A polyglycolated calixarene compound, and a preparation method and application thereof

Abstract

The application discloses a kind of polyethylene glycol cupped aromatic compounds and its preparation method and application, the polyethylene glycol cupped aromatic compound is connected by diazotization reaction by skeleton molecule and the diamino benzoic acid with metaposition protection, and then by upper end free carboxyl modification hydrophilic polyethylene glycol molecule to construct amphiphilic molecule;The compound can prepare nanomicelle assembly by film dispersion method, and simultaneously in macrocyclic cavity, anti-inflammatory drug can be loaded by host-guest interaction;The compound can realize drug release and nitric oxide absorption in anoxic environment, as a kind of cupped aromatic nanodrug system, in drug loading and nitric oxide absorption synergistic treatment anoxic related inflammation has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of polyglycolated calixarene compound, also relates to the preparation method and application of above-mentioned compound. BACKGROUND

[0002] Nitric oxide (NO) is one of the members of endogenous signaling molecules, involved in physiological pathways such as immune response, vasodilation and apoptosis, NO plays different roles in vivo depending on concentration and exposure time; low concentration can promote cell proliferation, can play an anti-inflammatory role; but high concentration is easy to cause apoptosis, play a pro-inflammatory role. Excess nitric oxide induces the production of inflammatory factors, leading to synovial inflammation, causing apoptosis. At the same time, excess NO induces strong nitrosative stress (RNS), recruits immune cells, and increases the number of osteoclasts, causing cartilage destruction; compared with the unexpected side effects of directly inhibiting nitric oxide synthase (iNOS), selective removal of excess NO is one of the effective strategies for treating inflammation.

[0003] Calixarene as the third generation of macrocyclic carriers has been widely concerned in recent years. Calixarene is a hollow ring structure synthesized from phenol and formaldehyde under strong acid or base, which is cup-shaped and its cavity size can be determined by the number of benzene rings. Its solubility in water or organic solvents is poor, but there are many methods for structural modification. Among them, azo-calixarene can be constructed by azo bond connection, which is often used as a low-oxygen-sensitive macrocyclic carrier to play a role in drug delivery, gene therapy, environment and food fields. Its reduction product, aminocalixarene, has natural active oxygen scavenging capacity and important application value in the treatment of related inflammatory diseases.

[0004] The existing drugs for treating inflammatory diseases have long treatment cycle, poor water solubility, gastrointestinal irritation, osteoporosis side effects, etc., and the reported nitric oxide absorption system is relatively single, which is difficult to achieve the expected therapeutic effect. SUMMARY

[0005] The purpose of the present application is to provide a polyglycolated calixarene compound for regulating nitric oxide absorption in a hypoxic environment, and to provide a preparation method of the above-mentioned polyglycolated calixarene compound and its application in preparing a drug for treating hypoxia-related inflammation.

[0006] Technical scheme: The present application discloses a kind of polyglycolated calixarene compound, structure is as shown in formula I:

[0007]

[0008] Wherein, n is an integer of 4-8, and m is an integer of 2-44.

[0009] The preparation method of the above polyethylene glycolized calixarene compound DAC4A comprises the following steps:

[0010] (1) taking 4-tert-butyl calixarene and phenol as raw materials, a calixarene is obtained by reverse Friedel-Crafts alkylation in a toluene solution containing aluminum chloride, that is, a macrocyclic molecular skeleton 1-1 is formed;

[0011] (2) taking 3,4-diaminobenzoic acid and 9-fluorenylmethyl-N-succinimidyl carbonate as raw materials, a single protection of the meta amino group is realized by a nucleophilic reaction, that is, an intermediate 2-1 is formed;

[0012] (3) the intermediate 3-1 is obtained by diazotization reaction of the macrocyclic molecular skeleton 1-1 and the intermediate 2-1;

[0013] (4) taking polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride as raw materials, an intermediate 4-1 is synthesized, and then the intermediate 4-2 is obtained by stirring in ammonia water;

[0014] (5) the protecting group of the intermediate 3-1 is removed under the action of 1,8-diazabicyclo[5.4.0]undec-7-ene, that is, DBU, the meta amino group is protected by Boc, and then the intermediate 4-2 is connected by an amidation reaction, and the Boc protection is removed, so that the polyethylene glycolized calixarene compound of the application is obtained.

[0015] Wherein, the synthesis route, step (1) is:

[0016]

[0017] Wherein, step (2) is:

[0018]

[0019] Wherein, step (3) is:

[0020]

[0021] Wherein, step (4) is:

[0022]

[0023] Wherein, step (5) is:

[0024]

[0025] The application further discloses a polyethylene glycolized calixarene nanomicelle, which is prepared from the polyethylene glycolized calixarene compound, has a hydrophilic chain end exposed to water and a hydrophobic end phenyl ring skeleton as a core, and forms a stable spherical structure micelle with multiple cavities on the surface.

[0026] The preparation method of the nanomicelles comprises the following steps:

[0027] The DACnA and the drug are weighed and placed in a glass bottle, and the organic phase is fully dissolved, and the solvent is removed by evaporation under reduced pressure to obtain a uniform thin film; the preheated water phase is added, and the ultrasonic treatment is continued, and the nanomicelles are obtained by membrane filtration.

[0028] The drug is an anti-inflammatory drug, the organic phase is chloroform, the water phase is a PB buffer solution, and the organic phase: water phase = 1:2-5 (v:v).

[0029] The ultrasonic treatment time is 5-20 min, and the 0.45 mu m membrane filtration is performed.

[0030] The polyglycolated calixarene compound or the polyglycolated calixarene nanomicelle can be applied to regulate the absorption of nitric oxide in a hypoxic environment.

[0031] The polyglycolated calixarene compound or the polyglycolated calixarene nanomicelle can also be applied to prepare an anti-inflammatory drug.

[0032] The application is that the polyglycolated calixarene compound or the polyglycolated calixarene nanomicelle is used for synergistically preparing an anti-inflammatory drug with curcumin.

[0033] Invention principle: the application discloses a polyglycolated calixarene compound DAC4A for regulating the absorption of nitric oxide in a hypoxic environment, the polyglycolated calixarene is first combined with a nitric oxide absorption and a calixarene drug-loaded molecule, a synergistic treatment platform is constructed, the polyglycolated calixarene compound is connected by diazotization reaction of a skeleton molecule and a meta-protected diaminobenzoic acid, and then a hydrophilic polyethylene glycol molecule is modified by a free carboxyl group at the upper end to construct an amphiphilic molecule; the compound can be used as a carrier, and the breaking of an azo bond is realized under the action of an overexpressed azo reductase in a hypoxic microenvironment, and the free ortho-phenylenediamine structure can react with nitric oxide, so that the effect of absorbing excess nitric oxide is achieved.

[0034] The compound can prepare nanomicelle assembly by thin film dispersion method, and the polyethylene glycol monomethyl ether is modified on the upper end of the azacalixarene through amidation reaction, which can provide sufficient hydrophilic segment, and the benzene ring skeleton at the lower edge can construct an amphiphilic macrocycle molecule together, so as to self-assemble to form nanomicelles. The nanomicelles are carriers and therapeutic agents, and the post-processing is simple, and anti-inflammatory drugs can be loaded in the macrocycle cavity through host-guest interaction for synergistic anti-inflammatory effect. The self-assembled nanomicelles based on the macrocycle amphiphilic reagent integrate multiple macrocycle cavities into a single nanoparticle, have the functions of realizing drug hypoxia release and quantitative loading of multiple drugs, and rely on the active oxygen scavenging and nitric oxide absorption effects of the macrocycle itself to achieve the purpose of synergistic treatment. The delivery system has important significance for expanding the new structure of macrocycle carriers in the aspect of anti-inflammatory, developing new drugs, and achieving the synergistic therapy of space consistency of carriers.

[0035] Advantages: Compared with the prior art, the polyethylene glycolated calixarene compound of the present application has the following remarkable advantages: (1) The polyethylene glycolated calixarene compound of the present application has accurate molecular weight, can integrate multiple macrocycle cavities into the same nanoparticle, and realize accurate loading of multiple drugs; the surface-modified polyethylene glycol hydrophilic segment has good biocompatibility, can prolong in vivo circulation, the macrocycle molecule can be reduced by overexpressed azo reductase in a hypoxic microenvironment, realize accurate release of drugs, and cause nitric oxide absorption effect; (2) The synthesis method and post-processing are simple and easy to operate, the micelles are prepared by thin film dispersion method, the operation is simple and easy to obtain, the prepared micelles have stable and uniform structure, and have good reproducibility; (3) In the preparation of anti-inflammatory drugs, the macrocycle molecule can load curcumin molecules in the cavity through host-guest interaction, which can solve the deficiency caused by single reliance on its own anti-inflammatory activity, and has the effect of synergistic treatment of inflammation. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a nuclear magnetic characterization diagram of intermediate compound 1-1 in example 1;

[0037] Figure 2 It is a nuclear magnetic characterization diagram of intermediate compound 2-1 in example 1;

[0038] Figure 3 It is a nuclear magnetic characterization diagram of intermediate compound 3-1 in example 1;

[0039] Figure 4 It is a nuclear magnetic characterization diagram of intermediate compound 4-2 in example 1;

[0040] Figure 5 It is a nuclear magnetic characterization diagram of intermediate compound 5-1 in example 1;

[0041] Figure 6 It is a nuclear magnetic characterization diagram of target product DAC4A in example 1;

[0042] Figure 7 The fitting curve diagram of the fluorescence titration curve and the binding constant of DAC4A and curcumin in Example 2, wherein A is the direct fluorescence titration curve of DAC4A and RhB, B is the fitting curve of direct fluorescence titration, C is the competitive fluorescence titration curve of DAC4A and curcumin, and D is the fitting curve of competitive fluorescence titration;

[0043] Figure 8 The particle size distribution diagram of DAC4A nanomicelles before and after drug loading in Example 3, wherein A is after drug loading, and B is before drug loading;

[0044] Figure 9 The Fourier infrared spectrum of DAC4A nanomicelles in Example 3;

[0045] Figure 10 The in vitro drug release curve diagram of DAC4A nanomicelles in Example 5;

[0046] Figure 11 The particle size change diagram of DAC4A nanomicelles in Example 6 under in vivo simulation environment;

[0047] Figure 12 The ultraviolet spectrum after SDT treatment and the absorbance value (A) and time relationship curve (B) under 365 nm in Example 7;

[0048] Figure 13 The ultraviolet spectrum of DAC4A under the change of NO concentration before and after SDT treatment in Example 8, wherein A is the initial ultraviolet spectrum of the sample group after SDT treatment, B is the change of absorption peak with the increase of NO concentration, and C is the control group;

[0049] Figure 14 The in vitro NO absorption of DAC4A by Griess reagent method in Example 8;

[0050] Figure 15 The cell survival rate diagram of DAC4A nanomicelles in RAW cell toxicity test in Example 9;

[0051] Figure 16 The intracellular NO absorption fluorescence image of DAC4A in Example 10;

[0052] Figure 17 The effect of PEG modification with different molecular weights on the hypoxia responsiveness of DAC4A in Example 11;

[0053] Figure 18 The effect of PEG modification with different molecular weights on the in vitro NO absorption of DAC4A in Example 11;

[0054] Figure 19Example 11. Effect of PEG modification with different molecular weight on the DPPH radical scavenging of DAC4A in vitro. DETAILED DESCRIPTION

[0055] The technical solutions of the present application are further described below in combination with examples. The test materials used in the examples can be purchased through conventional channels.

[0056] Example 1

[0057] The PEGylated calix[4]arene for regulating hypoxic absorption of nitric oxide of the present application has the following synthetic route:

[0058]

[0059]

[0060] Synthesis and structural characterization of intermediate compound 1-1:

[0061] 4-tert-butyl calix[4]arene (4.4 g, 6.78 mmol) was dissolved in a reaction flask containing toluene (100 mL), phenol (4.5 g, 47.8 mmol) was weighed and slowly dropped into the flask, toluene (10 mL) was added, and then aluminum chloride (8 g, 60 mmol) was added, and stirred at 60°C overnight. After cooling to room temperature, hydrochloric acid (3%, v / v, 100 mL) was added and stirred for 30 min, the aqueous phase was washed once with toluene, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the toluene was rotary evaporated, the semi-solid residue was slurried with methanol (35 mL), the precipitate was filtered and vacuum dried, to obtain a crude white solid powder, then the crude product was dissolved in chloroform (10 mL) at 55°C, and the above crude solution was added to methanol (50-80 mL) to precipitate a white solid powder (2.38 g, yield 82%), which was intermediate compound 1-1.

[0062] 1 H NMR (400 MHz, DMSO-d6) δ 9.77 (s, 4H), 7.12 (d, J = 7.5 Hz, 8H), 6.65 (t, J = 7.5 Hz, 4H), 3.88 (s, 8H).

[0063] Synthesis and structural characterization of intermediate compound 2-1:

[0064] Dissolve 3,4-diaminobenzoic acid (2 g, 13.1 mmol) in 0.1 M sodium bicarbonate solution: acetonitrile (1 : 1, v / v, 50 mL) to get a brown slurry, add 9-fluorenylmethyl-N-succinimidyl carbonate (4.4 g, 13.1 mmol) in small portions to the slurry over 5 min, stir at room temperature for 16-18 h, monitor by TLC (ethyl acetate: petroleum ether = 1 : 1), the solution color becomes lighter and clear. Acidify the reaction mixture with 1 M hydrochloric acid (15 mL), the solution becomes tan, filter the precipitate and wash with cold water, cold diethyl ether, n-hexane and methanol in sequence, and dry under vacuum to get a grey solid (4.65 g, yield 95%) which is intermediate compound 2-1

[0065] 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 7.99-7.21 (m, 9H), 6.72 (d, J = 8.4 Hz, 2H), 5.70 (s, 2H), 4.52-4.25 (m, 3H).

[0066] Synthesis and structural characterization of intermediate compound 3-1:

[0067] Dissolve calix[4]arene (0.4 g, 0.94 mmol) and sodium acetate (0.98 g, 12 mmol) in methanol: N,N-dimethylformamide (5:8, v / v, 13 mL) to get solution A (PH 7-10). Dissolve intermediate 2-1 (1.58 g, 4.23 mmol) and hydrochloric acid (37%, v / v, 2 mL) in N,N-dimethylformamide solution (24 mL), then place the solution below -5 °C, slowly drop sodium nitrite aqueous solution (5 mL, 6%, w / w) into the above solution, react at low temperature for 30 min to get solution B.

[0068] Slowly drop solution B into solution A at -5 °C to get a suspension, stir at room temperature for 2-3 h, then adjust PH = 1 with hydrochloric acid (37%, v / v, 15 mL), continue to stir for 30 min, filter the mixture, wash with water and methanol repeatedly, and dry to get a yellow powder (1.4 g, yield 75%) which is intermediate compound 3-1.

[0069] 1H NMR (300 MHz, DMSO-d6) δ 8.50 - 8.44 (m, 4H), 8.34 (d, J = 8.6 Hz, 4H), 8.09 (dd, J = 8.6, 1.5 Hz, 4H), 7.89 (dd, J = 29.9, 7.5 Hz, 20H), 7.50 - 7.30 (m, 20H), 7.10 (d, J = 7.5 Hz, 8H), 5.01 (d, J = 6.7 Hz, 8H), 4.60 (d, J = 6.7 Hz, 4H), 3.86 (s, 8H).

[0070] Synthesis and structural characterization of intermediate compound 4-2:

[0071] Polyethylene glycol monomethyl ether (MW = 1000, 15 g, 15 mmol) was dissolved in a reaction flask containing dichloromethane (200 mL), followed by the addition of p-toluenesulfonyl chloride (7.15 g, 37.5 mmol), potassium hydroxide (8.4 g, 150 mmol), sealed with a balloon, and reacted at 28 °C for 12 h. TLC monitoring (dichloromethane:methanol = 10:1). The organic phase was washed with 2M hydrochloric acid (100 mL x 2), the lower organic phase was removed, dried over anhydrous sodium sulfate, filtered, and the organic phase was rotary evaporated to obtain a viscous liquid, which was then precipitated with cold anhydrous ether, the precipitate was filtered and dried by oil pump extraction to obtain a white powder (13.5 g, yield 90%).

[0072] Intermediate 4-1 (10 g, 10 mmol) was dissolved in ammonia water (50 mL), followed by the addition of ammonium chloride (5.35 g, 100 mmol), and reacted at 40 °C for 72 h, with TLC monitoring (dichloromethane:methanol = 10:1). The reaction solution was saturated with sodium chloride, extracted with dichloromethane (100 mL x 3) three times, the organic phases were combined, and rotary evaporated to a viscous state, which was then precipitated with cold ether, filtered and dried to obtain a white solid powder (8.7 g, yield 87%).

[0073] 1 H NMR (400 MHz, Chloroform-d) δ 3.88 - 3.46 (m, 88H), 3.40 (s, 3H), 2.92 (t, J = 5.2 Hz, 2H).

[0074] Synthesis and structural characterization of intermediate compound 5-1:

[0075] Intermediate compound 3-1 (400 mg, 0.2 mmol) was dissolved in N,N- dimethylformamide (10 mL), then 1,8-diazabicyclo[5.4.0]undec-7-ene, DBU (200 μΐ, 2.4 mmol) was added dropwise into the reaction flask, the solution changed from yellow orange to red, 25 °C reaction for 5-30 min, TLC monitoring (dichloromethane:methanol = 5:1) to the end point. A large amount of cold water was injected into the reaction solution until a large amount of precipitate was precipitated, then the mixture was loaded into a centrifuge tube and centrifuged (6000 rpm, 10 min), the red precipitate was collected, swabbed with petroleum ether, filtered and dried to obtain a red solid powder (116 mg, 54%).

[0076] 1 H NMR (400 MHz, DMSO-d6) δ 7.93 - 7.82 (m, 4H), 7.70 (s, 8H), 7.07 (d, J = 7.5 Hz, 8H), 6.29 (s, 1H), 5.76 (s, 1H), 3.80 (d, J = 35.9 Hz, 8H).

[0077] Synthesis and structural characterization of the target product (DAC4A):

[0078] GuHCl (2.66 mg, 0.028 mmol) and di-tert-butyl dicarbonate (44.65 mg, 0.205 mmol) were added to absolute ethanol, the reaction was warmed to 38 °C to dissolve, then intermediate compound 5-1 was added, and reacted at 38 °C overnight. TLC monitoring (dichloromethane:methanol = 5:1) to the end point. The solvent ethanol was removed by distillation under reduced pressure, and the residue was swabbed with petroleum ether to obtain a red powder. The precipitate was washed with saturated NaCl solution and dried to obtain intermediate compound 5-2. Intermediate 5-2 (50 mg, 0.03 mmol) was dissolved in tetrahydrofuran (8 mL), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91 mg, 0.24 mmol) and N,N-diisopropylethylamine (63 μΐ, 0.36 mmol) were added, and activated at 60 °C for 20 min. Polyethylene glycol monomethyl ether amino (180 mg, 0.18 mmol) was added, and reacted at 60 °C for 12 h. The solution was transferred into a dialysis bag (MWCO: 1500 Da), dialyzed in high-purity water for 24 h to remove impurities, and then freeze-dried to obtain a red powder. The powder was dissolved in 25 mL dichloromethane, 5 mL trifluoroacetic acid was added, and reacted at room temperature for 2 h. The solvent was removed by distillation under reduced pressure, the crude product was dissolved in 20 mL saturated sodium bicarbonate solution, extracted with dichloromethane (50 mL x 2), the organic phases were combined and the solvent was removed by distillation under reduced pressure to obtain a red powder, which was the target product (DAC4A) (63 mg, yield 46%).

[0079] 1H NMR(400MHz,Chloroform-d)δ8.83(s,4H),8.50(s,8H),8.10(d,J=7.5Hz,4H), 7.70(s,8H),7.62(s,4H),3.82(s,8H),3.67(s,352H),3.37(d,J=22.2Hz,12H).

[0080] Example 2

[0081] Determination of the binding constant of azocalixarene (DAC4A) and curcumin

[0082] Testing tools: Quartz cuvettes were used as sample cells, the excitation wavelength was 365nm, the excitation slit was 5nm, the emission slit was 10nm, and the instrument was a Hitachi fluorescence spectrophotometer (F-2710FL fluorescence).

[0083] The binding constant of azocalixarene (DAC4A) and rhodamine B was determined by direct fluorescence titration. Stock solutions of DAC4A and RhB were prepared at a concentration of 10 μM using PBS buffer (pH 7.4). For testing, RhB was diluted to 0.5 μM and added to a fluorescence cuvette. The volume was then adjusted to 2 mL with PBS (final RhB concentration: 0.375 μM). The initial fluorescence intensity of the RhB solution was measured and recorded. Subsequently, a predetermined volume of DAC4A solution was added dropwise to the fluorescence cell, maintaining a constant final RhB concentration. The binding constant K between DAC4A and RhB was determined using the changes in fluorescence intensity and corresponding host concentration. a Data were fitted according to a 1:1 stoichiometric ratio. The binding constant of DAC4A and Cur was determined using competitive fluorescence titration. Competitive fluorescence titration was performed by gradually adding the competing substance Cur to an RhB@DAC4A solution. The fluorescence intensity at the emission wavelength of RhB and the concentration of Cur were fitted according to a 1:1 competitive binding model. Each titration experiment was performed in triplicate. a The average value of the three test results is taken, and the fluorescence titration spectrum results are attached. Figure 7 As shown in Figures A and C. Curve fitting of fluorescence intensity with corresponding host and curcumin concentrations was performed using Origin, and the results are shown in the appendix. Figure 7 As shown in B and D, the binding constant K of DAC4A with RhB and Cur is... a They are (1.01±0.08)×10 7 M -1 and (5.0±0.4)×10 5 M -1 .

[0084] Example 3

[0085] Preparation and characterization of azocryptophane (DAC4A) drug-loaded nanomicelles:

[0086] Precisely weigh DAC4A (10 mg, 2 μmol) and curcumin (1.84 mg, 10 μmol) into a glass bottle, dissolve with 3 mL of chloroform, and evaporate the solvent under reduced pressure to obtain a uniform thin film; add 10 mL of PB buffer solution (pH 7.4, 10 mM) preheated to 37°C, continue to ultrasonic for 5-10 min, filter with a 0.45 μm microporous filter to obtain nanomicelles loaded with curcumin, and the particle size is measured by DLS as shown in FIG. 1A, with an average particle size of about 150 nm and a PDI of 11.64%. Figure 13

[0087] Precisely weigh DAC4A (10 mg, 2 μmol) into a glass bottle, dissolve with 3 mL of chloroform, and evaporate the solvent under reduced pressure to obtain a uniform thin film; add 10 mL of PB buffer solution (pH 7.4, 10 mM) preheated to 37°C, continue to ultrasonic for 5-10 min, filter with a 0.45 μm microporous filter to obtain empty nanodrug delivery system, and the particle size is shown in FIG. 1B, with an average particle size of about 200 nm and a PDI of 19.39%. Figure 13

[0088] From the results Figure 8 it can be seen that compared with the empty nanodrug delivery system, the particle size and polydispersity index of the cryptophane nanomicelles after loading curcumin decrease significantly, indicating that the combination of curcumin and the hydrophobic cavity further increases the overall hydrophobicity of the material, making the internal hydrophobic region more compact and forming smaller particle size.

[0089] Fourier infrared spectroscopic characterization of azocryptophane (DAC4A) nanomicelles:

[0090] An infrared spectrometer with an attenuated total reflection ATR accessory was used to analyze the chemical structure of the nanodrug delivery system, and free curcumin Cur, freeze-dried empty and drug-loaded nanodrug delivery systems, and DAC4A and Cur physical mixture were scanned in the range of 4000-400 cm -1 -1, respectively, and the results are shown in FIG. 2. Figure 9 Cur is near 3500 cm -1 -1 is the stretching vibration peak of O-H bond on phenolic hydroxyl group, 1600-1750 cm -1 -1 is the stretching vibration peak of C=O carbonyl group, 1550 cm -1 -1 is the stretching vibration peak of C=C, 1000-1250 cm -1 -1 is the characteristic absorption peak of -C-O-C- ether bond; DAC4A is near 3250 cm -1 -1 is the stretching vibration peak of N-H on primary amine, 2800 cm​​-1 near the characteristic absorption peak of C-H on the polyethylene glycol chain, 1650 cm -1 near the N-H bending vibration absorption peak of primary amine, 1450 cm -1 and 1550 cm -1 near the C=O stretching vibration peak and N=N stretching vibration, respectively, 1250-1350 cm -1 is the aromatic amine C-N stretching vibration peak, 1150 cm -1 near the C-O-C stretching vibration peak on the polyethylene glycol chain.

[0091] Compared with the blank and the drug-loaded nanodelivery system, due to the influence of hydrogen bond and other non-covalent interactions between DAC4A and Cur molecules on the bond force constant of hydrogen atom and its connected functional group, the characteristic absorption peaks of curcumin Cur, 1650 cm -1 , 1550 cm -1 , and 3500 cm -1 disappeared obviously, proving that the drug was successfully encapsulated.

[0092] Example 4

[0093] Encapsulation efficiency and drug loading of azacalixarene (DAC4A) nanomicelles:

[0094] The encapsulation efficiency and drug loading of the nanodelivery system were determined by reverse dialysis. The drug-loaded nanomicelles were prepared by the method in Example 3, and 5 mL of the prepared drug-loaded nanomicelle suspension was dispersed in 150 mL of 15% ethanol. 5 mL of 15% ethanol was placed in the dialysis bag (MWCO: 1000 Da), and 1 mL of dialysate was taken out from the dialysis bag every 2 h. The absorbance value was determined by ultraviolet spectrophotometry, and the same volume of medium was added. The free drug concentration was calculated by using the standard curve of curcumin. The encapsulation efficiency of the drug-loaded nanodelivery system was 75.23 ± 1.14% and the drug loading was 11.62 ± 0.8% by using formula (1-3).

[0095]

[0096] wherein

[0097] M: total drug mass (mg) added;

[0098] m: free drug mass (mg);

[0099] Example 5

[0100] In vitro drug release of azacalixarene (DAC4A) nanomicelles:

[0101] Drug-loaded DAC4A nanomicelles were prepared using the nanomicelle preparation method in Example 3, and the prepared drug-loaded micelles were transferred to a dialysis bag and placed in a PB buffer solution (pH 7.4, 10 mM) and a PB solution containing 0.2% SDS, respectively, and then placed in a 37°C constant temperature shaker. The release medium was taken out every 1 h, and the same volume of medium was supplemented. The content of curcumin was then determined by a fluorescence spectrophotometer, with an excitation wavelength of 425 nm, an excitation slit of 5 nm, and an emission slit of 10 nm. The results are shown in FIG. 8. Figure 10 As shown in FIG. 8, the drug-loaded micelles in the PB solution containing 0.2% SDS had a release efficiency of more than 80% within 10 h, and the control group had a release efficiency of less than 40% within 24 h.

[0102] Example 6

[0103] Stability test of azacalixarene (DAC4A) nanomicelles:

[0104] The drug-loaded and unloaded DAC4A nanomicelles were prepared using the nanomicelle preparation method in Example 3. The prepared drug-loaded micelles were placed in a 10% FBS PBS solution to simulate the body fluid environment, and then placed in a 37°C constant temperature shaker. The particle size change was detected by DLS at 1, 2, 3, 4, and 5 days, respectively. The results are shown in FIG. 9. Figure 11 It can be proved that the particle size of the drug-loaded micelles remains basically unchanged, indicating that the prepared nanodrug delivery system has good stability in the simulated body fluid circulation.

[0105] Example 7

[0106] In vitro simulation of azacalixarene (DAC4A) hypoxia cleavage:

[0107] In vitro hypoxia cleavage of DAC4A was simulated by using sodium dithionite (SDT) to simulate azo reductase to achieve cleavage of the azo bond. The cleavage was characterized by scanning the ultraviolet absorption spectrum and detecting the change in the absorption peak of the azo bond at 365 nm. A solution of DAC4A (1 mg / mL) was prepared using 5 mL of PBS at pH 7.4 as a solvent as a sample solution. Sodium dithionite (0.17 g, 1 mmol) was weighed and dissolved in 5 mL of PBS to obtain an SDT solution. 1 mL of the sample solution was taken and gradually added with the SDT solution to keep the sample concentration unchanged. The sample was placed in a 37°C water bath, and the ultraviolet absorbance was measured at 365 nm every 2 min, with three parallel measurements and an average value. The average value was plotted against time to obtain a curve. The results are shown in FIG. 10. Figure 12 As shown in FIG. 10, with the continuous addition of the SDT solution, the ultraviolet absorption spectrum of DAC4A changed obviously, and the absorbance value at 365 nm gradually decreased. When the addition amount was 1 mL, the absorbance value was basically the lowest. Figure 12The azo bond absorption peak in the middle B shown after adding 1 mL of SDT solution rapidly dropped to the minimum within 2 min, indicating that DAC4A has good anaerobic cleavage ability.

[0108] Example 8

[0109] In vitro nitric oxide absorption test of azocyclophane (DAC4A):

[0110] (1) Determination of in vitro nitric oxide absorption by ultraviolet absorption method:

[0111] A saturated NO solution (1.8 mM at 20°C) was prepared as a stock solution by slowly dropping 2M H2SO4 into a glass flask containing a saturated NaNO2 aqueous solution to generate NO.

[0112] The principle of this method is that NO will react with o-phenylenediamine structure to generate benzotriazole and hydrolyze, and the structural change will result in a significant ultraviolet absorption at around 350 nm.

[0113] A stock solution of DAC4A with a concentration of 50 μM was prepared using PBS with a pH of 7.4 as the solvent, 1 mL of the solution was taken and placed in an EP tube, and sodium dithionite (8.75 mg, 0.05 mmol) was added to obtain a sample solution. Another 1 mL of the stock solution was taken in an EP tube without any treatment as a control group. The NO stock solution (1.8 mM) was diluted to a series of concentration gradient solutions of 0-180 μM, 1 mL of the above different concentration gradient solutions was taken in the sample tube and the control tube, and after incubation for 30 min, the absorption curve of each mixed solution at 200-600 nm was measured by ultraviolet spectrophotometry. The results are shown in the following figure: Figure 13 As shown in the figure, A is the initial ultraviolet spectrum of the sample group after SDT treatment, and from B, it can be seen that with the increase of NO concentration, the absorption peak at 350 nm increases significantly, while C has no obvious change. It shows that the material after SDT cleavage can show obvious NO absorption effect.

[0114] (2) Determination of in vitro nitric oxide absorption by Griess method:

[0115] The principle of this method is that NO is oxidized to nitrite under Griess reagent, which promotes the generation of azo dye, and the ultraviolet absorption value of the dye at 540 nm is proportional to the amount of NO.

[0116] DAC4A and SDT stock solutions were prepared using PBS (pH 7.4, 10 mM) buffer, and the concentration of each was 10 mM. Several EP tubes were taken and numbered as A, B, C, and D, and each group was prepared according to the following formula:

[0117] A: 80 μl DAC4A solution (10 mM) + 100 μl SDT solution (10 mM) + 20 μl 37% hydrochloric acid;

[0118] B: 80 μΐ PBS + 100 μΐ SDT solution (10 mM) + 20 μΐ 37% hydrochloric acid;

[0119] C: 80 μΐ DAC4A solution (10 mM) + 100 μΐ PBS + 20 μΐ 37% hydrochloric acid;

[0120] D: 180 μΐ PBS + 20 μΐ 37% hydrochloric acid;

[0121] Group A is the experimental group, and groups B, C, and D are the control groups;

[0122] Take 50 μΐ of the above different solutions into a 96-well plate, set three parallel holes for each group, add 100 μΐ of NO stock solution and 100 μΐ of Griess reagent to each hole, mix well, incubate at 37°C for 10 min, use the enzyme marker to measure the absorbance value at 540 nm under ABS (ultraviolet absorption) mode, and draw the curve as shown in the accompanying Figure 14 As shown in the accompanying

[0123] Example 9

[0124] Cytotoxicity experiment (MTT) of azacrown nanodrug delivery system (DAC4A):

[0125] The MTT method was used to evaluate the toxicity of DAC4A nanodrug delivery system on macrophages (RAW cells). RAW cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator. First, 100 μΐ of cell 1640 suspension was plated in a 96-well culture plate, and the number of cells was controlled to be 5000 cells / well, then 10 μΐ of different concentrations of drug-loaded nanomicelles (DAC4A@Cur), blank nanomicelles (blank@DAC4A), and curcumin (Cur) solution were added to each well, so that the final concentration of the drug in the cell well was 10, 25, 50, 100, 200, and 500 μg / mL. After incubation for 24 h, the culture medium was replaced and 10 μΐ of MTT solution (5 mg / mL) was added to each well, and then placed in the incubator for further incubation for 4 h. Subsequently, the MTT-containing culture solution was removed, 150 μΐ of DMSO was added to each well, and after the purple crystals were completely dissolved, the absorbance was measured at 570 nm using an enzyme marker.

[0126] As shown in the accompanying Figure 15As shown, the cytotoxicity results show that as the concentration of DAC4A increases, there is no obvious cell inhibition effect for the empty nanomicelles, and compared with the free drug group, the wrapping of DAC4A can significantly improve the biological toxicity of the drug, and when the concentration is as high as 500 μg / mL, it still maintains more than 85% cell survival rate. It is proved that the azocyclophane has good biocompatibility.

[0127] Example 10

[0128] Intracellular NO absorption test of azocyclophane DAC4A

[0129] The NO level in RAW cells was labeled using DAF-FM, and the intracellular NO absorption characteristics of DAC4A were tested by observing the change of NO level. The principle of this method is that DA can pass through the cell membrane, enter the cell and be catalyzed by intracellular esterase to form DAF-FM which cannot pass through the cell membrane. DAF-FM itself has only weak fluorescence, but can produce strong fluorescence after reacting with NO, with an excitation wavelength of 495 nm and an emission wavelength of 515 nm.

[0130] 500 μl of 1640 suspension of RAW cells was plated in a 24-well culture plate containing cell slides, and the cell number was controlled to be 50000 cells per well. After the cells adhered, LPS (final concentration 1 μg / mL) was added to stimulate the RAW cells to polarize into M1 type for 12 h. Then 10 μl of DAC4A and CAC4A (control group, without NO absorption) were added to make the final concentration of the drug to be 500 μg / mL. The culture medium was aspirated, 200 μl of PBS was washed, 300 μl of 5 μM DAF-FM DA diluted was added, and the volume was appropriate to cover the cells completely. After 37°C incubation for 20 min, the culture medium was aspirated, and PBS was washed for three times. 200 μl of 4% paraformaldehyde was added for room temperature fixation for 20 min, and PBS was washed for three times. Finally, 200 μl of DAPI staining solution (10 μg / mL) was added to the cells for 15 min of incubation in the dark, and PBS was washed for three times. The cell slides were clamped so that the cell adhering side contacted with the glass slide with anti-fluorescence quencher dropped thereon. The edges were sealed with nail polish, and after drying, the fluorescence microscope was used to observe and take pictures of the fluorescence.

[0131] As shown in the accompanying Figure 16 RAW cells after adding DAC4A, compared with the control group CAC4A and PBS group, the green fluorescence signal was obviously reduced, which indicated that DAC4A could realize the absorption effect of NO in the inflammatory cells under the excessive expression of azo reductase in the cells.

[0132] Example 11

[0133] Performance evaluation of cuprophan DAC4A modified by different molecular weight polyethylene glycol

[0134] (1) The influence of different molecular weight polyethylene glycol modification on encapsulation, drug loading and particle size

[0135] According to the synthesis method of the target product in Implementation 1, amino polyethylene glycol monomethyl ether (MW = 200), amino polyethylene glycol monomethyl ether (MW = 550), and amino polyethylene glycol monomethyl ether (MW = 1000) were selected to modify calixarene, respectively, to obtain products PEG4-DAC4A, PEG12-DAC4A, and PEG22-DAC4A, and a control group PEG22-CAC4A was added. Drug-loaded nanomicelles were prepared by the film dispersion method in Example 3, and the particle size and PDI were characterized; the encapsulation rate (DLE) and drug loading capacity (DLC) were determined by the reverse dialysis method in Example 4, and the results are shown in Table 1:

[0136] Table 1, the influence of different molecular weight PEG modification on the encapsulation rate, drug loading capacity and particle size of DAC4A

[0137]

[0138] As can be seen from Table 1, with the increase of molecular weight, the micelle particle size gradually increases and the PDI becomes narrower, which benefits from the increase of the proportion of hydrophilic part due to the increase of molecular weight, and the thickening of the nanomicelle hydration layer; and with the growth of the molecular chain, it tends to form larger particle size in the spatial scale. It is also observed that the encapsulation rate and drug loading capacity gradually increase, which benefits from the large hydrophobic cavity that can accommodate more hydrophobic drugs.

[0139] (2) The influence of different molecular weight polyethylene glycol modification on hypoxia response

[0140] Precisely weigh sodium dithionite (174 mg, 1 mmol) and dissolve it in 2 ml of PBS to obtain a 500 mM SDT solution. Precisely weigh PEG4-DAC4A (1.8 mg), PEG12-DAC4A (3.2 mg), and PEG22-DAC4A (5 mg), respectively, and dissolve them in 1 ml of PBS to obtain a 1 mM sample solution. Dilute the SDT solution to different concentration gradients, and then add the above sample solutions. The absorbance value at 365 nm was determined by ultraviolet spectrophotometry, and the concentration-absorbance curve was drawn, as shown in the accompanying Figure 17 .

[0141] From Figure 17It can be seen that, compared with PEG4-DAC4A, the other two polyethylene glycol modifications have no obvious influence on hypoxia response, and when the SDT concentration reaches 80 mM, the cleavage of the azo bond can be obviously realized, proving that PEG12-DAC4A and PEG22-DAC4A have good hypoxia responsiveness, while PEG4-DAC4A has insufficient hypoxia responsiveness, which is related to the low proportion of hydrophilic in the molecule, leading to poor dispersibility.

[0142] (3) Influence of polyethylene glycol modification with different molecular weights on in vitro NO absorption

[0143] Using the sample solution prepared in (2) above, 100 mM of SDT solution was added to simulate in vivo hypoxia cleavage. Then, the in vitro NO absorption was determined by the Griess method in Example 8. The results are shown in FIG. 6. Figure 18 As shown in FIG. 6, with the extension of the action time, the NO residual amount of the sample group showed a downward trend to varying degrees compared with the control group PBS and PEG22-CAC4A, and the PEG12 and PEG22 modification showed a significant decrease, and the NO absorption rate reached about 75% at 25 min, while the NO absorption rate of PEG4 modification was less than 40%. It is proved that the degree of hypoxia response is positively correlated with NO absorption, and the modification of PEG12 and PEG22 has no obvious difference in NO absorption, and the modification of PEG4 will make the NO absorption effect worse, which is due to the lack of hydrophilic segment in the molecule, leading to poor dispersibility and solubility, and cannot fully react with NO molecules.

[0144] (4) Influence of polyethylene glycol modification with different molecular weights on DPPH radical scavenging

[0145] DPPH radical scavenging rate as an important indicator of the level of reactive oxygen species scavenging plays a key role in the screening of anti-inflammatory active molecules. Determining the influence of different molecular weight modifications on the DPPH radical scavenging of the material can further optimize the material.

[0146] The specific operation is as follows: prepare a DPPH solution with a concentration of 0.2 mmol and a sample solution (PEG4-DAC4A, PEG12-DAC4A, PEG22-DAC4A), and the sample solution is treated with SDT. Prepare a positive control group, 0.2 mmol of VC solution; and a negative control PBS group. In a 96-well plate, add 50 μL of DPPH solution and 150 μL of sample solution, respectively, and set three replicates. After mixing, the absorbance value is determined by a microplate reader at a wavelength of 517 nm, and the absorbance value is Aj, and the control group is only DPPH solution. The absorbance value is Ac, and the DPPH radical scavenging rate is calculated according to the following formula:

[0147]

[0148] K - radical scavenging rate

[0149] A i - absorbance value determined immediately

[0150] A j - absorbance value after a period of time

[0151] A c - absorbance value of the control group

[0152] Results as shown in the accompanying Figure 19 PEG12-DAC4A has the optimal DPPH scavenging rate compared with the VC group, reaching about 75% in 20 min, and PEG22-DAC4A is about 60%. PEG4-DAC4A is only less than 50%. The results prove that different PEG modification has obvious influence on DPPH, and good DPPH scavenging rate needs sufficient hydrophilic segment modification, so that the molecule has better dispersibility in solution, and is convenient for scavenging. Insufficient hydrophilic segment modification can significantly reduce the DPPH scavenging capacity.

[0153] Therefore, the polyglycolated calixarene compound of the application can realize drug release and nitric oxide absorption in a hypoxic environment, and as a calixarene nanodrug delivery system, has important application value in drug loading and nitric oxide absorption synergistic treatment of hypoxia-related inflammation.

Claims

1. A polyethylene glycol-modified calixarene compound, characterized in that, The structure of the compound is shown in Formula I: ； Where n is an integer from 4 to 8, and m is an integer from 2 to 44.

2. A method for preparing the polyethylene glycol-modified calixarene compound according to claim 1, characterized in that, Includes the following steps: (1) Using 4-tert-butylcalixarene and phenol as raw materials, calixarene is obtained by reverse Friedel-Crafts alkylation in a toluene solution containing aluminum trichloride, thus forming a macrocyclic molecular skeleton 1-1; ； (2) Using 3,4-diaminobenzoic acid and 9-fluorenylmethyl-N-succinimide carbonate as raw materials, the meta-amino group is monoprotected by nucleophilic reaction, thus forming intermediate 2-1; ； (3) The macrocyclic molecular skeleton 1-1 and intermediate 2-1 are subjected to a diazotization reaction to obtain intermediate 3-1; ； (4) Using polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride as raw materials, intermediate 4-1 was synthesized and then stirred in ammonia water to obtain intermediate 4-2; ； (5) Intermediate 3-1 is deprotected by 1,8-diazabicyclo[5.4.0]undec-7-ene, i.e. DBU, and the meta-amino group is protected by Boc. Then, it is connected to intermediate 4-2 by amidation reaction to remove Boc protection, thus obtaining polyethylene glycolated calixarene compound. 。 3. A polyethylene glycol-modified calixarene nanomicelle, characterized in that, The nanomicelles are prepared from the polyethylene glycol-modified calixarene compound of claim 1, with their hydrophilic chain ends exposed in the aqueous phase and their hydrophobic benzene ring skeletons facing inward to form the core, thus forming a stable spherical structure with multiple cavities on the surface of the micelles.

4. A method for preparing the nanomicelles according to claim 3, characterized in that, The process includes the following steps: weighing polyethylene glycol-modified calixarene compounds and drugs, fully dissolving them in the organic phase, removing the solvent by vacuum evaporation to obtain a uniform thin film; adding preheated aqueous phase, continuing sonication, and filtering through a filter membrane to obtain nanomicelles.

5. The preparation method according to claim 4, characterized in that, The organic phase is chloroform, the aqueous phase is PB buffer solution, and the volume ratio of the organic phase to the aqueous phase is 1:2~5.

6. The preparation method according to claim 4, characterized in that, Ultrasonication time is 5-20 minutes, and filtration is performed using a 0.45μm filter membrane.

7. The application of the polyethylene glycol-modified calixarene compound of claim 1 or the polyethylene glycol-modified calixarene nanomicelles of claim 3 in regulating nitric oxide absorption under hypoxic conditions.

8. The use of the polyethylene glycol-modified calixarene compound of claim 1 or the polyethylene glycol-modified calixarene nanomicelles of claim 3 in the preparation of anti-inflammatory drugs.

9. The application according to claim 8, characterized in that, The application is to prepare anti-inflammatory drugs by synergistically combining the polyethylene glycol-modified calixarene compound of claim 1 or the polyethylene glycol-modified calixarene nanomicelles of claim 3 with curcumin.

Images