Sulfonyl quaterphenyl [3] arene sulfate macrocyclic compound as well as preparation method and application thereof

Abstract

The invention provides a sulfuryl quaterphenyl [3] arene sulfate macrocyclic compound as shown in a formula IV as well as a preparation method and an application of the sulfuryl quaterphenyl [3] arene sulfate macrocyclic compound. The preparation method of the compound comprises four steps of reaction: Suzuki coupling, cyclization, demethylation and sulfation, the process is simple, the total yield is high, and the yield of each step of reaction is superior to 70%. The compound has excellent water solubility and biocompatibility and can form a stable host-guest inclusion complex with an anti-cancer drug terpyridyl ruthenium through hydrogen bonds, pi-pi accumulation and other supramolecular acting forces, and the binding constant of the compound is as high as (3.59 + / -1.48) * 10 < 6 > M <-1 >. A cytotoxicity experiment shows that the half inhibitory concentration (IC50) of the inclusion complex to human colon cancer cells HCT116 is 2.95 mu M, and compared with a free terpyridyl ruthenium drug (IC50 = 64.93 mu M), the anticancer activity of the inclusion complex is improved by about 22 times. The invention provides an efficient and convenient new strategy for drug delivery, the bioavailability and the treatment effect of the drug can be remarkably improved without covalent modification of drug molecules, and the drug delivery system has a good industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the fields of organic synthesis, supramolecular chemistry and biomedicine, specifically involving a water-soluble sulfone-tetraphenyl[3] aromatic hydrocarbon sulfate macrocyclic compound, its efficient preparation method, and its application as a supramolecular carrier in the delivery of terpyridine ruthenium drugs. Background Technology

[0002] Tri-pyridine ruthenium ([Ru(bpy)3)) 2+ ) is a classic octahedral metal complex that has attracted widespread attention in photodynamic therapy, biosensing, and catalysis due to its unique photophysical properties, electrochemiluminescence activity, and antitumor potential. However, this compound has significant drawbacks under physiological conditions: firstly, it suffers from insufficient photostability and chemical stability, making it prone to ligand dissociation; secondly, its lack of targeting leads to low bioavailability and may produce systemic toxic side effects.

[0003] To overcome these obstacles, researchers have explored various nanodelivery systems, such as metal-organic frameworks and polymer micelles. However, most of these strategies rely on physical adsorption or simple encapsulation, which suffers from problems such as low drug loading capacity, easy drug leakage, and uncontrollable biodegradation of the carrier material.

[0004] Supramolecular chemistry offers a new approach to solving these problems. Constructing inclusion compounds using the weak interactions between macrocyclic hosts (such as crown ethers and columnar aromatics) and guest drug molecules is a promising strategy. However, existing macrocyclic hosts generally suffer from cavity size mismatch or insufficient interaction forces when facing large-sized, octahedral guest molecules like ruthenium terpyridine, resulting in low binding constants (typically Ka < 10). 4 M -1 The inclusion of ruthenium terpyridine (R) is only localized, which can easily lead to drug burst release and even disrupt the active configuration of R, resulting in fluorescence quenching and reduced activity. Furthermore, many biphenyl aromatic compounds with large cavities have poor water solubility, severely limiting their biological applications.

[0005] Therefore, there is an urgent need in this field to develop a novel macrocyclic host molecule that simultaneously meets the following conditions: (1) has a sufficiently large rigid cavity to achieve the overall inclusion of ruthenium terpyridine; (2) possesses high water solubility and biocompatibility; and (3) can generate strong and stable interactions with drug molecules to achieve efficient delivery. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sulfone-tetraphenyl[3] aromatic hydrocarbon sulfate macrocyclic compound that can efficiently encapsulate and deliver terpyridine ruthenium drugs, its simple and efficient preparation method and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a sulfone-based tetraphenyl[3] aromatic hydrocarbon sulfate macrocyclic compound represented by Formula IV.

[0008]

[0009] Secondly, the present invention provides a method for preparing the compound, characterized by comprising the following steps: (1) Suzuki coupling reaction: 4,4'-dibromodiphenyl sulfone, 2,5-dimethoxyphenylboronic acid and potassium carbonate were dissolved in a mixed solvent of 1,4-dioxane and water in a molar ratio of 1:2.5:4. The mixture was stirred and refluxed at 90°C for 12 hours under the catalysis of Pd(dppf)Cl2. After post-treatment, the sulfone-based tetraphenyl monomer shown in Formula I was obtained.

[0010]

[0011] (2) Cycling reaction: Compound I and paraformaldehyde were dissolved in 1,2-dichloroethane at a molar ratio of 1:2.5, boron trichloride ether catalyst was added, and the reaction was carried out at room temperature for 40 minutes. After post-treatment, sulfone-based tetraphenyl[3] aromatic methoxy macrocycles of Formula II were obtained.

[0012]

[0013] (3) Demethylation reaction: Dissolve compound II in dichloromethane, add BBr3 dropwise, stir at room temperature for 24 hours, quench and filter to obtain sulfone tetraphenyl[3] aromatic macrocyclic ring as shown in formula III.

[0014] (4) Sulphation reaction: Compound III and sulfur trioxide pyridine were dissolved in anhydrous pyridine at a molar ratio of 1:3. The reaction was carried out at 50°C for 4 hours under nitrogen protection, and then the temperature was raised to 75°C for 12 hours. After post-treatment and ion exchange, the sulfone-based tetraphenyl[3] aromatic sulfate macrocyclic compound shown in Formula IV was obtained.

[0015]

[0016] Thirdly, this invention provides the application of the sulfone-tetraphenyl[3] aryl sulfate macrocyclic compound as a terpyridine ruthenium drug delivery carrier. Experimental results show that the inclusion complex prepared in this invention has inhibitory activity (IC50) against HCT116 human colon cancer cells. 50 = 2.95 μM) compared to free terpyridine ruthenium (IC50) 50The concentration (64.93 μM) was increased by 22-fold, resulting in an order-of-magnitude increase in anticancer activity. Co-localization experiments demonstrated that this carrier can promote drug entry into the cell nucleus, thereby significantly enhancing the therapeutic effect.

[0017] This invention further discloses an anticancer drug composition of an inclusion complex formed by a sulfone tetraphenyl[3] aryl sulfate macrocyclic compound and a terpyridine ruthenium drug. The compositions of this invention are effective over a fairly wide dosage range. For example, the daily dose can be in the range of about 0.1 mg to 1000 mg per kilogram of body weight. In adult treatment, the dosage range is preferably 1 mg / kg to 10 mg / kg, administered once or several times. The actual dosage of this invention should be determined by the physician based on relevant circumstances, including the patient's physical condition, age, weight, individual response to the drug, severity of the patient's symptoms, etc., and therefore the above dosage range is not intended to limit the scope of this invention in any way.

[0018] When administered orally, the composition can be formulated into tablets, dispersible tablets, sugar-coated tablets, granules, dry powders, solutions, or capsules. Lactose or starch can be used as a carrier for preparing oral pharmaceutical compositions, with gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone, etc., being suitable binders. Starch or microcrystalline cellulose can be used as disintegrants, and talc, colloidal silica gel, glyceryl stearate, calcium or magnesium stearate, polyethylene glycol-4000, polyethylene glycol-6000, sodium metabisulfite, etc., are commonly used as anti-adhesives and lubricants. For example, tablets can be prepared by compressing wet granules. The active ingredient is mixed with a carrier and, selectively, a disintegrant additive. This mixture, along with an aqueous, alcoholic, or aqueous-alcoholic solution of the binder, is granulated in a suitable device. The dried granules are then compressed into tablets with the addition of other disintegrants, lubricants, and anti-adhesives.

[0019] Compared with the prior art, the beneficial effects of the sulfone-tetraphenyl[3] aromatic sulfate macrocyclic compound, its preparation method and application disclosed in this invention are as follows: 1. Innovative and efficient synthesis route: This invention is the first to design and realize a complete synthesis route for tetraphenyl[3] aromatic hydrocarbons with sulfone group as the core and sodium sulfate as the water-soluble modifying group. The route is ingeniously designed, requiring only four steps, and the yield of each step is higher than 70%. It is easy to operate, has a high overall yield, and has significant industrialization advantages.

[0020] 2. Breakthrough solution to the water solubility problem: By introducing a strongly hydrophilic sodium sulfate group into the molecular side chain, the key problem of poor water solubility of traditional biphenyl aromatics is fundamentally solved, laying the foundation for its application in the biomedical field.

[0021] 3. Achieving potent and stable drug inclusion: The compound of this invention can undergo multiple π-π stacking with ruthenium terpyridine through its rigid cavity, while the side chain sulfate group forms an extensive hydrogen bond network with the hydrogen atoms on the bipyridine, resulting in an extremely high binding constant ((3.59 ± 1.48) × 10⁻⁶). 6 M -1 This achieves overall inclusion of the terpyridine ruthenium molecule, rather than localized effects, which effectively avoids drug conformational damage and fluorescence quenching.

[0022] 4. Providing a universal design strategy: This invention not only provides a specific and efficient drug carrier, but also offers a universal new strategy and approach to solving the water solubility problem of biphenyl aromatic macrocyclic compounds and designing high-performance supramolecular delivery systems. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Structural formula and 1H NMR spectrum of sulfone-tetraphenyl monomer; Figure 2 Structural formula and 1H NMR spectrum of sulfone-tetraphenyl[3] aryl methoxy macrocycle; Figure 3 Structural formula and 1H NMR spectrum of sulfone-tetraphenyl[3] aromatic hydroxy macrocycle; Figure 4 The structural formula and 1H NMR spectrum of sulfone tetraphenyl[3] aromatic sulfate; Figure 5 Host-guest NMR shift characterization of sulfone tetraphenyl[3] aromatic sulfate and terpyridine ruthenium; Figure 6 Fluorescence titration curves and isothermal calorimetric titration curves of sulfone tetraphenyl[3] aromatic sulfate and terpyridine ruthenium; where a is the continuous titration of a concentration of 1×10 -6 The emission spectrum of ruthenium terpyridine (excitation wavelength 456 nm) when sulfone tetraphenyl[3] aromatic sulfate was added dropwise to PBS buffer (pH=7.4) of ruthenium terpyridine. b is the bonding constant between the fluorescence intensity value at 603 nm in a and the concentration of sulfone tetraphenyl[3] aromatic sulfate according to the 1:1 binding model; c is the isothermal calorimetric titration experimental curve of sulfone tetraphenyl[3] aromatic sulfate and ruthenium terpyridine. Figure 7The half-maximal inhibitory concentration (IC50) of the inclusion complex formed by sulfone tetraphenyl[3] aryl sulfate and ruthenium terpyridine against human colon cancer cells (HCT116) was 60%. 50 ) Spectrum and confocal laser scanning microscope image; where a is the half-maximal inhibitory concentration (IC50) of the inclusion complex formed by free terpyridine ruthenium and sulfone tetraphenyl[3] aryl sulfate and terpyridine ruthenium against human colon cancer cells (HCT116) as detected by the CCK-8 kit. 50 (b) is a cellular colocalization photograph of human colon cancer cells (HCT116) formed by the inclusion complex of terpyridine ruthenium and sulfone tetraphenyl[3] aromatic hydrocarbon sulfate and terpyridine ruthenium under a confocal laser scanning microscope. Detailed Implementation

[0025] The present invention will now be described in detail through specific embodiments, the purpose of which is to better understand the technical content of the present invention, rather than to limit the scope of protection of the present invention. Any changes or adjustments made based on the above-described concept of the present invention should be included within the scope of the claims of the present invention.

[0026] All raw materials and reagents used in this invention, such as 4,4'-dibromodiphenyl sulfone, 2,5-dimethoxyphenylboronic acid, potassium carbonate, 1,2-dichloroethane, boron trifluoride ether, paraformaldehyde, sulfur trioxide pyridine complex, anhydrous pyridine, sodium bicarbonate, anhydrous ethanol, dichloromethane, ethyl acetate, petroleum ether, DMEM-H complete culture medium, fetal bovine serum, paraformaldehyde, Triton X-100, DAPI staining solution, and human colon cancer cells (HCT116), are commercially available products unless otherwise specified.

[0027] Example 1

[0028] (1) Preparation of sulfone-based tetraphenyl monomer In a 250 mL round-bottom flask, 4,4'-dibromodiphenyl sulfone (7.5 g, 20.0 mmol), 2,5-dimethoxyphenylboronic acid (9.0 g, 50.0 mmol), potassium carbonate (K₂CO₃, 11.0 g, 80.0 mmol), and catalyst Pd(dppf)Cl₂ (1.50 g, 2.0 mmol) were added sequentially. The mixture was dissolved in 150 mL of a mixed solvent of 1,4-dioxane and water (V / V = 4:1). The reaction was refluxed in an oil bath at 90 °C with magnetic stirring for 12 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation. The resulting residue was dissolved in dichloromethane and extracted with water 2–3 times. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 1:2, v / v) to give a white solid sulfone tetraphenyl monomer (7.5 g, yield 93%).

[0029] Characterization data: 1 H NMR (400 MHz, CDCl3, 298K): δ [ppm] = 7.96 (d, J = 8.5 Hz,3H), 7.65 (s, 2H), 6.81 (d, J = 3.2 Hz, 4H), 4.01 (s, 2H), 3.83 (s, 5H), 3.63 (s, 6H).

[0030] (2) Preparation of sulfone-tetraphenyl[3]arene methoxy macrocycle In a 250 mL round-bottom flask, sulfone tetraphenyl monomer (0.50 g, 1.00 mmol) and paraformaldehyde (0.08 g, 2.50 mmol) were added and stirred until completely dissolved in 100 mL of 1,2-dichloroethane. Then boron trifluoride ether (1.0 mL, 0.10 mmol) was added as a catalyst and the mixture was reacted at room temperature for 40 minutes (TLC monitoring). After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate solution (3 × 100 mL) and washed. The organic phase was collected after separation, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was separated by silica gel column chromatography (elution gradient: petroleum ether / dichloromethane = 1:3 → pure dichloromethane) to obtain a white solid sulfone tetraphenyl[3] aryl methoxy macrocycle (0.16 g, yield 72%).

[0031] Characterization data: 1H NMR (400 MHz, CDCl3, 298K): δ [ppm] = 7.98-7.95 (m, 4H),7.66-7.62 (m, 4H), 6.81 (d, J = 3.2 Hz, 4H), 4.01 (s, 2H), 3.83 (s, 6H), 3.63(s, 6H). (3) Preparation of sulfone-based tetraphenyl[3] aromatic perhydroxy macrocycle In a 250 mL round-bottom flask, 0.50 g (0.32 mmol) of sulfone tetraphenyl[3] aryl methoxy macrocycle was dissolved in 100 mL of dichloromethane. Boron tribromide (BBr3, 0.8 mL, 3.93 mmol) was slowly added dropwise to the system with stirring at room temperature, and the reaction was continued for 24 hours (monitored by TLC). After the reaction was completed, the mixture was carefully quenched with an ice-water mixture, and the precipitate was collected by vacuum filtration to obtain a brick-red solid sulfone tetraphenyl[3] aryl hydroxy macrocycle (0.36 g, yield 82%).

[0032] Characterization data: 1 H NMR (400 MHz, Acetone-d6, 298 K): δ [ppm] = 8.00-7.97 (m,4H), 7.79-7.76 (m, 4H), 6.85 (s, 2H), 6.77 (s, 2H), 3.85 (s, 2H). (4) Preparation of sulfone tetraphenyl[3] aromatic sulfate In a 50 mL double-necked round-bottom flask, add sulfone tetraphenyl[3] aromatic perhydroxy macrocyclic ring (0.30 g, 0.22 mmol) and sulfur trioxide pyridine complex (1.08 g, 6.60 mmol). Under nitrogen protection, inject 10 mL of anhydrous pyridine as solvent. Place the reaction system in an oil bath, stir at 50 °C for 4 hours, and then heat to 75 °C for 12 hours. After the reaction is complete, cool to room temperature and pour off most of the solvent. Rinse the bottom of the flask with ethyl acetate and dichloromethane (twice each). Then add saturated sodium bicarbonate solution to dissolve the oil and adjust the pH of the aqueous phase to 7.5 (if necessary, a small amount of sodium bicarbonate solid can be added). Add 30 mL of ethanol to the solution, transfer the entire mixture to a centrifuge tube, and centrifuge at 3500 rpm for 10 minutes. Collect the supernatant, wash the lower solid with ethanol and extract twice by sonication, and combine all the supernatant liquids. Finally, the solvent was removed by vacuum distillation using a rotary evaporator to obtain the target product, sulfone tetraphenyl[3] aromatic sulfate (0.41 g, yield 73%), which is a brown solid.

[0033] Characterization data:1 H NMR (400 MHz, Methanol-d4, 298 K): δ [ppm] = 7.95 (dt, J =6.7, 2.4 Hz, 5H), 7.87-7.84 (m, 4H), 7.54-7.51 (m, 4H), 4.34 (s, 2H).

[0034] Example 2

[0035] To verify the interaction strength between the carrier and the drug of this invention, a fluorescence titration experiment was performed: (1) Solution preparation: Accurately weigh 1.28 mg of sulfone tetraphenyl[3] aryl sulfate (main body), dissolve it in PBS buffer and bring the volume to 0.5 mL to prepare a solution with a concentration of 5.0 × 10⁻⁶. -5 Stock solution of M. Accurately weigh 0.75 mg of ruthenium tripyridine (guest), dissolve in PBS buffer and bring to a final volume of 1.0 mL to prepare a solution with a concentration of 5.0 × 10⁻⁶. -6 M's stock solution.

[0036] (2) Titration and testing: Add 2.0 mL of guest stock solution (5.0 × 10⁻⁶) to the cuvette. -6 M). Subsequently, using a microsyringe, 0, 5, 10, 15, 20, 40, 60, 80, 120, 180, 360, 440, and 540 μL of bulk stock solution (5.0 × 10⁻⁶) were sequentially added to the cuvettes. -5 M). After each addition, at the excitation wavelength λ ex = 456 nm, emission wavelength λ em The fluorescence spectrum of the system was measured at 603 nm.

[0037] (3) such as Figure 6 As shown, with the addition of the host compound, the fluorescence intensity of terpyridine ruthenium is significantly enhanced, indicating that the host successfully encapsulates the guest molecule, resulting in an inclusion-induced fluorescence enhancement effect. Through nonlinear fitting calculation, the binding constant between the sulfone tetraphenyl[3] aryl sulfate and terpyridine ruthenium is as high as (3.59 ± 1.48) ×10 6 M -1 This result demonstrates that the compound of the present invention is a highly efficient supramolecular carrier of ruthenium terpyridine, and its strong bonding effect is expected to significantly improve the water solubility and in vivo stability of the drug.

[0038] Example 3

[0039] To evaluate the bioactivity of the inclusion complex, cytotoxicity and colocalization assays were performed: (1) Cell culture and drug administration: Human colon cancer cells (HCT116) were cultured at 8 × 10⁸ cells per well. 3 Cells were seeded at a density of 1,000 μL of DMEM-H complete medium in each well and pre-cultured at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, the medium was replaced with fresh medium containing different concentrations of samples (free terpyridine ruthenium and inclusion complexes prepared in this invention) and incubated for another 24 hours.

[0040] (2) Cytotoxicity assay (CCK-8 assay): 10% (v / v) CCK-8 solution was added to each well and incubated for 4 hours. The optical density (OD) of each well was measured at 450 nm using a microplate reader (ALL SHENG AMR-100) and the cell viability was calculated.

[0041] (3) Intracellular localization observation (confocal microscopy): HCT116 cells were seeded in confocal culture dishes and incubated with 200 μM free ruthenium tripyridine and the inclusion compound prepared in this invention for 4 hours. After incubation, the culture medium was discarded, and the cells were gently rinsed with PBS. Subsequently, the cell nuclei were stained with DAPI nuclear dye for 15 minutes, rinsed three times with PBS, and finally observed and imaged using a laser confocal scanning microscope (CLSM; Olympus FV1000).

[0042] Results: Cytotoxicity experiments showed that the inclusion complex prepared in this invention inhibited the half-maximal inhibitory concentration (IC50) of HCT116 cells. 50 The concentration was 2.95 μM, compared to free ruthenium triple pyridine (IC50) drug. 50 = 64.93 μM), its anticancer activity was increased by approximately 22 times. Confocal imaging results ( Figure 7 The study clearly showed that, compared with the free drug, the inclusion complex could deliver ruthenium tripyridine to the cell nucleus more effectively, demonstrating its superior nuclear-targeted delivery capability and thus significantly enhancing the anticancer effect.

[0043] Example 4 (Sulfone-based tetraphenyl[3] aromatic sulfate prepared in Example 1) Preparation of tablets containing 100 mg of sulfone tetraphenyl[3] aromatic sulfate per tablet:

[0044] The active ingredients, lactose, starch, and microcrystalline cellulose, are passed through a 100-mesh sieve and thoroughly mixed. A 2% aqueous solution of hydroxymethyl cellulose is added to the above mixed powder and mixed. The mixture is then passed through a 20-mesh sieve to form a soft mass. The resulting wet granules are dried at 45-55°C. Sodium carboxymethyl starch and magnesium stearate are added to the dried granules and compressed into tablets.

[0045] Example 5 The preparation of capsules containing 100 mg of sulfone tetraphenyl[3] aryl sulfate per capsule is as follows: .

[0046] The above embodiments fully illustrate the beneficial effects and implementation methods of the present invention. However, it should be understood that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these equivalent forms also fall within the scope defined by the claims of the present invention.

Claims

1. A sulfone-tetraphenyl[3] aromatic sulfate macrocyclic compound of formula IV, characterized in that... It has the following structural features: .

2. The method for preparing the sulfone-tetraphenyl[3] aromatic sulfate macrocyclic compound as described in claim 1, characterized in that, Includes the following steps: (1) Using 4,4'-dibromodiphenyl sulfone and 2,5-dimethoxyphenylboronic acid as raw materials, a Suzuki coupling reaction was carried out in an alkaline mixed solvent under the catalysis of Pd(dppf)Cl2 to obtain the sulfone-based tetraphenyl monomer shown in Formula I. ； (2) The compound shown in Formula I was cyclized with paraformaldehyde in 1,2-dichloroethane under Lewis acid catalysis to obtain the sulfone-based tetraphenyl[3] aryl methoxy macrocycle shown in Formula II; ； (3) The compound shown in Formula II was subjected to demethylation reaction with boron tribromide (BBr3) in dichloromethane to obtain the sulfone-tetraphenyl[3] aromatic macrocyclic ring shown in Formula III; ； (4) The compound shown in Formula III was subjected to a sulfation reaction with a sulfur trioxide pyridine complex in anhydrous pyridine, and the result was post-treated to obtain the sulfone-tetraphenyl[3] aromatic sulfate macrocyclic compound shown in Formula IV. 。 3. The application of the sulfone-tetraphenyl[3] aromatic sulfate macrocyclic compound as described in claim 1 in the preparation of a terpyridine ruthenium drug delivery system; wherein the terpyridine ruthenium drug delivery system refers to the inhibitory activity against colon cancer cells.

4. The application as described in claim 3, characterized in that, The binding constant between the sulfone-tetraphenyl[3] aryl sulfate macrocyclic compound and the terpyridine ruthenium drug is not less than 10. 6 M -1 .

5. An anticancer drug composition, characterized in that, The inclusion complex formed by the sulfone tetraphenyl[3] aromatic sulfate macrocyclic compound of claim 1 and the terpyridine ruthenium drug.

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