A calix[3]carbazole derivative modified with a cross-methylene group and its preparation method and application

Abstract

The present invention discloses a methylidene-modified calix[3]carbazole derivative and its preparation method and application, belonging to the field of medical technology. The methylidene-modified calix[3]carbazole derivative of the present invention has a general structural formula as shown in formula (II); in formula (II), R4, R5, and R6 are each independently selected from one of hydroxyl, C1-C3 alkoxy, amino-substituted C1-C5 alkoxy, benzylamino, hydrazine, fatty amine containing O atoms, fatty amine containing N atoms, or ethoxy. The methylidene-modified calix[3]carbazole derivative of the present invention has good activity in inhibiting tumor cell proliferation and has good development prospects in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a methylene-modified calix[3]carbazole derivative and a preparation method and application thereof. Background Art

[0002] With the vigorous development of supramolecular chemistry, technologies related to host-guest recognition based on macrocycles such as crown ethers, cyclodextrins, cucurbiturils, calixarene and pillararene have played an extremely important role in the fields of life medicine, materials science and pharmacy. The creation of new supramolecular macrocyclic host compounds with excellent performance has always been a research hotspot in this field. In recent years, a macrocyclic compound with carbazole as the basic unit has been discovered. Due to its advantages of easy synthesis, large cavity, rich electrons, and its wide range of potential uses, such as ion fluorescence sensing, carriers of pharmaceutical molecules and pharmaceutical excipients, it has become the focus of many researchers. Calix[3]carbazole is a calixcarbazole, a new type of calixarene with carbazole as the basic unit. Compared with traditional calixarene, this molecule has a larger cavity and rich π electron system. It can not only encapsulate drugs with larger molecular size, but also has good ultraviolet absorption and fluorescence coloration properties. Spectroscopic methods can be used to study its performance in drug recognition, detection and interaction. Due to the low synthesis yield of the calix[3]carbazole skeleton, it has a great impact on the subsequent molecular synthesis. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a calix[3]carbazole derivative modified with a cross-methylene group and its preparation method and application. First, the present invention uses carbazole as a raw material and synthesizes a calix[3]carbazole mother nucleus through nucleophilic substitution, esterification, cyclization and other reactions; then, the cross-methylene group and the upper edge of the calix[3]carbazole derivative are subjected to structural derivatization by oxidation, aminolysis, hydrazinolysis, aldehyde amine condensation and other reactions to synthesize a novel cross-methylene group-modified cross-methylene group-modified calix[3]carbazole derivative. The calix[3]carbazole derivative modified in this form belongs to a supramolecular macrocyclic main compound, and its molecule has a large cavity and a rich π electron system. It can not only encapsulate drugs with larger molecular size, but also has good ultraviolet absorption and fluorescence coloring properties. Spectral means can be used to study its encapsulation performance for drugs. Taking calix[3]carbazole as the research object, the structure of the calix[3]carbazole is modified, and the types and ranges of its conformations are explored and broadened. The recognition and encapsulation performance of drugs such as hydroxycamptothecin are explored and discovered.

[0004] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0005] In the first aspect, the present invention provides a calix[3]carbazole derivative, the general structural formula of which is shown in formula (I):

[0006]

[0007] In formula (I), R1, R2, and R3 are each independently selected from one of 2-ethyl propionate, allyl, amino, and cyanoethyl groups.

[0008] Preferably, the calix[3]carbazole derivative is one of compound I-1, compound I-2, compound I-3 or compound I-4:

[0009]

[0010] In a second aspect, the present invention provides a calix[3]carbazole derivative modified with a cross-methylene group, with the calix[3]carbazole derivative as a core, and the general structural formula of the cross-methylene group modified calix[3]carbazole derivative is shown in Formula (II):

[0011]

[0012] In formula (II), R4, R5, and R6 are each independently selected from one of a hydroxyl group, a C1-C3 alkoxy group, an amino-substituted C1-C5 alkoxy group, a benzylamino group, a hydrazine group, an O-containing fatty amine group, a N-containing fatty amine group, and an ethoxy group.

[0013] Preferably, the methylene-modified calix[3]carbazole derivative is one of compound II-5, compound II-6, compound II-7, compound II-8, compound II-9, compound II-10, compound II-11, compound II-12 or compound II-13:

[0014]

[0015]

[0016]

[0017] In a third aspect, the present invention provides a method for preparing a calix[3]carbazole derivative, comprising the following steps:

[0018] S1. Synthesis of calix[3]carbazole derivatives: using carbazole monomer as raw material, adding Lewis acid, reacting at room temperature for 0.5 to 1 hour; then adding paraformaldehyde, and continuing the reaction for 0.5 to 2 hours to obtain calix[3]carbazole derivatives;

[0019] S2. Synthesis of calix[3]carbazole derivatives modified with cross-methylene groups: the calix[3]carbazole derivatives obtained in step S1 are mixed with o-phthalic alcohol and reacted in an ice bath for a certain period of time; then an oxidant is added for oxidation reaction to obtain the product;

[0020] Alternatively, the calix[3]carbazole derivative obtained in step S1 is mixed with o-phthalic alcohol and reacted in an ice bath for a certain period of time; then an oxidant is added for oxidation reaction, and the oxidation reaction product is further reacted with the amine compound at 80-120° C. to obtain the product.

[0021] Furthermore, in the above technical solution, in step S1, the molar ratio of carbazole monomer to Lewis acid is 1:0.3-0.5;

[0022] The Lewis acid is ferric chloride.

[0023] Furthermore, in the above technical solution, in step S2, the oxidant is 2,3-dichloro-5,6-dicyanobenzoquinone;

[0024] The molar ratio of calix[3]carbazole derivative: o-phthalic alcohol: oxidant is 1:3-5:5-10;

[0025] The reaction time in the ice bath is 0.5-1h.

[0026] Furthermore, in the above technical solution, the solid-liquid ratio of the product of the oxidation reaction to the amine compound is 100 mg: (5-10) mL;

[0027] The amine compound is one of N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, hydrazine hydrate, 3-aminopropanol, ethanolamine, benzylamine or diglycolamine, and the reaction time is 6-12 hours.

[0028] In a fourth aspect, the present invention provides an anti-tumor drug comprising the methylene-modified calix[3]carbazole derivative and a pharmaceutically acceptable carrier thereof.

[0029] In a fifth aspect, the present invention provides the use of the methylene-modified calix[3]carbazole derivative or the anti-tumor drug in the preparation of a drug containing drug molecules or biomacromolecules that is identified by ultraviolet fluorescence.

[0030] Furthermore, in the above technical solution, the drug is at least one of cytarabine or hydroxycamptothecin.

[0031] In a sixth aspect, the present invention provides the use of the methylene-modified calix[3]carbazole derivatives in the preparation of fluorescent molecular probes.

[0032] The beneficial effects of the present invention are:

[0033] Specifically, it involves the synthesis of a cup[3]carbazole skeleton, oxidation of a bridge methylene group to form a ketal structure, and further substitution products and their synthesis methods to form a macrocyclic compound with strong fluorescence and rigid fixation, which has a wide range of potential uses, such as drug diagnostic reagents, ion fluorescence sensing, carriers of medical molecules, and identification of biological macromolecules.

[0034] Using the modified cup[3]carbazole with the above-mentioned edge and bridge methylene modification as the main macrocyclic compound, its molecular rigidity was fixed, and its host-guest recognition effect on hydroxycamptothecin was explored and discovered. Its potential application value in molecular carriers of hydroxycamptothecin drugs, fluorescent probes, and identification detection was preliminarily explored.

[0035] Fluorescence detection: Set the spectrum to 300nm~650nm, excitation wavelength to 300nm, dissolve the compound in the test solution and add a certain proportion of drug solution to obtain its fluorescence absorption spectrum. Substitute the data into the 1:2 binding equation to calculate its binding energy K. Take C drug as the horizontal axis and F as the horizontal axis. 549nm / F 364nm With λ as the vertical axis, the fluorescence change rate graph can be obtained, and the detection limit is 11.7 nM.

[0036] The methylene-modified calix[3]carbazole derivative of the present invention has good activity in inhibiting tumor cell proliferation and has good development prospects in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the reverse fluorescence titration spectrum of hydroxycamptothecin and compound II-7.

[0038] Figure 2 F 549nm / F 364nm Linear fitting equation curve.

[0039] Figure 3 is the Job curve.

[0040] Figure 4 The fitting equation curve is 1:2. DETAILED DESCRIPTION

[0041] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0042] In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0043] Example 1

[0044] A method for preparing a calix[3]carbazole derivative (Compound I-1) comprises:

[0045]

[0046] Compound a (1.00 g, 3.75 mmol) was placed in a 250 mL round-bottom flask. 200 mL of dichloroethane was added as the reaction solvent and stirred thoroughly. Then, 0.182 g (1.13 mmol) of ferric chloride was added and stirring continued for 60 min. The reaction solution turned from clear to dark green. 0.562 g (18.7 mmol) of paraformaldehyde was then added and the reaction continued for 3 h. TLC monitoring was performed until the starting material spot disappeared and only one main spot remained. Post-treatment: After the reaction was completed, 5 mL of aqueous ammonia was added for quenching. Stirring was continued for 10 min, and the reaction solution transformed into a clear yellow solution. After quenching, the reaction solution was extracted three times with water in batches. The combined organic phases were then rotary evaporated under reduced pressure to obtain the crude product. Purification by column chromatography (dichloromethane:petroleum ether = 4:1) yielded 0.481 g of the pure product as a white solid with an Rf = 0.5 (TLC: petroleum ether:dichloromethane = 10:1, v / v), yielding approximately 46.5%.

[0047] Compound I-1: 1 H-NMR (600MHz, DMSO-d6) δ8.35(d,J=1.7Hz,6H),7.41(dd,J=8.3,1.7Hz,6H),7.29(d,J=8.4Hz,6H),5. 70(q,J=7.1Hz,3H),4.19(s,6H),4.11–3.99(m,6H),1.55(d,J=7.1Hz,9H),1.00(tt,J=7.1,2.1Hz,9H).

[0048] HRMS(ESI):m / z:M + :calcd for:C 54 H 51 N3O6 837.3778; found:837.3745.

[0049] Example 2

[0050] A method for preparing a calix[3]carbazole derivative (Compound I-2) comprises:

[0051]

[0052] 1.00 g (5.1 mmol) of compound b was placed in a 250 mL round-bottom flask. 200 mL of dichloroethane was added as the reaction solvent and stirred thoroughly. Then, 0.434 g (2.4 mmol) of ferric chloride was added and stirring continued for 30 minutes. The reaction solution turned from clear to dark green. 0.727 g (24.3 mmol) of paraformaldehyde was then added to the reaction flask. The reaction was continued for 1 hour, monitored by TLC, until the starting material spot disappeared. After completion of the reaction, 1 mL of ammonia was added to quench the reaction. Stirring occurred for 10 minutes, and the reaction solution transformed into a clear yellow solution. After quenching, the reaction solution was extracted three times with water in batches. The combined organic phases were then rotary evaporated under reduced pressure to obtain the crude product, which was then purified by column chromatography (dichloromethane:petroleum ether = 4:1). 0.16 g of the pure product was obtained as a white solid with an Rf = 0.5 (TLC: dichloromethane:petroleum ether = 1:1, v / v), yielding approximately 12%.

[0053] Compound I-2: 1 H-NMR (600MHz, DMSO-d6) δ8.36(s,6H),7.41(d,J=8.5Hz,6H),7.34(d,J=8.3Hz,6H),5.83(td,J= 11.3,5.5Hz,3H),4.95(d,J=10.2Hz,3H),4.92–4.87(m,6H),4.81(d,J=17.1Hz,3H),4.20(s,6H).

[0054] HRMS(ESI):m / z:[M+Na] + calcd for C 48 H 39 N3 680.3042; found:680.3038.

[0055] Example 3

[0056] A method for preparing a calix[3]carbazole derivative (Compound I-3) comprises:

[0057]

[0058] (1) In a 200 mL stirring flask, add 2.00 g of 9-aminocarbazole, 120 mL of anhydrous dichloromethane, and 5 mL of pyridine. Under stirring at room temperature, gradually add dropwise 2.83 g of 9-fluorenylmethyl chloroformate (Fmoc-Cl) dissolved in 10 mL of dichloromethane. After addition, continue stirring and react for 2 h. TLC monitoring indicates that the reaction of the starting materials is complete. Extract three times with 200 mL of dichloromethane and evaporate under reduced pressure to obtain 4.2 g of (9H-fluorenyl)methyl (9H-carbazole)carbamate as a white solid, with a yield of 95%.

[0059] (2) In a 1000 mL flask with stirring, add 2.00 g of (9H-fluorenyl)methyl (9H-carbazole) carbamate, 500 mL of dichloroethane, and 300 mg of paraformaldehyde. Then, add 0.5 mL of boron trifluoride etherate solution dropwise. The solution turns from a colorless clear solution to a light blue. After the addition is complete, continue stirring and react for 2 hours. TLC monitoring shows that the reaction of the raw materials is complete. Add 30 mL of water and stir again for 30 minutes until the solution turns from a blue solution to a light yellow solution. Wash once with water, and remove the organic phase by vacuum distillation. Column chromatography gives 200 mg of a white solid with a yield of 11% and Rf = 0.4 (TLC: methanol: dichloromethane = 1:20, V / V).

[0060] (3) In a stirred 100 mL flask, 100 mg of the solid obtained in step (2) and 50 mL of dichloromethane were added, followed by dropwise addition of 0.5 mL of the piperidine solution. After addition, the mixture was stirred and reacted for 30 min. TLC monitoring indicated that the reaction of the starting material was complete. The mixture was washed three times with water, and the organic phase was removed by distillation under reduced pressure. Column chromatography afforded 35 mg of a gray solid, with a yield of 75%, and an Rf of 0.4 (TLC: methanol: dichloromethane = 1:5, V / V).

[0061] Compound I-3: 1 H-NMR (600MHz, DMSO-d6) δ8.17(s,6H),7.42–7.38(m,12H),5.68(s,6H),4.20(s,6H).

[0062] HRMS(ESI):m / z:[M+H] + calcd for C 39 H 30 N6 583.2610; found:583.2612.

[0063] Example 4

[0064] A method for preparing a calix[3]carbazole derivative (Compound I-4) comprises:

[0065]

[0066] 1.0 g (4.9 mmol) of compound c was placed in a 250 mL round-bottom flask. 200 mL of dichloroethane was added as the reaction solvent and stirred thoroughly. Then, 0.474 g (2.4 mmol) of ferric chloride was added and stirring continued for 30 minutes. The reaction solution turned from clear to dark green. 0.782 g (24.3 mmol) of paraformaldehyde was then added. The reaction was continued for 1 hour, monitored by TLC, until the starting material spot disappeared. After completion of the reaction, 1 mL of ammonia was added to quench the reaction. Stirring for 10 minutes transformed the reaction solution into a clear yellow solution. After quenching, the reaction solution was extracted three times with water in batches. The combined organic phases were then rotary evaporated under reduced pressure to obtain the crude product. This was then purified by column chromatography (dichloromethane:petroleum ether = 4:1) to yield 0.21 g of the pure product as a white solid with an Rf = 0.5 (TLC: dichloromethane:petroleum ether = 1:1, v / v), yielding approximately 19.5%.

[0067] Compound I-4: 1 H-NMR (600MHz, DMSO-d6) δ8.35(s,6H),7.56(d,J=8.3Hz,6H),7.53(dd,J=8.4,1.6Hz,6H),5.65(s,5H),4.24(s,6H).

[0068] HRMS(ESI):m / z:[M+H] + :calcd for C 45 H 30 N6 677.2430; found:677.2422.

[0069] Example 5

[0070] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-5) comprises:

[0071]

[0072] 1.5 g (0.0018 mol) of compound I-1 was placed in a 200 mL round-bottom flask. 100 mL of anhydrous dichloromethane was added, along with 2.5 g (0.0181 mol) of o-phthalic methanol. The mixture was stirred in an ice bath under nitrogen for 10 minutes. Then, 4.09 g (0.0181 mol) of 2,3-dichloro-5,6-dicyano-p-benzoquinone was added. The mixture was magnetically stirred for 1 hour and monitored by TLC. After the reaction, the reaction solution was extracted 7-10 times with dichloromethane / water until the aqueous layer was colorless. The combined reaction solutions were distilled under reduced pressure and purified by column chromatography (dichloromethane:methanol = 500:1) to yield 1.65 g of a white solid with an Rf = 0.4 (TLC: methanol:dichloromethane = 1:20, v / v), yielding approximately 76%.

[0073] Compound II-5: 1 H-NMR(600MHz,DMSO-d6)δ8.94(s,6H),7.71(t,J=6.8Hz,6H),7.37(ddd,J=44.3,8.9,3.2Hz,7H),7.22(s,12H),5 .75(d,J=7.2Hz,3H),5.03(d,J=14.1Hz,12H),4.11–3.92(m,6H),1.54(dd,J=7.2,2.2Hz,9H),1.03–0.92(m,9H).

[0074] HRMS(ESI):m / z:[M+Na] + calcd for C 78 H 69 N3O1 1262.4779; found:1262.4772.

[0075] Example 6

[0076] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-6) comprises:

[0077]

[0078] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 0.5 mL (0.1 mol / L) of aqueous sodium hydroxide solution was added, and the mixture was stirred in an oil bath at 80°C for 8 h. The reaction process was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water, and dilute sulfuric acid was added to adjust the pH to 2-3. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively, and dried to obtain 84.8 mg of a white solid with Rf = 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), a yield of 91%.

[0079] Compound II-6: 1 H-NMR (600MHz, DMSO-d6) δ8.89 (s, 6H), 7.68 (t, J = 10.3Hz, 6H), 7.37 (dd, J = 48.6, 8.8Hz, 6H), 7.22 (s, 13H), 5.41 (s, 2H), 5.11–4.94 (m, 12H), 1.44 (d, J = 7.0Hz, 9H).

[0080] HRMS(ESI):m / z:[M+Na] + calcd for C 72 H 57 N3O 121178.3840; found:1178.3851.

[0081] Example 7

[0082] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-7) comprises:

[0083]

[0084] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of ethanolamine was added, and the mixture was stirred in an oil bath at 120°C for 8 h, monitored by TLC. After the reaction of the raw materials was complete, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively, and dried to obtain 88.0 mg of a white solid with Rf = 0.4 (TLC: dichloromethane: methanol = 20:1, V / V). The yield was 85%.

[0085] Compound II-7: 1 H-NMR(600MHz,DMSO-d6)δ8.93(d,J=3.6Hz,6H),7.84(dq,J=9.0,5.2Hz,3H),7.70(dd,J =12.5,8.7Hz,6H),7.48–7.41(m,6H),7.23(s,12H),5.40(q,J=7.5Hz,3H),5.03(dd,J=2 2.0,4.0Hz,12H),4.41(s,2H),3.26(ddd,J=8.9,6.3,3.1Hz,3H),3.19(dt,J=11.0,5.8H z,3H),3.10(dt,J=12.5,6.1Hz,3H),2.95(dq,J=12.7,6.0Hz,3H),1.41(d,J=7.3Hz,9H).

[0086] 13 C NMR(150MHz,DMSO-d6)δ169.98,139.79,139.42,138.46,138.29,135.33,127.1 0,126.62,123.84,118.99,110.21,107.22,66.55,59.76,52.93,42.08,15.28.

[0087] HRMS(ESI):m / z:[M+Na] + calcd for C 78 H 72 N6O 121307.5106; found:1307.5105.

[0088] Example 8

[0089] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-8) comprises:

[0090]

[0091] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of 3-aminopropanol was then added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to yield 85.6 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, v / v), representing an 80% yield.

[0092] Compound II-8: 1 H-NMR (600MHz, DMSO-d6) δ8.93(s,6H),7.86(t,J=6.0Hz,3H),7.70(q,J=7.3,6.4Hz,6H),7.43(qd,J=8.7,3.0Hz,6H),7.23(s,12H),5.4 1–5.35(m,3H),5.03(d,J=17.5Hz,12H),3.17(dddd,J=23.9,10.9,4.8,1.9Hz,6H),3.06–2.92(m,6H),1.39(td,J=9.0,8.0,4.8Hz,15H).

[0093] HRMS(ESI):m / z:[M+Na] + calcd for C 81 H 78 N6O 12 1349.5575; found:1349.5576.

[0094] Example 9

[0095] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-9) comprises:

[0096]

[0097] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of diglycolamine was then added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to give 99.4 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), a yield of 87%.

[0098] Compound II-9: 1 H-NMR (600MHz, DMSO-d6) δ8.93(s,6H),7.95(t,J=5.9Hz,3H),7.70(t,J=8.2Hz,6H),7.49–7.40(m,6H),7.23(s,12H),5.41(q,J=7. 2Hz, 3H), 5.03 (d, J = 16.7Hz, 12H), 4.50 (t, J = 5.5Hz, 3H), 3.34–3.19 (m, 18H), 3.10 (hept, J = 6.7, 6.1Hz, 6H), 1.40 (t, J = 5.6Hz, 9H).

[0099] HRMS(ESI):m / z:[M+Na] + calcd for C 84 H 84 N6O 15 1439.5892; found:1439.5879.

[0100] Example 10

[0101] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-10) comprises:

[0102]

[0103] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of N,N-dimethylethylenediamine was added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to give 85.6 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), resulting in an 80% yield.

[0104] Compound II-10: 1H-NMR (600MHz, DMSO-d6) δ8.92 (s, 6H), 7.74 (d, J = 6.9Hz, 3H), 7.69 (d, J = 8.3Hz, 6H), 7.48–7.41 (m, 6H), 7.23 (s, 12H), 5.40 (q, J = 7 .0Hz,3H),5.03(d,J=13.7Hz,12H),3.01(ddd,J=14.2,10.7,7.0Hz,6H),2.11–2.01(m,6H),1.92(dd,J=6.2,3.3Hz,18H),1.42–1.36(m,9H).

[0105] HRMS(ESI):m / z:[M+Na] + calcd for C 84 H 87 N9O9 1388.6524; found:1388.6561.

[0106] Example 11

[0107] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-11) comprises:

[0108]

[0109] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of N,N-dimethylpropylenediamine was added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to give 85.6 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), resulting in an 80% yield.

[0110] Compound II-11: 1 H-NMR(600MHz,DMSO-d6)δ8.93(s,6H),7.91(s,3H),7.68(s,6H),7.48–7.31(m,6H),7.21(d,J=16.4Hz,12H),5.38 (s,3H),5.02(d,J=21.6Hz,12H),2.94(d,J=53.9Hz,6H),1.88(s,6H),1.79–1.62(m,18H),1.34(d,J=57.3Hz,18H).

[0111] HRMS(ESI):m / z:[M+H] +calcd for C 87 H 93 N9O9 1408.7175; found:1408.7165.

[0112] Example 12

[0113] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-12) comprises:

[0114]

[0115] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of benzylamine was then added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to give 85.6 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), resulting in an 80% yield.

[0116] Compound II-12: 1 H-NMR (600MHz, DMSO-d6) δ8.93(d,J=8.5Hz,6H),8.41(d,J=6.2Hz,1H),8.36(d,J=7.9 Hz,2H),7.70(d,J=24.0Hz,6H),7.53–7.32(m,6H),7.24(dd,J=11.5,7.0Hz,12H),7.1 2–6.98(m,12H),6.92(d,J=7.5Hz,3H),5.50(dt,J=13.0,6.6Hz,3H),5.09–4.97(m,12 H), 4.13 (d, J = 21.9Hz, 4H), 4.00 (d, J = 14.8Hz, 2H), 1.43 (dq, J = 11.2, 6.7, 4.9Hz, 9H).

[0117] HRMS(ESI):m / z:[M+K] + calcd for C 93 H 78 N6O9 1462.5501; found:1462.5496.

[0118] Example 13

[0119] A method for preparing a methylene-modified calix[3]carbazole derivative (Compound II-13) comprises:

[0120]

[0121] 100 mg of compound II-5 was placed in a 25 mL round-bottom flask, 5 mL of DMF was added, and the mixture was stirred until dissolved. 1 mL of 80% hydrazine hydrate was then added, and the mixture was stirred in an oil bath at 120°C for 8 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was poured into 10 mL of water. A large amount of solid precipitated, which was filtered and washed twice with water, methanol, and n-hexane, respectively. The solid was then dried to yield 85.6 mg of a white solid with an Rf of 0.4 (TLC: dichloromethane:methanol = 20:1, V / V), representing an 80% yield.

[0122] Compound II-13: 1 H-NMR(600MHz,DMSO-d6)δ9.22(dd,J=18.8,9.5Hz,3H),8.90(s,6H),7.69(d,J=8.9Hz,6H),7.54–7.45(m,6H) ,7.23(s,13H),5.34(qd,J=7.1,2.7Hz,3H),5.03(d,J=15.3Hz,12H),4.23(s,6H),1.46(dt,J=6.6,3.1Hz,9H).

[0123] HRMS(ESI):m / z:[M+H] + calcd for C 72 H 63 N9O9 1198.4827; found:1198.4818.

[0124] Example 14 Recognition of Hydroxycamptothecin by Methylene-Modified Calix[3]carbazole Derivatives

[0125] This embodiment uses fluorescence spectroscopy for testing, the solvent environment is pure water, λ ex =340nm,λ em =350~600nm, slit width is 5nm

[0126] Reverse titration fluorescence spectrum: Using hydroxycamptothecin as the test substrate, the excitation wavelength is 300nm, and compound II-7 is gradually added dropwise to measure the fluorescence spectrum of the composite system (see Figure 1 The emission wavelength of hydroxycamptothecin gradually decreased at 552 nm, while the emission wavelength of compound II-7 gradually increased at 400-500 nm. A clear isoemission point was observed at 502 nm.

[0127] This example uses Job's Plot test, setting C 主体 +C 客体= 50 μM, and the ratio of the two is constantly changed. The fluorescence intensity at a wavelength of ~380 nm is selected for fitting, and the highest inflection point is selected as a vertical dotted line. Figure 2 The corresponding horizontal coordinate is ~0.33, and the host-guest binding ratio is determined to be 2:1.

[0128] The reverse titration fluorescence spectrum data, the 428nm fluorescence data were selected, and the nonlinear calibration curve was fitted using a 1:2 equation (see Figure 3 ), and the binding constant was found to be 1.26×10 14 M -2 , the adjusted determination coefficient was 0.9975.

[0129] Select F 549nm / F 364nm Fluorescence data, linear fitting, see attached Figure 4 , HCPT concentration was 0.5~10μM, F 549nm / F 364nm There is a good linear relationship, and the standard regression equation is

[0130] F 549nm / F 364nm =-1.9807+2.6245×10 -7 *C,(R 2 =0.9964), and the limit of detection (LOD) was 11.7 nmol / L.

[0131] Compound II-7 can recognize and bind to hydroxycamptothecin in water at a binding ratio of 2:1, and within the concentration range of 0.5μM to 10μM, compound II-7 can achieve ratiometric fluorescence detection of hydroxycamptothecin with a detection limit of 11.7nmol / L.

[0132] Example 15 In vitro antitumor activity test of methylene-modified calix[3]carbazole derivatives

[0133] The preparation method of the composition comprises the following steps: using compound II-7 to encapsulate hydroxycamptothecin to prepare the composition, and then using ultraviolet spectrophotometry to test the drug loading amount and encapsulation efficiency of compound II-7 encapsulating hydroxycamptothecin.

[0134] The experimental process is as follows:

[0135] Six hydroxycamptothecin solutions with concentrations of 1.8220 mg / L, 3.6440 mg / L, 5.4650 mg / L, 7.2870 mg / L, 9.1090 mg / L, and 10.9310 mg / L were selected for quantitative UV absorbance testing at 378 nm. The results showed that Abs were 0.183, 0.368, 0.597, 0.747, 0.880, and 1.026, respectively. The calibration curve equation was: Abs = 0.09255*C + 0.04341, R 2 =0.9950. Add 5 mL of pure water to a watch glass, add 7 μL of compound II-1 (20 mmol / L stock solution) and 2.5 μL of hydroxycamptothecin, shake, and let stand for 2 hours. Transfer the solution to a pretreated dialysis bag and place it in dialysate. Replace the dialysate every 4 hours, repeat twice. Take 1 mL of the solution and test the UV absorbance in the organic reagent DMSO three times. The results are Abs of 0.197, 0.214, and 0.195, respectively, and the concentrations are 1.6600 mg / L, 1.6430 mg / L, and 1.6380 mg / L, respectively. Take the average value, calculate the M3 value, and substitute it into the formula to obtain the encapsulation efficiency and drug loading.

[0136] Encapsulation efficiency (%) = (encapsulated drug mass M3) / (initial drug mass M1) × 100%

[0137] Drug loading (%) = (loaded drug mass M3) / (initial drug mass M1 + drug carrier mass M2) × 100%

[0138] The final calculated drug encapsulation efficiency was 45.2% and the drug loading was 5.6%.

[0139] The in vitro activity test method and results are as follows: the anti-tumor drug hydroxycamptothecin was used as the positive control experimental group.

[0140] Screening method: MTT reduction method

[0141] Cell line: human breast cancer cells (MCF-7)

[0142] Effect time: 48h

[0143] The half-maximal inhibitory concentration IC of compound II-7 and compound II-7 / hydroxycamptothecin entrapped on tumor cell growth 50 (%, μM) see Table 1.

[0144] Table 1 In vitro inhibition test data of tumor cell proliferation by compound II-7 and compound II-7 / hydroxycamptothecin loading (IC 50 (μM)

[0145]

[0146] By IC 50 The inhibitory effect of the composition on MCF-7 cells was enhanced by nearly 2 orders of magnitude, indicating that compound II-7 can enhance the inhibitory effect of hydroxycamptothecin on MCF-7 cells.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A methylene-modified calix[3]carbazole derivative, characterized in that: Its general structural formula is shown in formula (II): (II) In formula (II), R4, R5, and R6 are each independently selected from one of a hydroxyl group, a C1-C3 alkoxy group, an amino-substituted C1-C5 alkoxy group, a benzylamino group, and a hydrazine group.

2. A calix[3]carbazole derivative modified with a cross-methylene group, characterized in that: The structural formula is as follows:

3. The method for preparing the methylene-modified calix[3]carbazole derivative according to claim 1 or 2, characterized in that: The steps include: S1. Synthesis of calix[3]carbazole derivatives: Carbazole monomer is used as a raw material, Lewis acid is added, and the Lewis acid is ferric chloride; the reaction is carried out at room temperature for 0.5 to 1 hour; then paraformaldehyde is added and the reaction is continued for 0.5 to 2 hours to obtain a calix[3]carbazole derivative, the general structural formula of which is shown in formula (I): （I） S2. Synthesis of methylidene-modified calix[3]carbazole derivatives: The calix[3]carbazole derivative obtained in step S1 is mixed with o-phthalic alcohol and reacted in an ice bath for a certain period of time; then an oxidant is added to carry out oxidation reaction to obtain compound II-5, compound II-6, and compound II-9: Alternatively, the calix[3]carbazole derivative obtained in step S1 is mixed with o-phthalic alcohol and reacted in an ice bath for a certain period of time; then an oxidant is added for oxidation reaction, and the oxidation reaction product is further reacted with an amine compound at 80-120° C. to obtain compound II-7, compound II-8, compound II-10, compound II-11, compound II-12, and compound II-13; In step S2, the oxidant is 2,3-dichloro-5,6-dicyanobenzoquinone.

4. The preparation method according to claim 3, characterized in that In step S1, the molar ratio of carbazole monomer to Lewis acid is 1:0.3-0.5; The molar ratio of calix[3]carbazole derivative: o-phthalic alcohol: oxidant is 1:3-5:5-10; The ice bath reaction time is 0.5-1 h; The solid-liquid ratio of the product of the oxidation reaction to the amine compound is 100 mg: (5-10) mL; The amine compound is one of N,N-dimethylethylenediamine, N,N-dimethylpropylenediamine, hydrazine hydrate, 3-aminopropanol, ethanolamine, benzylamine or diglycolamine, and the reaction time is 6-12 hours.

5. Use of the methylene-modified calix[3]carbazole derivative according to claim 1 or 2 in the preparation of drugs containing drug molecules or biomacromolecules that are identified by ultraviolet fluorescence.

6. The use according to claim 5, characterized in that The drug is hydroxycamptothecin.

7. Use of the methylene-modified calix[3]carbazole derivative according to claim 1 or 2 in the preparation of a fluorescent molecular probe for recognizing hydroxycamptothecin.

Images