SU763344A1 - Trans-2,3,11,12-(4',4"-diamyl)-dibenzo-18-corone-6 as selective indicator of potassium penetrability of biologically active and synthetic membranes - Google Patents

Description

The invention relates to a new chemical compound, trans-2,3, 11,12- (4, 4-diamyl) -dibenzo-18-crown-6, which can be used 5 in biology and medicine as a selective identifier for potassium membrane permeability.

Natural inductors of ion permeability of membranes are known, in particular 10 antibiotics of the naktin group, valinomycin, gramicidin, and, however, natural ionophores, despite a number of advantages, are chemically resistant and difficult to access. five

Synthetic ionophores are also known - diacyl derivatives of 2,3,11,12-18-crown-6, in particular trans-2, 3,11,12- (4, 4 -divaleryl) -dibenzo-18-crown-6 2. 20

The indicated compound is mainly an inducer of the permeability of biological membranes by divalent cations (Ca, Md), and, besides, is characterized by low selectivity with respect to potassium ion, induces ion transport only through biological membranes, less active by the effective concentration (100 times) and poorly dissolved

ROME in alcohol, which limits its scope.

A new compound, tr1ance-2, 3, 11, 12- (4, 4-diamyl) -dibenzo-18-crown-6 of general formula 1, is capable of effectively and selectively increasing the potassium permeability of both biological and artificial membranes under Clemmenson recovery. .

The structure of the obtained compound is proved by IR, NMR and mass spectroscopy.

The synthesized compound of formula-1 is capable of producing complexes with alkali metal ions and selectively induces the transport of potassium ions through both biological and artificial membranes.

Example 1. Synthesis of trans-2,3, 11D2- (4,4-diamyl) -dibenzo-18-crown-6 .I ,,

Take 1 g of trans-2,3,11 D2- (4.4 divaleryl) dibeno-18-crown-6, 1.3 g of amalgamated zinc, 2.4 ml of concentrated hydrochloric acid, 0.5 ml of water and 50 ml ethyl alcohol, is boiled for 40 hours, adding 0.2 ml of concentrated hydrochloric acid every hour, the mixture is decanted from unreacted zinc and the alcohol is distilled off, by adding water. The oil that is separated out is extracted with chloroform, washed with water until neutral, dried with CaSbt and the solvent is removed.

The reaction product is purified by column chromatography in a hexane-chloroform-acetone system — 3: 2: 0.5 and successive crystallization from hexane and acetone. Yield 0.5 g (53%), mp. 111-113 ° C.

Found,% C 72.18, H 9.24, Mv.BOO,

. io 44 t Calculated,% C 71.97; H 8.86; Mv 500.6.

NMR spectrum (m, d.): 6.5-6.8 (bN, m, ArN); 3.8-4.2 (16H, m, OSC); 2.48 (4H, T, -CHj); l, 00-i; 70 (12H, m, C-CH4); 0.82 (6H, T, C-CH).

IR spectrum, cm: 15 95.1520); 2950-2870, 1270-1150 (-C-O-C-); 870, 810 (1,2,4 - substituted benzene).

Example 2. The specificity of permeability induced by trans-2, 3, 11, 12 (4,4-diamyl) -dibenzo-18-crown-6 on biological and artificial membrane systems is examined for mitochondria and bilayer phospholipid membranes (BFM), which are classic objects for testing the membrane-active properties of various compounds.

Rat liver mitochondria were isolated by differential centrifugation. The permeability of the internal membranes of these subcellular organelles is determined by the rate of decrease in the optical density of the suspension at 520 nm on an LKF-69 photometer, the output of which was connected to a QH-102 recorder. The measurements were carried out with stirring in glass cuvettes with an optical path length of 1 cm. Incubation media 3 ml contain in all cases respiratory inhibitors — rotenone and antimycin (0.33 µg / ml each) and 10 mm trisnitrate (pH 7.45); in addition to these components of the medium, they contain 130 mm of potassium or sodium nitrate, or ammonium (when measuring permeability by H), or 86 mm of calcium nitrate, or magnesium or barium.

Thus, the volatile onset of mitochondria possessing the properties of osmometers is limited only by the transmembrane shift speed of the cation under study, since it is an anion penetrating through the mitochondrial membrane. The used preparations of these organelles are characterized by low permeability by semen cations, excluding Ca, which transports mitochondria at high speed using specific endogenous calcium p.pv oschik.

Permeability to various cations is estimated by the swelling rate of mitochondria, which is measured in L. mitochondria (1.1-1.2 mg of protein per 3 ml of medium) are added to the cuvette with the incubation medium, and the rate of decrease in optical density is recorded for 1 min. Then, depending on the concentration studied, 15-150 µl of the alcohol solution of cyclopolyether is introduced into the cuvette and continue recording for another 2-3 minutes. Samples in which only the corresponding volume of the standard is added are used as controls.

The research results show (Table 1) that trans-2,3,11,12- (4,4-diamyl) -dibenzo-18-crown-6 in low concentrations (2, -IxlCf M) induces the permeability of mitochondrial membranes. mainly for K ions, to a lesser extent, for Na, it does not act even when using significantly higher concentrations (5-1010 m) on the transfer rate of divalent cations; in the presence of M compound 1, the permeability of mitochondria by Na increases by 1.3 times, 2 times, while in K - 20 times. Higher concentrations of cyclopolyether (- () induce an overall increase in mitochondrial permeability for monovalent cations, however, K permeability still remains several times higher than for H and Na. Thus, the relative permeability of mitochondria for potassium, hydrogen and sodium is induced 1 "10-5m trans-2, 3, 11,12 (4, 4-diamyl) dibenzo-18-crown-6 is 1.0.46 and 0.075 respectively (the increase in potassium permeability is taken as 1). by increasing the concentration of compound 1 in the .2 range, I-lCf M of potassium permeability of mitocondrial membranes lichivaets linearly.

Table 1

The effect of different concentrations of trans-2, 3,11,12- (4, 4-diamyl) -dibenzo-18-crown-6 on the permeability of mitochondrial membranes for some mono- and divalent cations (A is permeability in the presence of cyclopolyether. Ad - in control)

0.25 4.7 1.0 1.0 1.0 1.0 1.0 0.5 7.0 1.0 1.0 1.0 1.0 1.0 i Continued from. 11 0.75 10.7 1.0 1.1 1.01.01.0 1.0 20.0 1.9 1.3 1.01.2l, q 5.0 42.4 15.6 2, 5 1.01.8.1.0 10.0 80.0 36.7, 6.2 1.12.81.6 50.0 - 49.8 14.6 0.5 2.8 1.0 100.0 - - - 0.5 1.7 1.0 The effects exerted by the synthesized cyclopolyester on the mitochondria correlate with the data obtained on the bilayer phospholipid membranes. The BFM from the lipids of the white matter of the brain of a bull is formed on a hole with a diameter of 1 mm in a teflon cup placed in a glass cell with a buffer solution: 25 mm Tris-NSB (pH 7.4). The membrane-forming solution contains 20 mg of brain phospholipids in 1 ml of decane. The parameters of BFC are measured using chlorine-silver electrodes connected to the OP-205 electrometer. Experiments performed on the Navy show that trans-2, 3, 11,12- (4, 4 diamyl) -dibenzo-18-crown-6 induces conductivity of bilayers consisting of potassium and proton components. When using the synthesized cyclopolyther, the conductivity of BFM along H increases 2.5 times, and along K increases 7.5 times, i.e. has a total decrease in resistance of 10 ra. When a 10-fold potassium gradient is created on the membrane, a membrane potential of 58 mV is generated, i.e. cyclopolyether gives the phospholipid mebrane properties of the potassium electrode. However, even when Compound 1 was used at a concentration of IxlO M, induction of conductivity of bilayers for calcium ions was not observed. The cation specificity of the proposed compound is estimated by potassium or sodium ions by adding, on the one hand, BFM 10 mM K, and on the other hand, 10 mM Na. In the presence of IxlO M trans-2,3,11,12- (4,4 diamyl) -dibenzo-18-crown-6 under these conditions, a potential difference is generated on the membrane, equal to 59 mV. It follows that the K, Na cationic constant of cationic equilibrium is 10. Example 3. The effective concentration of trans-2, 3,11,12- (4,4-diamyl) -dibenzo-18-crown-6, which induces thorough potassium permeability, is determined by are on biological membranes (mitochondria) and model systems (liposomes and BFM). The permeability of mitochondria and BFM at K is measured as in Example 2. Liposomes are prepared from a 10% alcohol solution of egg lecithin by dialysis against an aqueous solution containing 30 mM CWS and 30 mM tris-chloride (pH 7.4) with a threefold change buffer for 24 hours. The kinetics of the release of potassium ions from liposomes are recorded using an ESL-51G-04 glass electrode in combination with a laboratory pH meter pH-340, to the output of which is connected a KSP-4 recorder. The scale of the recorder is xsrc with a KCE solution of a known concentration. The data presented in Table 2 show that there is a direct proportionality between the concentration of cyclopolyether used and the potassium permeability induced by it in all the objects studied. A 5–10-fold increase in conductivity along K is achieved by adding to the membrane structures the proposed compound at a final concentration of 5) 10 M, which is 100 times less than the concentration in which the membrane-active properties of its prototype appear. Table 2 The effect of different concentrations of trans-2,3,11,12- (4, 4-diamyl) -dibenzo-18-crown-b on the potassium permeability of mitochondria, liposome and bilayer phospholipid membranes Conducted BFM in the presence of 10 mM K. Based on the results obtained, trans-2,3,11,12- (4, 4-diamyl) -dibenzo-18-crown-6 is a selective inducer of the potassium permeability of all types of membranes, 10b times more active than the known (2) current concentration, highly soluble in alcohol, therefore it is more convenient for the study of biological objects and can yt used as a tool in the study of ion transport mechanisms through kgshi

biological and artificial membranes, as well as for the manufacture of potassium-sensitive electrodes on its basis for measuring the activity of xypi ions in liquid media.

Claims (2)

1.Ovchinnikov Yu.A., Ivanov V.T., Shkrob A.M. Membrane active complexones. M., Science of 1974. 2.Assignment of the USSR on the application 2393378 / 23-04, cl. C 07 O 323/00, 02.08.76,