Patent Application
20240125786
This application claims priority to Japanese Patent Application No. 2022-157405, filed Sep. 30, 2022, the disclosure of which is hereby incorporated by reference in its entirety.
The present invention relates to an indole carboxylic acid compound, a mixture of two or more indole carboxylic acid compounds, and an analytical method. The present invention specifically relates to a stable isotope-labeled indole carboxylic acid compound, a mixture of two or more stable isotope-labeled indole carboxylic acid compounds, and an analytical method using the compound or the mixture.
Melanin is a pigment synthesized in melanocytes. Melanin is a pigment synthesized in melanocytes and includes two types of melanin, eumelanin and pheomelanin. In the biosynthesis of them, various metabolites are produced. To measure such metabolites, some techniques have been developed.
For example, Non-Patent Document 1 discloses synthesis of deuterium-labeled eumelanin (precursor) metabolites. Non-Patent Document 2 discloses analysis of eumelanin-related indolic compounds in urine by HPLC with fluorometric detection. Non-Patent Document 3 discloses normal values of urinary excretion and serum concentration of 5-S-cysteinyldopa (hereinafter also referred to as 5-S-CD) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (hereinafter also referred to as 6H5MI2C), biomarkers of melanoma progression.
Various metabolites involved in the above melanin synthesis may be usable as biomarkers for diseases or nutritional conditions. For example, these metabolites are probably used to understand the pathological condition of malignant melanoma, a type of skin cancer. For example, Non-Patent Document 3 discloses use of 5-S-CD and 6H5MI2C as biomarkers for melanoma, and a quantitative method by HPLC has been developed.
If such a metabolite can be detected or quantified with high sensitivity and high resolution, the metabolite probably has a higher utility value as a biomarker. Mass spectrometry, specifically, LC-MS/MS is a technique having high sensitivity and high resolution for compounds. To carry out the analytical method, an internal standard is required.
The present invention is therefore intended to provide a novel compound that can be used to detect or quantify a melanin-relating metabolite.
The present invention provides a novel indole carboxylic acid compound. The compound can be used as an internal standard, for example, in mass spectrometry for detecting or quantifying melanin-relating metabolites.
The present invention provides the following aspects.
where, in Formula (I), X1 to X8 are each independently C or 13C; Y is N or 15N; M is H, Na, K, Li, CH3, or C2H5; R1 is H or D; R2 is H, CH3, 13CH3, C2H5, or 13C2H5; R3 is H, CH3, 13CH3, C2H5, or 13C2H5; R4 is H or D; and R5 is H or D.
According to the present invention, a standard usable to detect or quantify, for example, a melanin-relating metabolite is provided. The compound according to the present invention can be used as an internal standard, for example, in mass spectrometry for detecting or quantifying a melanin-relating metabolite. This enables detection or quantification of the metabolite with high sensitivity and high resolution.
The effect of the invention is not limited to that described in this paragraph and may be any of the effect described in the present specification.
Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following preferred embodiments, which can be arbitrarily changed within the scope of the invention.
As described above, various metabolites are produced in the biosynthesis of melanin. The biosynthetic pathway of melanin is shown in
It has been proposed that some of these melanin-relating metabolites are used as a tumor marker for melanoma. For example, 5-S-CD may be clinically used as a tumor marker as disclosed in Non-Patent Document 3. Depending on the conditions of a human to be tested, however, the 5-S-CD value may not be abnormal even in a patient with tumor or may be false positive in some cases. In addition, 5-S-CD is rarely measured outside Japan. Improving the reliability of a tumor marker for melanoma should be useful.
The inventors of the present invention have developed a novel compound that can be used in mass spectrometry for detecting or quantifying a melanin-relating metabolite. Mass spectrometry using the novel compound enables detection or quantification of a melanin-relating metabolite with high sensitivity and high resolution.
Detecting or quantifying a melanin-relating metabolite with high sensitivity and high resolution as above contributes to the improvement in reliability of a melanoma examination technique.
Non-Patent Documents 2 and 3 disclose the detection of a melanin-relating metabolite by HPLC. However, each detection method disclosed in these documents detects fluorescence or electric conductivity derived from a catechol structure, and in the reliability on quantification, mass spectrometry is superior.
With the compound according to the present invention, a melanin-relating metabolite can be quantified by mass spectrometry. By mass spectrometry using the compound according to the present invention, a melanin-relating metabolite can be quantified with high accuracy. Such accurate quantification contributes to the improvement in reliability of a melanoma examination technique.
Non-Patent Document 1 discloses a stable isotope-labeled compound, but the total number of deuterium atoms introduced to one compound is 5. To synthesize the compound, a large amount of an expensive deuterium reagent is needed.
According to the present invention, a stable isotope-labeled compound to which a smaller number of deuterium atoms are introduced can be produced. In other words, in the production of the compound according to the present invention, an expensive deuterium reagent can be saved, and accordingly, a stable isotope-labeled compound can be synthesized at low cost.
According to the present invention, for example, in the clinical examination field, a trace metabolite can be detected by mass spectrometry with high sensitivity and can be quantified with high sensitivity. In addition, the present invention is applicable to any type of mass spectrometer. For example, to widely spread the quantification of a trace metabolite by mass spectrometry in the clinical examination field, it is desirable to yield the same value regardless of the time, the place, and the apparatus from any manufacturer. The compound according to the invention can be used in mass spectrometry regardless of the difference between apparatuses from various manufacturers. This can help the spread of quantification of a trace metabolite by mass spectrometry.
The present invention will next be described in further detail.
The present invention provides an indole carboxylic acid compound represented by Formula (I):
Components of Formula (I) will next be described.
In Formula (I), X1 to X8 are each independently C or 13C.
In other words, X1 is C or 13C and is preferably C. X2 is C or 13C and is preferably C. X3 is C or 13C and is preferably C. X4 is C or 13C and is preferably C. X5 is C or 13C and is preferably C. X6 is C or 13C and is preferably C. X7 is C or 13C and is preferably C. X8 is C or 13C and is preferably C.
From the viewpoint of the production cost, preferably, each of X1 to X8 may be C.
In Formula (I), Y is N or 15N and is preferably N. From the viewpoint of the production cost, Y is preferably N.
In Formula (I), M is H, Na, K, Li, CH3, or C2H5.
In an embodiment, M is H. In this case, examples of the compound include stable isotope-labeled 6H5MI2C or stable isotope-labeled 5H6MI2C. The structures of 6H5MI2C and 5H6MI2C are shown in
In another embodiment, M is an alkali metal such as Na, K, and Li, and M is preferably Na or K. In the embodiment, M is a counter cation and can, for example, improve the solubility of the compound.
In yet another embodiment, M may be CH3 or C2H5.
In Formula (I), R1 is H or D; R4 is H or D; and R5 is H or D.
Preferably, one or two of R1, R4, and R5 may be D, and the rest may be H. All of R1, R4, and R5 may be D.
In an embodiment, in Formula (I), one or both of R1 and R4 may be D. For example, in Formula (I), R1 is D; R4 is D; and R5 is H. Alternatively, R1 is D; R4 is H; and R5 is H. Alternatively, R1 is H; R4 is D; and R5 is H.
In Formula (I), R2 is H, CH3, 13CH3, C2H5, or 13C2H5, and R3 is H, CH3, 13CH3, C2H5, or 13C2H5. Preferably, one or both of R2 and R3 are H.
In an embodiment, R2 is H, CH3, or C2H5, and R3 is H, CH3, or C2H5. More preferably, R2 is H or CH3, and R3 is H or CH3.
For example, R2 is H, CH3, or C2H5; R3 is H, CH3, or C2H5; and one of R2 and R3 may be H.
In a preferred embodiment, R2 is H, and R3 is CH3.
In another preferred embodiment, R2 is CH3, and R3 is H.
In yet another preferred embodiment, R2 is H, and R3 is H.
The compound of the present invention has at least one stable isotope.
For example, the compound of the present invention may have at least one stable isotope, D (deuterium).
For example, the compound of the present invention may have at least one stable isotope, 13C.
For example, the compound of the present invention may have at least one stable isotope, 15N.
The position of such a stable isotope in the compound is as described above.
In a preferred embodiment, the number of stable isotopes, D, of the compound of the present invention is, for example, 3, 2, or 1. The stable isotope D may be present as R1, R4, or R5 described above.
The number of stable isotopes, 13C, of the compound of the present invention is, for example, 12 or less, preferably 10 or less, and more preferably 8 or less. The stable isotope 13C may be present as X1 to X8 described above and/or as a component of R2 and/or R3.
In an embodiment, one or more of X1 to X8 in Formula (I) may be a stable isotope 13C, and neither R2 nor R3 has a stable isotope 13C (that is, R2 and R3 are H, CH3, or C2H5).
In another embodiment, the compound may have no stable isotope, 13C. In other words, X1 to X8 may each be C, and R2 and R3 may each be H, CH3, or C2H5.
The number of stable isotopes, 15N, of the compound of the present invention is 1 or less. The stable isotope, 15N, may be present as Y described above.
In an embodiment, the stable isotope-labeled indole carboxylic acid compound of the present invention may be a compound represented by Formula (II).
In Formula (II), two Rs are each independently H or CH3 as described above. At least one of Hs and/or Cs constituting the indole ring may be a stable isotope D or a stable isotope 13C. Preferably, two Ds may be bonded to the six-membered ring (specifically at 4-position and 7-position) of the indole ring.
Preferably, the stable isotope-labeled indole carboxylic acid compound of the present invention may be a compound represented by Formula (III) or (IV). In the present specification, the compound of Formula (III) is also referred to as 6H5MI2C-4,7-d2 or 6H5MI2C-d2 as described later, and the compound of Formula (IV) is also referred to as 5H6MI2C-4,7-d2 or 5H6MI2C-d2.
Of the melanin-relating metabolites, 6H5MI2C and 5H6MI2C are useful as tumor markers for melanoma. By mass spectrometry using the stable isotope-labeled compounds of Formulae (III) and (IV) as the internal standards, 6H5MI2C and 5H6MI2C can be accurately quantified, and the accurate quantification contributes to the usability of 6H5MI2C and 5H6MI2C as the tumor markers.
The compound of the present invention can be synthesized, for example, from a stable isotope-labeled
The stable isotope-labeled
Of the compounds used in the synthesis of
For the pyruvic acid and the ammonia of the compounds used in the synthesis of
As described above, the carbons or the nitrogen constituting the skeleton of the compound of the Formula (I) may be stable isotope-labeled by appropriately using, as the material compounds, compounds that are stable isotope-labeled at corresponding elements.
As for deuterium labeling (R1, R4, and R5) in Formula (I), H-D exchange with deuterium oxide may be carried out, for example, as shown in
For example, as shown in
The present invention also provides a mixture of two or more different indole carboxylic acid compounds of the stable isotope-labeled indole carboxylic acid compounds according to the present invention described in the above section 2.
In other words, the present invention also provides a mixture of two or more different compounds of the stable isotope-labeled indole carboxylic acid compounds represented by Formula (I):
where, in Formula (I),
The description for Formula (I) in the section 2 is also applied to the present embodiment.
The mixture can also be used as an internal standard in mass spectrometry as described above. The two or more compounds contained in the mixture preferably have the same molecular weight. For example, the two or more compounds contained in the mixture may differ only in the structures of R2 and R3 and have the same molecular weight.
Two or more indole carboxylic acid compounds having similar chemical structures (especially, two or more compounds having the same molecular weight) in a biological sample may not need to be detected or quantified separately and independently. In such a case, the internal standard may be such a mixture as described above to reduce cost.
The mixture may contain at least an indole carboxylic acid compound of Formula (I) in which R2 is H and an indole carboxylic acid compound of Formula (I) in which R3 is H. For example, the mixture may be a mixture containing two compounds, a stable isotope-labeled 5H6MI2C and a stable isotope-labeled 6H5MI2C. For example, the mixture may be a mixture containing the compound of Formula (III) (6H5MIC-4,7-d2) and the compound of Formula (IV) (5H6MI2C-4,7-d2) and may specifically contain only these two compounds.
The present invention also provides an analytical method using the indole carboxylic acid compound described in the above section 2 or using the mixture described in the above section 3. In the analytical method, the indole carboxylic acid compound or the mixture may be used as a standard and may specifically be used as an internal standard.
The analytical method may be an analytical method targeting an indole carboxylic acid compound identical with the indole carboxylic acid compound of the present invention described in the above section 2 except that the target compound is not labeled with any stable isotope. For example, the analysis target may be an indole carboxylic acid compound having, in place of the stable isotopes (D, 13C, and 15N) contained in the above indole carboxylic acid compound of the present invention, corresponding atoms (H, C, and N).
When the mixture is used, the analytical method may be an analytical method targeting an indole carboxylic acid compound identical with the indole carboxylic acid compound of the present invention described in the above section 2 except that the target compound is not labeled with any stable isotope. The analytical method may specifically be a mass spectrometry method. When the mixture is used, the analysis target may be at least one of the two or more indole carboxylic acid compounds according to the present disclosure contained in the mixture except that the target compound is not labeled with any stable isotope.
For example, when the mixture is a mixture containing the compound of Formula (III) (6H5MIC-4,7-d2) and the compound of Formula (IV) (5H6MI2C-4,7-d2), the analysis target may be one or both of 5H6MI2C and 6H5MI2C. In the analytical method, 5H6MI2C and 6H5MI2C may be analyzed without distinction.
The analytical method may comprise carrying out mass spectrometry. In the mass spectrometry, the analysis target may be detected or quantified while the indole carboxylic acid compound according to the present invention or the mixture according to the present invention is used as a standard (specifically an internal standard).
The mass spectrometry may detect or quantify an indole carboxylic acid compound, for example, in a biological sample. In particular, the mass spectrometry may be for detecting or quantifying an indole carboxylic acid compound labeled with no stable isotope in a biological sample. The indole carboxylic acid compound in a biological sample may, for example, be one or both of 5H6MI2C and 6H5MI2C.
In an embodiment, the indole carboxylic acid compound in a biological sample may be a tumor marker for melanoma. For example, 5H6MI2C and 6H5MI2C are promising as a tumor marker for melanoma. The analytical method of the present invention may comprise detecting or quantifying such a tumor marker for melanoma and may specifically comprise detecting or quantifying such a tumor marker for melanoma by mass spectrometry.
The analytical method of the present invention may comprise generating, on the basis of the detection result or the quantification result, data on the risk of melanoma in a human from which the biological sample is derived, data on the presence or absence of melanoma in a human from which the biological sample is derived, or data on the progression of melanoma in a human from which the biological sample is derived.
The mass spectrometry may comprise ionizing an analysis target. The ionization may be carried out, for example, by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI) and may be carried out by any ionization other than these ionizations.
The mass spectrometry may be carried out with a mass spectrometer used in the art and may be carried out, for example, with a liquid chromatography tandem mass spectrometer (LC-MS/MS) or a matrix-assisted laser desorption-ionization mass spectrometer (MALDI-MS), but the apparatus for carrying out the mass spectrometry is not limited to them.
A specific procedure of the mass spectrometry may be appropriately designed by a person skilled in the art, for example, according to the type of a sample.
The present invention will next be described in more detail with reference to examples, but the present invention is not limited to these examples.
Deuterium-labeled 6H5MI2C and 5H6MI2C (i.e., the compounds of Formula (III) and Formula (IV)) were synthesized by the following procedure. The reaction pathway is shown in
In a 100-mL eggplant flask, 0.1 g (0.5 mmol) of
Under an inert gas atmosphere, the reaction mixture was extracted with 25 mL of ethyl acetate three times. The obtained extract was washed with 10 mL of brine containing 19 mg of sodium pyrosulfite (Na2S2O5) twice and was dried over magnesium sulfate. The magnesium sulfate was filtered off, and the filtrate was concentrated by using a rotary evaporator. The residue was dissolved in 2.5 mL of acetone, and 10 mL of hexane was added. After the removal of a brown oil, 15 mL of hexane was further added, followed by decantation to give a white precipitate. The resultant product was washed with hexane several times, and was dried under reduced pressure to give about 25 mg of 5,6-dihydroxyindole-2-carboxylic acid-d2 (DHICA-d2) (Compound iv in the figure).
About 20 mg of DHICA-d2 was dissolved in 4 mL of a mixed solution of methanol and ethyl acetate (1:1). To the solution, 0.8 mL of trimethylsilyldiazomethane (10% hexane solution, ca 0.6M) was added twice, and the reaction mixture was allowed to stand in a sealed, light-shielded container under an inert gas atmosphere at room temperature for about 4 hours. A trace amount the reaction mixture was sampled every one hour, and the progress of methylation was monitored by LC-MS. The reaction was quenched by adding 0.1 mL of a mixed solution of methanol and formic acid (1:1), and the reaction mixture was transferred to a plurality of PP tubes and was dried by using a centrifugal evaporator. The solid obtained here is a mixture of methyl esters such as methyl 5,6-dihydroxyindole-2-carboxylate-d2, methyl 5-hydroxy-6-methoxy-indole-2-carboxylate-d2, methyl 6-hydroxy-5-methoxy-indole-2-carboxylate-d2, and methyl 5,6-dimethoxyindole-2-carboxylate-d2.
In the example, trimethylsilyldiazomethane was used as a methylating agent due to easy availability and handling, but another methylating agent such as diazomethane may be used.
The mixture was dissolved in ethyl acetate and was subjected to preparative thin-layer chromatography with chloroform as an eluent. To sufficiently separate bands corresponding to components, multiple development about five times with chloroform is required. Each component is a fluorescent substance and thus can be easily observed under an UV lamp. Each component is easily oxidized into dark brown, and thus the mixture is preferably developed in a light-shielded developing chamber purged with an inert gas. In addition, when the developing solvent is volatilized, inert gas purge is preferably carried out.
Identical component bands collected from a plurality of plates for thin-layer chromatography were extracted with ethyl acetate, and the extract was concentrated by using a rotary evaporator. Without further purification, methyl esters were hydrolyzed to give carboxylic acids. The methyl ester in this step is methyl 5-hydroxy-6-methoxy-indole-2-carboxylate-d2, methyl 6-hydroxy-5-methoxy-indole-2-carboxylate-d2, or a mixture of these two esters at any ratio. To hydrolyze about 10 to 30 mg of the methyl esters, the methyl esters were placed in a 100-mL eggplant flask, and a solution of 380 mg of Na2S2O5 in 6 mL of 70% ethanol was added. The mixture was stirred under an inert gas atmosphere at 37° C. for 90 minutes, and then about 10 mL of 1M hydrochloric acid was added to adjust the pH to 1. The reaction mixture was extracted with 10 mL of ethyl acetate three times, and the combined extract was washed with 4 mL of brine twice. The washed liquid was dried over magnesium sulfate, and the magnesium sulfate was filtered off. The filtrate was concentrated by using a rotary evaporator. The product was recrystallized with acetone-hexane to give target compounds, 5-hydroxy-6-methoxy-indole-2-carboxylic acid-d2 (Compound vii, X=D, in the figure) and 6-hydroxy-5-methoxy-indole-2-carboxylic acid-d2 (Compound viii, X=D, in the figure).
Methylation with trimethylsilyldiazomethane or diazomethane yielded 5H6MI2C (5H6MI2C-d2) two to three times more than 6H5MI2C (6H5MI2C-d2). In some final applications (for example, for mass spectrometry), a mixture of 5H6MI2C-d2 and 6H5MI2C-d2 may be used, or the mixture may not interfere with an intended purpose. Such applications, for example, include a case in which the total amount of 5H6MI2C and 6H5MI2C in a sample is required to be determined and a case in which it is sufficient to detect the presence of one or both of 5H6MI2C and 6H5MI2C in a sample, but it is not necessary to specify which one is present.
In such a case, the methyl esters may not be exactly separated by thin-layer chromatography. In another case, a mixture of the methyl esters (optionally adjusted at an appropriate mixing ratio of, for example, 1:1 before hydrolysis) may be hydrolyzed in a single reaction container.
Authentic 5H6MI2C and 6H5MI2C were added to MSG3000 (Golden West Biologicals), which was a hormone-free serum, to prepare calibrators at concentrations shown in
A high concentration solution of 5H6MI2C-d2 and 6H5MI2C-d2 (1 μg/mL, 30% acetonitrile solution) was diluted with 4% phosphoric acid to prepare an internal standard solution at 5 ng/mL.
Samples were pretreated in the following procedure.
LC analysis conditions were as follows:
MS/MS analysis conditions were as follows:
On the basis of the above analytical results, the concentration of 5H6MI2C and the concentration of 6H5MI2C in the pooled serum were quantified to be 0.41 ng/mL and 0.59 ng/mL, respectively.
In this manner, 5H6MI2C and 6H5MI2C can be quantified by mass spectrometry using the stable isotope-labeled indole carboxylic acid compounds according to the present invention. 5H6MI2C and 6H5MI2C are contained in extremely small amounts in a biological sample. Mass spectrometry using the stable isotope-labeled indole carboxylic acid compounds according to the present invention enables detection and quantification of such extremely small amounts of melanin-relating metabolites.
For about 80 clinical samples (samples with melanoma), 5H6MI2C and 6H5MI2C in a serum were quantified by the method described in Example 2, and the suitability of 5H6MI2C and 6H5MI2C as tumor markers was evaluated. The result revealed that these tumor markers increased as the disease progressed and reflect the disease status, and the compounds were sufficiently useful as the tumor markers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-157405 | Sep 2022 | JP | national |
