Compound based on cyanine scaffold for diagnosis sepsis by selectively detect glutathione
09951226 · 2018-04-24
Assignee
Inventors
Cpc classification
C09B23/0066
CHEMISTRY; METALLURGY
C07D209/14
CHEMISTRY; METALLURGY
International classification
C07D209/14
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a compound based on cyanine scaffold for diagnosing sepsis by selectively detecting glutathione. The compound based on cyanine scaffold according to the present invention has the advantages of maintaining its structure in intracellular environment and of reacting selectively to glutathione only among many amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione, to produce changes in absorption or fluorescence spectrum, making the compound useful for the detection of in vivo glutathione in biosamples and also for the diagnosis of sepsis characteristically displaying the changes of glutathione concentration.
Claims
1. A compound represented by the below formula 1: ##STR00021## wherein, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently H, OH, halogen, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy; ##STR00022## R.sup.7, R.sup.8, and R.sup.9 are independently H, OH, CN, NO.sub.2, halogen, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy; R.sup.10 and R.sup.11 are independently H, OH, CN, NO.sub.2, halogen, C.sub.1-10 straight or branched alkyl, C.sub.1-10 straight or branched alkoxy, or NR.sup.12R.sup.13 wherein R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl; or R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, wherein the substituted 6-atom heterocycloalkyl is one wherein one or more substituents selected from the group consisting of OH, CN, NO.sub.2, halogen, O, C.sub.1-10 straight or branched alkyl, and C.sub.1-10 straight or branched alkoxy are substituted.
2. The compound according to claim 1, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently H, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy; ##STR00023## R.sup.7, R.sup.8, and R.sup.9 are independently H, NO.sub.2, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy; R.sup.10 and R.sup.11 are independently H, NO.sub.2, C.sub.1-10 straight or branched alkyl, C.sub.1-10 straight or branched alkoxy, or NR.sup.12R.sup.13, wherein R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl; or R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S, wherein the substituted 6-atom heterocycloalkyl is the one wherein one or more substituents selected from the group consisting of NO.sub.2, halogen, O, C.sub.1-10 straight or branched alkyl, and C.sub.1-10 straight or branched alkoxy are substituted.
3. The compound according to claim 1, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently C.sub.1-10 straight or branched alkyl; ##STR00024## R.sup.7, R.sup.8, and R.sup.9 are independently H or NO.sub.2; R.sup.10 and R.sup.11 are independently H or NR.sup.12R.sup.13 wherein R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl; or R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more Ns, wherein the substituted 6-atom heterocycloalkyl is the one wherein one or more substituents selected from the group consisting of O and C.sub.1-10 straight or branched alkyl are substituted.
4. A method for preparing a compound represented by formula 1 comprising the following steps as shown in the below reaction formula 1: preparing a compound represented by formula 3 by replacing the halogen (X) of a compound represented by formula 2 with piperazine (step 1); and preparing a compound represented by formula 1 by reacting the compound represented by formula 3 prepared in step 1) with a compound represented by formula 4 (step 2), ##STR00025## wherein, X is halogen; ##STR00026## R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in formula 1 of claim 1.
5. A method for detecting an amino acid containing thiol group comprising contacting the compound of claim 1 with a biosample.
6. The method for detecting an amino acid containing thiol group according to claim 5, wherein the amino acid containing thiol group is one or more amino acids selected from the group consisting of cysteine, homocysteine, and glutathione.
7. The method for detecting an amino acid containing thiol group according to claim 6, wherein the amino acid containing thiol group is glutathione.
8. A method for diagnosing a bacterial disease comprising contacting the compound of claim 1 with a biosample.
9. The method for diagnosing bacterial disease according to claim 8, wherein the bacterial disease is sepsis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
(52) Hereinafter, the present invention is described in detail.
(53) The present invention provides a compound represented by the below formula 1.
(54) ##STR00005##
(55) In the formula 1,
(56) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently H, OH, halogen, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy;
(57) ##STR00006##
(58) R.sup.7, R.sup.8, and R.sup.9 are independently H, OH, CN, NO.sub.2, halogen, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy;
(59) R.sup.10 and R.sup.11 are independently H, OH, CN, NO.sub.2, halogen, C.sub.1-10 straight or branched alkyl, C.sub.1-10 straight or branched alkoxy, or NR.sup.12R.sup.13,
(60) R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl;
(61) R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S;
(62) And, the said substituted 6-atom heterocycloalkyl is the one wherein one or more substituents selected from the group consisting of OH, CN, NO.sub.2, halogen, O, C.sub.1-10 straight or branched alkyl, and C.sub.1-10 straight or branched alkoxy are substituted.
(63) Preferably,
(64) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently H, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy;
(65) ##STR00007##
(66) R.sup.7, R.sup.8, and R.sup.9 are independently H, NO.sub.2, C.sub.1-10 straight or branched alkyl, or C.sub.1-10 straight or branched alkoxy;
(67) R.sup.10 and R.sup.11 are independently H, NO.sub.2, C.sub.1-10 straight or branched alkyl, C.sub.1-10 straight or branched alkoxy, or NR.sup.12R.sup.13,
(68) R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl;
(69) R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O, and S;
(70) And, the said substituted 6-atom heterocycloalkyl is the one wherein one or more substituents selected from the group consisting of NO.sub.2, halogen, O, C.sub.1-10 straight or branched alkyl, and C.sub.1-10 straight or branched alkoxy are substituted.
(71) More preferably,
(72) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently C.sub.1-10 straight or branched alkyl;
(73) ##STR00008##
(74) R.sup.7, R.sup.8, and R.sup.9 are independently H or NO.sub.2;
(75) R.sup.10 and R.sup.11 are independently H or NR.sup.12R.sup.13,
(76) R.sup.12 and R.sup.13 are independently C.sub.1-5 straight or branched alkyl;
(77) R.sup.10 and R.sup.11 can be linked with neighboring carbon atoms and fused with two phenyls, and they can also form non-substituted or substituted 6-atom heterocycloalkyl containing one or more Ns;
(78) And, the said substituted 6-atom heterocycloalkyl is the one wherein one or more substituents selected from the group consisting of O and C.sub.1-10 straight or branched alkyl are substituted.
(79) The present invention also provides a method for preparing the compound represented by formula 1 comprising the following steps as shown in the below reaction formula 1:
(80) preparing the compound represented by formula 3 by replacing the halogen (X) of the compound represented by formula 2 with piperazine (step 1); and
(81) preparing the compound represented by formula 1 by reacting the compound represented by formula 3 prepared in step 1) with the compound represented by formula 4 (step 2).
(82) ##STR00009##
(83) In the reaction formula 1,
(84) X is halogen;
(85) ##STR00010##
(86) R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are as defined in formula 1.
(87) The method for preparing the compound represented by formula 1 of the invention is described in more detail hereinafter.
(88) In the method for preparing the compound represented by formula 1 according to the present invention, step 1) is to prepare the compound represented by formula 3 by replacing the halogen (X) of the compound represented by formula 2 with piperazine. More precisely, the compound represented by formula 2 was dissolved in a solvent, to which piperazine was added, followed by stirring. Column chromatography was performed, through which the purified compound represented by formula 3 was prepared.
(89) At this time, the solvent was preferably selected from the group consisting of tetrahydrofuran; dioxane; ether solvents including ethylether and 1,2-dimethoxyethane; lower alcohols including methanol, ethanol, propanol and butanol; dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonigensulfonate, toluenesulfonate, chlorobenzenesulfonate, xylensulfonate, phenylacetate, phenylpropionate, phenylbutylate, citrate, lactate, -hydroxybutylate, glycolate, malate, tartlate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate, and dimethylformamide was more preferred as the solvent.
(90) The reaction temperature was preferably set in the range of 0 C.boiling point of the selected solvent, and the reaction time was not limited, but 0.510 hours were preferred.
(91) In the method for preparing the compound represented by formula 1 according to the present invention, step 2) is to prepare the compound represented by formula 1 by reacting the compound represented by formula 3 prepared in step 1) with the compound represented by formula 4. More precisely, a base was added to the compound represented by formula that was dissolved in a solvent, followed by stirring. The compound represented by formula 4 which was dissolved in a solvent was added to the above drop by drop, followed by stirring. Silica gel chromatography was performed to obtain the purified compound represented by formula 3.
(92) At this time, the solvent was preferably selected from the group consisting of tetrahydrofuran; dioxane; ether solvents including ethylether and 1,2-dimethoxyethane; lower alcohols including methanol, ethanol, propanol and butanol; dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonigensulfonate, toluenesulfonate, chlorobenzenesulfonate, xylensulfonate, phenylacetate, phenylpropionate, phenylbutylate, citrate, lactate, -hydroxybutylate, glycolate, malate, tartlate, methanesulfonate, prpanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate, and dichloromethane was more preferred as the solvent.
(93) The base herein was preferably selected from the group consisting of triethylamine (TEA), potassium hydroxide (KOH), sodium hydroxide (NaOH), lithium hydroxide (LiOH), and cesium carbonate (Cs.sub.2CO.sub.3), and triethylamine (TEA) was more preferred as the base.
(94) The reaction temperature was preferably set in the range of 0 C.boiling point of the selected solvent, and the reaction time was not limited, but 0.510 hours were preferred.
(95) The present invention also provides a chemical sensor for detecting the amino acid containing thiol group that comprises the compound represented by formula 1. Herein, the amino acid containing thiol group is selected from the group consisting of cysteine, homocysteine, and glutathione, and particularly glutathione is preferred.
(96) The present invention also provides a method for detecting the amino acid containing thiol group using the said chemical sensor. Herein, the amino acid containing thiol group is preferably selected from the group consisting of cysteine, homocysteine, and glutathione, and particularly glutathione is more preferred.
(97) The mechanism of the detection above is as follows: When the compound represented by formula 1 reacts to glutathione, the sulfonyl-phenyl derivative substituted in the compound represented by formula 1 is fallen apart, resulting in the cyanine derivative compound having free piperazine. Among the sulfonyl-phenyl derivatives fallen apart therefrom, the phenyl derivative is conjugated with thiol group in glutathione, during which absorption or fluorescence is changed. So, the changes of absorption or fluorescence alone or together can be screened, leading to the detection of the amino acid containing thiol group.
(98) The present invention also provides a composition for diagnosing bacterial disease comprising the compound represented by formula 1. Herein, the bacterial disease is sepsis.
(99) In addition, the present invention provides a method for diagnosing bacterial disease using the said composition. Herein, the bacterial disease is sepsis.
(100) In general, once caught sepsis, neutrophils, a kind of leucocytes, produce excessive amount of ROS (Reactive Oxygen Species) because of oxidative stress. This excessive ROS causes disorders in neuron, skin, and digestive system, etc. Glutathione that is an antioxidant capable of regulating oxidation/reduction potential reacts to the excessive ROS generated by sepsis, so that it can be converted to reversible glutathione disulfide (GSSG), that is an oxidized glutathione. Thus, the in vivo glutathione is down-regulated accordingly, by which sepsis can be diagnosed.
(101) To investigate the changes of absorption or fluorescence, which is the principle of glutathione detection for the chemical sensor, and the usability thereof for the diagnosis of sepsis, the following experiments were performed.
(102) First, the following experiment was performed to investigate the usability of the compound prepared in Example 1 for the detection of cysteine, homocysteine, and glutathione, among many biothiols which are the amino acids containing thiol group (RSH). As a result, the compound prepared in Example 1 reacted to such amino acids containing thiol group (RSH) as cysteine, homocysteine, and glutathione in biosamples, to cause changes in absorption and fluorescence spectrum (see
(103) In the meantime, the following experiment was also performed to investigate the usability of the compound prepared in Example 1 for the detection of intracellular biothiols which are the amino acids containing thiol group (RSH), for example for the detection of cysteine, homocysteine, and glutathione. As a result, when it was introduced in HeLa cells, the compound prepared in Example 1 reacted to such amino acids containing thiol group (RSH) as cysteine, homocysteine, and glutathione, which exist in cytoplasm, so that a strong red fluorescence was observed under confocal laser scanning microscope (see
(104) To evaluate the glutathione selectivity of the compound prepared in Example 2, the following experiment was performed. As a result, the compound prepared in Example 2 selectively reacted to glutathione alone among many amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione, with showing glutathione specific fluorescence spectrum (see
(105) To investigate the changes of absorption or fluorescence spectrum of the compound prepared in Example 2 according to the concentration of glutathione (GSH), the following experiment was performed. As a result, the reaction of the compound prepared in Example 2 depended on the concentration of glutathione (see
(106) Further, the reaction between glutathione (GSH) and the compound prepared in Example 2 was induced. And then the changes of absorption or fluorescence spectrum over the time were investigated by the following experiment. As a result, the compound prepared in Example 2 reacted to glutathione to change absorption or fluorescence spectrum fast (see
(107) To understand the reaction mechanism between glutathione and the compound prepared in Example 2, matrix assisted laser desorption/ionization time-off-flight mass spectrometry was performed, in which glutathione and the compound prepared in Example 2 were reacted. As a result, the changes of absorption or fluorescence spectrum according to the reaction between glutathione and the compound based on cyanine scaffold of the present invention were confirmed to be attributed to the reaction presented in
(108) To evaluate the selection specificity of the compound prepared in Example 2 to cysteine, homocysteine, and glutathione, among many biothiols that are the intracellular amino acids containing thiol group (RSH), the following experiment was performed. As a result, when the compound prepared in Example 2 was introduced in HeLa cells, it selectively reacted to glutathione alone among many intracellular amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione, by which a strong red fluorescence was observed under confocal laser scanning microscope (see
(109) It was further investigated whether glutathione could be down-or up-regulated by an oxidant. To do so, HeLa cells containing the antioxidant glutathione that had been prepared in Experimental Example 2 were treated with 100 M of H.sub.2O.sub.2, the oxidant, at 37 C. for minutes to induce the changes of glutathione concentration. 20 M of the compound prepared in Example 2 was also added thereto, followed by culture at 37 C. for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) three times to eliminate the remaining compound, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 653 nm and the band-path (BP) of 655755 nm was used. The concentration of the intracellular glutathione could be measured by the strength of the red fluorescence emitted from the compound prepared in Example 2. Also, since the compound prepared in Example 2 did not reacted to the oxidized glutathione disulfide (GSSG), it could be concluded that the compound had excellent selectivity to glutathione (see
(110) To observe the changes of glutathione level in the presence of an oxidant, RAW 264.7 cells (macrophage cell line, Korea Cell Line Bank) were cultured in RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in a 37 C. 5% CO.sub.2 incubator. The RAW 264.7 cells were treated with lipopolysaccharide (LPS) for 20 minutes at the concentration of 1 g/mL, and with interferon- for 16 hours at the concentration of 50 ng/mL, and with phorbol 12-myristate 13-acetate (PMA) for 20 minutes at the concentration of 20 M to induce the generation of endogenous feroxynitrite having the characteristics of an oxidant. Then, the cells were seeded in 35-mm glass bottomed dishes at the density of 310.sup.5 cells/dish in 1640 medium. 24 hours later, the compound prepared in Example 2 was treated thereto at the concentration of 20 M for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) three times, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The concentration of the intracellular glutathione could be measured by the strength of the red fluorescence emitted from the compound prepared in Example 2. Also, since the compound prepared in Example 2 did not reacted to the oxidized glutathione disulfide (GSSG), it could be concluded that the compound had excellent selectivity to glutathione (see
(111) To evaluate whether or not the compound prepared in Example 2 could be used for the detection of in vivo glutathione in the mouse model, the following experiment was performed. As a result, the compound prepared in Example 2 was confirmed to be efficiently used for the observation of the in vivo distribution of glutathione with detecting the strength of red fluorescence on In Vivo Imaging System (IVIS) spectrum (see
(112) The following experiment was also performed to investigate whether or not the compound prepared in Example 2 could be used for the diagnosis of sepsis. As a result, it was confirmed that the compound prepared in Example 2 could be efficiently used for the diagnosis of sepsis by measuring the concentration of glutathione (see
(113) Further, the following experiment was performed to analyze the cause of the selectivity of the compound prepared in Example 2. As a result, the selectivity of the compound prepared in Example 2 was attributed to the shape of piperazine in the active site where the reaction between the compound and glutathione happened, the shape of dansyl group that was binding to piperazine, SN binding length, and Band-gap energy (see
(114) The following experiment was performed to evaluate the glutathione (GSH) specific selectivity of the compound prepared in Example 3. As a result, the compound prepared in Example 3 reacted to glutathione alone, among many endogenous amino acids in biosamples, with displaying the changes of absorption or fluorescence spectrum, so that the compound was confirmed to be efficiently used for the detection of glutathione in biosamples (see
(115) Further, the following experiment was performed to investigate whether or not the absorption or fluorescence spectrum produced by the compound prepared in Example 3 could be affected by the concentration of glutathione. As a result, the compound prepared in Example 3 reacted only with glutathione dose-dependently (see
(116) Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
(117) However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Preparative Example 1: Preparation of 2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-propyl-2H-indole-2-ylidene)ethylidene]-1-cyclohexene-1-yl]ethenyl]-3,3-dimethyl-1-propylindolium iodide (IR-780 iodide)
(118) ##STR00011##
(119) IR-780 iodide was purchased from Sigma-Aldrich Co.
Example 1: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolium-2-ylidene)ethylidene)-2-(4-(2,4-dinitrophenylsulfonyl)piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
Step 1: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(120) ##STR00012##
(121) 69 mg of piperazine was added to the compound prepared in Preparative Example 1 dissolved in 10 mL of anhydrous DMF (dimethylformamide) in argon environment. The reaction mixture was stirred at 85 C. for 4 hours, followed by cooling at room temperature. The solvent was eliminated under reduced pressure. The mixture was purified by silica gel chromatography using dichloromethane:methanol (40:1) as a moving phase. As a result, 136 mg of the target compound was obtained as a blue solid (yield: 85%).
(122) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.66 (d, J=15.0 Hz, 2H), 7.26-7.32 (m, 4H), 7.09 (t, J=6.0 Hz, 2H), 6.97 (d, J=6.0 Hz, 2H), 5.80 (d, J=15.0 Hz, 2H), 3.88 (m, 8H), 3.26 (t, J=6.0 Hz, 4H), 2.44 (t, J=6.0 Hz, 4H), 1.84 (m, 6H), 1.68 (s, 12H), 1.02 (t, J=6.0 Hz, 6H).
(123) .sup.13C NMR (75 MHz, CDCl.sub.3) 173.3, 169.1, 142.8, 141.0, 140.3, 128.3, 123.7, 123.5, 122.2, 109.4, 85.9, 55.3, 48.2, 47.2, 45.2, 29.2, 25.0, 21.8, 20.3, 11.8. ESI MS m/z=589.6 [MI.sup.].sup.+; calcd exact mass 716.3.
Step 2: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(4-(2,4-dinitrophenylsulfonyl)piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(124) ##STR00013##
(125) 30 mg of Et.sub.3N was added to the compound prepared in step 1) dissolved in 10 mL of anhydrous dichloromethane in argon environment. After stirring the reaction mixture for 5 minutes, 53 mg of 2,4-dinitobenzene-1-sulfonyl chloride dissolved in 5 mL of dichloromethane was added to the mixture drop by drop at 0 C. After stirring the mixture at room temperature for 4 hours, the solvent was eliminated from the mixture under reduced pressure. The mixture was purified by silica gel chromatography using dichloromethane:methanol (30:1) as a moving phase. As a result, 167 mg of the target compound was obtained as a blue solid (yield: 75%).
(126) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.77 (m, 2H), 8.52 (s, 1H), 7.80 (d, J=15.0 Hz, 2H), 7.35 (m, 4H), 7.21 (m, 2H), 7.06 (d, J=9.0 Hz, 2H), 5.95 (d, J=15.0 Hz, 2H), 3.96 (t, J=6.0 Hz, 4H), 3.76 (d, J=6.0 Hz, 4H), 3.69 (d, J=6.0 Hz, 4H), 2.49 (t, J=6.0 Hz, 4H), 1.86 (m, 6H), 1.66 (s, 12H), 1.05 (t, J=6.0 Hz, 6H).
(127) .sup.13C NMR (75 MHz, CDCl.sub.3) 170.4, 169.9, 149.8, 147.9, 142.5, 141.9, 140.5, 136.8, 134.9, 128.6, 127.8, 126.3, 124.5, 122.2, 119.4, 110.0, 98.2, 54.0, 48.6, 47.8, 45.6, 28.9, 25.3, 21.5, 20.6, 11.7. MALDI-TOF MS m/z=820.5 [MI.sup.].sup.+; calcd exact mass 946.3.
Example 2: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(4-(5-(dimethylamino)naphthalene-1-ylsulfonyl)piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
Step 1: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(128) ##STR00014##
(129) The target compound was obtained by the same manner as described in step 1) of Example 1.
Step 2: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(4-(5-(dimethylamino)naphthalene-1-ylsulfonyl)piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(130) ##STR00015##
(131) 30 mg of Et.sub.3N was added to the compound prepared in step 1) dissolved in 10 mL of anhydrous dichloromethane in argon environment. After stirring the reaction mixture for 5 minutes, 53 mg of 5-(dimethylamino)naphthalene-1-sulfonyl chloride dissolved in 5 mL of dichloromethane was added to the mixture drop by drop at 0 C. After stirring the mixture at room temperature for 4 hours, the solvent was eliminated from the mixture under reduced pressure. The mixture was purified by silica gel chromatography using dichloromethane:methanol (30:1) as a moving phase. As a result, 167 mg of the target compound was obtained as a blue solid (yield: 82%).
(132) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.76 (d, J=9.0 Hz, 1H), 8.60 (d, J=9.0 Hz, 1H), 8.32 (d, J=9.0 Hz, 1H), 7.59-7.70 (m, 5H), 7.36 (m, 2H), 7.18-7.28 (m, 4H), 7.08 (d, J=9.0 Hz, 2H), 5.95 (d, J=15.0 Hz, 2H), 3.99 (t, J=6.0 Hz, 4H), 3.55 (d, J=6.0 Hz, 4H), 3.41 (d, J=6.0 Hz, 4H), 2.96 (s, 6H), 2.47 (t, J=6.0 Hz, 4H), 1.83 (m, 6H), 1.29 (s, 12H), 1.02 (t, J=6.0 Hz, 6H).
(133) .sup.13C NMR (75 MHz, CDCl.sub.3) 170.4, 168.9, 152.0, 142.5, 141.7, 140.5, 132.0, 130.9, 130.1, 128.7, 128.6, 127.2, 124.4, 123.6, 122.0, 110.3, 98.8, 53.3, 48.3, 47.6, 45.8, 45.6, 28.2, 25.4, 21.5, 20.6, 11.7. MALDI-TOF MS m/z=822.7 [MI.sup.].sup.+; calcd exact mass 949.4.
Example 3: Preparation of 2-((E)-2-((E)-2-(4-((2-butyl-1,3-dioxo-2,3-dihydro-1H-benzeneisoquinoline-6-yl)sulfonyl)piperazine-1-yl)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
Step 1: Preparation of 2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)-2-(piperazine-1-yl)cyclohex-1-enyl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(134) ##STR00016##
(135) The target compound was obtained by the same manner as described in step 1) of Example 1.
Step 2: Preparation of 2-((E)-2-((E)-2-(4-((2-butyl-1,3-dioxo-2,3-dihydro-1H-benzeneisoquinoline-6-yl)sulfonyl)piperazine-1-yl)-3-((E)-2-(3,3-dimethyl-1-propylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)binyl)-3,3-dimethyl-1-propyl-3H-indolium iodide
(136) ##STR00017##
(137) 30 mg of Et.sub.3N was added to the compound prepared in step 1) dissolved in 10 mL of anhydrous dichloromethane in argon environment. After stirring the reaction mixture for 5 minutes, 53 mg of 2-butyl-1,3-dioxo-2,3-dihydro-1H-benzoisoquinoline-6-sulfonyl chloride dissolved in 5 mL of dichloromethane was added to the mixture drop by drop at 0 C. After stirring the mixture at room temperature for 4 hours, the solvent was eliminated from the mixture under reduced pressure. The mixture was purified by silica gel chromatography using dichloromethane:methanol (30:1) as a moving phase. As a result, 41.5 mg of the target compound was obtained as a blue solid (yield: 27%).
(138) .sup.1H NMR (400 MHz, CDCl.sub.3) (ppm): 9.30 (d, J=8 Hz, 1H, ArH), 8.82 (t, J=8 Hz, 2H, ArH), 8.55 (d, J=8 Hz, 1H, ArH), 8.12 (t, J=8 Hz, 1H, ArH), 7.63 (t, J=12 Hz, 2H, ArH), 7.37-7.34 (m, 2H, ArH), 7.21 (d, J=4 Hz, 4H, ArH), 7.07 (d, J=8 Hz, 2H, CH.sub.2CH.sub.2), 5.95 (d, J=16 Hz, 2H, CH.sub.2CH.sub.2), 4.25 (t, J=8 Hz, 2H, CH.sub.2), 4.0 (t, J=8 Hz, 4H, CH.sub.2), 3.60 (t, J=8 Hz, 4H, CH.sub.2), 3.46 (t, J=8 Hz, 4H, CH.sub.2), 2.49 (t, J=8 Hz, 4H, CH.sub.2), 1.85-1.80 (m, 6H, CH.sub.2), 1.75 (t, J=8 Hz, 2H, CH.sub.2), 1.47 (t, J=8 Hz, 2H, CH.sub.2), 1.36 (s, 12H, CH.sub.3), 1.04 (t, J=8 Hz, 6H, CH.sub.3), 0.97 (t, J=8 Hz, 3H, CH.sub.3).
(139) .sup.13C NMR (75 MHz, CDCl.sub.3) (100 MHz, CDCl.sub.3): (ppm): 170.42, 169.03, 163.50, 162.92, 142.70, 141.79, 140.52, 129.87, 129.33, 128.89, 127.50, 127.40, 12 4.74, 122.11, 110.49, 99.12, 53.53, 48.51, 47.79, 46.03, 40.88, 30.36, 28.70, 25.64, 20.81, 20.54, 11.92. ESIm/z=1031.78. [M]; calculated exact mass=1031.39. ESI m/z=1031.78 [M]; calculated exact mass=1031.39.
(140) Chemical structures of the compounds prepared in Example 1Example 3 are presented in Table 1.
(141) TABLE-US-00001 TABLE 1 Example Chemical Structure 1
Experimental Example 1: Evaluation of the Detection of Biothiol
(142) The following experiment was performed to investigate the capability of the compound prepared in Example 1 of detecting cysteine, homocysteine, and glutathione, among many biothiols which are the amino acids containing thiol group (RSH).
(143) <1-1> Observation of Absorption Spectrum
(144) 10 M of the compound prepared in Example 1 and 100 M of each endogenous amino acid (glutathione, cysteine, homocysteine, dithiothreitol, phenylalanine, histidine, glutamine, lysine, glutamate, glycine, serine, alanine, arginine, methionine, tyrosine) were added to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% dimethylsulfoxide (DMSO), followed by observation of absorption spectrum presented through UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) at 25 C. The results are shown in
(145)
(146) As shown in
(147) <1-2> Observation of Fluorescence Spectrum
(148) The following experiment was performed by the same manner as described in Experimental Example <1-1> except that fluorescence spectrum was observed by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell) instead of observing absorption spectrum via UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) (.sub.ex=730 nm, .sub.em=736 nm, slit:10/10 nm). The results are shown in
(149)
(150) As shown in
(151) Therefore, it was confirmed that the compound prepared in Example 1 reacted to such amino acids containing thiol group (RSH) as cysteine, homocysteine, and glutathione in biosamples, to cause changes in absorption and fluorescence spectrum, so that it can be efficiently used for the detection of such amino acids containing thiol group (RSH) in biosamples.
Experimental Example 2: Evaluation of the Detection of Intracellular Biothiol 1
(152) The following experiment was performed to evaluate the selective detection capacity of the compound prepared in Example 1 for cysteine, homocysteine, and glutathione, among many biothiols that are the amino acids containing thiol group (RSH).
(153) HeLa cells (human adenocarcinoma cells, Korea Cell Line Bank) were cultured in RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in a 37 C. 5% CO.sub.2 incubator. The cells were either treated or not treated with 1 mM of N-methylmaleimide (NMM) known as a thiol blocker for 20 minutes. 100 M of each amino acid containing thiol group (RSH) such as cysteine, homocysteine, and glutathione was added thereto. Then, the cells were seeded in 35-mm glass bottomed dishes at the density of 310.sup.5 cells/dish in 1640 medium. 24 hours later, 10 M of the compound prepared in Example 1 was added thereto, followed by culture at 37 C. for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) twice to eliminate the remaining compound, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The results are shown in
(154)
(155)
(156)
(157)
(158)
(159)
(160) As shown in
(161) As shown in
(162) Therefore, the compound prepared in Example 1 was confirmed to be able to react to those amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione, in the cytoplasm of HeLa cells, so that it could be effectively used for the detection of the intracellular amino acids containing thiol group (RSH).
Experimental Example 3: Evaluation of the Glutathione Selectivity 1
(163) The following experiment was performed to evaluate the glutathione selectivity of the compound prepared in Example 2.
(164) 10 M of the compound prepared in Example 2 was treated to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% DMSO and 100 M of each glutathione, cysteine, homocysteine, dithiothreitol, phenylalanine, histidine, glutamine, lysine, glutamate, glycine, serine, alanine, arginine, methionine, and tyrosine, followed by observation of fluorescence spectrum at 25 C. by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell). The results are shown in
(165)
(166)
(167) As shown in
(168) As shown in
(169) Therefore, the compound prepared in Example 2 could be efficiently used for the detection of glutathione in biosamples because the compound could selectively react to glutathione only among other amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione.
Experimental Example 4: Observation of Absorption and Fluorescence Spectrum According to the Concentration of Glutathione 1
(170) The following experiment was performed to observe absorption and fluorescence produced by the compound prepared in Example 2 according to the concentration of glutathione (GSH).
(171) <4-1> Observation of Absorption Spectrum
(172) 10 M of the compound prepared in Example 2 and glutathione were added to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% dimethylsulfoxide (DMSO) with raising slowly the concentration of glutathione from 0 to 50 M, during which the changes of absorption spectrum were observed using UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) at 25 C. The results are shown in
(173)
(174) As shown in
(175) <4-2> Observation of Fluorescence Spectrum
(176) The following experiment was performed by the same manner as described in Experimental Example <4-1> except that fluorescence spectrum was observed by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell) instead of observing absorption spectrum via UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) (.sub.ex=730 nm, .sub.em=736 nm, slit:10/10 nm). The results are shown in
(177)
(178) As shown in
(179) Therefore, it was confirmed that the reaction of the compound prepared in Example 2 was glutathione dose-dependent.
Experimental Example 5: Observation of Absorption and Fluorescence Spectrum Over the time
(180) The reaction between glutathione and the compound prepared in Example 2 was induced. Then, the following experiment was performed to observe absorption and fluorescence spectrum over the time.
(181) <5-1> Observation of Absorption Spectrum
(182) 10 M of the compound prepared in Example 2 and 100 M of glutathione were added to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% dimethylsulfoxide (DMSO), followed by observation of absorption spectrum at 25 C. using UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) over the time. The results are shown in
(183)
(184) As shown in
(185) <5-2> Observation of Fluorescence Spectrum
(186) The following experiment was performed by the same manner as described in Experimental Example <5-1> except that fluorescence spectrum was observed by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell) instead of observing absorption spectrum via UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) (.sub.ex=730 nm, .sub.em=736 nm, slit:10/10 nm). The results are shown in
(187)
(188) As shown in
(189) Therefore, it was confirmed that the compound prepared in Example 2 reacted to glutathione so as to produce absorption or fluorescence spectrum fast.
Experimental Example 6: Mechanism of the Reaction with Glutathione
(190) To understand the reaction mechanism between the compound prepared in Example 2 and glutathione, matrix assisted laser desorption/ionization time-of-flight mass spectrometry was performed to induce the reaction between glutathione and the compound prepared in Example 2. The results are shown in
(191)
(192) As shown in
(193) From the above results, it was confirmed that the changes of absorption or fluorescence spectrum produced by the reaction between the compound of the invention based on cyanine scaffold and glutathione were attributed to the reaction presented in
Experimental Example 7: Evaluation of the Detection of Intracellular Biothiol 2
(194) The following experiment was performed to evaluate the selective detection capacity of the compound prepared in Example 2 for cysteine, homocysteine, and glutathione, among many biothiols that are the amino acids containing thiol group (RSH).
(195) HeLa cells (human adenocarcinoma cells, Korea Cell Line Bank) were cultured in RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in a 37 C. 5% CO.sub.2 incubator. The cells were either treated or not treated with 1 mM of N-methylmaleimide (NMM) known as a thiol blocker for 20 minutes. 100 M of each amino acid containing thiol group (RSH) such as cysteine, homocysteine, and glutathione was added thereto. Then, the cells were seeded in 35-mm glass bottomed dishes at the density of 310.sup.5 cells/dish in 1640 medium. 24 hours later, 10 M of the compound prepared in Example 1 was added thereto, followed by culture at 37 C. for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) twice to eliminate the remaining compound, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The results are shown in
(196)
(197)
(198)
(199)
(200)
(201)
(202) As shown in
(203) As shown in
(204) The compound prepared in Example 2 reacted selectively to glutathione alone among the amino acids containing thiol group (RSH) such as cysteine, homocysteine, and glutathione, etc, in the cytoplasm of HeLa cells, confirmed by the strong red fluorescence detected under confocal laser scanning microscope. Therefore, the compound of Example 2 can be efficiently used for the selective detection of glutathione.
Experimental Example 8: Observation of the Changes of the Concentration of Glutathione According to the Presence of an Oxidant 1
(205) 100 M of H.sub.2O.sub.2, the oxidant, was treated to HeLa cells prepared in Experimental Example 2 containing glutathione, the anti-oxidant, at 37 C. for 30 minutes to induce the changes of glutathione level. 20 M of the compound prepared in Example 2 was added thereto, followed by culture at 37 C. for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) three times to eliminate the remaining compound, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The results are shown in
(206)
(207)
(208)
(209) As shown in
(210) Also, as shown in
(211) Therefore, the compound prepared in Example 2 could be used for measuring the concentration of glutathione by detecting the strength of red fluorescence and was also confirmed to have excellent selectivity to glutathione since it did not react to the oxidized glutathione, glutathione disulfide (GSSG).
Experimental Example 9: Observation of the Changes of the Concentration of Glutathione According to the Presence of an Oxidant 2
(212) RAW 264.7 cells (macrophage cell line, Korea Cell Line Bank) were cultured in RPMI 1640 (Roswell Park Memorial Institute) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in a 37 C. 5% CO.sub.2 incubator.
(213) RAW 264.7 cells displaying the characteristics of generating ROS (Reactive Oxygen Species) or RNS (Reactive Nitrogen Species) were treated with 1 g/mL of lipopolysaccharide (LPS) for 20 minutes, 50 ng/mL of interferon- for 16 hours, and 20 M of phorbol 12-myristate 13-acetate (PMA) for 20 minutes in order to induce the generation of endogenous feroxynitrite having the characteristics of an oxidant. Then, the cells were seeded in 35-mm glass bottomed dishes at the density of 310.sup.5 cells/dish in 1640 medium. 24 hours later, the compound prepared in Example 2 was treated thereto at the concentration of 20 M for 20 minutes. The cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) three times, followed by imaging with confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The results are shown in
(214)
(215)
(216)
(217) As shown in
(218) Also, as shown in
(219) Therefore, the compound prepared in Example 2 could be used for measuring the concentration of glutathione by detecting the strength of red fluorescence and was also confirmed to have excellent selectivity to glutathione since it did not react to the oxidized glutathione, glutathione disulfide (GSSG).
Experimental Example 10: Evaluation of the Detection of Glutathione by Using a Mouse Model 1
(220) The following experiment was performed to evaluate the glutathione detection capacity of the compound prepared in Example 2 by using a mouse model.
(221) 200 L of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 20 mM of N-methylmaleimide (NMM) known as a thiol blocker was administered to each C57BL/6 mouse (78 weeks old, female, Jackson Laboratory) via intravenous injection. 20 minutes later, 200 L of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 50 mM of the compound prepared in Example 2 was administered to the mouse via intravenous injection. 20 minutes later, the strength of fluorescence was observed by using the epifluorescence mode of In Vivo Imaging System (IVIS) spectrum (Caliper LifeSciences, USA) equipped with the filter having the wavelengths of 675 nm and 720 nm [the strength of fluorescence was measured by Living Image Software 4.3.1 (Caliper Life Sciences, USA)]. The results are shown in
(222)
(223)
(224)
(225)
(226)
(227)
(228)
(229)
(230) As shown in
(231) As shown in
(232) Therefore, it was confirmed that the compound prepared in Example 2 could be efficiently used for the observation of in vivo glutathione distribution by detecting the red fluorescence observed from In Vivo Imaging System (IVIS) spectrum.
Experimental Example 10: Evaluation of the Detection of Glutathione by Using a Mouse Model 2
(233) The following experiment was performed to evaluate the glutathione detection capacity of the compound prepared in Example 2 by using a mouse model.
(234) 200 L of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 300 mg/kg of acetaminophen (APAP) known as a pain killer was administered to each C57BL/6 mouse (78 weeks old, female, Jackson Laboratory) via intravenous injection. The said APAP works as a pain killer in vivo when administered at a proper dose, but when treated at over-dose, it can damage organs and also reduce in vivo glutathione. 20 minutes later, 200 L of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 50 mM of the compound prepared in Example 2 was administered to the mouse via intravenous injection. 20 minutes later, the strength of fluorescence was observed by using the epifluorescence mode of In Vivo Imaging System (IVIS) spectrum (Caliper LifeSciences, USA) equipped with the filter having the wavelengths of 675 nm and 720 nm [the strength of fluorescence was measured by Living Image Software 4.3.1 (Caliper Life Sciences, USA)]. The results are shown in
(235)
(236)
(237)
(238)
(239)
(240)
(241) As shown in
(242) As shown in
(243) Therefore, it was confirmed that the compound prepared in Example 2 could be efficiently used for the observation of in vivo glutathione distribution by detecting the red fluorescence observed from In Vivo Imaging System (IVIS) spectrum.
Experimental Example 12: Evaluation of the Capacity of Diagnosing Sepsis 1
(244) The following experiment was performed to evaluate the capacity of diagnosing sepsis of the compound prepared in Example 2.
(245) According to a reference (J Vis Exp. 2011 May 7; (51). pii: 2860. doi: 10.3791/2860), C57BL/6 mouse (78 weeks old, female, Jackson Laboratory) was induced with sepsis by performing CLP (cecal ligation and puncture). 8 hours later, peritoneal cells were extracted from the mouse. The cells were centrifuged and reacted to the neutrophil marker Ly6G (eFluor450, eBioscience, Cat No. 48-5931) and 10 M of the compound prepared in Example 2 at room temperature. 25 minutes later, 1 mL of 3% fetal bovine serum (FBS) was added thereto, followed by centrifugation at 3000 rpm for 5 minutes to wash the cells. The cells were resuspended in 200 mL of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4), followed by FACS (Fluorescence Activated Cell Sorting) using the data analysis software FlowJo to observe the peaks (The setting for Alexa flour 700 fluorescence was hired, BD FACSVerse). The results are shown in
(246)
(247) As shown in
(248) From the above experiment, it was confirmed that the compound prepared in Example 2 could be efficiently used for the diagnosis of sepsis by measuring the concentration of glutathione.
Experimental Example 13: Evaluation of the Capacity of Diagnosing Sepsis 2
(249) The following experiment was performed to evaluate the capacity of diagnosing sepsis of the compound prepared in Example 2.
(250) Neutrophils were extracted from the bone marrow of C57BL/6 mouse (78 weeks old, female, Jackson Laboratory). The cells were infected with green fluorescent protein (GFP) tagged Pseudomonas Aeruginosa 01 (PA01) that is the causing bacteria of sepsis. 24 hours later, 10 M of the compound prepared in Example 2 was added thereto, followed by reaction at 37 C. for 20 minutes. At this time, the emitting filter with the excitation wavelength of 635 nm and the band-path (BP) of 655755 nm was used. The results are shown in
(251)
(252) As shown in
(253) After infecting the neutrophils with green fluorescent protein tagged PA01, the neutrophils were treated with the compound prepared in Example 2. At this time, the neutrophils got oxidative stress so that the cells produced ROS (Reactive Oxygen Species) excessively. As described in Experimental Example 12, glutathione reacted to ROS to generate glutathione disulfide (GSSG), resulting in the decrease of glutathione. Therefore, a red fluorescence indicating glutathione level was reduced in the red fluorescence channel.
(254) Therefore, it was confirmed that the compound prepared in Example 2 could be efficiently used for the diagnosis of sepsis by measuring the concentration of glutathione.
Experimental Example 14: Investigation of the Structure of the Compound and Measurement of Band-Gap
(255) To investigate the glutathione specific selectivity of the compound prepared in Example 2, the following experiment was performed.
(256) First, as shown in
(257)
(258) The fragment 1 of
(259) An experiment was performed by the same manner as described in Experimental Example 3 except that the fragment 1 or the fragment 2 was used instead of the compound prepared in Example 2. As a result, no changes in absorption or fluorescence spectrum were detected. From the above results, it was confirmed that the fragment 1 or the fragment 2 originated from the compound prepared in Example 2 did not have the capacity of detecting glutathione. To disclose the reason in relation to the above, the 3-dimensional structure of each of the compound of Example 1, the compound of Example 2, and the fragment 1 or the fragment 2, was investigated along with band-gap.
(260) To investigate the 3-dimensional structure and band-gap of the compounds, an experiment was performed based on Density Function Theory (DFT) at the level of B3LYP/6-31G* using Gaussian 09 program. The results are shown in
(261)
(262)
(263) As shown in
(264) Unlike the expectation that the conformation of piperazine could affect the selectivity, piperazine of the compound prepared in Example 2 showing glutathione specific selectivity and piperazine of the fragment 2 compound having no glutathione specific selectivity (compound of step 1 of Example 1) were equally in the form of chair conformation.
(265) When the compound prepared in Example 2 reacted to glutathione, as the reaction progressed as shown in
(266) Under the presumption that the length of SN bond in the compounds of Example 1, Example 2, and the fragment 1 could affect the glutathione specific selectivity, the length of SN bond was measured. As a result, the length of SN bond of the compound of Example 1 and the fragment 1 compound was respectively 1,667 and 1,660 . In the meantime, the length of SN bond in the compound prepared in Example 2 was 1,727 , suggesting that the binding energy of the compound prepared in Example 2 was smaller than that of the compound prepared in Example 1 and the fragment 1 compound, so that the bond in the compound of Example 2 could be broken more easily.
(267) Further, as shown in
(268) In conclusion, the selectivity of the compound prepared in Example 2 was attributed to the conformation of piperazine in the active site where the reaction to glutathione progressed, the conformation of dansyl group that is binding to piperazine, the length of SN bond, and the band-gap energy.
Experimental Example 15: Evaluation of the Glutathione Selectivity 2
(269) The following experiment was performed to evaluate the glutathione (GSH) selectivity of the compound prepared in Example 3.
(270) <15-1> Observation of Absorption Spectrum
(271) 10 M of the compound prepared in Example 3 and 100 M of each endogenous amino acid (alanine, arginine, cysteine, glutamate, glycine, glutathione, homocysteine, histidine, lysine, methionine, serine, and tyrosine) were added to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% dimethylsulfoxide (DMSO), followed by observation of absorption spectrum presented through UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) at 25 C. The results are shown in
(272)
(273) As shown in
(274) <15-2> Observation of Fluorescence Spectrum
(275) The following experiment was performed by the same manner as described in Experimental Example <15-1> except that fluorescence spectrum was observed by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell) instead of observing absorption spectrum via UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) (.sub.ex=730 nm, .sub.em=736 nm, slit:10/10 nm). The results are shown in
(276)
(277) As shown in
(278) Therefore, the compound prepared in Example 3 was confirmed to be efficiently used for the detection of glutathione in biosamples because the compound could selectively react to glutathione only among many other endogenous amino acids to make changes in absorption or fluorescence spectrum.
Experimental Example 16: Observation of Absorption and Fluorescence Spectrum According to the Concentration of Glutathione 2
(279) The following experiment was performed to observe absorption and fluorescence produced by the compound prepared in Example 3 according to the concentration of glutathione (GSH).
(280) <16-1> Observation of Absorption Spectrum
(281) 10 M of the compound prepared in Example 3 and glutathione were added to HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer (10 mM, pH=7.4) containing 10% dimethylsulfoxide (DMSO) with raising slowly the concentration of glutathione from 0 to 100 M, during which the changes of absorption spectrum were observed using UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) at 25 C. The results are shown in
(282)
(283) As shown in
(284) <16-2> Observation of Fluorescence Spectrum
(285) The following experiment was performed by the same manner as described in Experimental Example <16-1> except that fluorescence spectrum was observed by using RF-5310/PC fluorescence spectrometer (Shimada, 1 cm quartz cell) instead of observing absorption spectrum via UV-vis sepctra (Scinco 3000 spectrophotometer, 1 cm quartz cell) (.sub.ex=730 nm, .sub.em=736 nm, slit:10/10 nm). The results are shown in
(286)
(287) As shown in
(288) Therefore, it was confirmed that the compound prepared in Example 3 react to glutathione dose-dependently.
(289) Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.