CN107213466A - A kind of post aromatic hydrocarbons compound, its preparation method, pharmaceutical composition and purposes - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to a pillar aromatic compound, its preparation method, pharmaceutical composition and application. Specifically, the present invention relates to the compound represented by formula I, its preparation method, its pharmaceutical composition and its use. The compound involved in the invention not only improves the stability of the drug, but also enhances the inhibitory effect of the drug on tumor cells. The preparation method of the complex is simple, and has very important significance for the redevelopment and utilization of medicines.

Description

A kind of pillar aromatic compound, its preparation method, pharmaceutical composition and application

technical field

The invention belongs to the technical field of medicine, and relates to a pillar aromatic compound, its preparation method, pharmaceutical composition and application. Specifically, the pillararene compound refers to a complex formed by a pillararene derivative and a drug. Specifically, the drug is an antineoplastic drug.

Background technique

With the continuous development of medicinal chemistry, more and more new drugs have been developed and marketed. However, some drugs have low bioavailability due to problems such as poor solubility, poor oral absorption, and poor biocompatibility.

Nitrogen mustard is the earliest anti-tumor drug used clinically and has achieved outstanding curative effect. It is mainly used for the treatment of malignant lymphoma, and it also has certain curative effects on ovarian cancer, breast cancer, and prostate cancer. Its anti-tumor mechanism is due to the existence of highly active dual Chloroethylamine alkylating agent, after entering the body, forms a highly active ethyleneimine ion through intramolecular ring formation, thereby quickly binding to nucleophilic groups such as proteins and nucleic acids for alkylation. Nitrogen mustard is a representative of alkylating agent, which is highly active, extremely unstable in the body, and acts quickly when it enters the body. With the development of medicinal chemistry, the process of research and development of new drugs has become slow, so the advocacy of new uses of old drugs has attracted people's attention. Nitrogen mustard, as a classic old anti-tumor drug, is gradually restricted in use (reasons include nitrogen mustard is extremely unstable, side effects are also relatively large, etc.), and the inventors have not retrieved any reports about enhancing the efficacy of nitrogen mustard.

Oxaliplatin is the third-generation platinum-based antineoplastic drug after cisplatin and carboplatin. As a stable, water-soluble platinum-based alkylating agent, it is the first one that is obviously effective against colon cancer and has been shown to be effective in vivo and in vitro. Platinum antitumor drugs with broad-spectrum antitumor activity are also effective against cisplatin-resistant tumor cells.

Nitrogen mustard and oxaliplatin have the following structures:

Supramolecular chemistry medicine is a new application of supramolecular chemistry in the field of pharmacy. This field develops rapidly and has a wide range of research. It is a dynamic emerging interdisciplinary subject and is gradually becoming a relatively independent research field. At present, many supramolecular complex drugs formed by two or more molecules through intermolecular supramolecular interactions have been used in clinical practice. Supramolecular chemical drugs have good safety, low toxicity, few adverse reactions, high bioavailability, strong drug targeting, low multidrug resistance, good biocompatibility, high curative effect, and low development cost and cycle time. It has attracted much attention due to its short length and many other advantages. Supramolecular chemical drugs have great development potential and prospects.

The use of water-soluble supramolecular macrocyclic hosts (such as crown ethers, cyclodextrins, calixarene, cucurbituril, etc.) is the most common method for inclusion of drug molecules based on molecular recognition in aqueous solution. With the continuous development of supramolecular chemistry, molecular recognition studies of several generations of macrocyclic host molecules such as crown ethers, cyclodextrins, calixarenes, cucurbiturils, etc. in aqueous phase have been reported.

Among them, cyclodextrin and its derivatives are used as drug carriers to encapsulate drug molecules to improve drug solubility, stability, and adjust drug release rate, thereby improving the bioavailability of drugs in vivo. Currently, the above listed drugs using cyclodextrin as a carrier or additive include itraconazole, cisapride, mitomycin, piroxicam, dexamethasone, nitroglycerin, alprostadil, etc. Supramolecular chemistry has developed rapidly, and scientists have been devoting themselves to the research and development of new supramolecular chemistry subjects.

Pillararenes, as a new generation of supramolecular macrocyclic hosts after crown ethers, cyclodextrins, cucurbiturils, and calixarenes, were reported by Japanese chemist Ogoshi in the Journal of the American Chemical Society in 2008 (J.AM.CHEM.SOC.2008, 130 ,5022-5023) reported its synthesis for the first time. A class of cyclic oligomers connected by hydroquinone or hydroquinone ether at the para-position of the benzene ring through a methylene bridge, and the columnarene is a calix with a cylindrical rather than a conical conformation in the spatial structure Aromatics, which have a more rigid backbone. However, although pillararene has a more rigid skeleton and a suitable cavity structure than other macrocyclic host molecules, it is suspected that it has a greater toxicity because it contains multiple benzene ring structures. In addition, at present, there have been reports on some pillararene derivatives, such as the compounds shown in the following formulas 1 to 5.

However, no work has been reported on the inclusion of drug molecules based on pillararene derivatives to improve drug bioavailability, stability or solubility.

Contents of the invention

After intensive research and creative work, the present inventors have prepared a complex of pillararene derivatives and drug molecules. The inventors have surprisingly found that this type of complex can better improve the bioavailability of the drug in the body, and can be made into injection preparations and oral dosage forms, improve drug efficacy, reduce the toxic and side effects of the drug, the water solubility and Good stability. The following inventions are thus provided:

One aspect of the present invention relates to a complex formed by a drug and a pyrarene derivative; preferably, the complex is formed by an intermolecular supramolecular interaction of the drug and a pyrarene derivative.

In some embodiments of the present invention, in the complex, the drug is an antineoplastic drug; preferably, it is nitrogen mustard, cisplatin, carboplatin or oxaliplatin.

In some embodiments of the present invention, the complex, wherein the pillar arene derivative is shown in the following formula I,

in,

n is 5, 6, 7, 8, 9 or 10;

R is selected from the following formula II, formula III or formula IV, and two R are the same;

In formula II, M ⁺ is ammonium ion, sodium ion or potassium ion, and a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

In formula III, Y is amino group, aldehyde group, hydroxyl group, guanidine group, acid group, quaternary ammonium salt or pyridinium salt, and b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In formula IV, c is 1, 2, 3, 4 or 5.

Without being limited by theory, n is a positive integer equal to at least 5. When n is less than 5, it is difficult to form a ring, and the tension is too large. No n greater than 10 has been reported so far. Using 1,4-diethoxy as a raw material for the one-pot synthesis of all-ethyl-substituted pillararene, 5-10-membered rings can be isolated, but the yields of 7-, 8-, 9-, and 10-membered rings are very low (Jun-Li Hou, Chem. Commun., 2012, 48, 10999-11001). In addition, the 8-, 9-, and 10-membered ring structures are two cavities with folded structures, and the cavities are smaller than the 5, 6, and 7-membered rings, so the research on them is also limited). Without being bound by theory, on the one hand, n equal to 5 or 6 has a cavity structure of appropriate size, which is similar to the cavity structure of cyclodextrin; on the other hand, the yield of n equal to 5 or 6 is relatively high.

In some embodiments of the present invention, the complex, wherein the pillar arenes are selected from the following compounds 1-5:

Preferably, the compound 1 is the following compound 6,

In the present invention, compound 6 is the fully carboxylated water-soluble pillar [6] arene (CP6A), and its structure is the following formula V:

The pillar arene derivatives described in the present invention are preferably water-soluble pillar arene derivatives, wherein the fully carboxylated water-soluble pillar [5] arene and water-soluble pillar [6] arene are preferred, and the fully carboxylated water-soluble Pillar[6]arene is the best choice.

In a particularly preferred embodiment of the present invention, the pillararene complex is a complex formed of water-soluble pillararene derivatives and nitrogen mustard or oxaliplatin.

A schematic diagram of the structure of the nitrogen mustard/CP6A complex is shown in Figure 1A. A schematic diagram of the structure of the oxaliplatin/CP6A complex is shown in Figure 1B.

Without being limited by theory, pillararene or its derivatives contain multiple benzene ring structures, and their host-guest recognition effect is different from that of cyclodextrin and cucurbituril. The compounding of cyclodextrin, cucurbituril and drugs relies on the hydrophobic interaction in the ring However, using pillararene or its derivatives as the carrier and compounding the drug has -C—H┄π, cation┄π, etc. in addition to the hydrophobic effect. In the Chinese patent publication CN104922688A, the compound of cyclodextrin and oxaliplatin Have not obtained its Ka value size, just can find out from the ¹ H-NMR chemical shift of its compound that the interaction between them is very weak, yet the compound of pillar arene derivative and oxaliplatin in the present invention has Strong effect, the ¹ H-NMR chemical shift of oxaliplatin in the complex changes greatly, and its Ka value is further obtained (3.25±0.36)×10 ³ M ^-1 , compared with the invention patent CN104922688A, this The inventive columnarene derivative and the oxaliplatin complex have a stronger effect, so they have a better sustained-release effect on the drug and can greatly improve the bioavailability of the drug.

Both the CP6A/nitrogen mustard and CP6A/oxaliplatin complexes formed in the present invention are proved by ¹ H-NMR at the host-guest ratio of 1:1 (eg, Example 1 and Example 2). Without being limited by theory, since nitrogen mustard and oxaliplatin can intersperse into the cavity of CP6A, due to the shielding effect, the hydrogen on nitrogen mustard and oxaliplatin obviously moves to the high field, and the peak shape broadens or even disappears.

Those skilled in the art know that nitrogen mustard is extremely unstable in aqueous solution, enters the body quickly and acts quickly, stays in the blood for only 0.5-1min, 90% disappears from the blood within 1min, and 50% is in the form of metabolites within 24 hours discharge. Surprisingly, the nitrogen mustard/CP6A complex of the present invention can enhance the stability of the nitrogen mustard molecule. Without being limited by theory, the present invention utilizes its hydrochloride structure. Nitrogen in nitrogen mustard hydrochloride has a positive charge, and there are multiple -CH ₂ structures, which can be obtained through host-guest-C┄H┄π, ion- π and -C┄H┄Cl supramolecular interactions penetrate into the cavity of CP6A to form a strongly bonded supramolecular complex.

In particular, the present invention also relates to complexes formed of water-soluble pillar[6]arene and oxaliplatin. Without being limited by theory, in the oxaliplatin drug involved in the present invention, the -CH structure in the cyclohexanediamine in the molecule can also form a _-C┄H┄π supramolecular effect with the benzene ring in CP6A, so that The cyclohexanediamine part of oxaliplatin penetrates into the cavity of CP6A to form a supramolecular complex, which can enhance the drug's action time in vivo. Those skilled in the art know that although oxaliplatin is relatively stable in aqueous solution, once it enters the body, it will rapidly hydrolyze and act on DNA to form intra-strand and inter-strand crosslinks, thereby inhibiting DNA synthesis. The cell experiment of the present invention proves that the complex can reduce the IC ₅₀ value, which also shows that the action time of the drug is prolonged.

Another aspect of the present invention relates to the method for preparing the compound described in any one of the present invention, comprises the following steps:

directly dissolving a certain molar ratio of the mixture of the drug and the pyrene derivative in a benign solvent and mixing them uniformly, then freeze-drying or vacuum-drying under reduced pressure to obtain a complex (the color of the prepared complex is a brownish-yellow solid);

Preferably, the following steps are included:

Dissolving a mixture of nitrogen mustards and pillararene derivatives in a certain molar ratio directly in a benign solvent and mixing them evenly, and then freeze-drying the solution of the mixture or vacuum-drying under reduced pressure within 10 minutes;

or

Preferably, the following steps are included:

Dissolve the mixture of oxaliplatin and pillararene derivatives in a certain molar ratio directly in a benign solvent at 30°C-60°C (preferably 40°C-60°C) and ultrasonically shake for a period of time (preferably 10-20min), to Saliplatin is completely dissolved, and then the solution of the mixture is freeze-dried or vacuum-dried under reduced pressure;

Preferably, the above-mentioned pillararene derivative is the above-mentioned compound 6.

In some embodiments of the present invention, in the preparation method, wherein, the molar ratio of the pyrarene derivative to the drug is 0.1-10; preferably 0.5-5 or 0.8-1.5; more preferably 1.

In some embodiments of the present invention, the preparation method, wherein, the good solvent is selected from water, buffer solution (such as phosphate buffer solution, carbonic acid buffer solution, etc.), methanol, ethanol, isopropanol or mixtures thereof .

Another aspect of the present invention relates to a pharmaceutical composition, which comprises any one of the complexes of the present invention, and optional pharmaceutically acceptable auxiliary materials, such as carriers or excipients.

Usually the pharmaceutical composition of the present invention contains 0.1-90% by weight of the compound. Pharmaceutical compositions can be prepared according to methods known in the art. When used for this purpose, the compound can be combined with one or more solid or liquid pharmaceutical excipients, if necessary, to make a suitable administration form or dosage form for human use.

The compound of the present invention or the pharmaceutical composition containing it can be administered in the form of unit dosage, and the route of administration can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc. Dosage forms such as tablets, capsules, drop pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, lyophilized powder injections Wait. It can be common preparations, sustained-release preparations, controlled-release preparations and various microparticle drug delivery systems. Various excipients known in the art can be widely used in order to make the unit dosage form into tablets. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin paste, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitan fatty acid esters, sodium lauryl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oils, etc.; absorption enhancers Agents, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to formulate a dosage unit into a pellet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecylsulfonate, methylcellulose, ethylcellulose, etc. In order to formulate the administration unit into a suppository, various carriers known in the art can be widely used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like. In order to form a dosage unit into a capsule, the complex is mixed with the above-mentioned various carriers, and the mixture thus obtained is placed in a hard capsule or a soft capsule. The complex can also be made into microcapsules, suspended in an aqueous medium to form a suspension, and can also be packed into hard capsules or made into injections for application. In order to prepare the dosage unit into injection preparations, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1,3 - Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In addition, in order to prepare isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin can be added to the preparation for injection, and in addition, conventional solubilizers, buffers, pH regulators, etc. can also be added.

In addition, colorants, preservatives, fragrances, correctives, sweeteners or other materials can also be added to the pharmaceutical preparations, if necessary.

The dosage of the compound of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight and individual response of the patient or animal, the specific drug used, the route of administration and the frequency of administration Wait. The above dose can be administered in a single dose or divided into several, eg two, three or four doses.

As used herein, the term "composition" is meant to include a product comprising the specified amounts of each of the specified ingredients, as well as any product resulting, directly or indirectly, from the combination of the specified amounts of each of the specified ingredients.

Actual dosage levels of each active ingredient (drug or complex) in the pharmaceutical compositions of this invention may be varied to provide an amount of active ingredient effective to obtain the desired therapeutic response for a particular patient, composition, and mode of administration. Dosage levels will be selected based on the activity of the particular drug or complex, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is practice in the art to start dosages of the complex at levels lower than that required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.

Another aspect of the present invention relates to the use of the complex of the present invention in the preparation of a drug selected from:

Drugs for the treatment and/or prevention of tumors, especially malignant tumors,

drugs that suppress tumor cells, or

Drugs to treat and/or prevent cancerous pleurisy, pericardial effusion, or peritoneal effusion;

Preferably, the tumor is selected from lung cancer, liver cancer, bladder cancer, stomach cancer, ovarian cancer, breast cancer, prostate cancer, malignant lymphoma and colon cancer;

Preferably, the tumor cells are selected from lung cancer cells, liver cancer cells, bladder cancer cells, gastric cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, malignant lymphoma cells and colon cancer cells.

Another aspect of the present invention relates to a method for inhibiting tumor cells in vivo or in vitro, comprising the step of using an effective amount of the compound of the present invention; preferably, the tumor cells are selected from lung cancer cells, liver cancer cells, bladder cancer cells , gastric cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, malignant lymphoma cells and colon cancer cells. In one embodiment of the invention, the method is non-therapeutic.

Another aspect of the present invention relates to a method for preventing and/or treating tumors, especially malignant tumors, comprising the step of administering an effective amount of the compound of the present invention to a subject.

When used for the above-mentioned treatment and/or prevention, a therapeutically and/or preventively effective amount of a compound of the present invention may be used in pure form, or the compound may be formulated as a compound containing the compound of interest and one or more drugs. The pharmaceutical composition that can accept excipients is administered. It should be recognized, however, that the total daily dosage of the complexes and compositions of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dosage level will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the particular compound or drug employed; the specific compound or drug employed; Composition; age, weight, general health, sex, and diet of the patient; timing of administration, route of administration, and rate of excretion of the particular compound employed; duration of treatment; use in combination or concurrently with the particular compound employed Medications used; and similar factors well known in the medical field. For example, it is practice in the art to start dosages of the complex at levels lower than that required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained. Generally speaking, the dose of the compound of the present invention for mammals, especially humans, can be between 0.001-1000 mg/kg body weight/day, for example between 0.01-100 mg/kg body weight/day, for example between 0.01-10 mg/kg body weight /sky.

Since nitrogen mustard has obvious local stimulating effect and can easily cause tissue necrosis, it is only available for injection, including intravenous injection, arterial injection, subcutaneous injection, intradermal injection and intracavitary injection.

In the present invention, the compound administration route involving oxaliplatin and CP6A is preferably intravenous infusion.

In the present invention,

The term "intermolecular supramolecular interaction" or "supramolecular interaction" refers to interactions between molecules, such as van der Waals forces, hydrogen bonds, hydrophobic interactions, electrostatic interactions, π-π stacking, etc., or a combination thereof. Intermolecular supramolecular interactions are the basis of supramolecular chemistry research.

The term "molecular recognition" is the interaction between two or more molecules through non-covalent bonding. The process of molecular recognition is actually a process in which molecules combine with each other through the synergy of intermolecular forces under specific conditions. This actually reveals three important components in the principle of molecular recognition. "Specific conditions" means that molecules must rely on pre-organization to achieve a complementary state, and "intermolecular interaction force" means that molecules exist between molecules. Non-covalent interactions, while "synergy" emphasizes that molecules need to rely on macrocyclic effects or chelation effects to produce consistent effects between various interactions. Complementarity and preorganization are two key principles that determine molecular recognition. The former determines the selectivity of the recognition process, and the latter determines the bonding ability of the recognition process. Complementarity between substrate and receptor includes complementarity in spatial structure and spatial electrical properties. Spatial complementarity was first described by Fisher's "lock-key" relationship. Pre-organization means that the receptor and substrate molecules organize the substrate-accommodating environment better and the lower their solvation ability before recognition, the better their recognition effect and the more stable the complex formed. Molecular recognition relies on supramolecular interactions.

The term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of the diseases or conditions described in the present invention in a subject.

The term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition of the present invention.

The term "subject" may refer to patients or other animals, especially mammals, such as humans, dogs, monkeys, cattle, , horse, etc.

Beneficial Effects of the Invention

The complex of the present invention improves the bioavailability of drugs (especially effectively inhibiting tumors), has simple preparation and mild reaction conditions, and is suitable for industrial production, especially for the preparation of injection forms of nitrogen mustard and oxalibe , improve drug stability and anti-tumor activity, and make the drug more medicinally valuable.

Description of drawings

Figure 1: Figure 1A, a schematic diagram of the structure of the nitrogen mustard/CP6A complex. Figure 1B, Schematic diagram of the structure of the oxaliplatin/CP6A complex.

Figure 2: Relative cell survival rates of lung cancer cell A549, liver cancer cell HepG2 and human breast cancer cell MCF-7 at different CP6A concentrations.

Figure 3: Relative cell viability of normal hepatic 3T3 cells at different CP6A concentrations.

Fig. 4: ¹ H-NMR spectra of CP6A and nitrogen mustard; (a) CP6A; (b) CP6A+nitrogen mustard; (c) nitrogen mustard; D ₂ O, 5mM.

Figure 5: ¹ H-NMR spectrum of the interaction between CP6A and oxaliplatin; (A) CP6A; (B) CP6A+oxaliplatin; (C) oxaliplatin; D ₂ O, 5mM.

Figure 6: Figure 6A, evaluation of the inhibitory activity of the CP6A/NM complex on the tumor cell MCF-7. Fig. 6B, evaluation of the inhibitory activity of CP6A/NM complex on tumor cell HepG2 cells. NM means nitrogen mustard.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Test Example 1: Cytotoxicity test of CP6A

1. Experimental samples

CP6A (M is ammonium ion), prepared with reference to J.Am.Chem.SOC.2012, 134, 13248-13251.

Lung cancer cell A549, liver cancer cell HepG2, human breast cancer cell MCF-7 and normal liver cell 3T3: all provided by Peking Union Medical College Cell Bank.

2. Experimental method

MTT method:

3T3, A549, and MCF-7 all used DMEM medium (containing 10% FBS, 1% penicillin/streptomycin), and HepG2 used MEM medium (containing 10% FBS, 1% penicillin/streptomycin) in 5% CO _2. Cultivate at a constant temperature of 37°C, and prepare a solution by dissolving CP6A in PBS

Collect logarithmic growth cells (A549, MCF-7, HepG-2 cells), adjust the concentration of the cell suspension, inoculate the cell suspension in a 96-well plate, and plate to adjust the density of the cells to be tested to about 10,000/well. Incubate 100 μL of cell suspension in 5% CO ₂ at a constant temperature of 37°C for 24 hours. Observe under a microscope that cells adhere to the wall and grow. Add 10 μL of CP6A to the culture plate, where the concentrations of CP6A are 3.2 mM, 1.6 mM, 0.8 mM, respectively. 0.4mM, 0.2mM, 0.1mM. Add 10 μL of the same concentration of CP6A to every 5 wells, and add PBS to the last 5 wells as a blank control. After gently shaking on a shaker for 5 minutes, the culture plate was placed in a 5% CO ₂ , 37° C. constant temperature incubator for cultivation. After 48 hours, take out the culture plate, add 10 μl of MTT (5 mg/mL) solution to each well under sterile conditions, continue to cultivate for 4 hours, terminate the culture, and carefully suck off the culture solution in the well. Then, 100 μl DMSO was added to each well, and placed on a shaker for 10 minutes at low speed to fully dissolve the purple crystals. The absorbance of each well was detected at 490 nm in an automatic microplate reader.

Collect 3T3 cells grown in logarithmic phase, adjust the concentration of the cell suspension, inoculate the cell suspension on a 96-well plate, and plate to adjust the density of the cells to be tested to about 10,000/well, 100 μL of the cell suspension per well, in 5% CO ₂ , Incubated at a constant temperature of 37°C for 24 hours, and observed under a microscope, the cells adhered to the wall and grew. Add 10 μL of CP6A to the culture plate, the concentrations of CP6A are 2.9mM, 0.74mM, 0.15mM, 0.03mM, 0.003mM, 0.0003mM respectively. Add 10 μL of the same concentration of CP6A to every 5 wells, and add PBS to the last 5 wells as a blank control. After gently shaking on a shaker for 5 minutes, the culture plate was placed in a 5% CO ₂ , 37° C. constant temperature incubator for cultivation. After 48 hours, take out the culture plate, add 10 μl of MTT (5 mg/mL) solution to each well under sterile conditions, continue to cultivate for 4 hours, terminate the culture, and carefully suck off the culture solution in the well. Then, 100 μl DMSO was added to each well, and placed on a shaker at low speed for 10 minutes to fully dissolve the purple crystals. The absorbance of each well was detected at 490 nm in an automatic microplate reader.

3. Experimental results

As shown in Figure 2 and Figure 3 below.

The results showed that CP6A had no inhibitory ability to A549, HepG2, and MCF-7 tumor cells, and had slight toxicity to normal liver cell 3T3 at high concentrations. It can be seen that the water-soluble pillararene derivatives have very low toxicity.

Example 1: Preparation and characterization of CP6A/nitrogen mustard complex

Preparation and characterization of CP6A/nitrogen mustard complex:

Accurately weigh 20mg nitrogen mustard (purchased from Beijing Yinuokai Co., Ltd.) (0.105mmol) and 171mg CP6A (M is ammonium ion, the preparation method can refer to J.Am.Chem.SOC.2012,134,13248-13251) (0.105mmol) was mixed and dissolved in 5mL water until it was fully and uniformly mixed, and then the solution of the mixture was vacuum freeze-dried within 10 minutes to obtain a nitrogen mustard/CP6A complex. The ¹ H-NMR of the complex and the ¹ H-NMR of the main body CP6A and the nitrogen mustard alone in D ₂ O are shown in Figure 4, and the chemical shift values of the nitrogen mustard in the complex are shown in Table 1 below:

Table 1

It can be clearly seen from Table 1 and Figure 4 that when the nitrogen _mustard drug is included by _CP6A , its chemical shift value changes significantly _. high field. The chemical shift values are Δδ(a)=-1.527, Δδ(b)=-1.859, Δδ(c)=-1.717, which indicates that the nitrogen mustard enters the cavity of CP6A and is shielded by the benzene ring, making nitrogen The NMR signal peak of mustard moves to the high field. The broadening of the peak basically disappears, indicating that it is completely in the cavity of the CP6A host molecule and shields the NMR signal. The chemical shifts of H _b and H _c change the most, which also shows that its position is just in the center of the CP6A cavity , with the largest shielding effect. The results showed that the nitrogen mustard entered the cavity of CP6A.

Example 2: Preparation and Characterization of CP6A/Oxaliplatin Complex

Accurately weigh 20mg of oxaliplatin (0.05mmol) (purchased from Beijing Yinuokai Co., Ltd.) and 82mg of CP6A (0.05mmol) and mix them in 10mL of water. The drug is completely dissolved and uniformly mixed, and the mixed solution is freeze-dried under vacuum to obtain the CP6A/oxaliplatin complex. The ¹ H-NMR of the complex and the ¹ H-NMR of the main body CP6A and the oxaliplatin alone in D ₂ O are shown in Figure 5, and the chemical shift values of oxaliplatin in the complex are shown in Table 2 below:

Table 2

It can be clearly seen from Table 2 and Figure 5 that after oxaliplatin and CP6A form a complex, the chemical shift value changes significantly, and the proton peaks of H _{a, a'} , H _{b, b'} and H _c are obviously broadened , nearly disappears and moves upfield. The change value of its chemical shift is shown in Table 2, which shows that the cyclohexane part of the oxaliplatin molecule acts as the head and forms a supramolecular interaction with the benzene ring—the C┄H┄π supramolecular interaction interpenetrates into the cavity of CP6A and is subjected to benzene The shielding effect of the ring makes the NMR signal peak move to the high field. This result indicated that oxaliplatin intercalated into the cavity of CP6A.

Furthermore, the bonding constant of CP6A and oxaliplatin (3.25±0.36)×10 ³ M ^-1 was obtained.

Example 3: Evaluation of the inhibitory effect of CP6A/nitrogen mustard complex on tumor cells

1. Experimental samples

CP6A/nitrogen mustard complex: prepared in Example 1.

Human breast cancer cell MCF-7 and liver cancer cell HepG2: both provided by Peking Union Medical College Cell Bank.

2. Experimental method

MTT method was used to evaluate the inhibitory effect of CP6A/nitrogen mustard complex on human breast cancer cell MCF-7 and liver cancer cell HepG2. Nitrogen mustard alone was used as the control group, and its toxicity was measured 48 hours after administration.

For each type of cell, collect logarithmic growth cells (MCF-7, HepG-2 cells), adjust the concentration of the cell suspension, inoculate the cell suspension in a 96-well plate, and plate to adjust the density of the cells to be tested to about 10,000 /well, 100 μL cell suspension per well, incubate at 5% CO ₂ , 37°C for 24 hours at a constant temperature, observe under the microscope that the cells adhere to the wall, and add 10 μL of the prepared nitrogen mustard hydrochloride aqueous solution or containing The ammonium hexaacetate of nitrogen mustard hydrochloride (prepared in Example 1) (molar ratio is 1:1), wherein according to the nitrogen mustard concentration calculation is respectively 0mM, 0.01mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM , 0.8mM, 1mM, each concentration set 5 duplicate wells in parallel, in which only PBS was added to the last well as a blank control. And a blank group was set as a control. After gently shaking on a shaker for 5 min, the culture plate was placed in an incubator for culture. After 48 hours, take out the culture plate, add 10 μl of MTT (5 mg/mL) solution to each well under sterile conditions, continue to cultivate for 4 hours, terminate the culture, and carefully suck off the culture solution in the well. Then, 100 μl DMSO was added to each well, and placed on a shaker at low speed for 10 minutes to fully dissolve the purple crystals. The absorbance of each well was detected at 490 nm in an automatic microplate reader.

3. Experimental results

As shown in Figure 6A and Figure 6B.

The results showed that the CP6A inclusion drug nitrogen mustard (NM) significantly improved the anti-tumor cell activity of the drug compared with the nitrogen mustard drug alone.

Example 4: Evaluation of the inhibitory effect of CP6A/oxaliplatin complex on tumor cells

1. Experimental samples

CP6A/oxaliplatin complex: prepared in Example 2.

Lung cancer cells A549, liver cancer cells HepG2, T24 cells (bladder cancer cells), NCI cells (lung cancer cells), MGC-803 cells (gastric cancer cells), and BEL-7404 cells (liver cancer cells): all provided by Peking Union Medical College Cell Bank.

2. Experimental method

The MTT method was used to evaluate the inhibitory effect of CP6A/oxaliplatin complex on lung cancer cell A549, liver cancer cell HepG2, bladder cancer T24, lung cancer NCI, gastric cancer MGC-803, and liver cancer BEL-7404. As a control group, the toxicity of the cells was measured 48h after administration.

For each type of cell, collect the cells grown in the logarithmic phase, adjust the concentration of the cell suspension, inoculate the cell suspension on a 96-well plate, and plate to adjust the density of the cells to be tested to about 10000/well, 100 μL of the cell suspension per well, Incubate at 5% CO ₂ at a constant temperature of 37°C for 24 hours. Observe under a microscope that the cells adhere to the wall and grow. Add 10 μL of the prepared oxaliplatin aqueous solution or column ammonium hexaacetate containing oxaliplatin to the culture plate (implementation Example 2 preparation) (molar ratio is 1:1), wherein, according to the oxaliplatin concentration calculation is respectively 0mg/ml, 25mg/ml, 50mg/ml, 100mg/ml, 200mg/ml, 400mg/ml, each Five replicate wells were set up in parallel for each concentration, and only PBS was added at the end as a blank control. And a blank group was set as a control. After gently shaking on a shaker for 5 min, the culture plate was placed in an incubator for culture. After 48 hours, take out the culture plate, add 10 μl of MTT (5 mg/mL) solution to each well under sterile conditions, continue to cultivate for 4 hours, terminate the culture, and carefully suck off the culture solution in the well. Then, 100 μl DMSO was added to each well, and placed on a shaker at low speed for 10 minutes to fully dissolve the purple crystals. The absorbance of each well was detected at 490 nm in an automatic microplate reader.

3. Experimental results

See Table 3 below.

table 3

The results show that the complex formed by oxaliplatin and CP6A can significantly improve the inhibitory effect of the drug on various tumor cells.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

CLAIMS 1. A complex formed by a drug and a pyrarene derivative; preferably, the complex is formed by an intermolecular supramolecular interaction of the drug and a pyrarene derivative. 2. The compound according to claim 1, wherein the drug is an antineoplastic drug; preferably, it is nitrogen mustard, cisplatin, carboplatin or oxaliplatin. 3. The complex according to claim 1, wherein the pillar arene derivative is as shown in the following formula I, in, n is 5, 6, 7, 8, 9 or 10; R is selected from the following formula II, formula III or formula IV, and two R are the same; In formula II, M ⁺ is ammonium ion, sodium ion or potassium ion, and a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; In formula III, Y is amino group, aldehyde group, hydroxyl group, guanidine group, acid group, quaternary ammonium salt or pyridinium salt, and b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In formula IV, c is 1, 2, 3, 4 or 5. 4. The compound according to any one of claims 1 to 3, wherein the pillararene is selected from the following compounds 1-5: Preferably, the compound 1 is the following compound 6, 5. The method for preparing the complex of any one of claims 1 to 4, comprising the steps of: directly dissolving a certain molar ratio of the mixture of the drug and the pyrene derivative in a benign solvent and mixing them evenly, and then freeze-drying or vacuum-drying under reduced pressure to obtain a complex; Preferably, the following steps are included: Dissolving a mixture of nitrogen mustards and pillararene derivatives in a certain molar ratio directly in a benign solvent and mixing them evenly, and then freeze-drying the solution of the mixture or vacuum-drying under reduced pressure within 10 minutes; or Preferably, the following steps are included: Dissolve the mixture of oxaliplatin and pillararene derivatives in a certain molar ratio directly in a benign solvent at 30°C-60°C (preferably 40°C-60°C) and ultrasonically shake for a period of time (preferably 10-20min), to Saliplatin is completely dissolved, and then the solution of the mixture is freeze-dried or vacuum-dried under reduced pressure; Preferably, the above-mentioned pillar arene derivative is the following compound 6, 6 . The preparation method according to claim 5 , wherein the molar ratio of the pillararene derivative to the drug is 0.1-10; preferably 0.5-5 or 0.8-1.5; more preferably 1. 7. The preparation method according to claim 6, wherein the good solvent is selected from water, buffer solution, methanol, ethanol, isopropanol or mixtures thereof. 8. A pharmaceutical composition, which comprises the complex according to any one of claims 1 to 4, and optional pharmaceutically acceptable auxiliary materials. 9. Use of the complex according to any one of claims 1 to 4 in the preparation of a medicament selected from: Drugs for the treatment and/or prevention of tumors, drugs that suppress tumor cells, or Drugs to treat and/or prevent cancerous pleurisy, pericardial effusion or peritoneal effusion; Preferably, the tumor is selected from lung cancer, liver cancer, bladder cancer, stomach cancer, ovarian cancer, breast cancer, prostate cancer, malignant lymphoma and colon cancer; Preferably, the tumor cells are selected from lung cancer cells, liver cancer cells, bladder cancer cells, gastric cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, malignant lymphoma cells and colon cancer cells. 10. A method for inhibiting tumor cells in vivo or in vitro, comprising the step of using an effective amount of the compound according to any one of claims 1 to 4; preferably, the tumor cells are selected from lung cancer cells, liver cancer cells cells, bladder cancer cells, gastric cancer cells, ovarian cancer cells, breast cancer cells, prostate cancer cells, malignant lymphoma cells, and colon cancer cells.