 2 ) was 2.63h, the area under the curve AUClast was 42ng / mL, AUCinf was 48.5ng / mL, the bioavailability F% was 0.986%, and the drug retention time was 3.80h.

[0173] Table 9

[0174]

[0175] After rats were intravenously injected with 10 mg / kg of WSW-E01-166P solution, the time-average concentration of WSW-E01-166-P in plasma samples was plotted as follows: Figure 19 The pharmacokinetic parameters of rat plasma samples are shown in Table 10. The initial concentration of WSW-E01-166-P solution C0 was 5975 ng / mL, and the elimination half-life (T 1 / 2) was 4.93h, the area under the curve AUClast was 2368ng / mL, AUClnf was 2377ng / mL, the clearance CL was 70.2mL / min / kg, the steady-state apparent distribution volume Vss was 4.64L / kg, and the drug retention time was 1.10h.

[0176] Table 10

[0177]

[0178] T max T is the time to reach the maximum drug concentration. 1 / 2 For the elimination half-life, C max is the maximum drug concentration, C0 is the initial plasma concentration, AUClast is the area under the curve from time 0 to the last time point selected, AUCinf is the area under the curve from time 0 to infinity, CL is the clearance rate (the plasma volume cleared by the body's clearance organs per unit time), F is the bioavailability, Vss is the steady-state apparent distribution volume, and MRT is the residence time of the drug.

[0179] In summary, this study preliminarily explored the pharmacokinetic parameters of WSW-EO1-166-P through two administration methods (oral and intravenous injection). After SD rats were given 20 mg / kg WSW-EO1-166-P by gavage, the Tmax was 0.667h, indicating that WSW-EO1-166-P can be quickly absorbed into the blood and reach the highest blood concentration. The T1 / 2 was 2.63h, indicating that WSW-EO1-166-P is metabolized quickly in the body. The F(%) was 0.986, indicating that WSW-EO1-166-P has a significant first-pass effect, poor oral bioavailability, and it is difficult to maintain a blood concentration above the effective concentration, resulting in poor drug efficacy in vivo and not suitable for oral administration in the future. After intravenous administration of 10 mg / kg WSW-EO1-166-P to SD rats, the T1 / 2 was 4.93 hours and the Vss (L / kg) was 4.64, indicating that WSW-EO1-166-P was primarily distributed in the plasma and rapidly metabolized. The CL (mL / min / kg) was 70.2, indicating a high clearance rate, a low risk of toxic accumulation, and a good safety profile.

[0180] This study was the first to use the HPLC-MS / MS method to conduct a pharmacokinetic study of WSW-E01-166P in rats, providing a theoretical basis for the development and clinical application of new drug preparations for the prevention and treatment of ventricular arrhythmias by WSW-E01-166-P.

[0181] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A propofol-metoprolol twin drug, characterized in that: It is composed of propofol and metoprolol, named WSW-E01-166-P, and its chemical structure is shown in the following formula (I):

2. The method for preparing the propofol-metoprolol twin drug according to claim 1, wherein: The steps include: Step 1: Propofol and NaOH are added to acetonitrile and stirred to dissolve, and then phthalic anhydride is slowly added to react at room temperature. After the reaction is completed, the pH is adjusted to 2-3, and post-treatment is performed to obtain compound 1-10; Step 2: Dissolve the compound 1-10 and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in dichloromethane, then add triethylamine dropwise and transfer to an ice bath with stirring, then add a dichloromethane solution of metoprolol dropwise. After the reaction is completed, purification is performed to obtain WSW-E01-166-P.

3. The preparation method according to claim 2, characterized in that The molar ratio of propofol to phthalic anhydride is 1:(1.1-3).

4. The preparation method according to claim 2, characterized in that In the step 1, the reaction time is 30 to 60 minutes.

5. The preparation method according to claim 2, characterized in that In the step 1, the post-treatment is specifically as follows: extraction with ethyl acetate three times, combining the organic phases, washing with saturated brine, drying the organic phase over anhydrous sodium sulfate, and distilling under reduced pressure to obtain a crude product, which is separated by silica gel column chromatography to obtain compound 1-10.

6. The preparation method according to claim 2, characterized in that In the step 2, the molar ratio of compound 1-10 to metoprolol is 1:(1-5).

7. The preparation method according to claim 2, characterized in that In the step 2, the purification treatment is specifically as follows: after the reaction is completed as monitored by TLC, H2O is added to the reaction solution, extracted three times with EA, the organic phases are combined, washed with brine, and the organic phases are dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain a crude product, which is separated by silica gel column chromatography to obtain a white solid 1-11, named WSW-E01-166-P.

8. Use of the propofol-metoprolol twin drug according to claim 1 in the preparation of a drug for treating idiopathic ventricular arrhythmia.

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