CN112125852B - Squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity and synthesis method thereof - Google Patents
Squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity and synthesis method thereof Download PDFInfo
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- CN112125852B CN112125852B CN202011082597.0A CN202011082597A CN112125852B CN 112125852 B CN112125852 B CN 112125852B CN 202011082597 A CN202011082597 A CN 202011082597A CN 112125852 B CN112125852 B CN 112125852B
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- C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
- C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
- C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
- C07D235/14—Radicals substituted by nitrogen atoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention relates to a squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity and a synthesis method thereof, wherein 13 squaramide-bis-benzimidazole conjugates are designed and synthesized, the squaramide-bis-benzimidazole conjugate synthesized by the invention has moderate-strength affinity for chloride ions and promotes the transmembrane transport of the chloride ions through an anion exchange mechanism, the chloride ion transport activity of each conjugate under the acidic pH condition is higher than that under the physiological pH condition, and the synthesized conjugate also shows moderate-strength toxicity to selected solid tumor cells.
Description
Technical Field
The invention relates to the field of antitumor micromolecule potential drugs, in particular to a squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity and a synthesis method thereof.
Background
Cells are the smallest functional unit of an organism, and the balance of anions between the inside and outside of the cells is a prerequisite for maintaining the physiological functions of the cells, and the balance of anions is broken to cause cell death.
The anion with the largest content in the extracellular fluid is chloride ion. Under the control of chloride channels on the cell membrane, the intracellular and extracellular chloride concentrations were 4-60mM and 120mM, respectively. Dysfunction of the anion transporter or mutation of a gene associated with the chloride transporter may lead to various life-threatening diseases such as myotonia congenita, cystic fibrosis, barter's syndrome, guillain-tropsch syndrome, dengue disease, tubular acidosis, and deafness. Therefore, the deep research on the ion transport performance exerted by the anion channel or transporter protein not only helps us to understand the occurrence and development processes of diseases, but also has potential application in the research and development of specific drugs for treating ion channel diseases.
In view of the important medical significance of anion channels and transporter proteins in physiology and pathology, some artificially synthesized organic small molecule compounds can effectively mediate the transmission of anions on phospholipid double-layer membranes, can be used for simulating the structure and function of natural ion channels, and arouse great interest. These compounds, i.e. synthetic anion transporters, on the one hand, are capable of exerting anion transport activity without relying on the supply of metabolic energy of the cell, and can be used as effective research tools for the study of the structure, activity and possible mechanism of action of natural anion channels or transporters. On the other hand, tumor cells, which are caused by metabolic abnormalities to have a slightly higher intracellular pH (pHi) and a slightly lower extracellular pH (pHe) than normal cells, can escape from apoptosis and promote cell migration and metastasis. Although some innovative drugs have been developed to utilize the low external pHe value of tumor cells for drug delivery at specific sites, such as nanogels, polymersomes, micelles, etc., most drugs are complex to synthesize and have large molecular weight, and can only help to improve the enrichment of existing drugs in tumor cells, and have low antitumor activity. Because the small molecule anion carrier has the capability of transporting anions through a transmembrane, the small molecule anion carrier can disturb the steady state of cells, change the polarization state of the membrane, influence the pH value in the cells, change the permeability of cell membranes, destroy the balance of cell anions and cause apoptosis. Therefore, the synthesized anion transporter has high potential in the research and development of antitumor drugs, and is expected to be developed into a novel antitumor drug.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity and a synthesis method thereof.
The technical scheme of the invention is as follows: a squaramide-bis-benzimidazole conjugate having pH-dependent anion transport across membranes, the squaramide-bis-benzimidazole conjugate having the formula:
the squaramide-bis-benzimidazole conjugates have moderate affinity for chloride ions and facilitate transport of chloride ions across membranes via an anion exchange mechanism, and each conjugate has higher chloride transport activity at acidic pH than at physiological pH, wherein a portion of the conjugates exhibit moderate toxicity to selected solid tumor cells.
The invention also provides a synthesis method of the squaramide-bis-benzimidazole conjugate with pH-dependent anion transmembrane transport activity, which comprises the following steps:
s1), adding certain amounts of N-Boc-gly, EDC, HOBt, and DMAP to a reaction flask, and then adding CH2Cl2Dissolving; stirring at room temperature for 8-15min, transferring the mixed solution into a dropping funnel, and slowly dropping the mixed solution into a solution of o-phenylenediamine and DMF;
s2), stirring the mixed solution obtained in the step S1) to react for 2-5 days at room temperature, removing DMF and dichloromethane under reduced pressure, adding ethyl acetate, and sequentially adding saturated NaCl aqueous solution and saturated NH4Aqueous Cl solution and saturated NaHCO3Washing with a saturated NaCl aqueous solution after the aqueous solution washing;
s3), then using Na2SO4Drying, removing ethyl acetate under reduced pressure, dissolving the obtained residue in glacial acetic acid, reacting at 70 deg.C for 10h, removing solvent under reduced pressure, adding diethyl ether, vacuum filtering to obtain filtrate, and separating the filtrate by column chromatography to obtain compound 21, wherein the reaction formula is as follows:
s4), using a certain amount of compound 21 with CH3Dissolving OH, adding HCl, stirring at room temperature for 1-5h, removing solvent under reduced pressure, adding ammonia water, stirring at room temperature for 15-20min, removing ammonia water under reduced pressure, adding methanol to dissolve, separating by column chromatography,after lyophilization, compound 34 is obtained according to the following reaction scheme:
s5), dissolving the compound 34 with ethanol, adding triethylamine, reacting for 30-45min at room temperature, adding ethanol (270 mu L) solution of 3, 4-diethoxy cyclobut-3-ene-1, 2-dione in portions, reacting for 20-23h at room temperature, and performing vacuum filtration to obtain a white solid, namely a compound 8, wherein the reaction formula is as follows:
preferably, the compound 8 can recognize and bind to chloride ions.
Preferably, said compound 8 promotes the transport of chloride ions across membranes by an anion exchange process and is more active at acidic pH than at physiological pH.
Preferably, said compound 8 exhibits moderate toxicity to selected solid tumor cells.
The invention has the beneficial effects that:
1. the invention can be synthesized under simpler and milder conditions, and the obtained target compound can identify and combine chloride ions;
2. the compounds of the invention promote transport of chloride ions across membranes by an anion exchange process and are more active at acidic pH than at physiological pH.
3. The compounds of the invention exhibit moderate toxicity to selected solid tumor cells.
Drawings
FIG. 1 is a drawing showing the preparation of the object Compound 1 of example 3 of the present invention1HNMR spectrogram;
FIG. 2 is a drawing showing the preparation of Compound 2, object of example 3 of the present invention1HNMR spectrogram;
FIG. 3 is a drawing showing the preparation of Compound 3, object of example 3 of the present invention1HNMR spectrogram;
FIG. 4 is a drawing showing the preparation of Compound 4, object of example 3 of the present invention1HNMR spectrogram;
FIG. 5 is a drawing showing the preparation of Compound No. 5, which is the object of example 3 of the present invention1HNMR spectrogram;
FIG. 6 shows the preparation of Compound 6, object of example 3 of the present invention1HNMR spectrogram;
FIG. 7 is a drawing showing the preparation of Compound 7, object of example 3 of the present invention1HNMR spectrogram;
FIG. 8 is a drawing showing the preparation of Compound 8, object of example 3 of the present invention1HNMR spectrogram;
FIG. 9 is a drawing showing the preparation of Compound 9, object of example 3 of the present invention1HNMR spectrogram;
FIG. 10 is a drawing of target compound 10 of example 3 of the present invention1HNMR spectrogram;
FIG. 11 is a drawing showing the preparation of Compound 11, object of example 3 of the present invention1HNMR spectrogram;
FIG. 12 is a drawing of example 3 of Compound 12 of interest1HNMR spectrogram;
FIG. 13 is a drawing showing the preparation of Compound No. 13, object of example 3 of the present invention1HNMR spectrogram;
FIG. 14 shows a spectrum of the product of example 4 of the present invention, wherein (a) the molecular weight distribution in 4:1CH3In CN-DMSO, Compound 1 (1.0X 10)- 3M) and TBACl (1.5X 10)-2M) mixed negative ESI MS spectra; (b) compound 1 (1.0X 10) in the presence of varying concentrations of TBACl-3M) of1H NMR Nuclear magnetic Spectrum (4:1 CD)3CN-DMSO-d6,500MHz).
FIG. 15 is a graph of the efflux ratio of the compound of example 5 of the present invention, wherein (a) the relative chloride ion efflux ratio in EYPC liposomes of compounds 1-13 (5% mol, pH 7.0); (b) relative chloride ion efflux of Compound 1 at different pH conditions.
FIG. 16 shows the inhibitory activity of compounds 1 to 13 (50. mu.L) prepared in example 6 of the present invention on (a) Hela, (b) A549, (c) MCF-7 and (d) HepG2 cells, respectively, after 48 hours.
Detailed Description
The following further describes embodiments of the present invention with reference to the accompanying drawings:
example 1
This example provides the synthesis of compounds 14-26
Compound 14: N-Boc-gly (805mg,4.59mmol), EDC (1.77g,9.23mmol), HOBt (936mg,6.92mmol) and DMAP (30mg,0.24mmol) were weighed into a 100mL reaction flask and charged with CH2Cl2(40mL) dissolution;
after stirring at room temperature for 10min, the mixture was transferred to a dropping funnel and slowly (1 drop/6 s) added dropwise to a solution of o-phenylenediamine (500mg,4.62mmol) and DMF (1.5 mL);
the resulting mixture was stirred at room temperature for 3 days, the mixture was removed under reduced pressure, ethyl acetate (20mL) was added, and saturated aqueous NaCl (20 mL. times.2) and saturated NH were added in that order4Aqueous Cl (20 mL. times.2) and saturated NaHCO3After washing with an aqueous solution (20 mL. times.2), the reaction mixture was washed with a saturated aqueous NaCl solution (20 mL). With Na2SO4After drying, ethyl acetate was removed under reduced pressure;
the obtained residue was dissolved in glacial acetic acid (5mL), reacted at 70 ℃ for 10 hours, the solvent was removed under reduced pressure, diethyl ether (20mL) was added, and the filtrate was suction-filtered under reduced pressure, and the filtrate was separated by column chromatography (petroleum ether/ethyl acetate, 2/1, v/v) to give compound 14(515mg, 45%).1H NMR(400MHz,CD3OD)δ7.44(s,2H),7.13(s,2H),4.41(s,2H),1.39(s,9H)and negative ESI-MS:m/z 245.96([M-H]–)。
Compound 15: a similar synthesis procedure to compound 14, 266mg, 25%.1H NMR(CD3OD,400MHz)δ7.29(d,J＝7.2Hz,1H),7.21(s,1H),6.94(d,J＝7.6Hz,1H),4.36(s,2H),2.34(s,4H),1.37(s,9H)and negative ESI-MS:m/z 259.83([M-H]–)。
Compound 16: similar synthesis of compound 14, 458mg, 47%.1H NMR(CD3OD,400MHz)δ7.29(d,J＝8.8Hz,1H),6.93(s,1H),6.74(d,J＝8.8Hz,1H),4.35(s,2H),3.72(s,3H),1.37(s,9H)and negative ESI-MS:m/z 275.83([M-H]–)。
Compound 17: analogous synthesis of compound 14, 302mg, 38%.1H NMR(CD3OD,400MHz)δ8.00(s,1H),7.67(s,1H),4.48(s,2H),1.38(s,9H)and negative ESI-MS:m/z 381.87([M-H]–)。
Compound 18: analogous synthesis of Compound 14, 287mg, 30%.1H NMR(CD3OD,400MHz)δ8.37(s,1H),8.08(d,J＝8.9Hz,1H),7.56(d,J＝7.1Hz,1H),4.44(s,2H),1.38(s,9H)；13C NMR(CD3OD,100MHz)δ157.2,79.6,38.4,33.8,30.4,27.3,13.4；negative ESI-MS:m/z 290.88([M-H]–)and negative HR-ESI-MS for C24H12F12N6O2([M-H]-)Calcd:291.10878,Found:291.10983。
Compound 19: analogous synthesis of compound 14, 342mg, 34%.1H NMR(CD3OD,400MHz)δ7.14(s,1H),7.09–6.96(m,2H),4.39(s,2H),1.37(s,9H)and negative ESI-MS:m/z 281.88([M-H]–)。
Compound 20: similar synthesis procedure for compound 14, 140mg, 14%.1H NMR(CD3OD,400MHz)δ7.51–7.39(m,2H),7.14(d,J＝8.5Hz,1H),4.42(s,2H),1.41(s,9H)and negative ESI-MS:m/z 280.12([M-H]–)。
Compound 21: similar synthesis procedure for compound 14, 221mg, 38%.1H NMR(CD3OD,400MHz)δ7.45–7.34(m,2H),7.10(d,J＝8.6Hz,1H),4.38(s,2H),1.37(s,9H)；13C NMR(CD3OD,100MHz)δ156.9,154.3,127.5,114.4,79.4,27.2；negativeESI-MS:m/z 323.81([M-H]-)andnegative HR-ESI-MS for C13H16BrN3O2([M-H]-)Calcd:324.03421,Found:324.03513。
Compound 22: analogous synthesis of compound 14, 365mg, 37%.1H NMR(CD3OD,400MHz)δ7.08(d,J＝8.3Hz,1H),6.84(t,J＝10.2Hz,1H),4.49(s,2H),1.48(s,9H)；13C NMR(CD3OD,100MHz)δ159.8,157.5,156.5,154.5,96.8,96.6,79.3,27.2；negativeESI-MS:m/z 281.91([M-H]–)andnegative HR-ESI-MS for C13H15F2N3O2([M-H]–)Calcd:282.10485,Found:282.10547。
Compound 23: analogous synthesis of compound 14, 143mg, 23%.1H NMR(CD3OD,400MHz)δ7.21(d,J＝7.3Hz,1H),7.07(d,J＝6.4Hz,1H),6.83(t,J＝9.2Hz,1H),4.40(s,2H),1.37(s,9H)；13C NMR(CD3OD,100MHz)δ156.8,153.4,122.5,106.9,79.4,26.9；negativeESI-MS:m/z 263.93([M-H]–)andnegative HR-ESI-MS for C13H16FN3O2([M-H]-)Calcd:264.11428,Found:264.11514。
Compound 24: analogous synthesis of compound 14, 246mg, 27.5%.1H NMR(CD3OD,400MHz)δ7.74(s,1H),7.58(d,J＝7.9Hz,1H),7.40(d,J＝8.3Hz,1H),4.43(s,2H),1.38(s,9H)andnegativeESI-MS:m/z 314.08([M-H]–)。
Compound 25: analogous synthesis of compound 14, 159mg, 16.2%.1H NMR(CD3OD,400MHz)δ7.28(s,2H),4.36(s,2H),1.37(s,9H)；13C NMR(CD3OD,100MHz)δ156.9,154.8,148.8,146.4,104.7,98.9,79.4,27.2；negativeESI-MS:m/z281.94([M-H]–)andnegative HR-ESI-MS for C13H15F2N3O2([M-H]–)Calcd:282.10485,Found:282.10583。
Compound 26: analogous synthesis of compound 14, 268mg, 29%.1H NMR(CD3OD,400MHz)δ7.39(s,1H),7.12(s,1H),6.89(t,J＝9.4Hz,1H),4.37(s,2H),1.37(s,9H)andnegativeESI-MS:m/z 263.93([M-H]-)。
Example 2
This example provides the synthesis of compounds 27-29
Compound 27: compound 14(60mg,0.24mmol) was weighed into a 25mL reaction flask with CH3OH (3mL) solutionAfter decomposition, HCl (2M,3mL) was added and stirred at room temperature for 1 h. After removing the solvent under reduced pressure, aqueous ammonia (3 mL. times.2) was added to the reaction flask, and the mixture was stirred at room temperature for 15min, after which the aqueous ammonia was removed under reduced pressure. After dissolving in methanol (2mL), the mixture was separated by column chromatography (dichloromethane/methanol/ammonia, 24/3/1, v/v/v), and lyophilized to give compound 27(39mg, 45%).1H NMR(500MHz,MeOD)δ7.54(dd,J＝6.0,3.2Hz,2H),7.22(dd,J＝6.0,3.2Hz,2H),4.05(s,2H)。
Compound 28: analogous synthesis of compound 27, 40mg, 72%.1H NMR(CD3OD,400MHz)δ7.30(d,J＝8.2Hz,1H),7.21(s,1H),6.95(d,J＝8.2Hz,1H),3.91(s,2H),2.34(s,3H)。
Compound 29: analogous synthesis of compound 27, 55mg, 71%.1H NMR(CD3OD,400MHz)δ7.30(d,J＝8.8Hz,1H),6.93(d,J＝2.3Hz,1H),6.75(dd,J＝8.8,2.4Hz,1H),3.89(s,2H),3.72(s,3H)。
Compound 30: analogous synthesis of compound 27, 69mg, 70%.1H NMR(CD3OD,400MHz)δ8.03(s,1H),7.68(s,1H),4.07(s,2H)。
Compound 31: analogous synthesis of compound 27, 62mg, 73%.1H NMR(CD3OD,400MHz)δ8.35(s,1H),8.07(d,J＝10.9Hz,1H),7.55(d,J＝8.9Hz,1H),4.00(s,2H)。
Compound 32: analogous synthesis of compound 27, 42mg, 75%.1H NMR(CD3OD,400MHz)δ7.17(dd,J＝9.4,3.1Hz,1H),7.08–6.96(m,1H),3.94(s,2H)。
Compound 33: analogous synthesis of compound 27, 72mg, 85%.1H NMR(CD3OD,400MHz)δ7.58(d,J＝1.4Hz,1H),7.34(d,J＝8.5Hz,1H),7.23(dd,J＝8.6,1.7Hz,1H),3.94(s,2H)。
Compound 34: analogous synthesis of compound 27, 144mg, 68%.1H NMR(CD3OD,400MHz)δ7.57–7.45(m,2H),7.22(dd,J＝8.6,1.9Hz,1H),4.05(s,2H)。
Compound 35: analogous synthesis of compound 27, 76mg, 70%.1H NMR(CD3OD,400MHz)δ6.98(d,J＝8.5Hz,1H),6.73(t,J＝10.4Hz,1H),3.93(s,2H)。
Compound 36: analogous synthesis of compound 27, 64mg, 73%.1H NMR(CD3OD,400MHz)δ7.22(d,J＝8.0Hz,1H),7.10–7.05(m,1H),6.84(dd,J＝10.8,8.1Hz,1H),3.95(s,2H)。
Compound 37: analogous synthesis of compound 27, 54mg, 72%.1H NMR(CD3OD,400MHz)δ7.77(s,1H),7.61(d,J＝8.5Hz,1H),7.44(d,J＝8.5Hz,1H),4.02(s,2H)。
Compound 38: analogous synthesis of compound 27, 70mg, 72%.1H NMR(CD3OD,400MHz)δ7.35(t,J＝8.8Hz,2H),4.13(s,2H)。
Compound 39: analogous synthesis of compound 27, 79mg, 73%.1H NMR(CD3OD,400MHz)δ7.42(dd,J＝8.8,4.7Hz,1H),7.15(dd,J＝9.1,2.2Hz,1H),6.92(td,J＝9.5,2.4Hz,1H),4.00(s,2H)。
Example 3
Synthesis of Compounds 1-13
Compound 1: compound 27(39mg) was dissolved in ethanol (3mL), triethylamine (300. mu.L) was added thereto, and after 30min at room temperature, a solution of 3, 4-diethoxycyclobut-3-ene-1, 2-dione (21mg,0.12mmol) in ethanol (270. mu.L) was added in portions (10. mu.L/10 min/portion). The reaction was carried out at room temperature for 23 hours, followed by suction filtration under reduced pressure to obtain Compound 1(12mg, 31%) as a white solid.1H NMR(DMSO-d6400MHz, see fig. 1) δ 12.57(s,2H),8.25(s,2H),7.54(s,4H),7.19(s,4H),5.03(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.4,168.2,151.9,122.2,115.9,115.4；negative ESI-MS:m/z 371.07([M-H]–)and negative HR-ESI-MS for C20H16N6O2([M-H]–)Calcd:371.12510,Found:371.12570。
compound 2: analogous synthesis of Compound 1, 35mg, 79%.1H NMR(DMSO-d6400MHz, see FIG. 2). delta.12.33 (s,2H),8.21(s,2H), 7.51-7.20 (g: (g) ((g))m,4H),7.00(s,2H),4.99(s,4H),2.40(s,6H)；13C NMR(DMSO-d6,100MHz)δ183.3,168.2,132.3,111.5,21.6；negativeESI-MS:m/z 399.06([M-H]–)and negative HR-ESI-MS for C22H20N6O2([M-H]-)Calcd:399.15640,Found:399.15680。
Compound 3: analogous synthesis of compound 1, 43mg, 71%.1H NMR(DMSO-d6400MHz, see fig. 3) δ 12.29(s,2H),8.19(s,2H),7.44(s,2H),7.04(s,2H),6.82(d, J ═ 7.9Hz,2H),5.00(s,4H),3.79(s, 6H);13C NMR(DMSO-d6,100MHz)δ183.1,168.1,155.9,119.4,111.8,110.9,95.1；negative ESI-MS:m/z 431.01([M-H]–)and negative HR-ESI-MS for C22H20N6O4([M-H]-)Calcd:431.14622,Found:431.14667。
compound 4: analogous synthesis of Compound 1, 33mg, 43.1%.1H NMR(DMSO-d6400MHz, see fig. 4) δ 13.51(s,2H),8.32(s,2H),8.22(s,2H),7.81(s,2H),5.15(s, 4H);13C NMR(DMSO-d6,100MHz)δ182.9,168.1,156.1,128.6,127.7,125.9,123.3；negativeESI-MS:m/z 642.89([M-H]–)and negative HR-ESI-MS for C24H12F12N6O2([M-H]–)Calcd:643.07463,Found:643.07489。
compound 5: analogous synthesis of compound 1, 45mg, 69%.1H NMR(DMSO-d6400MHz, see fig. 5) δ 11.76(s,2H),8.45(s,2H),8.31(s,2H),8.12(d, J ═ 8.7Hz,2H),7.73(d, J ═ 8.9Hz,2H),5.11(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.5,168.3,157.1,142.9,118.2；negative ESI-MS:m/z 460.99([M-H]–)andnegative HR-ESI-MS for C20H14N8O6([M-H]–)Calcd:461.09525,Found:461.09579。
compound 6: analogous synthesis of compound 1, 30mg, 70%.1H NMR (DMSO-d6,400MHz, see FIG. 6) Δ 12.77(s,2H),8.22(s,2H), 7.40-7.13 (m,4H),5.02(s, 4H);13CNMR(DMSO-d6,100MHz)δ183.8,167.7,154.8,146.9,144.6,111.5,99.8；negative ESI-MS:m/z 443.02([M-H]–)andnegative HR-ESI-MS for C20H12F4N6O2([M-H]–)Calcd:443.08741,Found:443.08801。
compound 7: analogous synthesis of Compound 1, 52mg, 71%.1H NMR(DMSO-d6400MHz, see fig. 7) δ 8.23(s,2H),7.76(s,2H),7.52(s,2H),7.34(dd, J ═ 8.5,1.7Hz,2H),5.04(s, 4H);13C NMR(DMSO-d6,100MHz)δ182.9,168.2,153.4,124.9,114.4；negativeESI-MS:m/z 438.89([M-H]–)andnegative HR-ESI-MS for C20H14Cl2N6O2([M-H]–)Calcd:439.04715,Found:439.04749。
compound 8: analogous synthesis of Compound 1, 96mg, 73%.1H NMR(DMSO-d6400MHz, see fig. 8) δ 12.62(s,2H),8.22(s,2H), 7.60-7.53 (m,2H),7.21(d, J ═ 10.1Hz,2H),5.02(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.5,168.3,153.3,126.7,125.2,122.3；negativeESI-MS:m/z 526.85([M-H]–)and negative HR-ESI-MS for C20H14Br2N6O2([M-H]–)Calcd:526.94612,Found:526.94629。
compound 9: similar synthesis procedure for compound 1, 58mg, 72%.1H NMR (DMSO-d6,400mhz, see fig. 9) δ 12.76(s,2H),8.23(s,2H),7.25(d, J ═ 9.9Hz,2H),7.07(t, J ═ 10.6Hz,2H),5.05(s, 4H);13C NMR(DMSO-d6,100MHz)；negativeESI-MS:m/z 442.99([M-H]–)and negative HR-ESI-MS for C20H12F4N6O2([M-H]–)Calcd:443.08741,Found:443.08789。
compound 10: analogous synthesis of Compound 1, 51mg, 73%.1H NMR(DMSO-d6400MHz, see fig. 10) δ 8.23(s,2H),7.35(d, J ═ 7.5Hz,2H),7.17(dd, J ═ 14.4,6.5Hz,2H), 7.07-6.86 (m,2H),5.05(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.4,168.2,152.4,137.9,131.6,123.1,108.5,110.3,69.3,56.4；negativeESI-MS:m/z 407.01([M-H]–)andnegative HR-ESI-MS for C20H14F2N6O2([M-H]–)Calcd:407.10625,Found:407.10687。
compound 11: analogous synthesis of compound 1, 42mg, 74%.1H NMR(DMSO-d6400MHz, see fig. 11) δ 12.91(s,2H),8.23(s,2H),7.88(s,2H),7.71(d, J ═ 8.3Hz,2H),7.49(d, J ═ 8.5Hz,2H),5.06(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.4,168.3,154.9,122.9(d,J＝31.4Hz),119.1；negativeESI-MS:m/z 507.03([M-H]–)and negative HR-ESI-MS for C22H14F6N6O2([M-H]–)Calcd:507.09986,Found:507.10004。
compound 12: analogous synthesis of compound 1, 54mg, 71%.1H NMR(DMSO-d6400MHz, see FIG. 12) Δ 8.16(s,2H), 7.70-7.54 (m,4H),5.02(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.3,168.2,154.1,148.2,145.8；negativeESI-MS:m/z 442.94([M-H]–)and negative HR-ESI-MS for C20H12F4N6O2([M-H]–)Calcd:443.08741,Found:443.08786。
compound 13: analogous synthesis of compound 1, 61mg, 72%.1H NMR(DMSO-d6400MHz, see fig. 13) δ 12.56(s,2H),8.17(s,2H), 7.65-7.42 (m,2H),7.34(d, J ═ 8.8Hz,2H),7.03(t, J ═ 9.3Hz,2H),5.01(s, 4H);13C NMR(DMSO-d6,100MHz)δ183.4,168.2,159.9,157.7,153.4,110.4,110.1；negativeESI-MS:m/z 407.04([M-H]–)andnegative HR-ESI-MS for C20H14F2N6O2([M-H]–)Calcd:407.10625,Found:407.10605。
example 4
Ion binding constant test
(1) Solution preparation
Solution A: the test compound was dissolved in deuterated acetonitrile to give a final concentration of 1 mM.
Solution B: weighing a certain amount of TBACl, and dissolving with a certain amount of A solution.
(2) Test procedure
First, the A solution (500. mu.L) was added to a nuclear magnetic tube, and the nuclear magnetic resonance hydrogen spectrum was measured. Then, a certain volume of the B solution is added into the nuclear magnetic tube, the nuclear magnetic resonance hydrogen spectrogram is tested after the solution is vortexed for a period of time, and the volume of the B solution added each time is recorded. This procedure was repeated until the observed chemical shift of hydrogen was essentially unchanged. Finally, calculating the final concentration (x) of TBACl in each solution system, taking the final concentration (x) as a horizontal coordinate and the chemical shift variation as a vertical coordinate, and obtaining a binding constant K through fitting calculationa. It can be seen from FIG. 14(a) that the compound is combined with chloride ions in a 1:1 manner, and in FIG. 14(b), with the continuous addition of TBACl, the chemical shifts of Ha and Hb of compound 1 gradually move to the low field direction, which shows that compound 1 mainly recognizes and combines chloride ions through Ha and Hb.
Example 5
Ion transport Activity assay
(1) Soybean lecithin (30mg) was weighed into a test tube (1 cm. times.10 cm), dissolved with redistilled chloroform (500. mu.L), blown dry with nitrogen gas to uniformly cover the inner surface of the tube with lecithin, and the residual solvent was removed under vacuum drying (>4 h).
(2) Add 1ml of internal solution (500mM NaCl,25mM HEPES, pH 6.0, 7.0) or (500mM NaCl,5mM citric acid, pH 4.0, 5.0) to the tube and vortex for 1min, vortex all liposomes coated on the tube, rest for 5min, vortex again for 1min, and rest for 20 min.
(3) Freeze-thaw cycling 8 times through liquid nitrogen and 50 deg.C water bath.
(4) The solution was squeezed back and forth 15 times through a 100nm PC membrane using an Avanti liposome preparation extruder.
(5) With external solution (500mM NaNO)325mM HEPES, pH 6.0, 7.0) was eluted, and non-encapsulated NaCl outside the liposome was removed from the Sephadex G-25 column to obtain liposomes having a particle size of 100nm and the volume thereof was accurately measured and used 15 times.
(6) The liposome solution was measured into a 5ml small beaker and an external buffer solution (500mM NaNO) was added325mM HEPES, pH 7.0) or (500mM NaNO)35mM citric acid, pH 4.0, 5.0) to a total volume of 1980. mu.L. The chloride selective electrode was immersed in the test solution and the test was started after rapid stirring by a stirrer. After video recording began, 20. mu.L of DMSO solution containing the test compound was added (pure DMSO as a blank) for 30s, and 50. mu.L of 10 wt% Triton X-100 solution was added after 5min for 30s to destroy the liposomes, so that all the chloride ions entrapped inside the liposomes were released.
(7) The relative chloride ion outflow rate is represented by the formula: relative Chloride efflux (C-C)0)/(Ctotal-C0) And (6) calculating. By the Hill equation: k is a radical ofin＝k0+kmax×[compound]n/([compound]n+[EC50]n) Fitting to obtain EC50A value and a value of n. As shown in fig. 15(a), 13 compounds can transport chloride ions in liposomes at pH 7.0, and 15(b) compound 1 has higher chloride ion transport activity at acidic pH. The anion binding affinities and chloride transport activities of compounds 1-13 are shown in table 1.
TABLE 1 anion binding affinities and chloride transport activities of Compounds 1-13
a is represented by the expression in CD3CN-DMSO-d6(4/1, v/v) 1H NMR titration;
b represents the test by chloride selective electrode technology under conditions of 500mM NaCl solution (25mM HEPES) in liposome and 500mM NaNO3 solution (25mM HEPES) in liposome;
cRA1represents the anion binding affinity of each compound relative to compound 1;
dRA2the chloride ion elution rate at pH 4 for each compound relative to itself at pH7 is shown.
Example 6
Cytotoxicity test
After the cells were cultured to a logarithmic growth phase, a cell suspension was prepared by digestion with pancreatin. 96-well plate plating (3000 cells per well) was performed. After 24 hours, the medium was aspirated and replaced with 100. mu.L of medium containing 50. mu.M of the test compound (DMSO content was maintained at 1% per well, 4 duplicate wells per compound) and after a further 48 hours of incubation 10. mu.L of MTT solution (5mg/mL) was added to each well, after 4 hours the medium was aspirated from the wells and 100. mu.L of DMSO was added to each well and after shaking the absorbance at 570nm was measured using a microplate reader. Fig. 16(a), 16(b), 16(c) and 16(d) show moderate toxicity to selected solid tumor cells.
It is to be understood that the starting materials employed in the present invention, unless otherwise specified, are prepared by conventional means or purchased commercially.
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
Claims (2)
1. A squaramide-bis-benzimidazole conjugate having pH-dependent anion transport across membranes, wherein the squaramide-bis-benzimidazole conjugate has the formula:
in the formula (I), the compound is shown in the specification,
2. a method for synthesizing a squaramide-bis-benzimidazole conjugate having pH-dependent anion transport activity across membranes, comprising the steps of:
s1), mixing a certain amountN-Boc-gly, EDC, HOBt, and DMAP were added to the reaction flask, followed by CH addition2Cl2Dissolving; stirring at room temperature for 8-15min, transferring the mixed solution into a dropping funnel, and slowly dropping the mixed solution into a solution of o-phenylenediamine and DMF;
s2), stirring the mixed solution obtained in the step S1) to react for 2-5 days at room temperature, removing DMF and dichloromethane under reduced pressure, adding ethyl acetate, and sequentially adding saturated NaCl aqueous solution and saturated NH4Aqueous Cl solution and saturated NaHCO3Washing with a saturated NaCl aqueous solution after the aqueous solution washing;
s3), then using Na2SO4Drying, removing ethyl acetate under reduced pressure, dissolving the obtained residue in glacial acetic acid, reacting at 70 deg.C for 10h, removing solvent under reduced pressure, adding diethyl ether, vacuum filtering to obtain filtrate, and separating the filtrate by column chromatography to obtain compound 14, wherein the reaction formula is as follows:
s4), applying a certain amount of compound 14 with CH3Dissolving OH, adding HCl, stirring at room temperature for 1-5h, removing the solvent under reduced pressure, adding ammonia water, stirring at room temperature for 15-20min, removing the ammonia water under reduced pressure, adding methanol to dissolve, separating by column chromatography, and freeze-drying to obtain a compound 27, wherein the reaction formula is as follows:
s5), dissolving the compound 27 in ethanol, adding triethylamine, reacting for 30-45min at room temperature, adding 270 mu L of ethanol solution of 3, 4-diethoxy cyclobutyl-3-ene-1, 2-dione, reacting for 20-23h at room temperature, and performing vacuum filtration to obtain a white solid, namely a compound 1, wherein the reaction formula is as follows:
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| WO2007014608A1 (en) * | 2005-07-29 | 2007-02-08 | Merck Patent Gmbh | Quadratic acid ii derivatives |
| WO2016029146A1 (en) * | 2014-08-22 | 2016-02-25 | University Of Washington | Specific inhibitors of methionyl-trna synthetase |
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| WO2007014608A1 (en) * | 2005-07-29 | 2007-02-08 | Merck Patent Gmbh | Quadratic acid ii derivatives |
| WO2016029146A1 (en) * | 2014-08-22 | 2016-02-25 | University Of Washington | Specific inhibitors of methionyl-trna synthetase |
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