WO2017002754A1

WO2017002754A1 - Simple test for periodontal disease using fluorescent dye

Info

Publication number: WO2017002754A1
Application number: PCT/JP2016/068991
Authority: WO
Inventors: 康祐難波; 中山　淳; 彬大谷
Original assignee: 国立大学法人徳島大学
Priority date: 2015-06-29
Filing date: 2016-06-27
Publication date: 2017-01-05
Also published as: JPWO2017002754A1; JP6782978B2

Abstract

The present invention provides: a diagnostic drug which can detect a disease associated with the increase in the concentration of methylmercaptan in the oral cavity, e.g., periodontal disease, simply and with high sensitivity; a kit including the diagnostic drug; and a diagnosis method using the diagnostic drug. Specifically, the present invention provides: a diagnostic drug for use in a test for a disease associated with the increase in the concentration of methylmercaptan in the oral cavity in a subject, said diagnostic drug comprising a compound represented by formula I [wherein R¹, R², R³ and R⁴ independently represent a substituent such as a hydrogen atom; R⁵ represents an alkenyl group which may be substituted; and X represents O or S], or an isomer of the compound, or a pharmaceutically acceptable salt, a hydrate or a solvate of the compound or the isomer; a kit including the diagnostic drug; and a diagnosis method using the diagnostic drug.

Description

Simple examination of periodontal disease using fluorescent dyes

The present invention relates to a simple examination of periodontal disease using a fluorescent dye. Specifically, diagnostic agents for examination of diseases such as periodontal diseases using triazapentalene derivatives as fluorescent dye compounds, kits containing the diagnostic agents, and periodontal diseases using the diagnostic agents The present invention relates to a method for diagnosing a disease.

Periodontal disease is an inflammatory disease caused by bacterial infection in the oral cavity, and it has been reported that in Japan it is over 30% at the age of 15 and over, and over 80% is over the age of 40. Periodontal disease can be cured with appropriate care if it is in the early stage of gingivitis, periodontitis, or alveolar osteomyelitis. The bones that support the teeth melt and eventually lose the teeth. Therefore, examination for early detection of periodontal disease is important. However, it is also known that early detection is difficult because periodontal disease has no subjective symptoms even if it is infected and often becomes severe without the patient's awareness. For this reason, development of diagnostic agents and diagnostic methods for early detection of periodontal disease has been made.

It is known that methyl mercaptan is generated by the metabolism of periodontal disease bacteria in the oral cavity of periodontal disease patients. Therefore, as a method for diagnosing periodontal disease, there is known a method for detecting the presence of methyl mercaptan in a sample such as saliva or expiration by a color reaction (Patent Documents 1 and 2). However, since the amount of methyl mercaptan in saliva or exhalation is very small, these conventional detection methods based on the color reaction have a problem in detection sensitivity. Therefore, a simpler test for diseases such as periodontal disease and a diagnostic agent for the test have been demanded.

Further, the triazapentalene skeleton is known to be an excellent fluorescent chromophore that emits strong fluorescence while having a compact structure as compared with a group known as a conventional fluorescent chromophore, such as fluorescein. (Patent Document 3).

JP 2004-309283 A Special table 2011-524526 WO2012 / 121356A1

The present invention has been made to solve the above-described problems in the prior art, and is capable of detecting easily and highly sensitive diseases that cause an increase in the concentration of methyl mercaptan in the oral cavity, such as periodontal disease. It is intended to provide a diagnostic agent, a kit containing the diagnostic agent, and a diagnostic method using the diagnostic agent.

As a result of diligent research to solve the above problems, the present inventors have selectively reacted with a thiol such as methyl mercaptan and the fluorescent color of the triazapentalene derivative represented by the following formula I is orange. The color was changed from green to green, and the fluorescence intensity was found to be greatly enhanced. In addition, the compound represented by the formula (I) of the present invention has an extremely compact structure and can be synthesized simply and efficiently. Furthermore, since it has a compact structure, it has high water solubility, and a functional group on the triazapentalene skeleton can be easily introduced, so that it can be applied to various fields. Furthermore, since the thiol detection sensitivity is high, the triazapentalene derivative was found useful as a thiol sensor such as methyl mercaptan, and the present invention was completed.

That is, the present invention relates to the following [1] to [18].
[1]
Formula I:

I
[Where:
R ¹ , R ² , R ³ , and R ⁴ are hydrogen, cyano, hydroxy, nitro, optionally substituted amino, optionally substituted alkyl, optionally substituted cycloalkyl, substituted Each independently selected from the group consisting of optionally heterocycloalkyl, optionally substituted alkoxy, optionally substituted aryl, and optionally substituted heteroaryl;
R ⁵ is an optionally substituted alkenyl;
X is O or S]
Diagnosis for examination of diseases that result in an increase in oral methyl mercaptan concentration in a subject, including a compound represented by the above, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof medicine;
[2]
The diagnostic agent according to [1] above, wherein X is O;
[3]
^{^{^{R 1, R 2, R 3}}} , and ^{R 4} is hydrogen, cyano, optionally substituted _C 1 -C ₆ alkyl, optionally substituted _C 1 -C ₆ alkoxy, and may be substituted The diagnostic agent according to [1] or [2] above, each independently selected from the group consisting of 6 to 10-membered monocyclic or bicyclic aryl;
[4]
The diagnostic agent according to any one of [1] to [3] above, wherein R ¹ , R ² , R ³ , and R ⁴ are hydrogen;
[5]
The diagnostic agent according to any one of [1] to [4] above, wherein R ⁵ is optionally substituted C ₂ -C ₁₂ alkenyl;
[6]
R ⁵ is represented by the following formula:

[In the formula, the wavy line indicates the bonding point with the rest of the molecule]
The diagnostic agent according to any one of the above [1] to [5], which is represented by the structure:
[7]
The compound has the following formula:

The diagnostic agent according to [1], which is represented by the structure of
[8]
The disease is selected from the group consisting of periodontal disease, dental caries, bad breath, stomatitis, oral cancer, blood disease, cardiovascular disease, metabolic disorder, respiratory disease, osteoporosis, and necrotic soft tissue disease, [1 ] The diagnostic agent according to any one of [7] to [7];
[9]
The diagnostic agent according to any one of [1] to [8] above, wherein the disease is selected from the group consisting of periodontal disease and bad breath;
[10]
The diagnostic agent according to any one of [1] to [9] above, wherein the concentration of methyl mercaptan is 20 ppb or less;
[11]
The diagnostic agent according to any one of the above [1] to [10];
A medium capable of absorbing or contacting the test sample;
Instructions for use;
A kit containing an appropriate base; and an appropriate pharmaceutically acceptable additive;
[12]
The kit according to [11] above, wherein the test sample is breath or oral fluid;
[13]
The kit according to [11] or [12] above, wherein the medium is a buffer solution, air, activated carbon, or a solid film; and [14]
(I) a step of bringing the diagnostic agent according to any one of [1] to [10] above into contact with a test sample in the presence of a base as appropriate; and (ii) irradiation with ultraviolet rays using a UV device, and fluorescence A method for diagnosing a disease that causes an increase in the concentration of methyl mercaptan in the oral cavity in a subject, comprising the step of examining methyl mercaptan in a test sample by examining the degree of color change.

Another aspect of the present invention provides:
[15]
The diagnostic agent according to any one of [1] to [10] above, further comprising a base;
[16]
The present invention relates to the kit according to any one of [11] to [13] above, or the diagnostic agent according to [15] above, wherein the base is a guanidine type organic base.

Yet another aspect of the present invention provides:
[17]
A compound represented by the above formula I, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvent thereof, for use in a test for a disease that causes an increase in oral methyl mercaptan concentration in a subject Japanese; and [18]
A compound represented by the above formula I, or an isomer thereof, or a pharmaceutically acceptable salt or hydrate thereof in the manufacture of a diagnostic agent for the examination of a disease that causes an increase in oral methyl mercaptan concentration in a subject Or the use of solvates.

In a preferred embodiment, the subject in the above [1] to [18] is a human or non-human animal.

The compound represented by the formula (I) of the present invention, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof selectively reacts only with a thiol such as methyl mercaptan. Since the fluorescence intensity is greatly enhanced, it has an extremely compact structure and high detection sensitivity, it is useful as a thiol sensor such as methyl mercaptan. Therefore, the compound, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is a diagnostic agent for testing for a disease that causes an increase in oral methyl mercaptan concentration in a subject. Can be used as

FIG. 1 shows the 1.60 × 10 ⁻⁷ M 1,3a, 6a-triazapentalene-vinylketone (R ⁵ = 2-methyl-1-propenyl) (TAP) prepared by the procedure described in Example 8. -VK1) 0%, 5% (8.00 × 10 ⁻⁹ M), 10% (1.60 × 10 ⁻⁸ M), 20% (3.20 × 10 ⁻⁸ M) methyl mercaptan adduct in the solution ), And 30% (4.80 × 10 ⁻⁸ M), UV was applied to the solution, and fluorescence was observed. This indicates that the change in fluorescence can be observed with the naked eye when the reaction between TAP-VK1 and methyl mercaptan has progressed by 5%. The concentration of the adduct (8.00 × 10 ⁻⁹ M) at the stage when the reaction has progressed by 5% is 8.00 ppb when converted to ppb units. FIG. 2 shows that 1.60 × 10 ⁻⁷ M TAP-VK1 solution prepared by the procedure described in Example 8 contains 0%, 5% (8.00 × 10 ⁻⁹ M) of methyl mercaptan adduct, 10% % (1.60 × 10 ⁻⁸ M), 20% (3.20 × 10 ⁻⁸ M), and 30% (4.80 × 10 ⁻⁸ M) of the fluorescence spectrum of the solution. Each curve in the figure shows the fluorescence spectrum of 0%, 5%, 10%, 20%, and 30% solution from the bottom. FIG. 3 shows a DMF solution (control) that was passed through activated carbon not ventilated with methyl mercaptan gas obtained in the experiment of Example 9 and a DMF solution that was passed through activated carbon ventilated with methyl mercaptan gas (after reaction). ) Shows the result of fluorescence observation.

Definitions As used herein, an active ingredient of a diagnostic agent of the present invention for the examination of diseases such as periodontal disease has the following formula I:

I
[Where:
R ¹ , R ² , R ³ , and R ⁴ are hydrogen, cyano, hydroxy, nitro, optionally substituted amino, optionally substituted alkyl, optionally substituted cycloalkyl, substituted Each independently selected from the group consisting of optionally heterocycloalkyl, optionally substituted alkoxy, optionally substituted aryl, and optionally substituted heteroaryl;
R ⁵ is an optionally substituted alkenyl;
X is O or S]
Or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

The structure of the compound of the present invention represented by the above formula I has a 1,3a, 6a-triazapentalene (TAP) skeleton, and a C (═X) group directly connected at the 2-position of the triazapentalene skeleton (for example, An oxo group or a thioxo group, preferably an oxo group), and an alkenyl group as R ⁵ linked to the oxo group or thioxo group.

In the present invention, a thiol such as methyl mercaptan is added on the alkenyl group as R ⁵ , and a fluorescence spectrum is observed when the product after the addition reaction is irradiated with ultraviolet rays (UV) or the like. Here, the compound represented by the formula I of the present invention does not react at all with molecules having other functional groups such as alcohol, amine, carboxylic acid, and sulfide, but selectively reacts only with thiol.

The substituents R ¹ , R ² , R ³ , and R ⁴ on the triazapentalene skeleton of the compound represented by the formula I of the present invention may all be hydrogen, or independently, other than hydrogen. It may have a substituent. The substituent may be either an electron donating group or an electron withdrawing group. Examples of the substituent include hydrogen, cyano, hydroxy, nitro, optionally substituted amino, optionally substituted alkyl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. , A group selected from the group consisting of optionally substituted alkoxy, optionally substituted aryl, and optionally substituted heteroaryl, but is not limited thereto.

In one embodiment, the substituents R ¹ , R ² , R ³ , and R ⁴ in Formula I are hydrogen, cyano, optionally substituted C ₁ -C ₆ alkyl, optionally substituted C Each is independently selected from the group consisting of ₁ -C ₆ alkoxy and optionally substituted 6-10 membered monocyclic or bicyclic aryl.

In addition, the terms “optionally substituted amino”, “optionally substituted alkyl”, “optionally substituted cycloalkyl”, “optionally substituted heterocycloalkyl”, “substituted” “Alkoxy”, “optionally substituted aryl”, and “optionally substituted heteroaryl” are unsubstituted or further substituted with one or more substituents. Such substituents include, but are not limited to, cyano, hydroxy, nitro, amino, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, and halogen (eg, fluorine, chlorine, bromine and Iodine) and the like.

As used herein, the term “cyano” refers to a substituent represented by —CN.

As used herein, the term “hydroxy” refers to a substituent represented by —OH.

As used herein, the term “nitro” refers to a substituent represented by —NO ₂ .

The term “amino” as used herein refers to a substituent represented by —NH ₂ .

The term “alkyl” as used herein refers to a saturated linear or branched hydrocarbon, preferably an alkyl having 1 to 6 carbon atoms (C ₁ -C ₆ alkyl), more preferably a carbon number. Refers to _1-3 alkyl (C ₁ -C ₃ alkyl). “Alkyl” used in the present invention includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like.

As used herein, the term “cycloalkyl” refers to an alicyclic saturated hydrocarbon, preferably a monocyclic cycloalkyl having 3 to 8 carbon atoms (C ₃ -C ₈ cycloalkyl), more preferably Refers to monocyclic cycloalkyl having 3 to 6 carbon atoms (C ₃ -C ₆ cycloalkyl). The “cycloalkyl” used in the present invention includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “heterocycloalkyl” as used herein refers to a cycloalkyl having one or more heteroatoms selected from the group consisting of nitrogen, carbon, and sulfur atoms as ring members, Refers to monocyclic 3-8 membered heterocycloalkyl, more preferably monocyclic 3-6 membered heterocycloalkyl. The “heterocycloalkyl” used in the present invention includes, but is not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholino and the like.

The term “alkoxy” as used herein refers to a group in which an oxygen atom is bonded to a linear or branched alkyl, preferably an alkoxy having 1 to 6 carbon atoms (C ₁ -C ₆ Alkoxy), more preferably alkoxy having 1 to 3 carbon atoms (C ₁ -C ₃ alkoxy). Examples of “alkoxy” used in the present invention include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, and isobutoxy.

As used herein, the term “aryl” refers to an aromatic hydrocarbon, preferably 6-10 membered monocyclic or bicyclic aryl. Examples of the “aryl” used in the present invention include, but are not limited to, phenyl, indenyl, naphthyl, and azulenyl.

As used herein, the term “heteroaryl” refers to an aryl having one or more heteroatoms selected from the group consisting of nitrogen, carbon, and sulfur atoms as ring members, preferably a single atom. Refers to cyclic or bicyclic 5- to 10-membered heteroaryl. Examples of the “heteroaryl” used in the present invention include, but are not limited to, furyl, pyrrolyl, imidazolyl, thienyl, indolyl, quinolyl and the like.

Specific examples of the triazapentalene skeleton include compounds described in Patent Document 3 (for example, [Table 4], [Table 5], and [FIG. 12]). A typical example is given below.

The R ⁵ group in formula I of the present invention is an optionally substituted alkenyl.

As used herein, the term “alkenyl” refers to at least one (eg, one, two, or three, preferably one) carbon-carbon double bond (the double bond is terminal). Linear or branched unsaturated hydrocarbon chain, preferably having 2 to 12 carbon atoms (C ₂ -C ₁₂ alkenyl), More preferably, it refers to alkenyl having 2 to 6 carbon atoms (C ₂ -C ₆ alkenyl). The “alkenyl” used in the present invention includes, but is not limited to, vinyl, propenyl, butenyl (eg, 3-butenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl), pentenyl ( Examples thereof include 3-methyl-1-butenyl, 3-methyl-2-butenyl), hexenyl, heptenyl, octenyl (for example, 2,2-dimethyl-4-methyl-3-pentenyl) and the like.

The term “optionally substituted alkenyl” in R ⁵ is unsubstituted or further substituted with one or more substituents. Such substituents include, but are not limited to, cyano, hydroxy, nitro, amino, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, halogen (eg fluorine, chlorine, bromine and iodine ), And alkoxycarbonyl.

For example, R ⁵ is represented by the following formula:

[In the formula, the wavy line indicates the point of attachment to the rest of the molecule]
The case of 2-methyl-1-propenyl represented by the structure

Typical specific examples of the substituent R ⁵ are listed below.

[In the formula, the wavy line indicates the bonding point with the rest of the molecule]

The compounds of formula I according to the invention may exist in the form of their isomers. Such isomers include various stereoisomers such as tautomers, geometric isomers, optical isomers, and mixtures thereof. Moreover, since the positive charge and the negative charge in the triazapentalene skeleton of the compound represented by the formula I of the present invention are delocalized, the formula I ′ can be described instead of the formula I. Compounds of any electronic state are encompassed by compounds of formula I.

“Pharmaceutically acceptable salts” of the compounds of formula I of the present invention include, but are not limited to, inorganic acid salts (eg, hydrochloride, hydrobromide, hydroiodide, Sulfates, nitrates and phosphates) and organic acid salts (eg formate, acetate, propionate, fumarate, oxalate, malonate, succinate, methanesulfonate, ethane Acid addition salts such as sulfonate, benzenesulfonate, maleate, lactate, malate, tartrate, citrate, and trifluoroacetate), metal salts (e.g., lithium, potassium, Calcium salts, magnesium salts, sodium salts, zinc salts, and aluminum salts), and inorganic bases (eg, ammonium salts, diethanolamine salts, ethylenediamine salts, triethanolamine salts) And organic bases (e.g., triethylamine salts) base addition salts such as.

The “hydrate” of the compound represented by Formula I of the present invention refers to a substance in which one or more water molecules are hydrated. A “solvate” of a compound of formula I of the present invention refers to a substance in which one or more solvent molecules are solvated. Such solvents include, but are not limited to, halogenated hydrocarbons (eg, dichloromethane), hydrocarbons (eg, cyclohexane, toluene), ethers (eg, diethyl ether, tetrahydrofuran), and alcohols ( For example, ethanol) etc. can be mentioned.

In an exemplary embodiment, the compound of formula I of the present invention has the formula:

(1,3a, 6a-triazapentalene-vinyl ketone body (R ⁵ = 2-methyl-1-propenyl)) (hereinafter referred to as TAP-VK1).

As used herein, the term “subject” is an organism that is to be diagnosed by the diagnostic agent, kit, or diagnostic method of the present invention, including but not limited to a mammal, such as a human or non-human. An animal etc. can be mentioned. Non-human animals include, but are not limited to, mice, rats, monkeys, dogs, cats, cows, horses, sheep, goats, rabbits, and pigs. In one embodiment, a “subject” in the present invention is a human. In another embodiment, the “subject” in the present invention is a non-human animal.

As used herein, the term “disease leading to an increase in methylmercaptan concentration in the oral cavity” is not limited, but blood such as periodontal disease, dental caries, halitosis, stomatitis, oral cancer, leukemia, etc. Examples include diseases, cardiovascular diseases such as heart disease, metabolic disorders such as diabetes, respiratory diseases such as pneumonia, osteoporosis, and necrotic soft tissue diseases. Preferably, it is a disease of periodontal tissue, for example, periodontal disease (for example, gingivitis, periodontitis, alveolar osteomyelitis, periodontitis, etc.) and bad breath.

The diagnostic agent of the present invention examines a disease by detecting oral thiols such as methyl mercaptan in a subject. When the methyl mercaptan concentration is high, for example, 500 ppb corresponding to exhalation of a patient with severe periodontal disease Not only in the case of the above concentrations, specifically 600 ppb, but also in low concentrations of methyl mercaptan, specifically about 1 ppm or less, about 100 ppb or less, about 50 ppb or less, about 20 ppb or less (eg Even a concentration of about 10 ppb or less and about 1 ppb or less) can be detected.

Another aspect of the invention includes a diagnostic agent of the invention; an appropriate base; a medium capable of absorbing or contacting the test sample; instructions for use; and an optionally pharmaceutically acceptable additive. Provide kit.

As used herein, the term “base” refers to a compound of formula I above, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof and a thiol such as methyl mercaptan. Are preferably blended in order to react. The base may be included in the diagnostic agent or may be included in the kit. Such bases include, but are not limited to, alkali metal amides such as lithium diisopropylamide, sodium amide, and lithium bistrimethylsilylamide; sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and cesium carbonate Alkali metal carbonates such as sodium phosphate and potassium phosphate; amines such as triethylamine, diisopropylethylamine, pyridine, and N-methylmorpholine; sodium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, Alkali metal phosphates such as potassium phosphate and tribasic potassium phosphate; alkali metal fluorides such as cesium fluoride and potassium fluoride; sodium methoxide, sodium ethoxide, potassium ester Alkali metal alkoxides such as xoxide, sodium t-butoxide and potassium t-butoxide; Alkali metal hydrides such as sodium hydride and potassium hydride; Alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; Calcium hydroxide and water Alkaline earth metal hydroxides such as barium oxide; diazabicycloundecene (DBU); diazabicyclononene (DBN); and tetramethylguanidine (TMG), methyltriazabicyclodecene (MTBD), and triazabicyclodecene Examples thereof include guanidine type organic bases such as (TBD), dicyclohexyloctylguanidine (DCOG), tricyclohexylguanidine (TCG), and pentamethylguanidine (PMG). These may be used alone or in combination of two or more. A typical base used in the present invention is a guanidine-type organic base, preferably TMG.

As used herein, the term “test sample” includes, but is not limited to, breath (exhalation), oral fluid (such as saliva and gingival crevicular fluid), and biofilms, Preferred are breath and oral fluid.

As used herein, the term “medium capable of absorbing or contacting a test sample” refers to a substance capable of absorbing the test sample therein or contact with the test sample on its surface. Refers to a substance that can. Examples of such a medium include, but are not limited to, a buffer solution, air, activated carbon, a solid film, and the like, and preferably activated carbon. The activated carbon is preferably treated with a base. Examples of such a base include the bases defined above, preferably an alkali metal hydroxide, more preferably potassium hydroxide.

The kit of the present invention includes instructions for carrying out the present invention by appropriately using each component included in the kit.

The kit of the present invention can be constructed in various forms. Such forms include, but are not limited to, a cylindrical form such as a straw containing a solid or a liquid containing the diagnostic agent of the present invention, a solid film containing or coated on the surface of the diagnostic agent of the present invention And the like, and the form of a sample tube such as a syringe containing the sheet.

The kit of the present invention appropriately contains a pharmaceutically acceptable additive depending on its form. Such additives include, but are not limited to, excipients used in the manufacture of pharmaceutical preparations (for example, general test / diagnostic drugs, and kits for the test / diagnosis), binding agents Agents, preservatives, stabilizers, buffering agents, pH adjusting agents, and fragrances.

The kit of the present invention brings about an increase in the methyl mercaptan concentration in the oral cavity, depending on the fluorescent color produced by the reaction between the diagnostic agent of the present invention and the test sample, or the numerical value such as the obtained fluorescence intensity or the calculated methyl mercaptan concentration. Means may be included that provide criteria for easily diagnosing a disease. Examples of such means include, but are not limited to, a diagram or a table in which the fluorescent color or numerical value is associated with a disease.

As a typical embodiment of the kit of the present invention, the following kits can be mentioned.
formula:

A diagnostic agent comprising a compound represented by:
Activated carbon treated with potassium hydroxide;
Instructions for use;
Optionally, a kit comprising a guanidine-type organic base (for example, tetramethylguanidine); and a pharmaceutically acceptable additive as appropriate.

The present invention further includes (i) contacting the diagnostic agent of the present invention with a test sample in the presence of a base as appropriate; and (ii) examining methyl mercaptan in the test sample. Methods for diagnosing diseases that result in elevated mercaptan levels are also provided. (I) In the step of bringing the diagnostic agent of the present invention and the test sample into contact with each other in the presence of a base as appropriate, the diagnostic agent of the present invention and the test sample may be brought into direct contact. After absorption or contact with a medium and separation and extraction of methyl mercaptan, it may be contacted with the diagnostic agent of the present invention. (Ii) The step of inspecting methyl mercaptan in the test sample is not limited, but is a step of simply inspecting by irradiating ultraviolet rays using a UV device and examining the degree of change in fluorescent color, In addition, the step of quantifying methyl mercaptan by measuring the fluorescence spectrum and precisely inspecting it can be mentioned.

The UV device may be a commercially available UV light or lamp, for example. A pen-type UV light that is generally commercially available at a low price can also be used. The usable UV wavelength is about 10 to 400 nm. For example, any of short wavelength (for example, about 254 nm), medium wavelength (for example, about 302 nm), and long wavelength (for example, about 365 nm) should be used. Typically, the long wavelength set for a commercially available pen-type UV light can be used.

Spectrophotometers and fluorometers used in laboratories or medical sites can be used for quantitative measurement of fluorescence spectra. Further, the fluorescence intensity can be expressed using the fluorescence quantum yield.

(Production method)
The triazapentalene derivative represented by the formula I used in the present invention can be produced, for example, according to the following production methods, but is not limited to these production methods.

Specifically, first, according to the method described in WO2012 / 121356A1 (for example, Example 7), an alkyne derivative and an azide derivative having an R substituent (corresponding to a substituent defined by R ¹ to R ⁴ ) are obtained. The intermediate I which is a triazapentalene derivative is produced by reacting in the presence of a catalytic amount of a copper iodide / amino ether complex. Alternatively, the triazapentalene derivative can be synthesized by other commonly known organic chemical synthesis methods or obtained commercially. Here, the substituent R on the triazapentalene skeleton is converted into a substituent R ¹ on the triazapentalene skeleton by using a substitution reaction on an aromatic ring known in ordinary organic chemistry after the production of the intermediate I. ~ R ⁴ may be introduced.

Next, the produced intermediate I is reacted with lithium hydroxide for deesterification, and then reacted with N, O-dimethylhydroxylamine to produce intermediate II. Next, the intermediate II is reacted with a Grignard reagent to obtain a desired target (production route 1).

Alternatively, the desired product is obtained by reacting the intermediate I produced above with a Grignard reagent (production route 2).

Typical specific examples of intermediate I that can be used in the present invention are listed below.

The present invention will be described more specifically with reference to the following examples. However, the following examples are provided for the purpose of explaining the present invention, and the present invention is limited to the following examples. is not. In the examples, the following abbreviations have the following meanings. If an abbreviation is not defined, each term shall have the meaning commonly used in the art.
CDCl ₃ = deuterated chloroform conv. = Conversion yield DMAP = Dimethylaminopyridine DMF = Dimethylformamide EDCI = 1-Ethyl-3- (3'-dimethylaminopropyl) carbodiimide equiv = Equivalent Me = Methyl MeOH = Methanol MeSH = Methyl mercaptan NMR = Nuclear magnetic resonance rt = Room temperature TEA = triethylamine Tf = trifluoromethylsulfonyl THF = tetrahydrofuran TLC = thin layer chromatography TMG = tetramethylguanidine

Production examples of the compound of the present invention will be shown.
Example 1
Preparation of 1,3a, 6a-triazapentalene-vinyl ketone body (R ⁵ = 2-methyl-1-propenyl) (TAP-VK1) (1) Preparation of intermediate I

Methyl propiolate (3.5 mL, 39.35 mmol) was added to a solution of azidoditriflate (10 g, 26.23 mmol) in tetrahydrofuran (874 mL, 0.03 M), and the mixture was stirred at room temperature. To this solution was added triethylamine (18.3 mL, 131.16 mmol) and CuI (0.25 g, 1.31 mmol) at the same temperature and stirred for an additional 3 hours. The completion of the reaction was confirmed by TLC, and then concentrated under reduced pressure. The residue was purified by flash column chromatography (ethyl acetate: hexane = 3: 7, 1% TEA) to obtain a methyl ester (intermediate I) as brown crystals (3.9 g, 23.59 mmol, yield). Rate: 90%).

(2) Production of intermediate II

Methanol (57.4 mL) and water (19.1 mL) were added to the methyl ester compound (2.53 g, 15.31 mmol) as intermediate I obtained in (1) above and stirred. To this solution was added lithium hydroxide monohydrate (674 mg, 16.08 mmol) at 0 ° C. After warming to room temperature, the mixture was further stirred for 24 hours. After confirming the disappearance of the raw material by TLC, it was concentrated under reduced pressure. Toluene was added to the residue and concentrated again to obtain a brown amorphous. To this was added DMF (76.6 mL) and stirred. Cool the solution to 0 ° C. and add EDCI (6.46 g, 33.69 mmol), DMAP (0.56 g, 4.59 mmol), and N, O-dimethylhydroxylamine (2.99 g, 30.62 mmol). did. The reaction solution was warmed to room temperature and stirred for 12 hours. To this reaction solution was added 5% aqueous citric acid solution at 0 ° C. This solution was extracted with ethyl acetate. The collected organic layer was dried over magnesium sulfate, filtered and concentrated under reduced pressure. The obtained residue was purified by flash column chromatography (ethyl acetate: hexane = 1.1, 1% TEA) to obtain brown crystals (2.83 g, 13.39 mmol, 2-step yield: 87%).

(3) Manufacture of TAP-VK1

Weinreb amide (10 mg, 50.97 μmol) as intermediate II obtained in (2) above was dissolved in tetrahydrofuran (101.9 μL), and the reaction solution was cooled to −78 ° C. and stirred. Isobutenyl magnesium bromide (203.9 μL, 101.93 μmol) was slowly added, and the mixture was further stirred at the same temperature for 2 hours. The disappearance of the raw material was confirmed by TLC. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the temperature was raised to 0 ° C. This solution was extracted with ethyl acetate and washed with saturated brine. The collected organic layer was dried over magnesium sulfate, filtered, and then concentrated under reduced pressure. The obtained residue was purified by flash column chromatography (ethyl acetate: hexane = 1: 4, 1% TEA) to obtain the desired target product TAP-VK1 as a yellow solid (8.1 mg, 42.81 μmol, Yield: 84%).
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.64 (1H, d, J = 1.2 Hz), δ 7.45 (1H, d, J = 1.2 Hz), 7.19 (1H, d, J = 2.8 Hz), δ6.96 (1H, t, J = 1.2 Hz), δ6.74 (1H, t, J = 2.8 Hz), δ2.30 (3H, d, J = 0.8 Hz) , Δ 2.04 (3H, d, J = 1.2 Hz)
LRMS: [M + H] ⁺ 190.3

Example 2
Production of 1,3a, 6a-triazapentalene-vinyl ketone body (R ⁵ = 3-butenyl) In the same manner as in the reaction of (3) in Example 1 above, intermediate I (50 mg) obtained in Example 1 above was prepared. , 0.303 mmol) and vinylmagnesium bromide (363 μL, 0.363 mmol) were reacted in the same manner to obtain the desired target product (12.5 mg, 66.1 μmol, yield: 22%).
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.62 (1H, d, J = 1.2 Hz), δ 7.45 (1 H, br-d, J = 2.0 Hz), 7.20 (1 H, d , J = 2.0 Hz), δ 6.76 (1 H, t, J = 2.8 Hz), δ 5.90 (1 H, m), δ 5.10 (1 H, dd, J = 17.2, 1.6 Hz) , Δ5.01 (1H, dd, J = 10.0, 1.6 Hz), δ3.11 (2H, t, J = 7.2 Hz), δ2.51 (2H, br-q, J = 6.8) )

Example 3
Production of 1,3a, 6a-triazapentalene-vinyl ketone body (R ⁵ = 2,2-dimethyl-4-methyl-3-pentenyl) In the same manner as in the reaction of (3) in Example 1 above, Intermediate I (20 mg, 0.121 mmol) obtained in 1 was reacted with isobutenyl magnesium bromide (291 μL, 0.145 mmol), and the desired reaction product was obtained in the same manner. (7.8 mg, 31.8 μmol, yield: 27%).
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.58 (1H, d, J = 1.2 Hz), δ 7.40 (1H, br-d, J = 2.8 Hz), 7.18 (1H, d , J = 2.8 Hz), δ 6.75 (1H, t, J = 2.8 Hz), δ 5.31 (1H, t, J = 1.2 Hz), δ 3.12 (2H, s), δ 1.74. (1H, d, J = 1.2 Hz), δ 1.65 (1 H, d, J = 1.2 Hz), δ 1.27 (6H, s)

Example 4
Fluorescence properties of TAP-VK1 The fluorescence properties were measured using the following equipment and method.
Equipment used Absorbance measurement: V-630 spectrophotometer (manufactured by JAS.CO)
Fluorescence measurement: FP-8200 Fluorometer (manufactured by JAS.CO)
Measurement method A sample (1-2 mg) for fluorescence measurement was dissolved in dichloromethane (10 mL). 1 mL of this solution was taken with a whole pipette and adjusted to 10 mL using a volumetric flask. The solution was transferred to a quartz cell and degassed by bubbling argon for 30 minutes. In the same manner, 9,10-diphenylanthracene (9,10-DPA, Φ _F = 0.91 (in cyclohexane)) was prepared as a reference substance. The absorption spectrum was measured in the range of 750 to 250 nm using a V-630 spectrophotometer. The fluorescence spectrum was measured in the range of 380 to 750 nm by irradiating light of 370 nm as an excitation wavelength.

The fluorescence characteristics of TAP-VK1 were an excitation wavelength λ _abs = 409 nm, a fluorescence wavelength λ _em = 574 nm, and a quantum yield Φ _F = 0.023, indicating weak orange fluorescence.

Example 5
Reaction of TAP-VK1 with methyl mercaptan

TAP-VK1 (1.6 mg, 8.314 μmol) was dissolved in dichloromethane (42.3 μL). The reaction solution was cooled to 0 ° C., tetramethylguanidine (1.04 μL, 8.314 μmol) and methyl mercaptan (2 mL, 1 μg / μL, 41.57 μmol) were added, and the mixture was stirred at the same temperature for 3 hours. After confirming the termination of the reaction by TLC, vacuum concentration was performed. The residue was purified by flash column chromatography (ethyl acetate: hexane = 1: 9, 1% TEA) to obtain a methyl mercaptan adduct as a yellow amorphous substance (1.2 mg, 4.989 μmol).

Note that TAP-VK1 did not react at all with other functional groups such as alcohol, amine, carboxylic acid, and sulfide. This suggests that the compound represented by Formula I of the present invention is an effective component of an excellent test / diagnostic reagent for thiols (particularly methyl mercaptan), which is an index of occurrence of periodontal disease and the like.

Example 6
Fluorescence properties of methyl mercaptan adduct of TAP-VK1 The methyl mercaptan adduct of TAP-VK1 (1.71 mg, 7.205 μmol) was dissolved in 10 mL of dichloromethane using a measuring flask. 1 mL of this solution was taken with a whole pipette and adjusted again to 10 mL using a volumetric flask. This solution (7.205 × 10 ⁻⁵ M) was subjected to argon bubbling for 30 minutes. Other operations were carried out in the same manner as in Example 4 and used for absorption spectrum and fluorescence spectrum measurement.

The fluorescence characteristics of the methyl mercaptan adduct of TAP-VK1 were excitation wavelength λ _abs = 388 nm, fluorescence wavelength λ _em = 516 nm, and quantum yield Φ _F = 0.49, indicating strong green fluorescence. That is, by adding methyl mercaptan to TAP-VK1, the fluorescence color changed from orange to green, and the fluorescence intensity increased more than 20 times.

Example 7
Reaction of TAP-VK1 and methyl mercaptan in solid state Tetramethylguanidine (132.6 μL, 1.057 mmol) was added to TAP-VK1 (20 mg, 105.70 μmol) and stirred. After 30 minutes, the solution was concentrated under reduced pressure. Methyl mercaptan gas (20 mL) was added to the obtained solid and allowed to stand at room temperature for 24 hours. By adding CDCl ₃ to this residue and measuring NMR, 50% conv. To confirm that the desired methyl mercaptan adduct was produced. When this solution was irradiated with UV, green fluorescence indicating the formation of a methyl mercaptan adduct was observed.

Example 8
Fluorescence intensity of TAP-VK1 methyl mercaptan adducts under dilution conditions In order to prove that the diagnostic agent of the present invention can be detected even at low concentrations of methyl mercaptan (≦ 20 ppb) actually contained in exhaled breath, Fluorescence observation was performed by changing the mixing ratio of TAP-VK1 and its methyl mercaptan adduct under the conditions.

Sample Preparation Method Preparation of TAP-VK1 Solution (Solution A) (1) 3 mg (15.9 μmol) of TAP-VK1 (molecular weight: 189.21) was weighed.
(2) Using a volumetric flask, it was dissolved in 10 mL of dichloromethane (final concentration: 1.59 × 10 ⁻³ M).
(3) 1 mL was weighed using a whole pipette and adjusted to 10 mL with a volumetric flask (final concentration: 1.59 × 10 ⁻⁴ M).

Preparation of methyl mercaptan adduct solution (solution B) (1) 3.8 mg (16.0 μmol) of the adduct (molecular weight: 237.32) was weighed.
(2) Using a volumetric flask, it was dissolved in 10 mL of dichloromethane (final concentration: 1.60 × 10 ⁻³ M).
(3) 1 mL was weighed using a whole pipette and adjusted to 10 mL with a volumetric flask (final concentration: 1.60 × 10 ⁻⁴ M).

Solution A and Solution B were mixed at the following ratios to obtain 0, 5%, 10%, 20%, and 30% solutions of 1.60 × 10 ⁻⁴ M methyl mercaptan adduct, and then each dilution. The solution was diluted 10-fold with dichloromethane to give 0%, 5%, 10%, 20%, and 30% solutions of 1.60 × 10 ⁻⁵ M methyl mercaptan adduct.

Based on each of the obtained diluted solutions, each diluted solution having the following concentration was produced.
1.60 × 10 ⁻⁶ M Diluent Each 250 μL of the above diluted solution was taken and diluted with 2.25 mL of dichloromethane to obtain 0%, 5%, 10% of 1.60 × 10 ⁻⁶ M methyl mercaptan adduct. %, 20%, and 30% solutions were prepared.
1.60 × 10 ⁻⁷ M diluted solution Take 25 μL of each of the above diluted solutions and dilute with 2.475 mL of dichloromethane to obtain 0%, 5%, 10% of 1.60 × 10 ⁻⁷ M methyl mercaptan adduct. %, 20%, and 30% solutions were prepared.

0%, 5% (8.00 × 10 ⁻⁹ M), 10% (1.60 × 10 ⁻⁸ M), 20% (3 of the resulting 1.60 × 10 ⁻⁷ M methyl mercaptan adduct .20 × 10 ⁻⁸ M) and 30% (4.80 × 10 ⁻⁸ M) solutions were measured for fluorescence in the same manner as in FIG. The results are shown in FIG.

As is clear from FIGS. 1 and 2, it was found that the diagnostic agent of the present invention can sufficiently observe fluorescence even with an adduct of methyl mercaptan (≦ 20 ppb, for example, 8.00 ppb) below the concentration in breath.

Therefore, since the diagnostic agent of the present invention can sufficiently detect a trace amount of methyl mercaptan contained in exhaled breath, it is included in the exhaled breath by inhaling the diagnostic agent of the present invention and irradiating UV to observe fluorescence. Methyl mercaptan can be easily detected.

Example 9
Capture of methyl mercaptan in air by medium and detection by TAP-VK1 The following experiment was conducted to prove that methyl mercaptan in air was captured by the medium and reacted with TAP-VK1. It was.

(1) Activated carbon (manufactured by Wako Pure Chemical Industries, Ltd.) was washed with DMF and 1M aqueous potassium hydroxide solution, and then vacuum-dried.
(2) Using a permeator (gas generator), 600 ppb of methyl mercaptan gas (corresponding to the concentration in exhaled breath of severe periodontal disease patients) was generated. Next, the gas generated from the permeator was connected to a Pasteur pipette with a hose and aerated (corresponding to 2 L of exhalation) for 10 minutes.
(3) A microtube in which TAP-VK1 (1.89 μg, 0.01 μmol) was previously placed was placed on the lower side of the Pasteur pipette, and 100 μL of DMF was allowed to flow from the upper side of the Pasteur pipette. DMF was collected with a microtube, and the microtube was capped and stirred vigorously.
(4) A UV lamp was applied to the microtube and the state of fluorescence was observed. The progress of the reaction was confirmed by HPLC analysis.

In the DMF solution (control) that was passed through activated carbon that was not ventilated with methyl mercaptan gas, very weak yellow fluorescence derived from TAP-VK1 was observed. On the other hand, in the DMF solution (after the reaction) that was passed through activated carbon in which methyl mercaptan gas was passed, strong green fluorescence derived from the methyl mercaptan adduct of TAP-VK1 was observed (FIG. 3).

From the above, it was proved that methyl mercaptan having a concentration corresponding to the concentration in the expired air of periodontal disease patients contained in the air was captured by an appropriate medium and detected by TAP-VK1. By using this method, it is expected that a very small amount of methyl mercaptan in the air can be efficiently captured and detected.

Since the diagnostic agent of the present invention, the kit containing the diagnostic agent, and the diagnostic method using the diagnostic agent can selectively detect methyl mercaptan in the oral cavity with high sensitivity, the methyl mercaptan concentration in the oral cavity can be detected. A disease causing an increase, for example, periodontal disease can be detected easily and with high sensitivity, and can be used as a periodontal disease sensor in various modes.

Claims

Formula I:

I
[Where:
R 1 , R 2 , R 3 , and R 4 are hydrogen, cyano, hydroxy, nitro, optionally substituted amino, optionally substituted alkyl, optionally substituted cycloalkyl, substituted Each independently selected from the group consisting of optionally heterocycloalkyl, optionally substituted alkoxy, optionally substituted aryl, and optionally substituted heteroaryl;
R 5 is an optionally substituted alkenyl;
X is O or S]
Diagnosis for examination of diseases that result in an increase in oral methyl mercaptan concentration in a subject, including a compound represented by the above, or an isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof medicine.
The diagnostic agent according to claim 1, wherein X is O.
R 1, R 2, R 3 , and R 4 is hydrogen, cyano, optionally substituted C 1 -C 6 alkyl, optionally substituted C 1 -C 6 alkoxy, and may be substituted The diagnostic agent according to claim 1 or 2, which is independently selected from the group consisting of 6 to 10-membered monocyclic or bicyclic aryl.
The diagnostic agent according to any one of claims 1 to 3, wherein R 1 , R 2 , R 3 , and R 4 are hydrogen.
The diagnostic agent according to any one of claims 1 to 4, wherein R 5 is optionally substituted C 2 -C 12 alkenyl.
R 5 is represented by the following formula:

[In the formula, the wavy line indicates the point of attachment to the rest of the molecule]
The diagnostic agent according to any one of claims 1 to 5, which is represented by the structure:
The compound has the following formula:

The diagnostic agent according to claim 1, which is represented by the structure of
The disease is selected from the group consisting of periodontal disease, dental caries, bad breath, stomatitis, oral cancer, blood disease, cardiovascular disease, metabolic disorder, respiratory disease, osteoporosis, and necrotic soft tissue disease. 8. The diagnostic agent according to any one of 1 to 7.
The diagnostic agent according to any one of claims 1 to 8, wherein the disease is selected from the group consisting of periodontal disease and bad breath.
The diagnostic agent according to any one of claims 1 to 9, wherein the concentration of methyl mercaptan is 20 ppb or less.
The diagnostic agent according to any one of claims 1 to 10;
A medium capable of absorbing or contacting the test sample;
Instructions for use;
A kit comprising an appropriate base; and an appropriate pharmaceutically acceptable additive.
The kit according to claim 11, wherein the test sample is breath or oral fluid.
The kit according to claim 11 or 12, wherein the medium is a buffer solution, air, activated carbon, or a solid film.
formula:

A diagnostic agent comprising a compound represented by
Activated carbon treated with potassium hydroxide;
Instructions for use;
A kit comprising, optionally, tetramethylguanidine; and, optionally, a pharmaceutically acceptable additive.
(I) a step of bringing the diagnostic agent according to any one of claims 1 to 10 into contact with a test sample in the presence of a base as appropriate; and (ii) a change in fluorescence color by irradiating ultraviolet rays using a UV device. A method for diagnosing a disease that causes an increase in oral methyl mercaptan concentration in a subject, comprising the step of examining methyl mercaptan in a test sample by examining the degree of the above.