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    • 专利摘要:
    The invention relates to heterocyclic compounds, in particular to the preparation of resorufin derivatives of the formula 1a or 16. SNL, C (0) CZ (where RI is Nili C1 in position 2; H or C | -Cd-alkyl; Ke - H, C ( -C4-alkyl or C1; Cd is H or R -fR -annulated phenyl ring; or 2; LI -N-CHu-CHu-N -CHu-CHfc, -N (CHs) -CHft-COO - or - C (0) -CH-CH, g-CHN-K-in free form 9 silt, -CH4-CHH; 1, c-bond, -C (0) - (where, 2 shtiZ, -NH- NHCHuCH 2 -, - C (0) CHaCHe- –C (0) -, –C (0) CH (OH) CH (OH) C (0) -; A is the radical formed from hydantoin, diphenylhydantoin, tert. -butyl-oxycarbonyl-b-thyroxine, phenobarbital, estradiol, 7-theophylline, digok of benzene, digoxigenin monodigitox sozid, 3-amino-3-deoxytetraioiodothyroacetic acid, thyroxine methyl ether or xanthine, and the radical is (CH $) mC (O) -L4-L $ -A when it is in position 4 and at - in position 2, and in the case of p 0,8-6,4 L N (CH a) CH «Ј (0) - or-C (O) - CH-CH-CHfc-N-CH-CHfc; Le - simple and A is a radical derived from immunoglobulin G, which are the fluorescent labels of low molecular weight or high molecular weight compounds. The goal is to develop a method for obtaining these compounds. The preparation is carried out from the compound of the formulas 1a and 16, where, instead of the LЈA substituent, there is a reactive radical, C1 or -O- -NC (0) -CH $ -CH2-C (0), and a compound of the formula X4L (jA, where L. jj A is ucaeno higher; X4 is H or (0) -CHg- (O), and the active radicals of the compounds may have protective groups which, if necessary, are removed with the release of the target product (L in free form. 9 Il, 1 table One 00 04
    公开号:SU1621811A3
    申请号:SU864027894
    申请日:1986-07-24
    公开日:1991-01-15
    发明作者:Кляйн Кристиан；Батц Ханс-Георг；Херрманн Руперт
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the chemistry of heterocyclic compounds, in particular, to a process for the preparation of novel resorufin derivatives of the general formula la. (Or 16,
    (sn2) ts (o) t2
    ABOUT
    /
    ra or xanthine, and the radical - (SNg) t-C-L1-1 -A with
     is in position 4 and with ha "2 in position 2, and in the case of and, 8-6,4
    Лг-Ґсн2о ш / |
    ch3 oo
    2- (СН2) daС (0) Ь1Ь2
     HE
    R "is H or C1 in position 2;
    R $ - AND or O-C 4ga ki;
    Ke-H, SC-Cd-alkyl or C1;
    R4 - H or Ra and R together form annelated phenyl ring,
     or 2;
    p-1
    The aim of the invention is to obtain novel resorufin derivatives of the general formula It which, as fluorescent labels of low molecular weight or high molecular weight compounds, increase the sensitivity and accuracy of determining the concentration of 30 components of blood samples.
    The goal is reached
    About 35
    , -J-,
    SNE.
    kt OTJ -GPGG pap-V-C- that compound of the general formula Ila UU, tWi / 1N / y or 116
    CH3
    L k-sh. - C - (SNO) l 21
    About where, 2SH,
    -NH -, - NHCH2CH2-1
    ABOUT
    40
    45
    (
    ABOUT
    he i
    o t o
    -CCN2CHN2S -, - ssN (he) sn (OH) s-R
    IIII 4o

    A is a radical formed from - 5 ° where R ' -R4, m and b, have the indicated dantoins, diphenylhydantoin, above values
    tert-butylox carbonyl-L-thyroxine, phenobarbital, estradiol, 7-theophylline, d-toxigenin, digoxigenin monodi-nitoxoside, 3-amino-3-deoxytitriodothyroacetic acid, thyroxine methyl ester L | - a simple bond and A - a radical derived from immunoglobulin G,
    which are fluorescent labels of low molecular weight or high molecular weight compounds and can be used in medicine in a fluorescent immunoassay to determine the concentration of haptec or antigen in a blood sample.
    The aim of the invention is to obtain novel resorufin derivatives of the general formula It which, as fluorescent labels of low molecular weight or high molecular weight compounds, increase the sensitivity and accuracy of determining the concentration of blood sample components.
    The goal is achieved by
    that a compound of general formula Ila or 116
    ABOUT
    40
    (,
    ABOUT
    WH CLjXj
    he i
    about
    subjected to interaction with the compound of General formula III
    jXal A
    where Lg and A are as defined above;
    ABOUT.
     H or
    -o0
    :
    moreover, the active radicals of the compounds Ila or 116 and III can have protective groups which, if necessary, are removed, with the release of the target product in free form.
    FIG. 1 through 9 are curves illustrating the method.
    Example 1. s- (1-Diphenylhydantoiyl) pronionyl piperazide resorufin-4-carboxylic acid.
    but. Resorufin-4-carboxylic acid.
    16 g of nitrosorezorin, 15.5 g of 2,6-dioxybenzoic acid and 8.6 pyrolusite are suspended in 200 ml of methyl alcohol, after which the suspension is cooled to. Then 10.6 ml of concentrated sulfuric acid is added dropwise to the suspension, after which the mixture is further stirred for 2 hours at room temperature. Separate in the form of a red colored precipitate, rapapurine-4-carboxylic acid is filtered off, washed with methyl alcohol and dried.
    The diazoresorcinol derivative is dissolved in 200 ml of water and 50 ml of a 25% ammonia solution and the solution is filtered. Under ice cooling, 50 g of zinc dust was added in portions to the blue colored filtrate, and the reaction mixture was allowed to warm to room temperature. It is easy to monitor the course of reduction using thin layer chromatography (eluent: methyl alcohol - ethyl acetate 1: 1; DC plates with silica gel). The reaction solution is filtered, and then the filtrate is acidified with glacial acetic acid and a small amount of concentrated hydrochloric acid. Resorufin-4-carboxylic acid precipitated out is filtered off and dried under vacuum over phosphorus pentoxide.
    Output 16.33 g.
    16218116
    Rf (silica gel, elto-agent: n-butyl alcohol - ice on acetic acid - water in the ratio 4: 1::, 4.
    0
    five
    c
    0
    50
    $ 0
    B.N., 0,0-Triacetyldihydroresorufin-4-carboxylic acid.
    12.9 g of resorufin-4-carboxylic acid is dissolved in 30 ml of glacial acetic acid and 30 ml of acetic anhydride, the solution is mixed with 27.6 g of tin (II) chloride, after which the mixture is stirred for 1 hour at 80 ° WITH. The reaction mixture is then poured onto 600 ml of ice-water, stirred for 1 hour, and then the precipitate is filtered off. After drying, the solid is dissolved in 500 ml of acetone. The solution is filtered and the filtrate is evaporated, whereby 11.3 g of product are obtained after drying.
     H-NMR (D 4-DMSO), (Y: 2.24, 2.26 and 2.29 (S, 9H); 6.94 (dd, 5 and 2.2 Hz, 1H); 6, 98 (d, 2 Hz, 1H); 7.04 (d, Hz, 1H); 7.61 (d, J 8.5 Hz, 1H); 7.67 (d, Hz, 1H).
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.46.
    C. Chloride H, 0,0-triacetyl-dihydroresorufin-4-carboxylic acid.
    38.5 g of the triacetate described in Example 16 are mixed with 54 ml of oxalyl chloride and the mixture is cooled to 0 ° C. A few drops of dimethylformamide were added to the cooled mixture, after which the mixture was allowed to warm to room temperature. In this case, the dissolution of the solid is accompanied by the evolution of gas. The reaction mixture is evaporated to dryness in vacuo, three times the product is dissolved in 200 ml of dry methylene chloride and the solution is again evaporated to dryness.
    Exit 41
    N-tert-Butyloxycarbonyl (BOC) - piperazine.
    12.61 g of N-benzhydrylpiperaein (EMPA-cheraie) are dissolved in 100 ml of a mixture of 1,4-dioxane and water, taken / in a ratio of 3: t. To the prepared solution is added dropwise a solution of 12.0 g of di-tert-butyl dicarbonate in
    50 ml of 1,4-dioxane. The mixture is stirred for 0.5 h, then 50 ml of oxen are added dropwise to it, filtered, after which the precipitate is collected.
    Yield 16.2 g of N-BOC-m-benzhydryl-piperazine.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.92.
    7 g of N-BOK-N -benehydrylpiperazine is dissolved in 100 ml of ethyl acetate and 5 ml of glacial acetic acid. Hydrogenation is carried out in the presence of 0.3 g of palladium on activated carbon, after which the catalyst is filtered off and the filtrate is evaporated. The resulting residue is mixed with 100 ml of water and 20 ml of 1N. hydrochloric acid solution is filtered, the filtrate is extracted twice with ethyl acetate, and then the aqueous phase is brought to the main reaction by adding sodium hydroxide solution. The oily product which precipitates out is extracted with dichloromethane. After drying the organic phase over sodium sulfate and evaporation, 3.2 g of N-BOC-piperazine are obtained in the form of an oily substance which after a few days is completely crystallized.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1). 0.05 with ninhydrin turns blue.
    D.N -Boc-piperazide L, 0,0-triacetyldihydroresorufin-4-carboxylic acid.
    To 25 g of the acid chloride described in example 1c, and 17.3 ml of triethylamine in 450 ml of dichloromethane at 0 ° C, a solution of 13.8 g of N-BOC piperazine in 50 ml of dichloromethane is added dropwise. The reaction mixture is further stirred for 1 hour without cooling, then extracted three times with water, after which the organic phase is evaporated.
    Exit 36.0 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.64.
    E.N.-BOC-piperazide N-acetyl dihydroresorufin-4-carboxylic acid,
    34.3 g of triacetate described in Example 1d and 17.1 g of sodium sulfite
    0
    five
    0
    5 Q
    0
    with
    0
    stirred for 1 hour at 500 ml of a mixture of 1,4-dioxane and water, taken in a 1: 1 ratio. After this, the reaction mixture is evaporated, the residue is dissolved in ethyl acetate, the solution is filtered off from the insoluble salt, after which the filtrate is chromatographed on 2 liters of silica gel (eluent: ethyl acetate - dichloromethane 4: 1 ratio as it starts wash the product, switch to pure ethyl acetate).
    Exit 14 g.
    Rf (silica gel, eluting agent: ethyl acetate - dichloromethane in the ratio 4: 1) 0.28.
    g.N -BOK-piperazide resorilelin-4-carboxylic acid.
    5 g of the N-acetyl compound described in Example 1e is dissolved in 200 ml of methyl alcohol and 600 ml of water. To the prepared solution was added 1.8 g of sodium hydrogen carbonate and 10.7 ml of 1N. sodium hydroxide solution, and then 14 g of potassium hexacyanoferrate. The reaction mixture is stirred for 0.5 hours at room temperature, after which the pH of the mixture is adjusted to 5. The precipitated product is filtered off using vacuum.
    Exit 2.72 g.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.28.
    h. Piperaeid resorufin-4-carboxylic acid trifluoroacetate.
    1 g of the BOC proivvodnogo described in example 1G, incubated for 15 min in 20 ml of triperoxonane acid. Then the reaction mixture is evaporated, the residue is treated with diethyl ether, after which the product is filtered off.
    Output 0.96 g.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.02.
    and. Reaction of piperazide resorufin-4-carboxylic acid with N-hydroxy-succinimide ester of 3- (1-difenyl-dantoinyl) propionic acid.
    191 mg of the trifluoroacetate described in Example 1h and 210 mg of N-hydroxysuccini9162181
    3- (1-diphenylhydantoynyl) propionic acid mid ester (obtained from diphenylhydantoin sodium salt and 3-bromopropionic acid ethyl ester) is stirred for 15 hours in 20 ml of dioxane and 20 ml of O, 1 M potassium phosphate buffer solution pH 8.5. The precipitated product is filtered off, the filtrate is evaporated ./, and the residue is chromatographed on RP 18 silica gel (eluent: isopropyl alcohol), resulting in an additional product. The product is crystallized 5 from a mixture of ethyl acetate and methyl alcohol, resulting in a total of 250 mg of the conjugate product.
    Rf (silica gel eluting with the following agents: chloroform-methanol ice / acetic acid 9: 1: 0.1) 0.61.
     n-NMR (d16-DMSO), d: 2.6-2.8 (t, 2H); 3.0-3.8 (t, YUN); 6.74 (d, J 25 2.2 Hz, 1H); 6.82 (d, 5 Hz, 1H); 6.91 (dd, 5 and 2.2 Hz); 7.25-7.33 (t, YUN), 7.55 and 7.66 (in d, 5 Hz, 2H) million
    UV / VIS (0.1 M potassium phosphate buffer solution with pH 7.5): flMQKC 576.8 nm.
    Emission of fluorescence: 592 nm.
    Example 2. Interaction of pipa-g of resorufin-4-carboxylic acid razid with 3- (1-diphenylhydantoinyl) acetic acid N-oxysuccinimide ester.
    By analogy with the example described in Example 1, from 365 mg of piperazi trifluoroacetate 40 and resorufin-4-carboxylic acid and 339 mg of 3- (1-diphenylhydantoinyl) acetic acid N-hydroxysuccinimide ester, 210 mg of (2-diphenylhydroxy-methylmethylcarbonyl) piperadaidaidemethyl anhydroxycarbonylpiperapirate peaididaidaide -4-carboxylic acid.
    Rf (silica gel, eluent: n-butanol - ice 4: 1: 1 acetic acid - water) 0.82.
    H-NMR (D16-LMSO), I: 3.2-4.5 (ha, sUN); 6.60 (d, 4 Hz, 1H) .; 6.71 and (d, 5 Hz, 1H); 6.80 (dd, 05 and 2.04 Hz, 1H); 7.39 (S, YUN); 7.52 (d, 5 Hz, 1H); 7.61 (d,, 0 Hz, 1H); 9.65 (s, 1H) million .55
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0):, 4 nm.
    Emission of fluorescence: fl max 592 nm.
    ten
    PRI me R 3. Reaction of resorufin-4-carboxylic acid piperazide with N-BOK -L-thyroxine N-oxysuccinimide ester.
    By analogy with the example described in Example 1, out of 212 mg of piperazide trifluoroacetate resofurin-4-carboxylic acid and 419 mg of N-BOK-L-thyroxine N-oxysuccinimide ester, 320 mg of product are obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.58.
    The N-BOK-L-thyroxine N-oxysuccinimide ester was prepared as follows.
    a) N-BOK-thyroxin.
    A solution of 10 g (12.5 mmol) of L-thyroxine sodium salt is a mixture consisting of 300 ml of a mixture of dioxane and water in a ratio of 2: 1 and 15 ml of 1N. The sodium hydroxide solution is mixed with 3 g (13.75 mmol) of di-tert-butyl dicarbonate (BOC) O, after which the reaction mixture is stirred for 2 hours at room temperature under conditions that exclude access of light.
    By adding 2 M KHSO solution, the pH of the mixture was adjusted to 2, the mixture was extracted with ethyl acetate, the extract, which was a solution of the product in ethyl acetate, was washed with water, dried over sodium sulfate and evaporated. The solid residue is triturated with petroleum ether, the product is filtered off using vacuum and dried in a desiccator.
    Yield 9.45 g (86% of theoretically calculated value).
    Rf (silica gel, eluent: chloroform - ligroin - acetic acid in the ratio 6: 3: 1) 0.6.
    b. N-Oxisuccinimide ester of N-BOC-thyroxine.
    1.2 g (9.5 mmol) of N-oxysuccinimide was added to a solution of 8.8 g of L-BOC-thyroxine in 200 ml of ethylene glycol dimethyl ether. The solution is cooled to 10 ° C, after which it is mixed dropwise with a solution of 2.3 g (9.9 mmol) of dicyclohexylcarbodiimide in 40 ml of ethylene glycol dimethyl ether. The reaction mixture is stirred for -. 2 hours at room temperature,. the dicyclohexylmoleum precipitated out is filtered off using vacuum, after which the filtrate is evaporated.
    vacuum under vacuum 40CC. The resulting residue is triturated with isopropyl alcohol and filtered using vacuum, and then dried in a desiccator at room temperature.
    Yield 9.19 g L- (N-BOC-L-thyroxin) piperazide resorufin-4-carboxylic acid (94% of the theoretically calculated value), total yield with respect to,. to thyroxin 81%.
    Rf (HPTLC-RP 18; eluting agent: nitromethane - ethanol 9: 1) 0.8 or (HPTLC-RP 18; eluting agent; acetonitrile - water in COOT-jj wearing 8: 2) 0.6.
     H-NMR f (U LMSO),): 1.36 (S, 9H); 2.81 (s, 4H); 2.9-3.2 (m, 2H); 4.5-4.9 (ta, 1H); 7.08 (s, 2H); 7.63 (d, Hz, 1H); 7.90 (s, 2H); 9.2 (s, 20 1H) ppm.
    Example4. Conjugation of resorufin-4-carboxylic acid piperazide with 3-0- 3- (M-succinimidoxycarbonyl) was examined by extradiol.25
    By analogy with Example 1 described, from 212 mg of reeofurin-4-carboxylic acid piperazide trifluoroacetate and 220 mg of 3-0-S3- (M-succinimidyloxycarbonyl) propyl} estradiol, 30 295 mg of (3-carbonyl) propyl estradiolyl piperazide resorufin-4 are obtained. - carboxylic acid.
    Rf (silica gel, eluting agent: chloroform - methanol - ice on hydrocarbonic acid in the ratio 9: 1: 0.1) 0.58.
    3-0 - {. 3- (s-Succinimidoxycarbonyl) propyl estradiol. is prepared in the usual way from 3-0-carboxypropyl-estra-40 diol (obtained from estradiol and bromobutyric acid), and N-oxysuccinimide in the presence of dicyclohexylcarbodiimide.
    Example 5. Conjugation of piperazide resorufin-4-carboxylic acid with N 3- (K-succinimidoxycarbonyl) propyl phenobarbital.
    By analogy with Example 1 described in Example 1, from 212 mg of piperazide trifluoroacetate, resorufin-4-carboxylic acid and 205 mg ((M-succinimidooxycarbonyl) propyl phenobarbital, 220 mg of m- (3-carbonyl) propyl phenobarbital resorufin-4-carboxylic acid ..
     55
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) -0.45.,
     five
    ,
    j
    0
    five
    0
    ,
    0
    e

    five
    112
    N-L3- (N-Succinimidoxycarbonyl) propyl phenobarbital is prepared in the usual way from phenobarbital-1-butyric acid and N-oxysuccinimide in the presence of dicyclohexylcarbodiimide.
    Example 6: Conjugation of resorufin-4-carboxylic acid piperazide with theophylline-7-propionic acid N-oxysuccinimide ester.
    From 212 mg of resorufin-4-carboxylic acid piperazide trifluoroacetate and 175 mg of theophylline-7-propionic acid N-oxysuccinimide ester are obtained by analogy with the described in example 1 and 200 mg of M- (theophyllin-7-propionyl) pi resorphine-4 piperazide carboxylic acid
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.44.
    Theophylline-7-propionic acid N-oxysuccinimide ester is prepared in the usual way from theophylline-7-propionic acid and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.
    Example 7. Conjugation of N-oxysuccinimide ester of L- (4-re-oorophinyl-carbonyl) sarcosine with 1- (2-amido-ethyl) -diphenylhydantoin.
    A. (tert-Butoxycarbonylmethyl) methylamide H, 0,0-triacetyl dihydroresorufin-4-carboxylic acid.
    10 tons of the acid chloride described in example 1c are introduced into the reaction by analogy with that described in example 1d with sarcosine t-butyl ester.
    Output 7.5 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.77.
    B. (tert-Butoxycarbonylmethyl) methylamide H-acetyl dihydroresorufin-4-carboxylic acid.
    7.5 g of the product described in example 7a was deacetylated by analogy with that described in example 1g.
    Output 5.2 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.56.
    B.- (tert-Butoxycarbonylmethyl) methylamide resorufin-4-carboxylic acid.
    4.5 g of the product described in pre-76, is introduced into tax interaction with that described in Example 1g.
    Output 2.6 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0, t) 0.64.
    N- (carboxymethyl) methylamide, reso1 uphin-4-carboxylic acid.
    0.55 g of the product described in prier 7c is kept for 1 hour at room temperature in 6 ml of trituroacetic acid. Then the reaction mixture is evaporated to dryness, the residue is triturated with diethyl ether, and then the product is filtered.
    Output 0.45 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) - -O, I.
    D.N-oxysuccinimide ester of N- (4-resorufinylcarbonyl) sarcoein.
    200 mg of the product from Example 7 are stirred for 14 hours with 72 mg of N-oxysuccinimide and 138 mg of dicyclohexyl-carbodiimide in 40 ml of tetrahydrofuran. The urea which is precipitated is filtered off, the filtrate is evaporated and the resulting residue is chromatographed on RP 18 silica gel (eluent : nitromethane - ethanol in a ratio of 4: 1).
    Yield 150 mg.
    Rf (silica gel RP-18, eluting agent: nitromethane - ethanol in the ratio 4: 1) 0.79.
    e. Conjugation of Kozin N- oxysuccinimide ester M- (4-resorufinylcarbonyl) sar with 1- (2-aminoethyl) diphenylhydantoin.
    125 mg of N-oxysuccinimide ester from Example 7d are stirred for 1 hour with 90 mg of 1- (2-aminoethyl) diphenylhydantoin in 40 ml of a mixture consisting of dioxane and a potassium phosphate buffer solution with a pH of 8.5, taken in the corresponding wearing 1: 1. Dioxane is distilled from the reaction mixture, ammonia is added to the residue until complete color change, filtration is performed, after which the product is precipitated from the filtrate by adding hydrochloric acid.
    Yield of n- (4-resorufincarbonyl) sarcosinyl-1- (2-aminoethyl) diphenylhydantoin 110 mg.
    five
    0
    five
    0
    five
    five
    0
    five
    Rf (silica gel, eluent: n-butanol - ice 4: 1: 1 acetic acid - water) 0.78.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): / lNvavt; 575 nm.
    Emission of fluorescence: fl nltss t 572 them.
    Try on Resorufin-4-carboxylic acid 2- (1-Diphenylhydroxy-vinyl) ethylamide.
    a.2- (1-Diphenylhydantoinyl) ethyl amide K, 0.0 triacetyl digidorvosorufin-4-carboxylic acid.
    By analogy with the one described in Example 1d, 1.37 g of 1- (2-aminoethyl) diphenylhydantoin are reacted with 1.2 g of acid chloride M, 0.0-triacetyl dihydroresorufincarboxylic acid. The result is 1.9 g of the product in the form of a weakly colored foamy substance.
    b.2- (1-Diphenylhydantoinyl) ethyl amide resorufin-4-carboxylic acid.
    The product obtained in Example 8a is oxidatively deacetylated by analogy with that described in Examples 1e and 1g. From 1.9 g of the substance, 600 mg of N-2t (1-diphenylhydantoinyl) ethyl resin of rufin-4-carboxylic acid are obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.68.
    H-NMR (-DMSO), Y: 3.2-4.6 (t, 4H); 6.73 (d, 2 Hz, 1H); 6.84 (d, 5 Hz, tH); 6.86 (dd, 5 and 2.2 Hz, 1H); 7.2-7.4 (t, YUN); 7.62 and 7.66 (d, 5 Hz, 2H); 8.66 (t, broad, Hz, 1H) j 9.58 (S, 1H) ppm.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): 575 nm.
    Emission of fluorescence: dk 591 nm.
    Example 9. Combination of piperazide 6-methylresorufin-4-carboxylic acid with N- oxnsuccinimide ester of 2- (1-diphenylhydantoinyl) acetic acid.
    but. 2-Methyl-4-nitrozorezoretsin.
    19.8 g of 2-methylresorcinol and 13.4 g of potassium hydroxide are dissolved in 120 ml of ethyl alcohol, after which the solution is cooled to 5 ° C. After this, 24 ml of isopentylnitrile is added dropwise to the cooled solution, the mixture
    stirred for 3 hours, after which the precipitate formed is filtered off. The yellow-colored solid is stirred in 200 ml 5 n. sulfuric acid solution. A light yellow product is deposited in the sediment.
    Exit 22 g.
    Rf (silica gel, eluting the medium tO in: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) "0,53.
    b.6-Methylresapurin-4-carboxylic acid.15
    15.3 g of 2-methyl-4-nitro-eoreorcin, 15.4 g of 2,6-dioxybenzoic acid, 8.8 g of pyrolusite and 11 ml of concentrated sulfuric acid are reacted by analogy with Example 1a described in Example 20 (20).
    Output 28.7 g.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 5 0.15.,
    c.H, 0,0-Triacetyl-6-methyldihydro-resorufin-4-carboxylic acid.
    From 10 g of 6-methylresazapurin-4-carboxylic acid, 19.8 g of tin chloride-. 30 Va (II), 20 ml of acetic anhydride and 150 ml of glacial acetic acid are obtained, by analogy with the one described in Example 16, directly a triacetylated leuko compound. The crude, 5 product is purified by treatment with boiling acetone.
    Output 7.3 g
    Rf (silica gel, eluent: chloroform - methanol - ice on 40 acetic acid in the ratio 9: 1:: 0.1) 0.51. Q
    H-NMR (d 6-DMSO), 0: 2.10; 2.25; 2.29; 2.33 (12H each); 7.00; 7.09; 7.50 and 7.74 (in d, 8 Hz, 4H). $
    N-BOK-piperaeid N1, 0,0-triacetyl-6-methyldihydroresorufin-4 carboxylic acid.
    By analogy with those described in examples 1g and 1d of 5 g of K, 0,0-triacetyl-6-me- JQ tildigdrorresorufin-4-carboxylic acid, 10.7 ml of oxalyl chloride and 2 g (N-BOC-piperazine gives 3 g of the product .
    Rf (silica gel, eluting medium 55: ethyl acetate) "0,57.
    e. Piperazide 6-methyl-esterufin-4-carboxylic acid trifluoroacetate.
    1 g of the triacetyl derivative described in Example 9g is introduced into the reaction by analogy with that described in Examples 1g and 1h.
    Output 0.43 g
    e. Interaction of piperazide 6-methylresorufin-4-carboxylic acid with N- oxysuccinimide ester of 2- (phenylhydantoinyl) acetic acid.
    222 mg of the compound obtained in Example 9d are reacted with 200 mg of 2- (1-diphenylhydantoinyl) acetic acid N-oxysuccinimide ester.
    Yield of N- (1-diphenylhydantoinyl) methylcarbonyl piperazide 6-methylresorufin-4-carboxylic acid 250 mg.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): $ waKC 584 nm.
    Emission of fluorescence: avtc 600 nm.
    Example 10. Interaction of piperazide 9-hydroxy-5-benzo-phenoxazone-8-carboxylic acid with 2- (1-diphenyl-hydantoynyl) acetic acid N-hydroxysuccinimide ester.
    a.12-9-hydroxy-5-benzoCa oxide phenoxazone-8-carboxylic acid.
    2.84 g of 1,3-dioxy-4-nitrosonaphthalene, 2.31 g of 2,6-dioxybenzoic acid, 1.29 g of pyrolusite and 1.6 ml of concentrated sulfuric acid are reacted by analogy with the one described in example 1a
    Output 2.8 g
    Rf (silica gel, eluent: n-butanol - ice 4: 1: 1 acetic acid - water) 0.63.
    b.12-Acetyl-5,9-diacetoxybenzo Ca3 phenoxazone-8-carboxylic acid.
    By analogy with that described in Example 9c, from 2.4 g of the 12-oxide E-hydroxy-5-benzo d of phenoxazone-8-carboxylic acid, 1.8 g of the triacetylated dihydro compound are obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.31.
    B.M.-BOC-piperazide 12-acetyl-5,9-diacetoxybenzo (a) phenoxazyl-8-carboxylic acid.
    1.6 g of the triacetyl compound described in Example 106 is reacted by analogy with Example-9g with oxalyl chloride and N-BOC-piperazine.
    Yield 1.2g.
    five
     H-NMR (CDC19) ,: 1.49 (s, 9H): 1.12, 2.27, 2.46 (s, 12H); 3.0-3.9 (t, 8H); 7.03 (d, Hz, 1H); 7.16-7.94 (t, 6H) .5
    gN — BOC-piperazide 9-hydroxy-5 benzo and 1-phenoxazone-8-carboxylic acid.
    By analogy with example 1e, from 0.93 g of the triacetyl compound obtained in Example 10c, 10 0.51 g of product are obtained.
    Rf (silica gel, eluting agent; chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.69.5
    e. Piperazide 9-oxo-5-benzo aliphenoxazone-8-carboxylic acid trifluoroacetate.
    From 0.5 g of the BOC-protected compound described in Example South, 20 each, by analogy with Example 1h, 0.5 g of product is obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.02.
    e. Conjugation of piperazide 9-hydroxy-5-benzo-phenoxazone-8-carboxylic acid with N-hydroxysuccinimide ester
    2- (1-diphenylhydantoinyl) acetic acid lots.
    From 50 mg of piperazide obtained in Example 10d and 150 mg of 2- (1-diphenylhydantoinyl) acetic acid N-oxysuccinimide ester, anoreania with Example 1 and 70 mg of N-Ј (1-diphenylhydantoinyl) methylcarbonylZpiperazide 9- is obtained by analogy with Example 1 and hydroxy-5 benzo a2Phenoxazone-8-carboxylic acid.
    40
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:; 0.1) -0.57.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): $ 560 nm.
    Emission of fluorescence: yi make 615 nm.-.
    H-NMR (Cd 6-LMSO), d: 3.0-4.5 (m, SQ YUN); 6.37 (s, 1H); 6.80 (d, Hz, and 1H); 7.2-7.35 (t, YUN); 7.35-8.0 (hectares, ЗН); 8.10 (dd, and 2 Hz, 1H); 8.56 (dd, Hz, 1H); 9.60 (s, 1H) .55
    Example 11. (1-Diphenylhydantoinylmethylcarbonyl) piperazide 8-ethylreo-eorufin-4-carboxylic acid.
    but. 6-Ethyl-4-nitrozorezoretsin.

    By analogy with Example 9a, from 7.5 g 4-ethylresorcinol, 4.5 potassium hydroxide, and 8 ml isopentyl nitrile, 6-ethyl-4-nitrosorosorcinol is obtained in the form of a yellow colored solid.
    Output 7.5 g
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1:: 0.1) 0.37.
    b.8-ethylresorufin-4-carboxylic acid.
    By analogy with that described in Example 1a, from 7.4 g of 6-ethyl-nitrosore-eorecin, 6.8 g of 2,6-dioxybenoic acid, 3.9 g of manganese dioxide and 5 ml of concentrated sulfuric acid are obtained after reduction using 8 g of zinc dust. 9.5 g of the product.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.05. t
    c.N -BOK-piperazid Y, 0,0-triacetyl-8-e-di-dihydride orurufin-4-carboxylic acid.
    By analogy with Example 16, 7.0 g of 7.7 g of 8-ethylresorufin-4-carboxylic acid, 15.4 g of tin (II) chloride, 30 ml of glacial acetic acid and 15.3 ml of acetic anhydride give N, 0, O-triacetyl-8-ethylres orufin-4-carboxylic acid, which as a crude product is further processed by analogy with example 1b, directly into acid chloride and, by analogy with example 1d, into BOC-piperazide.
    Exit 4g.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.86.
    N -BOC-piperazide 8-ethylresorucin-4-carboxylic acid.
    From 4 g of N -BOK-piperazidy, 0,0-trna-acetnl-8-ethyldihydro-esorufin-4-carboxylic acid, by analogy with examples 1e and 1x, N -BOC-piperazide of the corresponding carboxylic acid is obtained.
    Output 0.5 g (
    Rf (silica gel, eluting agent: chloroform - methanol - 4: 1) 0.67.
    e. Piperazide 8-ethyl resorufin-4-carboxylic acid trifluoroacetate.
    330 mg of the corresponding BOC piperizide is incubated for 1.5 hours in 35 ml of a mixture consisting of dichloromethane and trifluoroacetic acid, in a ratio of 6: 1. The residue obtained after evaporation is taken up in diethyl ether, the product is filtered off using vacuum and dried ..
    Exit 350 mg.
    Rf (silica gel, eluirkuu means: butanol - ice on acetic acid - water in the ratio 4: 1: 1) 0.33,
    e. Interaction with N- oxysuccin-imide ester of 2- (1-diphenylhydantoyl) acetic acid.
    By analogy with example 1 and from 325 mg of piperazide trifluoroacetate 8-ethyl resorufin-4-carboxylic acid 2 and 435 mg of 2- (1-diphenylhydantoinyl) acetic acid N-oxysuccinimide ester, 120 mg of product is obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice for 2 (acetic acid in a ratio of 9: 1:
    : 0.1) 0.43. .
     n-NMR (d6-DMSO), d: 1.15 (t, J 7.2 Hz, 3N); 2.52 d, 2 Hz, 2H); 3.1-4.0 (t, 8H); 4.25-4.45 (t, 2H); 3 6.42 (s, broad, 1H); 6.94 (d, J 9.0 Hz, 1H); 7.3-7.5 (s, 11H); 7.68 (d,, 0 Hz, 1H); 9.54 (s, 1H); 11.2 (S, broad, 1H) million
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): Pit) C (575 nm.
    Emission of fluorescence: $ MOW nm.
    Example 12. (1-Diphenylhydrotoinylmethylcarbonyl) piperazide 8-chloro resorufin-4-carboxylic acid.
    a.8-Chlororeapurin-4-carboxylic acid.
    From 17.3 g of 4-chloro-6-nitrosorezorecine, 15.4 g of 2,6-dioxybenzoic acid, 8.6 g of pyrolusite and 10.7 ml of concentrated sulfuric acid are prepared by analogy with Example 1 a. resapurin-4-carboxylic acid.
    Exit 17.1g.
    Rf (silica gel, eluent: butanol - ice 4: 1: 1 acetic acid - water) 0.58.
    B.M., 0,0-Triacetyl-8-chlorodihydro-resorufinic carboxylic acid. e
    16.3 g of 8-chlororesazapurin-4-carboxylic acid and 18.9 g of tin (II) chloride are heated for 0.5 h in 100 ml of a mixture of glacial acetic acid
    and acetic anhydride, taken in a 1: 1 ratio at 80 ° C, after which the reaction mixture was poured into 500 ml of ice-water. The mixture is stirred for 2 hours, the precipitate is filtered off and dried over Sicapent. The solid is dissolved in 500 ml of acetone, after which the insoluble material remains by filtration from the solution. The filtrate is evaporated, and after drying the residue, 12.3 g of product are obtained.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.39 l
    GN-NMR (d16-LMSO), O: 2.25 (S, 3N); (S MO; 7.16 (d, 8 Hz, W) 7.30 (S, 1H); 7.76 (d, 8 Hz, 1H); 7.90 (S, 1H) mln.
    c.N -BOC-piperazide 8-chlorosorufin-4-carboxylic acid.
    By analogy with examples 16, 1b, 1d and 1g, 5 g of M, 0,0-triacetyl-8-chloro dihydroresorufin-4-carboxylic acid gives 0.8 g of product.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.7.
    g. Piperazide 8-chlorosorufin-4-carboxylic acid trifluoroacetate.
    By analogy with example 1h, 0.81 g of the product is obtained from 0.8 g of the BOC-protected compound obtained in Example 12c.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in rotogenia 9: 1: 0.1) 0.07.
    e. Conjugation of piperazide 8-chloro-eorufin-4-carboxylic acid with N-oxysuccinimide ester of 2- (1-diphenylhydantoinyl) acetic acid.
    By analogy with Example 1, 400 mg of piperazide 8-chlororesorufin-4-carboxylic acid trifluoroacetate and 410 mg of 2- (1-diphenylhydantoinyl) acetic acid N-oxysuccinimide ester are obtained from 400 mg of the trifluoroacetate acetate.
    Rf (silica gel, eluent: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.38.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.0): nm.
    Emission of fluorescence: $ kaks 597 nm.
    Example 13. Conjugation of piperae 8-chlorosorufin-1-carboxylic acid with (2-aminoethyl) amide theophylline-7-propionic acid.
    a.8-Chlorresorufin-1-carboxylic acid.
    8.7 g of 4-chloro-6-nitrozorezorecin and 7.71 g of 3,5-dioxybenzoic acid are dissolved in 200 ml of methyl alcohol. To the prepared solution was added 4.8 g of pyrolusite and portions of 5.3 ml of concentrated sulfuric acid. The reaction mixture is stirred for 2 hours at room temperature, filtered and ammonia is added to the solution until the color changes to blue, and then 200 ml of water. The solution is filtered, the filtrate is mixed with 25 ml of concentrated ammonia solution and 20 g of zinc dust with cooling, after which the mixture is further stirred without cooling for about 15 minutes. Then, 200 mg of activated carbon is added to the mixture, 2 are filtered, the filtrate is acidified to pH 2, after which the precipitated orophosphine derivative is separated by centrifugation. 3
    Output 3.9 g
    Rf (silica gel, eluent: n-butanol - ice 4: 1: 1 acetic acid - water) 0.88.
    b.N, 0,0-Triacetyl-8-chlorohydric o-resorufin-1-carboxylic acid
    From 3.5 g of 8-chlororesorphine-1-carboxylic acid, by analogy with Example 16, 3.2 g of product are obtained. t Rf (silica gel eluting medium: 4: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.43.
    c. Piperaeid trifluoroacetate 8-chlorosorufin-1-carboxylic acid.
    From 3 g of the triacetyl compound obtained in Example 116, by analogy with Examples 1c-1c, 1.4 g of product are obtained.
    Rf (silica gel, eluting agent: 5 in: chloroform - methanol - ice on acetic acid in the ratio 9: 1: 0.1) 0.08.
    Interaction of piperazide 8-chlororesorphine-1-carboxylic acid with theophylline-7-propionic acid with (2-aminoethyl) amide.
    By analogy with example 8 of 420 mg. piperazide K, 0,0-triacetyl-8-chlori3
    Q 5 0 5 0
    d
    five
    ABOUT
    g
    five
    hydroresorufin-1-carboxylic acid and 300 mg of (2-aminoethyl) amide, theophylline-7-propionic acid; 190 mg of N-C (theophylline-7-propionyl) -2-aminoethyl amide, 8-chloroororufin-1-carboxylic acid .
    Example 14. Labeling of immunoglobulin G N-oxysuccinimide ester of N- (4-resorufinylcarbonyl) sarcosine.
    100 mg of human IgG is dissolved in 10 ml of a 0.1 M potassium phosphate buffer solution with a pH of 8.0, after which the prepared solution is mixed with 5 mg of N- (4-pe-eorophinylcarbonyl) sarcosine H-oxysuccinimide ester (Example 7e). The reaction mixture is incubated for 12 hours at room temperature, and then the chromatograph on Ultrogal ASA 202 (LKB) H- (4-re-euphrenylcarbonyl) sarcosinyl immunoglobulin. In this case, the tagged protein is washed out to free low molecular weight resurifin. The degree of labeling is determined by measuring the extinction. It is 3, i.e. An IgG molecule is associated with three molecules of the resorufin derivative.
    PRI me R 15. Labeling of immunoglobulin G with N-oxysuccinimide ester of (4-resorufinylcarbonyl) piperidine-4-carboxylic acid.
    a.K1- (4-Resorufinylcarbonyl) piperidin-4-carboxylic acid.
    2.0 g of the acid chloride H, 0,0-triacetyldihydroresorufin-4-carboxylic acid described in example 1c, by analogy with example 1e, is brought into interaction with 0.9 g of methyl piperidine-4-carboxylic acid hydrochloride, the analogies with examples 1e and 1g are deacetylated and oxidized, after which the product is washed with sodium hydroxide solution to N- (4-resorufinylcarbonyl) -piperidine-4-carboxylic acid.
    Output 0.9 g
    Rf (silica gel RP-18, eluting agent: nitromethane - ethanol in the ratio 4: 1) 0.44.
    UV / VIS (0.1 M potassium phosphate buffer solution, pH 8.5): l / cs.576.2 nm.
    b.N-oxysuccinimide ester of N- (4-p e zor, ufinyl carbonyl) pip-yperidine-4-carboxylic acid.
    Out of 200 mg of No- (4-re-oorufinylcarbyl) piperidine-4-carboxylic acid, 240 mg (N-oxysuccinimide and 468 mg of dicyclohexidcarbodiimide) are obtained
    analogy with example 7d 190 mg of the product.
    Rf (silica gel RP-18, eluting agent: nitromethane - ethanol in the ratio 4: 1) 0.7.
    IR (sample pressed with potassium bromide): 3415 (ha, broad), 1814 (t), 1773 (t), 1734 (S), 1626 (t), 1214 (ha),
    at. Tagging of rabbit IgG with N-hydroxy succinimide ester of No.- (4-resorufinyl carbonyl) piperidine-4-carboxylic acid.
    10 mg of rabbit IgG in 1 ml of O, 1 M potassium phosphate buffer solution with a pH of 8.5 is mixed with 100 μl of a solution of 1.9 mg of N- oxysuccinimide ester I- (4-resorufinylcarbonyl) piperidine-4-carboxylic acid in 1 ml 1, 4-dioxane, after which the prepared mixture is incubated for 2 hours at room temperature. This corresponds to the molar ratio of the rubber-fin derivative and rabbit IgG 6.4: 1. 2
    After chromatography on ACA 202 (eluent: 0.1 M potassium phosphate buffer solution, pH 8.5), the protein fraction is obtained, which has an absorbance ratio A578 / A of $ 70 0.97, C, respectively
    3.4 mol of resorufin per 1 mol of IgG. If in a similar experience 10 mg IgG
    rabbits are mixed with 20 μl of an activated resorufin solution, then at
    1.05 mol of the indicated dye per 1 mol of IgG receive a load of 0.8.
    The absorption maximum of resorubin-labeled IgG is about 578 nm. The solution gives a strong fluorescence of light red.
    If the solution of resorufin-labeled IgG is exposed to daylight for a month, the fluorescence intensity decreases to 59% of the initial value, while the similarly obtained, but fluorescent-labeled IgG decreases the intensity to 16%, and the labeled Texas Rot IgG - up to 12%.
    Example 16. Determination of diphenylhydantoin in human serum using FNIA.
    1950 μl (0.1 M sodium phosphate buffer solution (pH 7.8) is mixed with 5 μl of sample (1), 25 μl of antibody solution (2) and 25 μl of diphenyl 5 solution
    - five
    about
    ABOUT
    five
    hydantoin-resorufin (3). The mixture was incubated for 5 min at 37 ° C, and then the fluorescence polarization was measured (the excitation wavelength was 575 nm, the radiated wavelength was 594 nm, and the measuring instrument was a 650-10 Hitachi fluorescence spectrometer).
    1. Test: donor human serum, supplemented with a known amount of diphenylhydantoin. To obtain a calibration curve, human donor serum was used, which contains diphenylhydantoin in concentrations, µg / ml: a-2.5; b-5; c-10; g - 20; d - 40.
    2. Antibody solution: 450 μg of antibody per 1 ml of 0.1 M sodium phosphate buffer solution (pH 7.8).
    Antibodies are produced in the usual way by immunizing sheep with diphenylhydantoin, which is associated with bovine serum albumin.
    The antiserum is purified by precipitation with ammonium sulfate and chromatography on DEAE-cellulose.
    3. A solution of diphenylhydantoinoreorofuin (10 M): a conjugate of diphenylhydantoin and resorufin from example 1 and in
    0.1 M sodium phosphate buffer solution (pH 7.8).
    The measurement results obtained with diphenylhydantoin solutions 1a, 16, 1b, 1d and 1d are shown in FIG. one.
    Using this calibration curve, the concentrations of diphenylhydantoin in samples with an unknown content of diphenylhydantoin can also be determined.
    Similar calibration curves can also be obtained in cases where the diphenylhydantoin conjugates from Examples 2.7.8 or 10e are used instead of the diphenylhydantoin conjugate from Example 1 used above.
    FIG. 2 — calibration curves obtained using the (1-diphenylhydantoinylmethylcarbonyl) conjugate resorufin-4-carboxylic acid conjugate (Example 2); in fig. 3 - the same, M- (4-resorufinylcarbonyl) sarcosinyl-1- (2-aminoethyl) -diphenylhydantoin conjugate (Example 7); in fig. 4 - the same, 2- (1-diphenylhydroan-ynyl) ethylamide conjugate, resorufin-4-carboxylic acid (Example 8); in fig. 5 - the same
    25162
    a conjugate of 2- (1-diphenylhydantoinyl) piperazidmethylcarbonyl. 9-hydroxy-5-benzoEaZ-phenoxazone-8-carboxylic acid piperazide (Example 10).
    Example 17, Determination of the activity of endoglycosidase resorufin-vysokomannoznym glycopeptide.
    A. Treatment of high-mannose glyco-peptide with Mg-oxysuccinimide ester of M- (4-resorufinylcarbonyl) -sarcosine.
    50 mg of a high mannose glycopeptide is mixed with 10 ml of a 0.1 M potassium phosphate buffer solution with a pH of 8.0. The solution is adjusted to pH 8.0. 25 mg of M- (4-resorufinylcarbonyl) sarcosine m-oxysuccinimide ester, dissolved in 3 ml of dioxane, are added to it, and after 1 hour, the same amount of N-oxysuccinimide dye ester in 3 ml of dioxane. The mixture is stirred for 14 hours at room temperature, then dioxane is distilled off in vacuo, after which the residue is diluted with water to 70 ml and then with buffer solution A (0.02 M tris-HCl, 2 mM magnesium chloride, 2 mM manganese chloride). (II), 2 mM calcium chloride, pH 7.2) to 140 ml. By adding an aqueous solution of ammonia, the pH was adjusted to 7.2. The precipitate thus formed is separated by centrifugation. The liquid above the precipitate is introduced into a column filled with Con A-Sepharose (1 x 15 cm). The free dye is washed out with buffer A. Once the flowing liquid loses its red color, the first fraction is washed out - re-orufin-high-mannose glycopeptide with 2% methylmannoside in buffer A as an eluent (about 100 ml). After that, the second fraction is eluted with a 2% aqueous solution of methyl mannose. Both fractions are dialysed against water and lyophilized. Both fractions are suitable for the determination of endoglycosidase activity as described below.
    b, Determination of endoglycosidase activity.
    Resorufin-high mannose glycopeptide is kept in a suitable buffer solution with endoglycosidase, for example, in citrate buffer solution with pH 5.5 with endoglycoeidase H (sample 1). In parallel, a sample is prepared which does not contain endoglycosidase (sample 2).
    eleven
    26
    .Q
    20 25, 30
    0

    40
    five
    After incubation, both samples are mixed with Con A-Sepharose, after which the samples are shaken in order to connect the resorufin-high mannose glycopeptide. Resorufin-labeled peptide, which, as a result of enzyme activity, a sugar fragment cleaved, does not bind. After 15 minutes, Con A-Sepharose is separated by centrifugation, the supernatant is adjusted to pH 7.5, after which the fluorescence (excitation, for example, 550 nm, emission 595 nm) is measured. The difference between sample 1 and the control value (sample 2) indicates the amount of cleaved resorufin-high mannose glycopeptide and is therefore a measure of the activity of the enzyme.
    Example 18. The compound digitoksinin-Z-hemisuccinate-Y-hydroxyisucimide ester with resorufin-4-piperazide of carboxylic acid.
    438 mg of reeorufin-4-piperazide carboxylic acid and 570 mg of digitoxigenin-3-hemisuccinate-N-hydroxysuccinimide ester are introduced into 160 ml of dioxane-water 1: 1 mixture. The solution was adjusted to pH 8.5 with a 5% aqueous solution of potassium carbonate and stirred for 3 hours at room temperature. The dioxane is evaporated, adjusted to pH 1 and the precipitated product is filtered off with suction.
    Yield 670 mg.
    Rf (silica gel-HPTLC, yield agent: butanol - glacial vinegar - water 4: 1: 1) 0.64.
    Rf resorufin-4-piperazide of kaboic acid 0.09.
    Example 19. Compound of resorubin-4-piperazide of carboxylic acid with digoxigenin monodigictoxoside- (3G-hemiglutarate) -N-hydroxysuccinimide ester.
    Analogously to example 18, out of 438 mg of resorufin-4-piperazide carboxylic acid and 731 mg of digoxigenin monodigitoxpoide- (3-hemglurate) -N-hydroxysuccinimide ester, 630 mg of raw material is obtained. After chromatography on silica gel RP 18 (eluent: isopropanol - water 2: 1), 34 mg of product are obtained.
    Rf (silica gel-HPTLC, yield agent: butanol - glacial vinegar - water 4: 1: 1) 0.61.
    Example 20. Compound of N- (4-resorufinylcarbonyl) piperidin-4-carboxylic acid-N -hydroxysuccinimide ester with 3-amnno-3-deoxydigoxigenogen.
    140 mg of 3 amnno-3-deoxydihydroxygeanin and 140 mg of N (4-resorufinylcarbonyl) ghyperidin-4-carboxylic acid-N-gndroxuccinimide ester from example 15 are mixed for 2 hours in a mixture of 80 ml of 0.1 M buffer solution potassium phosphate pH 8.5 and 120 ml of a mixture of dioxane-. methylformamide 1; 1. It is evaporated at room temperature to dryness and purified on RP 18 silica gel (eluent: gradient from 0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in isopropanol — water 65:35).
    30 mg of product is obtained.
    Rf (cp 18 RP, the means of those - of the range: isopropanol - water 1: 1) 0.60.
    Rf aminodigoxigenin 0.55. 20
    Rf resorufin-N-hydroxysuccinimide ester 0.71.
    Example 21. Preparation of N- (3- (resorufin-2-yl) tefopionyl) -tyroxyn methyl ester by combining resor-25 fnn-2-propionic acid with thyroxyn methyl ether,
    A. Hydrogenation of 7-hydroxycoumarin to dihydroxyphenylpropionic acid,
    6.5 g of 7-hydroxycoumarin -30 solution in 25 ml of 5 M sodium hydroxide solution, 100 ml of water and 20 ml of ethanol, and after adding 2 g of Pd-activated carbon, are hydrogenated at room temperature and normal pressure. Filter, acidify and centrifuge the solution.
    Yield 90%
    NG-NMR (nMCO-de, 2.4-2.9 (ha, 4H); 6.16-6.91 (m, 4H) ppm-1.40
    b.Resorufnk-2-propionic acid. 40 mmol dihydroxyphenylpropionic
    acids and 40 mmol of nitrosoreeorcin are dissolved in DMF, 3 g of manganese dioxide are added and 5 4 ml of sulfuric acid are added dropwise. The derivative of the resorphins is filtered, dissolved in ammonia and reduced with zinc. The product was purified by chromatography on silica gel (solvents: ethyl acetate - methanol 8: 1).
    Yield 25%.
    MS (product TM S 2, EDM + 429.
    v. Conjugate reaction.
    30 mg of reeorufin-2-propionic acid lots are converted with N-hydroxysuccinicide and morpholinoethylisocyanide into DMF B active ether. Then, 00 mg of roxin methyl ether and triethyl 50 are added.
    $
    0
    five
    0 s
    0
    five
    0
    amine. Upon completion, the reaction is acidified and purified through Sephadex 1 H 20 / LMF.
    Yield 20 mg.
    Example 22. Preparation of N- (3-sal-zorufin-2-yl-propionyl) -aminoethylhydantoin by conjugation of resorufin-2-propionic acid with aminoethylhydantoin.
    Similarly to the preceding conversion reaction, respectively, from 150 mg of adduct, 100 mg of the desired product are obtained.
     H-NMR (LMSO-a6), 0: 6.27-7.95 (m, 5H); 7.34 (S, YUN),.
    Example 23. Preparation of resorufin-4-carboxylic acid (digoxigeninsuccinoyl) piperazide by conjugation of resorufin-4-carboxylic acid-piperazide trifluoroacetate with digoxig .HHH-CyK4HHOHn-N-rHflpOKCHCYKUHHHKHKKHCHKHHHHKHKKHKHHHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHHKHKHHKHHKH
    1 mmol of the starting compounds are converted as in Example 1. 170 mg of the desired product is obtained.
    Rf (cshkagel, solvent: chloroform - methanol - ice on acetic acid 9: 1: 0,, 19.
    Example 24. Preparation of M- (reoorufin-4-carbonyl) aminodigoxigen-sarcosin by combining resorufin-4-carboxylic sarcosin-N-oxysuccinimide ester with aminodigoxigenogen.
    0.174 mmol of each substance is converted to the desired product under the condition of example 7e.
    Rf (silica gel, chloroform - methanol - ice on acetic acid 9: 1:: 0.1) 0.28.
    Example 25. Conjugation of 2-chloro-resorufin-4-carboxylic acid-piperazide-trifluoroacetate-with diphenylhydantoinoxacetic acid-N-hydroxysuccinimide.
    The transformation is carried out as in Example 1. From 270 mg of 2-chloro-resorufin-4-carboxylic acid-piperazide trifluoroacetate and 173 mg of diphenylhydroxy-vinyl acetic acid-N-hydroxysuccinimide ester, 20 mg of N- (diphenylhydroantoinylmethylcarbonyl) are obtained ) piposide-2-chlororezorufin-4-carboxylic acid.
    Rf (silica gel, solvent: chloroform - methanol - ice on acetic acid 9: 1: 0.1) 0,380
    Example 26. Preparation of resorufin-4-carbonyl-tetraiodiodothylacetyl-piperazide by combining tetraiodoacetic acid N-oxysuccinimide ester with piperazide-trifluoroacetate-4-carboxylic acid.
    a.Tetradiodothyroacetic acid - N-hydroxysuccinimide ester.
    In the usual manner (for example, as in Example 21), an active ester is obtained from an acid (500 mg) by transformation with N-hydrosuccinimide and 10 morpholinoethyl isocyanide.
    Yield 300 mg.
    Rf (silica gel, solvent: chloroform - methanol - ice on acetic acid 15 9: 1: 0,, 87.
    b. Conjugate reaction.
    0.18 mmol of the starting compounds are converted, as described in Example 1, into a dioxane buffer solution with a pH value of 8.5 and purified through preparative HPLC. ,
    Yield 20 mg. Q
    1 H-NMR (SD3OD), 0: 7.06, 7.11, 7.80, 7.85 (4S, 4H); 6.35-7.58 (m, 5H) .and
    Example 27. Conjugation of resorufin-4-carboxylic acid-piperazide trifluoroacetate with 1-methyl-3- (M-succinimidocarbonylpropyl) xanthine.
    The transformation is carried out in the well known example 1.
    From 194 mg of 1-methyl-3- (N-succinimido-carbonyl) propylxanthine and 245 mg of Resorufin-4-carboxylic acid-piperazide-trifluoroacetate, 110 mg of 35 (1-methyl-3-S-succinimidocarbonyl propyl ) xanthinyl piperaeid resorufin-4-carboxylic acid.
    Rf (silica gel, chloroform - methanol - ice on acetic acid 9: 1: s: 0.1) 0.43.
    Example 28. Determination of theophylline in human serum using FPIA.
    Experiments were carried out in the same manner as described in 45 in Example 16, but a solution of 7.5 µg / ml polyclonal antibody with respect to theophylline is used as an antibody solution (2), and a JQ solution 2.14 nmol is used as resorufin solution (3) / l (theophylline-7-propionic acid) piperazide reeorufin-4-carboxylic acid in accordance with Example 16, and as a sample - human donor serum with the following 55 mg of theophylline, µg / ml: a) 2.5 ; B) 5; c) 10; d) 20; e) 40.
    The result is a calibration curve shown in FIG. 6
    Using this curve, it is possible to determine the concentration of theophylline in the test samples.
    Example 29. Determination of thyroxin in human serum using FPIA.
    The experiments were carried out in the same way as described in example 16, but a solution of 376 kg / ml polyclonal antibody with respect to thyroxin is used as an antibody solution (2), a solution of 1.56 nmol / l is used as a solution of re-oruphine (3) (L -tyroxin) -piperazide resorufin-4-carboxylic acid, obtained from the compound in accordance with Example 3 after cleavage of the protective BOC group from it with trifluoroacetic acid, and as a sample - human donor serum with the following thyroxine additives, µg / 0.1 l: a) 3; (B) 6; c) 12; d) 18; e) 30.
    The result is a calibration curve shown in Fig.7. Using this curve, it is possible to determine the concentration of thyroxin in the test samples with an unknown concentration.
    Example 30. Determination of digoxin in human serum using FPIA.
    The experiments were carried out in the same way as described in Example 16, but a solution of 440 ng / ml polyclonal antibody with respect to digoxin is used as an antibody solution (2), a solution of 0.25 nmol / ml M- as a solution of resorufin (3) (4-resorufinyl-carbonyl) sarcosinyl-3-aminodigoxigenin (Example 24). and as samples, human donor serum with the following digoxin additives, ng / ml: a) 0.5; B) 1; c) 2; d) 3; e) -5.
    The result is a calibration curve depicted in Fig.8. Using this curve, one can determine the concentration of digoxin in the test samples with an unknown concentration.
    In a heterogeneous immunoassay, before determining the concentration of free or bound ligand, it is necessary to separate the ligand bound to the antibody and the free ligand1 by precipitating with suitable substances or by using an antibody bound to a solid carrier. In a homogeneous immunoassay, the formation of an antibody-ligand complex in a sample without such separation is investigated. The methods of homogeneous immunoassay can be ranked, for example, the fluorescent Qnenching method, the fluorescent Enhancement method and the fluorescent polarization method, in which the fluorescent substance is used as a label.
    Since the compounds corresponding to general formulas 1a or 16 have absorption and emission maxima that largely lie outside the range of biological material that interferes with the determination due to its own fluorescence, they are particularly suitable for use in fluorescence polarization immunoassay (FPIA). In addition, the proposed compounds with a suitable substitution have a particularly high Stokes concentration of approximately 70 nm.
    Basically, the FPIA method is based on the principle of conventional fluorescent immunoassay.
    If a suitable fluorescently labeled laggand is excited by flat polarized light before fluorescence, then as a result of the slight time delays between the excitation and emission, the molecule rotates before the radiation is emitted. Thus, the plane of plane-polarized light is equally rotated by a certain angle. The set of molecules within this short period of time can result in the rotational diffusion result in the known depolarization of fluorescent emission. In order to polarize emitter fluorescence, it is characteristic that it is the larger, the larger the molecule, and, therefore, the slower the rotation. This relationship can be used to measure the binding of a ligand to an antibody, since the free labeled ligand has a smaller molecule volume than the complex consisting of the labeled ligand bound to the antibody. Polarization is inversely proportional to the concentration of the detected ligand found in the sample.
    The concentration of a labeled ligand or ligand analog and the antibody required for such an immunological method is determined depending on
    c 0
    five
    about 0 5 0 5
    five
    applied measuring device, as well as on the characteristic properties of the applied labeled ligand or ligand analog and the antibody itself. This concentration also depends on the ligand to be determined and therefore must be established empirically. This determination can be made through simple optimization.
    The concentration of the ligand, which must be determined, in most cases fluctuates in the region of 10-mole. Preferred for measuring the concentration of ligand in the test sample is 10-10 mol, most preferred 10-10 / 0 mol. A higher concentration of ligand can be measured after diluting the original sample.
    The measurement is carried out at a specific pH value, which may be in the range of 3 to 12. Typically, the pH value lies in the range of 5 to 10, preferably 6 to 9. Various buffer solutions can be used to establish and maintain a certain pH value, for example borate, phosphate or carbonate or tris buffer. When using the proposed compounds, it is not critical which buffer solution is used. The choice is determined, first of all, depending on the antibody used, the ligand to be determined, as well as the fluorescent label used.
    The FPIA method is preferably carried out at a constant temperature, usually at 10-40 ° C.
    The exact relationship between polarization and concentration of the ligand or ligand analogue to be determined can be determined from a calibration curve. It is obtained by measuring the polarization values of solutions with different but known concentrations of the corresponding substance. The unknown concentration of ligand in the test sample can then be determined from such a calibration curve.
    Compounds according to general formulas 1a and 1b can be widely used as a fluorescent label.
    For example, in immunofluorescence microscopy as an antigen, proteins or whole cells can be made
    33
    visible with a fluorescently labeled antibody. In the corresponding methods, the distribution of the hapten or antigen in the cell can also be directly observed when the corresponding compound is introduced as a fluorescent label into the cell and then observed, for example, under a microscope. Moreover, in contrast to the homogeneous fluorescent immunoassay described above, the fluorescent properties of the resorufin derivatives do not change.
    A disadvantage of known dyes that are used as a fluorescent label, such as fluoresceins such as fluorescein isothiocyanate, or rhodamine dyes like Texas Rot, is that they fluoresce at relatively short wavelengths. In addition, the yield in the reaction of combination with an appropriate carrier is often low and the products formed as a result of the reaction have poor color stability.
    The proposed compounds, corresponding to the general formulas 1a and 16, fluoresce longwave with good color stability, and they can be obtained in good yield from the compound corresponding to the general formula Ha or 116, and the corresponding partner,
    used for the coupling reaction.
    i.
    The proposed compounds can be used for labeling substances with other fluorescent immunological methods. Thus, it is possible to label these reactive compounds with resorufin and the reagent of another complex-forming system. In this case, the complexing system refers to all those combinations of substances which, taking into account the specific strength of the exchange interaction, are capable of forming complexes. Known combinations are, for example, hormone / spec, receptor, biotin / avidin, carbohydrate derivative / lectin, etc. For example, a biotype-labeled protein can be determined using a combination product obtained from avidin and a reactive compound corresponding to the general formula of Ha or Hb. In addition, compounds that meet the general formulas 1a and 16 can be used in determining the reagent
    162
    Jq
    15 20 25
    thirty
    ze
    d "50 ,,
    181134
    system lectin - a derivative of carbohydrate.
    When screening cells in Fluorescent Activateo Cell Sorter, fluorescent latex particles are used. Such particles may be well fluorescently labeled, for example, by reacting latex particles containing hydroxyl groups, amino groups, or carboxyl or sulfonic acid groups, with reactive rheofurin compounds having the general formulas Ha or 116.
    Compounds of general formulas 1a and 16 may be used to determine enzymes. For this purpose, a resorufin derivative corresponding to the general formula of Na or Nb may be bound to a substrate that is capable of being cleaved by a detectable enzyme. This coupling product is also a compound that meets the general formula 1a or 16. After reacting a substrate labeled with resorufin, which did not enter into interaction, the activity of the enzyme can be determined. For example, a reactive derivative of re-oruphine corresponding to the general formula Na or Nb may be associated with a glycopeptide, and the resultant combination product may be used as a substrate to determine the activity of endoglycosidase. For the determination of endoglycosidase, labeling of dansyl compounds is known. Resorufin derivatives are more sensitive than such compounds.
    A disadvantage of the known compounds is that the wavelength of their fluorescent radiation is in the region where the radiation of the blood sample components lies, which leads to a distortion of the measurement results. This follows from curve 1 in FIG. 9.
    Obviously, at a wavelength of 525 nm, corresponding to the maximum fluorescence of a known compound, the sample itself emits fluorescent radiation, which has an interfering effect. The maximum radiation emitted by the proposed ipPeA1 compound is at least 590 nm. At this wavelength, the background fluorescence of a blood sample is significantly lower than at
    351621811
    525 nm (emission maximum of known compounds).
    Thus, using the compounds obtained by the proposed method, one can determine the content of ligands without interference caused by the components of the samples.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    The method of obtaining derivatives of resorphine of general formula 1a or 16,
    1 snd, with number 4
    about
    jKCH2) mC (OM., Lj
    de RI is H or C1 in position 2} RQ is H or C-Cdgalkyl; R3 is H, C-Cd-alkyl or C1; Cd - H or RJ and RA together form annelated phenyl ring; m 0 or 2; n - 1,
      - - CH2-C °°
    CH3
    Shi-W V-c-: II O
    L 0 СЬ ЗЬ, С (СНо) /). 2,
    ABOUT
    where p is 1.2 or 3,
    -NH -, - NHCH2CH2-,
    -SSNoSNoS-,
    Iiii
    oo
    one
    36
    -CCH (OH) CH (OH) C-,
    oo
    A is a radical derived from hydotoin, diphenylhydantoin, tert-butyloxycarbonyl-tec ythyroxine, phenobarbital, est-radiol, 7-theophylline, digoxigenin, digoxigenin monodigitoxoside, 3-amino-3-deoxytetraodes, 3-amino-3-deoxyterate, and 3-amino-digoxin, digoxigenin monodigitotoxoside, 3-amino-3-deoxytetohydrate, 3-amino-dihydrogen, digoxigenin monodigitotoxoside, 3-amino-3-deoxytetrahydrate, 3-amino-dihydrogen, digoxigenin monodigitoxoside, 3-amino-3-deoxytetraiodot, 3-amino-dihydrogen, digoxigenin, ether or xanthine, and the radical - (CHg -CL -Lg-A
    when it is in position 4 and when in position 2, and in the case of p 0.8 ... 6.4
    25
    H
    -NSN9S- w / I 2 I
    CH o
    ABOUT
    L (j is simple
    bond and A is a radical formed from immunoglobulin G,
    characterized in that the compound of the general formula Na or 116
    R,
    35
    I
    (
    about
    CH2)
    he is 4
    50 where Rj is R, m and L (have the indicated meanings;
    ABOUT
    X4 - H, C1 or - O -
    subjected to interaction with the compound of General formula III
    de L and A have
    X - H or
    37t621811
    XftLflA, indicated values;
    OK
    ABOUT
    Have
    pry
    250
    i
    I
    §150100
    :
    4t
    Yu 15 20 Z5 30 35 Concentration of diphenylshdantoin 6 sample
    1
    38
    moreover, the active radicals of the compounds (On or 116 and III can have protecting groups that are removed if necessary, with the release of the target product in free form.
    J I I I I I H, I I ..... ,,, |
    5 a a x l
    Youth center IifenagiEatyuina FIG. 2
    50
    ABOUT U / OJAYAJAJ Konisnpratsi Nifenididon yuly 8 sample CE 3
    50
    J5 20 25 JO I Concentration DFG b sample FIG A
    iff
    yu ago
    Concentrators DFG and araOe Figure 9
    9 V IS X 25 30 K "O Koi & the strength of theofaplin
    Fie.6
    100-1
     11 i-l I I I 11 I i I I I I I I 11 I 11 I 11 I
     -g - g
    Standard buffer
    Fie.7
    t g
    Koiemtratsi Lnvpina Al
    650 6 5600SSO $ 2S / n500
    9
    Compiled by N.Naryshkova
    Proofreader M. Demchik
    Editor L. Veselovska Tehred M. Didyk
    Order 4257 Circulation: Subscription
    VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5
    Proofreader M. Demchik
    G 1
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    同族专利:
    公开号 | 公开日
    ES2002110A6|1988-07-16|
    CS273617B2|1991-03-12|
    JPS62174066A|1987-07-30|
    CS549286A2|1990-08-14|
    JPS6236368A|1987-02-17|
    EP0209875B1|1990-08-29|
    CA1338320C|1996-05-07|
    KR870001184A|1987-03-12|
    US4954630A|1990-09-04|
    US5304645A|1994-04-19|
    JPS62174067A|1987-07-30|
    EP0209875A1|1987-01-28|
    AT55998T|1990-09-15|
    YU45348B|1992-05-28|
    DE3526565A1|1987-02-05|
    DE3673715D1|1990-10-04|
    YU130286A|1987-12-31|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DE19853526565|DE3526565A1|1985-07-25|1985-07-25|RESORUFIN DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN FLUORESCENT IMMUNOASSAYS|
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