JPH0353300B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a dihydroperoxide compound and a method for producing the same. The dihydroperoxide compounds provided by the present invention are effectively utilized in the detection of peroxide-activated substances such as blood or hemoglobin. If blood or hemoglobin is contained in urine, feces, or vomit, it can be assumed that some disease such as inflammation or ulcer is progressing in the urinary or digestive system, such as the kidneys, stomach, or intestines. Therefore, in order to promptly diagnose and treat these diseases, it is important to accurately detect blood or hemoglobin (occult blood) in the urine, feces, vomit, etc. The dihydroperoxide compound of the present invention is suitably used as a reagent for testing occult blood. [Prior art and its problems] A test device for detecting occult blood consists of a carrier impregnated with an organic hydroperoxide, a color indicator, a buffer, a wetting agent, an activator, and a stabilizer. The peroxide is activated to produce nascent oxygen, which oxidizes the indicator and produces color. Conventionally, 2,5-dimethylhexane-2,
5-dihydroperoxide and cumene hydroperoxide are known. Although these peroxides have been put into practical use, they are not stable over time, resulting in a significant drop in detection sensitivity.False negative results may occur if vitamin C is included in the urine sample. In the case of a multinomial test piece that detects medium components, it may cause discoloration of other adjacent test pieces, resulting in a decrease in performance;
It has drawbacks such as low color sensitivity. In recent years, compounds in which the benzene ring of cumene hydroperoxide is substituted with a C _1-6 alkyl group, Cl, Br, I, NO ₂ or a carboxyl group have been proposed as hydroperoxides that improve these drawbacks (Japanese Unexamined Patent Application Publication No. 1987-1999).
Publication No. 190663). Although this peroxide was considerably improved over conventional ones, its stability over time was not sufficiently satisfactory. [Means for Solving the Problems] The present invention aims to provide a peroxide free from the above-mentioned drawbacks, and the present invention comprises the following dihydroperoxide compound and its production method. (1) General formula [In the formula, R represents a hydrogen atom, a halogen atom, or a 2,4,7-trioxaoctyl group, and n
represents an integer from 1 to 4. ] A dihydroperoxide compound represented by: (2) The compound according to item 1, wherein n represents 2. (3) 2,5-diphenylhexane-2,5-dihydroperoxide, 2,5-bis(4-bromophenyl)hexane-2,5-dihydroperoxide or 2,5-bis[4-(2,4, 7-Trioxaoctyl)phenyl]hexane-2,5-dihydroperoxide. (4) General formula [In the formula, R represents a hydrogen atom, a halogen atom, or a 2,4,7-trioxaoctyl group, and n
represents an integer from 1 to 4. ] A general formula characterized by oxidizing the dihydroxy compound represented by with an aqueous hydrogen peroxide solution under acidic conditions. [In the formula, R has the same meaning as described above.] A method for producing a dihydroperoxide compound represented by the following. In the above formula, R represents a hydrogen atom, a halogen atom, or a 2,4,7-trioxaoctyl group as described above. The halogen atom may be any atom, but chlorine, bromine, and iodine are preferable. In the above formula, n represents an integer of 1 to 4, particularly n is 2
It is preferable that Typical dihydroperoxide compounds of the present invention include 2,5-diphenylhexane-2,5-dihydroperoxide, 2,5-bis(4-bromophenyl)hexane-2,5-dihydroxide, or 2,5-dihydroperoxide. Examples include 5-bis[4-(2,4,7-trioxaoctyl)phenyl]hexane-2,5-dihydroperoxide. The dihydroperoxide compound of the present invention represented by the above formula () is a new compound, and is produced by oxidizing the dihydroxy compound represented by the above formula () with an aqueous hydrogen peroxide solution under acidic conditions. Preferably a dihydroxy compound ()
is dissolved in a suitable organic solvent such as ether, a 30% or 50% aqueous hydrogen peroxide solution and a small amount of mineral acid such as sulfuric acid or hydrochloric acid are added to this solution, and the mixture is allowed to react at room temperature for more than ten hours. After the reaction is complete, the desired product is collected from the reaction mixture in a conventional manner. For example, the desired product is obtained by adding water to the reaction mixture, extracting with a suitable organic solvent such as ethyl acetate, distilling off the solvent from the extract, and purifying the residue by column chromatography etc. be able to. Dihydroxy compound () has the general formula () [In the formula, R is a hydrogen atom, a halogen atom, or 2,
represents a 4,7-trioxaoctyl group, and X represents a halogen atom] After reacting the phenyl halide derivative represented by the following with n-butyllithium (or magnesium), the general formula (n represents an integer of 1 to 4) by reacting with a diketone body. The reaction between the phenyl halide derivative and n-butyllithium or magnesium is carried out under the same conditions as for the usual Grignard reaction. For example, by reacting both compounds in a suitable organic solvent such as tetrahydrofuran or diethyl ether at -78°C (in the case of n-butyllithium) or at room temperature to reflux (in the case of magnesium), and then adding the diketone body. Compound () is obtained. As mentioned above, the dihydroperoxide compound () of the present invention is used as a peroxide in the measurement of peroxide-activated substances, and is particularly advantageously used for detecting occult blood in urine, feces, and vomit. Such a test device for detecting occult blood comprises a carrier impregnated with a composition consisting of the dihydroperoxide compound () of the present invention, a color indicator and, if necessary, a buffer, a wetting agent, an activator, a stabilizer and a solvent. As the indicator, so-called oxidized indicators that change color when oxidized are used, examples of which include orthotolidine, benzidine, leucomalachide green, and the like. Buffers are used to keep the PH value on the test device constant, such as citrate, malate, succinate, etc. When the test device is immersed in the sample, the pH value is between 4 and 8. Preferably one that can be maintained within this range. Wetting agents are used to uniformly wet the test device with the sample liquid when the test device is immersed in the sample, and suitable examples include sodium uralyl sulfate, sodium dodecylbenzenesulfonate, sodium dioctyl sulfosuccinate, and surfactants. used for. The activator is used to increase the sensitivity of the color reaction on the test device, and 3-aminoquinoline, quinine, isoquinoline, etc. are preferred. The stabilizer is a thickening agent used to prevent the test drug from flowing out of the test device, and is preferably a polymer such as polyvinyl alcohol, polyvinylpyrrolidone, or polyethylene glycol, or preferably gelatin or gum arabic.The solvent is a mixture of the above drugs. Any material may be used as long as it is easily soluble, and ethyl alcohol, acetone, benzene, toluene, chloroform and the like are advantageously used.
The carrier is preferably a nonwoven fabric made of paper, glass fiber, or plastic material, as long as it does not dissolve or react with the solvent and absorbs the composition. The amounts of the dihydroperoxide compound and other reagents used in the above test composition and test device are not particularly important, and are appropriately determined according to conventional methods. That is, an amount sufficient to react with the peroxide-activated substance to be detected and cause a color reaction is selected. Next, the present invention will be explained in more detail by showing Examples, Reference Examples, and Test Examples. Example 1 2,5-diphenylhexane-2,5-dihydroperoxide 2,5-hexanedione under argon atmosphere
5.70 g (50 mmol) of 2M phenylmagnesium bromide in 50 ml dry tetrahydrofuran solution
Tetrahydrolane solution (62.5ml, 125mmol)
was added at 0°C, and then stirred at room temperature overnight.
Water was added to the reaction solution and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (CH ₂ Cl ₂・MeOH,
20:1) to produce 8.00g (29.6m) of 2,5-diphenylhexane-2,5-diol
mol) was obtained. To a solution of 8.00 g (29.6 mmol) of the above alcohol compound in 7 ml of diethyl ether, 15 ml of a 30% aqueous hydrogen peroxide solution and 0.372 ml of concentrated sulfuric acid were added at 0°C, and the mixture was reacted overnight at room temperature. After the reaction, water was added and extraction was performed with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. 2,5-diphenylhexane-2,5-dihydroperoxide by elution with dichloromethane-methanol (20:1)
6.27g (20.7mmol) was obtained. NMR (ppm, CDCl ₃ ) 7.93 (s, 2H), 7.28 (s, 10H), 1.90 (s,
4H), 1.52 (s, 6H) IR (νcm ^-1 , CHCl ₃ ) 3520, 3320 Example 2 2,5-bis(4-bromophenyl)hexane-2,5-dihydroperoxide 1,4-dibromobenzene under argon atmosphere
972 mg (40.0 mmol) of magnesium in a solution of 9.40 g (39.8 mmol) in dry tetrahydrofuran (30 ml)
added. Furthermore, 1.20 ml (10.2 mmol) of 2,5-hexanedione was added to the reaction solution and stirred overnight.
A saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. By developing with dichloromethane-methanol (20:1), 2.88 g (6.726 mmol) of 2,5-bis(4-bromophenyl)hexane-2,5-diol was obtained. After adding 5 ml of ether, 20 ml of 50% aqueous hydrogen peroxide solution and 0.500 ml of concentrated sulfuric acid to 2.88 g (6.726 mmol) of the above alcohol compound and reacting in a greenhouse for 16 hours,
Water was added and extraction was performed with ethyl acetate. After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. Elution with ethyl acetate-hexane (2:1) gave 2.07 g (4.50 mmol) of 2,5-bis(4-bromophenyl)hexane-2,5-dihydroperoxide. NMR (ppm, _CDCl3 ) 8.03 (s, 2H), 7.33 (d, 4H, J-9Hz),
7.10 (d, 4H.J-9Hz) IR (νcm ^-1 , CHCl ₃ ) 3520, 3320 Example 3 2,5-bis[4-(2,4,7-,trioxaoctyl)phenyl]hexane-2 ,5-dihydropuroxide Under an argon atmosphere, 2.70 ml (23.6 mmol) of β-methoxyethoxymethyl chloride and 5.21 ml (29.9 mmol) of N,N-diisopropylethylamine were added to a solution of 3.71 g (19.8 mmol) of 4-bromobenzyl alcohol in dry dichloromethane (37 ml). The reaction was allowed to proceed at room temperature for 18 hours. Water was added to the solution and extracted with dichloromethane, and the organic layer was washed with water and concentrated under reduced pressure. The resulting residue was separated and purified using silica gel column chromatography. 4-promo-1-(2,4,7-trioxaoctyl) by elution with dichloromethane
4.87 g (17.7 mmol) of benzene was obtained. Under argon atmosphere, 4.87 g of the above bromo compound
(17.7 mmol) of dry tetrahydrofuran (50 ml)
460 mg (18.9 mmol) of magnesium pieces were added to the solution and refluxed for 4 hours. After cooling the reaction solution to 0°C, 671 mg (5.88 mmol) of 2,5-hexanedione was added.
A solution of in dry tetrahydrofuran (7 ml) was added. After reacting in a greenhouse for 16 hours, a saturated aqueous ammonium chloride solution was added and the mixture was extracted with ethyl acetate.
The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography. Elution with dichloromethane-methanol (20:1) gave 1.56 g (3.08 mmol) of 2,5-bis[4-(2,4,7-trioxaoctyl)phenyl]hexane 2,5-diol. After adding 4 ml of ether, 16 ml of 50% aqueous hydrogen peroxide solution and 0.400 ml of concentrated sulfuric acid to 1.56 g (3.08 mmol) of the above alcohol compound and reacting at room temperature for 19 hours,
Water was added and extracted with ethyl acetate. After washing the organic layer with water, it was concentrated under reduced pressure, and the resulting residue was separated and purified using silica gel column chromatography. 2,5-bis[4-(2,4,7
-trioxaoctyl)phenyl]hexane-
2,5-dihydroperoxide 1.19g (2.12m
mol) was obtained. NMR (ppm, _CDCl3 ) 8.06 (s, 2H), 7.50-7.14 (m, 8H), 4.76 (s,
4H), 4.54 (s.4H), 3.85-3.47 (m, 8H), 3.41
(s, 6H), 1.94 (s, 4H), 1.53 (s, 6H) IR (νcm ^-1 , CHCl ₃ ) 3520, 3320 Reference example Test paper manufacturing method Solution dihydroperoxide compound ()
Molecular weight x 2.24 x 10 ^-3 g p-toluenesulfonyl-N-diethylamide
5.0g Sodium dioctyl sulfosuccinate 1.5g Ethanol 100ml Wet the paper thoroughly with the solution and dry it in a drying oven at 40°C for 20 minutes. Solution Acrylamide 10g Polyethylene glycol 10g Trisodium citrate dihydrate 9g Citric acid monohydrate 1g Saponin 100mg EDTA-2Na 30mg Water 100ml Thoroughly wet the dried paper with the solution.
Dry in a drying oven at 40°C for 50 minutes. Solution Orthotolidine 1.20g 3-Aminoquinoline 0.5g Benzene 100ml Soak the paper that has been dried in the solution thoroughly in the solution.
Dry in a drying oven at 40°C for 10 minutes. this,
Used as a test paper for performance evaluation. Test Example 1 A test piece obtained as shown in the above test paper manufacturing example is immersed in a sample for 1 second. The color development of the test piece is compared with a predetermined color tone table over time, and the judgment code written on the color tone table is visually read. The concentration of occult blood in the sample is determined from the degree of coloration using the color tone table. The correlation between the determination code and hemoglobin concentration is shown below.

[Table] The color tone that correlates with the hemoglobin concentration in the color tone table is based on peroxide 2,5-dimethylhexane-2,
This figure shows the color of a test piece produced using 5-dihydroperoxide by the method shown in the above production example after a determination time of 60 seconds. As a result, 2,5-diphenylhexane-2,
5-dihydroperoxide had a coloration corresponding to the color chart in a determination time of about 30 seconds. The 2,5-dimethylhexane used to create the color table
2,5-dihydroperoxide requires 60 seconds. From the above, the dihydroperoxide compound () of the present invention has higher sensitivity than the peroxide 2,5-dimethylhexane-2,5-dihydroperoxide used in commercially available test strips for measuring urinary occult blood. It was found that it has Next, Tables 2 and 3 show the results of the test conducted after storage at 60°C.

【table】

[Table] Tables 2 and 3 show that 2,5-diphenylhexane-2,5-dihydroperoxide has excellent stability against changes over time. Test Example 2 It is known that when a test piece for measuring occult blood and a test piece for measuring glucose are adjacent to each other on a stake, the glucose test paper becomes discolored. 2,5-diphenylhexane as peroxide
A test piece prepared by the method shown in the production example above using 2,5-dihydroperoxide and 2,5-
A test piece using dimethylhexane-2,5-dihydroperoxide and a test piece using dimethylhexane-2,5-dihydroperoxide were pasted on separate stakes, and a glucose test piece was pasted on an adjacent portion of each stake and stored at 40°C for one month. The results showed that while there was no discoloration of the glucose test piece in the former case, discoloration occurred in the latter case. From this, the peroxide () of the present invention has little effect on other adjacent urine test items. [Effects of the Invention] The dihydroperoxide compound () of the present invention can be effectively used for detecting peroxide-activated substances, particularly blood or hemoglobin. That is, when the dihydroperoxide compound () of the present invention is used as the organic hydroperoxide in a peroxide-activated substance measuring composition comprising an organic hydroperoxide and a color indicator, the measuring composition having the following features can be obtained. (1) It is stable over time and can maintain good detection sensitivity even after long-term storage. (2) In the case of a multi-item test strip for detecting components in urine, it does not discolor adjacent test strips such as glucose test strips, and does not cause performance deterioration. (3) Faster color reaction rate and higher color sensitivity than conventional measurement compositions. As described above, the dihydroperoxide compound () of the present invention has very excellent properties as an organic hydroperoxide for measuring peroxide-activated substances. Furthermore, the present invention provides an advantageous method for producing such dihydroperoxide compounds ().

Claims (1)

[Claims] 1. General formula (In the formula, R is a hydrogen atom, a halogen atom, or 2,
4,7-trioxaoctyl group, n is 1-
(representing an integer of 4) A dihydroperoxide compound represented by: 2. The compound according to claim 1, wherein n represents 2. 3 2,5-diphenylhexane-2,5-dihydroperoxide, 2,5-bis(4-bromophenyl)hexane-2,5-dihydroperoxide or 2,5-bis[4-(2,4,7- The compound according to claim 1, which is trioxaoctyl)phenyl]hexane-2,5-dihydroperoxide. 4 General formula (In the formula, R is a hydrogen atom, a halogen atom, or 2,
4,7-trioxaoctyl group, n is 1-
(representing an integer of 4) is oxidized with an aqueous hydrogen peroxide solution under acidic conditions, A method for producing a dihydroperoxide compound represented by the formula (wherein R has the same meaning as described above).