JP3388141B2 - Oxamil hapten compounds, antibodies and methods of measurement - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to N, N-dimethyl-2-methylcarbamoyloxyimino-2- (methylthio) acetamide (hereinafter referred to as “oxamyl” in the present specification).
Said) hapten compounds, antigens, antibodies and fragments thereof.

The present invention further relates to an immunological assay method using the above-mentioned antigen, antibody and fragment thereof.

[0003]

Oxamyl is represented by the following formula (5):

[Chemical 6] It is a carbamate insecticide having a structure represented by: The carbamate insecticide is a group of compounds having an insecticidal action, which does not contain Cl or P as a constituent element of the compound and is composed of C, H, O and N. The main carbamate insecticides are structurally classified into three types. Firstly, naphthyl or substituted phenyl-N-methyl type, secondly heterocyclic N, N-disubstituent type, and thirdly oxime (-C = N
It is an oxime type having a -O-) bond in the molecule.

Of these, oxamyl is an oxime-type carbamate insecticide and is used as an agricultural chemical having an insecticidal and nematicidal action. It has a unique osmotic and insecticidal action that the drug, which is highly water-soluble, is applied to the aerial part of the crop and moves to the underground part to exert nematicidal power. Oxamyl is effective against thrips (including Thrips palmi), aphids, and nematodes by contact and normal osmotic insecticidal activity, and by being absorbed from underground and aboveground (Pesticide Handbook No. 62). P.-p. 79).

In recent years, there has been great social interest in residual pesticide residues in the environment such as soil, water, and the atmosphere, and in recent years, particularly in post-harvest pesticide residues of imported agricultural products. Regarding oxamyl, based on the Food Sanitation Law, the residual standard values are cereals (0.02 ppm) and fruits (0.5-5.
0 ppm), vegetables (0.05-5 ppm), beans and potatoes (0.1 ppm), etc. ("Latest pesticide residue analysis method", above). In order to ensure the safety of the environment and foods, it is necessary to measure the amount of oxamyl contained in them quickly and accurately.

[0006] Conventionally, oxamyl has been analyzed by gas chromatography, high performance liquid column chromatography (HPLC), etc. after extraction and purification from samples of fruits, vegetables and the like. For example, a method has been adopted in which a sample is extracted with acetone, purified by Florisil column chromatography, and then analyzed by gas chromatography.
These methods have problems that the sample preparation is complicated and requires a lot of labor and time, has a skill in analysis, and requires a high cost for measuring devices and equipment. Oxamyl measurement needs to produce analysis results of a huge number of samples in a short time, especially in the analysis of pesticide residues such as imported agricultural products, which is not only accurate but also easy, quick and economical. There is a demand for new measurement methods that are provided.

The immunological measurement method is a method for detecting an antigen based on an antigen-antibody reaction in which the antibody specifically recognizes the antigen, and in recent years, due to its excellent accuracy, convenience, rapidity and economical efficiency. It is getting attention. In immunoassays, a wide variety of labels have been applied as detection methods, such as enzymes, radiotracers, chemiluminescent or fluorescent substances, metal atoms, sols, latices and bacteriophages.

Among immunological measurement methods, the enzyme immunoassay method (EIA) using an enzyme has come to be widely used as a particularly excellent method. An excellent review of enzyme immunoassays is available in Tijssen P, “Practice and theory of e
nzyme immunoassays "in Laboratory techniques in bi
ochemistry and molecular biology, Elsevier Amsterd
am New York, Oxford ISBN 0-7204-4200-1 (1990).

Generally, for a molecule having a large molecular weight,
By inoculating animals without further modification,
An antibody that recognizes an antigen can be produced by inducing an appropriate immune reaction. However, low molecular weight compounds such as oxamyl cannot normally elicit an immune response when inoculated into animals. These molecules act as a group of epitopes for the first time by binding to a high molecular compound having immunogenicity, and elicit an immune response in the presence of T cell receptors, resulting in the production of antibodies by a group of B lymphocytes. To be done. Molecules that produce immunogenicity only when bound to a high molecular compound in this way are collectively called "haptens".

However, even when a low-molecular compound bound to a high-molecular compound is used as an antigen, the obtained antibody often does not recognize the desired molecule or has a very low affinity. Therefore, it is generally necessary to use not the low molecular weight compound itself but the one having a spacer arm (bonding hand) introduced together with a functional group that can be used for binding as a hapten. However, in that case, a preferable antibody cannot be obtained unless an appropriately introduced one is used in consideration of all problems such as the bond / functional group arrangement and the size of the bond. Appropriate introduction must be devised according to each molecule.

Japanese Unexamined Patent Publication No. 8-231591 discloses a general method for producing an antibody against a carbamate compound. However, with respect to oxamyl, only the compound having na = 3 in the above formula (1) was synthesized (Example 4). Thus, despite its very high need, oxamyl antibodies have not been obtained prior to the present invention.

[0012]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a derivative of a hapten compound which constitutes an antigen for producing a novel antibody having reactivity with oxamyl.

Another object of the present invention is to provide a conjugate of the oxamyl derivative with a polymer compound or a labeling substance. The conjugate becomes an antigen for producing an antibody reactive with oxamyl.

Another object of the present invention is to provide a novel antibody or fragment thereof having a strong affinity for oxamyl, and a method for producing the same. In the present specification, the "fragment" of the antibody means a part of the antibody capable of binding to the antigen, such as a _Fab fragment.

The present invention, in one aspect thereof, provides a monoclonal antibody reactive with oxamyl.

A further object of the present invention is to provide a hybridoma which produces the above-mentioned antibody or fragment thereof.

The present invention further aims at providing an immunological assay method for oxamyl, which comprises using the above-mentioned antibody or a fragment thereof.

[0018]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a derivative of oxamyl in which a functional group that can be used for bonding with a spacer arm and a polymer is introduced into oxamyl is used as a hapten. As a result, an antibody reactive with the compound was successfully obtained, and the present invention was completed.

Oxamyl which is the object of the present invention has the following formula (5):

[Chemical 7] Is a compound represented by.

Derivatives used as haptens for antibody production are represented by the following formulas (1)-(4):

[Chemical 8] [In the formulas (1) to (4), R ₁ is, if desired, 1 or 2 halogen atoms (in the present specification, the halogen atom is F,
Cl, Br or I) or carbon number 1-3
The following formula (R _1a ) _optionally substituted with an alkyl group of
-(R _1d ):

[Chemical 9] R ₂ has a structure selected from the group consisting of:
H ₂ ) _nc , CH ₂ CONH (CH ₂ ) _nd , or CO (C
H ₂ ) _ne , where nc is an integer of 1-3, nd is an integer of 1-5, ne is an integer of 1-5; R ₃ is a carbon number of 1-6, preferably Is an alkyl group of 1-3, na is an integer of 5-15; and nb
Is an integer of 1-10].

In the compound of formula (2), R ₁ is covalently bonded to the adjacent carbon atom or nitrogen atom by 1,2-position substitution, 1,3-position substitution or 1,4-position substitution on the 6-membered ring. . Preferably, when R ₁ is a cyclohexane ring, 1,3
Position substitution or 1,4-position substitution, and in the case of a benzene ring, it is 1,4-position substitution.

In the compound of formula (3), nc is preferably 1.

Derivatives (1)-(4) in which a spacer arm and a functional group usable for binding are bound to oxamyl (or a portion thereof) of the present invention are used as haptens and those bound to a suitable polymer compound are used as an antigen. By using it, an antibody reactive with oxamyl can be obtained.

The present invention relates to the hapten compound, a conjugate of a hapten compound and a polymer compound, an antibody that reacts with oxamyl and a method for producing the same, and a method for immunologically measuring oxamyl using the hapten compound or the antibody.

Preparation of Oxamyl Derivative The oxamyl derivative represented by any of the formulas (1) to (4) can be prepared by a known method.
For example, there is a method described below.

The oxamyl derivative represented by any one of the formulas (1) to (4) is obtained by removing a carboxyl protecting group from an ester compound in which a carboxyl protecting group is covalently bonded to the carboxyl terminal of each compound by a known method. It can be manufactured by

The carboxyl protecting group may be a known one,
Specific examples thereof include a methyl group, an ethyl group, a benzyl group, a 4-methoxybenzyl group, a 3,4-dimethoxybenzyl group, a tert-butyl group, a phenyl group, and a tert-group.
Examples thereof include a butyldimethylsilyl group, a tert-butyldiphenylsilyl group, a triisopropylsilyl group and a 2-trimethylsilylethoxy group.

The protective group for the carboxyl group can be deprotected in the presence of acid or alkali. A lower alkyl group such as a methyl group and an ethyl group, and a benzyl group, a phenyl group and the like are mixed with water and an organic solvent such as methanol, ethanol and THF in a mixed solution of lithium hydroxide, sodium hydroxide, potassium hydroxide and the like. 60 ° C in the presence of alkali
It can be removed by stirring at 0 ° C., preferably 0 ° C. to 10 ° C., 5 minutes to 20 hours, preferably 5 minutes to 1 hour. Methyl group, benzyl group, 4-methoxybenzyl group,
3,4-methoxybenzyl group, tert-butyl group, te
rt-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triisopropylsilyl group, 2-trimethylsilylethoxy group etc. are dichloromethane, 1,2-
In a suitable organic solvent such as dichloroethane, ethyl acetate, benzene, toluene, acetonitrile, THF, diethyl ether, mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, etc., or organic acids such as acetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, etc. It can be removed by stirring in the presence of −10 ° C. to 60 ° C., preferably 0 ° C. to 25 ° C., 1 hour to 20 hours, preferably 2 hours to 5 hours. Further, benzyl group, 4-methoxybenzyl group, and 3,4-methoxybenzyl group are subjected to hydrogenolysis, tert-butyldimethylsilyl group, tert-butyldiphenylsilyl group, triisopropylsilyl group, silyl group such as 2-trimethylsilylethoxy group. Can be removed with pyridinium fluoride, tetrabutylammonium fluoride (TBAF) or the like.

I. Described in formula (1), (2) or (4)
Of the compound represented by the formula (1), (2) or (4) is, for example, a compound represented by the following formula (6):

[Chemical 10] [In formula (6), R ₄ is —SCH ₃ or —CH ₂ SR ₃ (wherein R ₃ is as defined in formula (4)); R ₅ is —CON (CH ₃ ) ₂ or -C (C
H ₃ ) ₃ ; R ₆ is (CH ₂ ) _na , R ₁ or (C
H ₂ ) _nb (wherein na, nb and R ₁ are as defined in formula (1), (2) or (4)); and R ₇ is a carboxyl protecting group] It can be produced by removing the protecting group for the carboxyl group represented by R ₇ from the ester compound.

As the method for producing the ester compound represented by the general formula (6), a method of reacting a mixture of an amino acid derivative and an oxime with a carbonylating agent (A), a method of reacting a carbonic acid ester derivative of the oxime with an amino acid ester. A known method such as (B) or a method (C) of reacting a carbamate derivative of an amino acid ester with an oxime can be used.

[0031](A) Mixture of amino acid derivative and oxime
Of reacting carbonyl with a carbonylating agent This method is used to produce an ester compound of formula (6)
If the following formula (7):

[Chemical 11] In the oxamyl oxime represented by the formula (7), R ₄ and R ₅ are as defined above, or the oxime of the analog thereof, the formula (8):

[Chemical 12] An amino acid ester derivative having a carboxyl group protected by a protecting group, represented by the formula (8), R ₆ and R ₇ are as defined above, is bonded via a carbonyl group.

The reaction is carried out by mixing the oxime of formula (7) and the amino acid ester derivative of formula (8) in a suitable solvent such as dichloromethane, 1,2-dichloroethane, benzene, toluene, THF, diethyl ether, ethyl acetate, acetonitrile and the like. , Phosgene, trichloromethyl chloroformate (TC
F), triphosgene, a carbonylating agent such as N, N-carbonyldiimidazole, and a basic catalyst such as triethylamine or pyridine in the presence of −10 ° C. to 60 ° C.
℃, preferably minus 0 ℃ to 30 ℃, 1 hour to 2
It is carried out by stirring for 0 hour, preferably for 2 hours to 5 hours.

The amino acid ester represented by the formula (8) is
For example, the following equation (9):

[Chemical 13] It can be obtained by introducing a protecting group R ₇ into the carboxyl group of the free amino acid represented by [in the formula (9), R ₆ is as defined above], and is known as a synthetic method thereof. Many methods are known. For example, in a solution or suspension of an alcohol corresponding to an amino acid, hydrogen chloride, p
-A method of dehydration condensation by adding an acid catalyst such as toluenesulfonic acid, an acid chloride obtained by reacting an amino acid with thionyl chloride or oxalyl chloride, etc. without solvent or with a suitable solvent such as benzene, toluene, dichloromethane, 1,2-dichloroethane Method of reacting with alcohol in the presence of solvent (Experimental Chemistry Course 22, pages 214-227,
The Chemical Society of Japan, Maruzen Co., Ltd.). Further, after protecting the amino group of the amino acid with a suitable protecting group, dichloromethane, 1,2-dichloroethane, tetrahydrofuran (THF), diethyl ether, benzene, toluene, xylene, N, N-dimethylformamide (DM
F) in the presence of a suitable solvent such as N, N-dicyclohexylcarbodiimide (DCC), N, N-diisopropylcarbodiimide (DIPC), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC) A method of dehydration condensation with an alcohol using a carbodiimide condensing agent or an azodicarboxylic acid ester such as triphenylphosphine and diethyl azodicarboxylate, a method of dehydration condensation using triphenylphosphine and 2,2′-dipyridyl disulfide, or BOP reagent,
The protecting group of the amino acid derivative obtained by the method of synthesizing an ester using another condensing agent such as DPPA reagent (Experimental Chemistry Course 22, p. 43-51, edited by The Chemical Society of Japan, Maruzen Co., Ltd.) is removed. Another method is to obtain an amino acid ester. Alternatively, a method of removing the protecting group of the amino acid derivative obtained by a method of reacting with an alkyl halide in the presence of a base can be used. Amino acid te
For rt-butyl ester, dichloromethane, 1,
Amino acid derivative obtained by stirring amino acid protected amino acid and isobutene in the presence of phosphoric acid, sulfuric acid, boron trifluoride diethyl ether complex, etc. in a suitable solvent such as 2-dichloroethane, benzene, toluene, THF, etc. The method of obtaining by removing the protecting group of is also mentioned.

Examples of the amino group-protecting group include a benzyloxycarbonyl group (Z group), a tert-butoxycarbonyl group (Boc group) and a 9-fluorenylmethoxycarbonyl group (Fmoc group). Examples of the method for removing the protecting group include a method by hydrogenolysis, a method using a mineral acid such as hydrochloric acid and sulfuric acid, an organic acid such as trifluoroacetic acid, a method using a base such as pyridine and piperidine, and the like.

(B) Carbonic acid ester derivative of oxime
Method of reacting mino acid ester When the ester compound of formula (6) is produced using this method, the following formula (10):

[Chemical 14] [In the formula (10), Y represents a hydrogen atom or a substituent of a phenyl group, preferably a nitro group, a methanesulfonyl group and the like; and R ₄ and R ₅ are as defined above. ] The carbonic acid ester derivative of oxime represented by the above formula is reacted with the amino acid ester of the above formula (8).

The reaction is carried out by using triethylamine, diisopropylethylamine, 1,8-diazabicyclo [5.4.0] in an organic solvent such as dichloromethane, 1,2-dichloroethane, ethyl acetate, benzene, toluene, acetonitrile, THF and diethyl ether. Undec-7-en (DB
U) In the presence of a base such as 1,5-diazabicyclo [4.3.0] non-5-ene (DBN) and 1,4-diazabicyclo [2.2.2] octane (DABCO), from -10 ° C. It is carried out by stirring at 50 ° C., preferably 0 ° C. to 25 ° C., 1 hour to 20 hours, preferably 2 hours to 5 hours.

The carbonic acid ester derivative of the oxime represented by the formula (10) includes, for example, the oxime represented by the above formula (7) and commercially available phenyl chlorocarbonates, dichloromethane, 1,2-dichloroethane, ethyl acetate and benzene. Minus 10 ° C in the presence of a base such as triethylamine or diisopropylethylamine in a suitable organic solvent such as toluene, acetonitrile, THF or diethyl ether.
To 50 ° C, preferably 0 ° C to 25 ° C, 1 hour to 2
It can be obtained by stirring for 0 hour, preferably for 1 hour to 5 hours.

(C) Induction of carbamate of amino acid ester
Method of reacting conductor with oxime When the ester compound of formula (6) is produced using this method, the following formula (11):

[Chemical 15] [In the formula (11), R ₆ , R ₇ and Y are as defined above] and the carbamate derivative of the amino acid ester represented by the formula (7) is reacted with the oxime represented by the formula (7).

The reaction is carried out in an organic solvent such as dichloromethane, 1,2-dichloroethane, ethyl acetate, benzene, toluene, acetonitrile, THF, diethyl ether, etc., with triethylamine, diisopropylethylamine, 1,8-.
Diazabicyclo [5.4.0] undec-7-ene (DB
U) From -10 ° C in the presence of a base such as 1,5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO). It is carried out by stirring at 50 ° C., preferably 0 ° C. to 25 ° C., 1 hour to 20 hours, preferably 2 hours to 5 hours.

The carbamate derivative of the amino acid ester represented by the formula (11) is obtained by, for example, adding the amino acid ester represented by the formula (8) and commercially available phenyl carbonates to dichloromethane, 1,2-dichloroethane, ethyl acetate,
Triethylamine in a suitable organic solvent such as benzene, toluene, acetonitrile, THF, diethyl ether,
It can be obtained by stirring in the presence of a base such as diisopropylethylamine at -10 ° C to 50 ° C, preferably 0 ° C to 15 ° C, 1 hour to 20 hours, preferably 1 hour to 3 hours.

II. Method for producing compound described in formula (3)
Example of Method The compound described in formula (3) can be prepared, for example, by the following formula (1
2):

[Chemical 16] It can be produced by removing the protecting group for the carboxyl group represented by R ₇ from the ester compound represented by [in the formula (12), R ₂ and R ₇ are as defined above].

(A) When R ₂ = (CH ₂ ) _{nc In} the formula (12), the compound of R ₂ = (CH ₂ ) _nc is
For example, the oxamyl oxime of the above formula (7) and the following formula (13):

[Chemical 17] [In the formula (13), X is a halogen atom; and nc
And R ₇ are as defined above] can be obtained by a known method such as a method of reacting with a halocarboxylic acid ester represented by

The reaction is carried out, for example, with dichloromethane, 1,2
-Dichloroethane, ethyl acetate, benzene, toluene,
Inorganic bases such as potassium carbonate and sodium carbonate in organic solvents such as acetonitrile, THF and diethyl ether,
Alternatively, in the presence of an organic base such as triethylamine or diisopropylethylamine, 0 to 100 ° C., preferably 5
It is carried out by stirring at 0 to 80 ° C. for 1 to 20 hours, preferably 1 to 3 hours.

[0044] _{(B) R 2 = CH 2} CONH (CH 2) a _nd
In the case where R ₂ ═CH ₂ CONH (CH ₂ ) _nd in the formula (12), the compound represented by the following formula (14):

[Chemical 18] And an oxamyl oxime acetic acid derivative represented by the following formula (15):

[Chemical 19] It can be produced by a known method such as a method of dehydration-condensing the amino acid ester represented by [in the formula (15), nd and R ₇ are as defined above].

When the dehydration condensation reaction is used, the oxamyl oxime acetic acid derivative of the formula (14) and the amino acid ester of the formula (15) can be prepared according to the method described above. The dehydration condensation is carried out by subjecting the oxamyl oxime acetic acid derivative and the amino acid ester to DC in the presence of a suitable solvent such as dichloromethane, 1,2-dichloroethane, THF, diethyl ether, benzene, toluene, xylene and DMF.
A method using a carbodiimide-based condensing agent such as C, DIPC, EDC, or an azodicarboxylic acid ester such as triphenylphosphine and diethyl azodicarboxylate, a method using triphenylphosphine and 2,2′-dipyridyl disulfide, or a BOP reagent , DPP
Method using other condensing agents such as A reagent (Experimental Chemistry Course 2
2, p. 43-51, edited by The Chemical Society of Japan, Maruzen Co., Ltd.) and the like.

(C) When R ₂ = CO (CH ₂ ) _{ne In} the formula (12), R ₂ = CO (CH ₂ ) _ne is obtained, for example, by dehydration condensation of dicarboxylic acid monoester with oxamyl oxime. It can be prepared by a known method such as a method of reacting a dicarboxylic acid monoester monoacid halide or a method of reacting a dicarboxylic acid dihalide and then reacting with an alcohol.

When the method of reacting dicarboxylic acid dihalide and then reacting with alcohol is used, the reaction is
For example, dichloromethane, 1,2-dichloroethane, ethyl acetate, benzene, toluene, acetonitrile, TH
In an organic solvent such as F or diethyl ether, in the presence of an inorganic base such as potassium carbonate or sodium carbonate or an organic base such as triethylamine or diisopropylethylamine, minus 10 to 100 ° C, preferably 0 to 2
It is carried out by mixing oxamyl oxime, carboxylic acid chloride and then alcohols at 5 ° C. and stirring for 1 hour to 30 hours, preferably 1 hour to 5 hours.

Alternatively, compounds with ne = 2 and R ₇ = H can also be obtained, for example, by reacting oxamyl oxime with succinic anhydride. In this case, the reaction is dichloromethane, 1,2-dichloroethane, ethyl acetate,
In an organic solvent such as benzene, toluene, acetonitrile, THF, diethyl ether, etc.
0 ° C, preferably 0 to 25 ° C, 1 to 40 hours,
It is preferably stirred for 5 to 10 hours. In this reaction, an inorganic base such as potassium carbonate or sodium carbonate or an organic base such as triethylamine or diisopropylethylamine may be allowed to coexist.

The compound obtained by the above production method is
If necessary, silica gel chromatography or recrystallization operation can be performed to obtain a highly purified product.

The preparation of the antigen and antibody of the present invention and the immunological measurement method will be described below. These can be prepared according to a known method, for example, a method described in a seminar on biochemistry experiments, immunobiochemistry research method (edited by the Japanese Biochemical Society), or the like.

Bonding of oxamyl derivative and polymer compound
Preparation of body The oxamyl derivative synthesized as described above is bound to an appropriate polymer compound and then used as an immunizing antigen.

Examples of preferable polymer compounds include keyhole limpet hemocyanin (hereinafter referred to as "KLH"), ovalbumin (hereinafter referred to as "OVA"), bovine serum albumin (hereinafter referred to as "BSA"), Rabbit serum albumin (hereinafter referred to as "RSA"), etc., but KLH
And BSA are preferred.

The bond between the oxamyl derivative and the high molecular compound is, for example, the activated ester method (AE KARU et al .: J.
Agric. Food Chem. 42 301-309 (1994)), or mixed acid anhydride method (BFErlanger et al .: J. Biol. Chem. 234 109).
0-1094 (1954)) and other known methods.

The activated ester method can generally be carried out as follows. First, a hapten compound is dissolved in an organic solvent and reacted with N-hydroxysuccinimide in the presence of a coupling agent to produce N-hydroxysuccinimide ester.

As the coupling agent, an ordinary coupling agent commonly used in condensation reaction can be used.
Dicyclohexylcarbodiimide, carbonyldiimidazole, water-soluble carbodiimide and the like are included. Examples of the organic solvent include dimethyl sulfoxide (hereinafter referred to as “D
MSO "), N, N-dimethylformamide (D
MF), dioxane and the like can be used. The molar ratio of the hapten compound and N-hydroxysuccinimide used in the reaction is preferably 1: 10-10: 1, more preferably,
It is 1: 1-1: 10, most preferably 1: 1. The reaction temperature is 0-100 ° C, preferably 5-50 ° C, more preferably 22-27 ° C, the reaction time is 5 minutes-24 hours,
It is preferably 30 minutes-6 hours, more preferably 1-2 hours. The reaction temperature can be a temperature above the melting point and below the boiling point.

After the coupling reaction, the reaction solution is centrifuged, and the supernatant solution is added to the solution in which the polymer compound is dissolved to cause a reaction.
For example, when the polymer compound has a free amino group, an acid amide bond is formed between the amino group and the carboxyl group of the hapten compound. The reaction temperature is 0-60 ° C, preferably 5-40 ° C, more preferably 22-27 ° C,
The reaction time is 5 minutes to 24 hours, preferably 1 to 16 hours,
More preferably, it is 1-2 hours. The reaction product can be purified by dialysis, a desalting column or the like to obtain a conjugate of the oxamyl derivative and the polymer compound.

On the other hand, the mixed acid anhydride used in the mixed acid anhydride method is obtained by the usual Schotten-Baumann reaction, and by reacting this with a polymer compound, the desired hapten-polymer compound conjugate is obtained. Manufactured. The Schotten-Baumann reaction is carried out in the presence of a basic compound. As the basic compound, a compound commonly used in the Schotten-Baumann reaction can be used. For example, tributylamine, triethylamine, trimethylamine, N-methylmorpholine, pyridine, N, N-dimethylaniline, DBN, DBU, DA
Organic base such as BCO, potassium carbonate, sodium carbonate,
Examples thereof include inorganic bases such as potassium hydrogen carbonate and sodium hydrogen carbonate. The reaction is usually minus 20 ° C-100.
C., preferably 0.degree. C.-50.degree. C., and the reaction time is 5 minutes-10 hours, preferably 5 minutes-2 hours. The reaction between the obtained mixed acid anhydride and the polymer compound is usually carried out at -20 ° C-150 ° C, preferably 0 ° C-100 ° C, and the reaction time is 5 minutes-10 hours, preferably 5 minutes-5. It's time. The mixed acid anhydride method is generally performed in a solvent. As the solvent, any solvent conventionally used in the mixed acid anhydride method can be used, and specifically, dioxane, diethyl ether, tetrahydrofuran, ethers such as dimethoxyethane, halogenated halogen compounds such as dichloromethane, chloroform and dichloroethane. Hydrocarbons, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as methyl acetate and ethyl acetate, aprotic polar solvents such as N, N-dimethylformamide, dimethyl sulfoxide and hexamethylphosphoric triamide. Is mentioned. Examples of the haloformate used in the mixed acid anhydride method include isobutyl chloroformate, methyl chloroformate, methyl bromoformate, ethyl chloroformate, ethyl bromoformate and the like. The use ratio of the hapten, the haloformic acid ester and the polymer compound in the method can be appropriately selected from a wide range.

In the immunological assay method, an oxamyl derivative to which a labeling substance such as an enzyme is bound can be used in the same manner as described above. As the labeling substance, horseradish peroxidase (hereinafter, referred to as “HR
P ”), enzymes such as alkaline phosphatase, fluorescein isothiocyanate, color-forming substances such as rhodamine, radioactive substances such as ³² P and ¹²⁵ I, and chemiluminescent substances.

Preparation of Polyclonal Antibody The conjugate of the oxamyl derivative and the polymer compound can be used to prepare the polyclonal antibody of the present invention by a conventional method. For example, an oxamyl derivative-KLH conjugate dissolved in a phosphate buffer (hereinafter referred to as "PBS") and mixed with Freund's complete or incomplete adjuvant or an auxiliary agent such as alum is used as an immunizing antigen. This is done by immunizing the animal. As the animal to be immunized, any animal commonly used in the art can be used, and examples thereof include mouse, rat, rabbit, goat, horse and the like.

The administration method for immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, subcutaneous injection and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred. The administration can be performed once or at appropriate intervals, preferably multiple times at intervals of 1 to 5 weeks.

Blood can be taken from the immunized animal and the serum separated therefrom can be used to assess the presence of polyclonal antibodies that react with oxamyl.

Preparation of Monoclonal Antibody The conjugate of the oxamyl derivative and the polymer compound can be used to prepare the monoclonal antibody of the present invention by a known method.

When preparing a monoclonal antibody,
At least the following work steps are required. (A) Preparation of a conjugate of an oxamyl derivative used as an immunizing antigen and a polymer compound (b) Immunization of animals (c) Collection of blood, assay, and preparation of antibody-producing cells (d) Preparation of myeloma cells (E) Cell fusion of antibody-producing cells and myeloma cells and selective culture of hybridomas (f) Screening and cell cloning of hybridomas producing the desired antibody (g) Monoclonal culture of hybridomas or transplantation of hybridomas into animals Preparation of antibody (h) Measurement of reactivity of prepared monoclonal antibody, etc.

A conventional method for producing a hybridoma producing a monoclonal antibody is described in, for example, Hybridoma Techniques, Cold.
Spring Harbor Laboratory
Or Laboratory), 1980 edition), Cell histochemistry (Shuji Yamashita et al., edited by The Society for Tissue and Cell Chemistry;
Year).

Hereinafter, a method for producing the above-described monoclonal antibody against oxamyl of the present invention will be described, but it will be apparent to those skilled in the art that the method is not limited thereto.

The steps (a)-(b) can be carried out by almost the same method as described for the polyclonal antibody.

The antibody-producing cells in step (c) are lymphocytes, which can be generally obtained from the spleen, thymus, lymph nodes, peripheral blood, or a combination thereof, but splenocytes are most commonly used. . Therefore, after the final immunization, a site where antibody-producing cells are present, for example, the spleen is excised from a mouse in which antibody production is confirmed, and splenocytes are prepared.

The myeloma cells that can be used in the step (d) include, for example, Balb / c mouse-derived myeloma cell line P3 / X63-Ag8 (X63) (Natur).
e, 25 6, 495-497 (1975 )), P3 / X63-Ag8. U
1 (P3U1) (Current Topics.in Microbiology and
Immunology, 81 1-7 (1987)), P3 / NSI-1-A
g4-1 (NS-1) (Eur.J.Immunol., 6 , 511-519
(1976)), Sp2 / 0-Ag14 (Sp2 / 0) (Natu
re 276 , 269-270 (1978)), FO (J. Immuno.Meth., 3
5 , 1-21 (1980)), MPC-11, X63.653, S
Myeloma cell line such as 194, or 21 derived from rat
0. RCY3. Ag1.2.3. (Y3) (Nature 277 , 1
31-133, (1979)) and the like can be used.

The above-mentioned cell lines are subcultured in Dulbecco's modified Eagle's medium (DMEM) or Iscove's modified Dulbecco's medium (IMDM) containing fetal bovine serum to secure a cell number of about 3 × 10 ³ or more on the day of fusion. .

The cell fusion in the step (e) is a known method, for example, the method of Milstein et al. (Methods in
Enzymology, 73 , 3 (1981)) and the like. The most commonly used method at present is to use polyethylene glycol (PEG). Regarding the PEG method, for example, cell histochemistry, Shuji Yamashita et al. (Supra)
It is described in. As another fusion method, a method by electroprocessing (electrofusion) can be adopted (Etsuko Okouchi et al., Experimental Medicine 5.1315-15, 198).
7). Other methods can also be adopted as appropriate. The ratio of cells to be used may be the same as in the known method, and for example, splenocytes may be used about 3-10 times as much as myeloma cells.

The selection of the hybridoma group in which the splenocytes and the myeloma cells are fused to obtain the antibody-producing ability and the proliferating ability can be selected, for example, when the hypoxanthine-anidin phosphoribosyltransferase-deficient strain is used as the myeloma cell line. It can be performed by using a HAT medium prepared by adding hypoxanthine / aminopterin / thymidine to DMEM or IMDM.

In the step (f), a part of the culture supernatant containing the selected hybridoma group is taken and, for example, E
The antibody activity against oxamyl is measured by the LISA method.

Further, cell cloning of the hybridoma, which was found to produce an antibody reactive with oxamyl by the measurement, is carried out. For this cell cloning method,
Method of diluting so that one hybridoma is contained in one well by limiting dilution "Limiting dilution method"; Method of picking colonies on soft agar medium; Method of taking out one cell by micromanipulator; Method by cell sorter The "sorter clone method" for separating cells of the above is mentioned. The limiting dilution method is simple and often used.

Regarding the wells in which the antibody titer is recognized, those in which the cell titer is stably obtained by repeating the cell cloning 1 to 4 times by the limiting dilution method, for example, are selected as the anti-oxamyl monoclonal antibody-producing hybridoma strain. Examples of the medium for culturing hybridomas include:
DMEM or IMDM containing 10% fetal bovine serum is used. Hybridomas are preferably cultivated, for example, at a carbon dioxide concentration of about 5-7% and at 37 ° C. (in a thermostat in 100% humidity).

The large-scale culture for preparing the antibody in the step (g) is carried out by a follow-fiber type culture device or the like. Alternatively, a mouse of the same strain (for example, Bal described above)
It is also possible to grow the hybridoma in the abdominal cavity of b / c) or Nu / Nu mouse and prepare the antibody from the ascites fluid.

The culture supernatant or ascites fluid obtained as described above can be used as an anti-oxamyl monoclonal antibody. Further, dialysis, salting out with ammonium sulfate, gel filtration, lyophilization, etc. are carried out to collect the antibody fractions. An anti-oxamyl monoclonal antibody can be obtained by purification. When a higher degree of purification is required, it can be carried out by combining commonly used methods such as ion exchange column chromatography, affinity chromatography, and high performance liquid chromatography (HPLC).

The subclass, antibody titer, etc. of the anti-oxamyl monoclonal antibody obtained as described above can be determined by using a known method such as the ELISA method described later.

Measurement of Oxamyl by Antibody As a method of measuring oxamyl by the antibody used in the present invention, a radioisotope immunoassay method (RIA method), EL
ISA method (Engvall, E., Methods in Enzymol., 70 , 41
9-439 (1980)), fluorescent antibody method, plaque method, spot method, agglutination method, Ouchterlony
Various methods generally used for the detection of antigens, such as the method ("Hybridoma Method and Monoclonal Antibody", published by R & D Planning Co., Ltd., pages 30-53, Showa 5
March 5, 7). The ELISA method is widely used from the viewpoint of sensitivity, simplicity, and the like.

Oxamyl can be measured by the following procedure, for example, by the indirect competitive inhibition ELISA method among various ELISA methods. (A) First, a conjugate of an oxamyl derivative as an antigen and a polymer compound is immobilized on a carrier. (B) The solid phase surface on which the antigen is not adsorbed is blocked with an antigen, for example, a protein unrelated to the antigen. (C) A sample containing various concentrations of oxamyl and an antibody are added thereto, and the antibody is allowed to react competitively with the solid-phased antigen and free oxamyl to form a solid-phased antigen-antibody complex and a free oxamyl-antibody complex. Generate the body. (D) By measuring the amount of the immobilized antigen-antibody complex, the amount of free oxamyl in the sample can be determined from the calibration curve prepared in advance.

In step (a), the carrier on which the antigen is immobilized is not particularly limited, and any carrier commonly used in the ELISA method can be used. For example, a 96-well microtiter plate made of polystyrene can be mentioned.

To immobilize the antigen on the carrier, for example,
A buffer solution containing the antigen may be placed on the carrier and incubated. Known buffers can be used, and examples thereof include Dulbecco's phosphate buffer. The concentration of the antigen in the buffer solution can be selected from a wide range, but is usually about 0.01-100 μg / ml, preferably 0.05-5 μg / ml. When a 96-well microtiter plate is used as a carrier, 300 μl / well or less is used as 20-150 μl / well.
Wells are preferable. Furthermore, the incubation conditions are not particularly limited, but usually overnight incubation at about 4 ° C. is suitable.

As the antigen immobilized on the carrier,
Not only the conjugate of the oxamyl derivative used for antibody preparation and the polymer compound, but also the conjugate of another derivative represented by any one of formulas (1) to (4) and the polymer compound can be used. . For example, when an antibody is prepared using a conjugate of the compound of formula (1) and a polymer compound as an antigen, a compound other than formula (1) can be used as the immobilized antigen. Further, in the compounds of the formulas (1) to (4), antigens having different numbers of na to ne can be used for antibody preparation and solid phase immobilization, respectively. Furthermore, other oxamyl-analogous compounds not included in formulas (1)-(4) can also be used as the immobilized antigen.

Blocking in the step (b) is carried out in the carrier having the antigen (the conjugate of the oxamyl derivative and the polymer compound) immobilized thereon, in addition to the oxamyl derivative portion, there is a portion to which an antibody to be added later can be adsorbed. In some cases, it is done with the sole purpose of preventing it. As the blocking agent, for example, BSA or skim milk solution can be used.
Or, Block Ace (“Block Ace”,
Code No. manufactured by Dainippon Pharmaceutical Co., Ltd. Commercially available blocking agents such as UK-25B) can also be used. Specifically, but not limited to, for example, a buffer solution containing a blocking agent [for example, 85 mM borate buffer solution (pH 8.0 containing 1% BSA and 60 mM NaCl) added to the portion on which the antigen is immobilized. )] Is added thereto, the mixture is incubated at about 4 ° C.-room temperature for 1 hour-5 hours, and then washed with a buffer solution. The washing solution is not particularly limited, but for example, 60 mM Na
A borate buffer solution containing Cl can be used.

Then, in the step (c), the sample containing oxamyl and the antibody are brought into contact with the solid-phased antigen, and the antibody is reacted with the solid-phased antigen and the free oxamyl to form the solid-phased antigen-antibody complex and the free solid. An oxamyl-antibody complex is formed.

At this time, as an antibody, an antibody against oxamyl of the present invention is added as a first antibody, and further an antibody against the first antibody having a labeling substance bound thereto is further added as a second antibody in order to react.

The first antibody is dissolved in a buffer and added. Although not limited, the reaction may be performed at about 37 ° C. for about 1 hour. After the reaction is completed, the carrier is washed with a buffer solution to remove unreacted primary antibody. As the cleaning liquid, for example,
A borate buffer supplemented with 60 mM NaCl can be used.

The second antibody is then added. For example, when a mouse monoclonal antibody is used as the first antibody, it is suitable to use an anti-mouse antibody-goat antibody to which an enzyme (eg, peroxidase or alkaline phosphatase) is linked. About 500-10 to the first antibody bound to the carrier
It is desirable to react with a second antibody diluted so that the final absorbance is 000 times, preferably 4 or less, more preferably 0.5-3.0. A buffer solution is used for dilution. Although not limited thereto, the reaction is performed at room temperature for about 1 hour, and after the reaction, it is washed with a buffer solution. Through the above reaction, the second antibody binds to the first antibody. Alternatively, a labeled first antibody may be used, in which case the second antibody is unnecessary.

Then, in step (d), a chromogenic substrate solution that reacts with the labeling substance of the second antibody bound to the carrier is added, and the absorbance is measured, whereby the amount of oxamyl can be calculated from the calibration curve.

When peroxidase is used as the enzyme that binds to the second antibody, for example, hydrogen peroxide is used as the substrate and 3,3 ′, 5,5′-tetramethylbenzidine or o-phenylenediamine (hereinafter "OP
"D"). But not limited to
After adding the coloring solution and reacting at room temperature for about 10 minutes, the enzymatic reaction is stopped by adding 1N sulfuric acid. Three,
When using 3 ', 5,5'-tetramethylbenzidine,
The absorbance at 450 nm is measured. When using OPD, the absorbance at 490 nm is measured. On the other hand, when alkaline phosphatase is used as the enzyme that binds to the second antibody, for example, p-nitrophenyl phosphate is used as a substrate for color development, and 2N NaOH is added to stop the enzymatic reaction.
The method of measuring the absorbance at 415 nm is suitable.

The inhibition rate is calculated as the rate of decrease in the absorbance of the solution in which oxamyl was added to react with the antibody, relative to the absorbance of the reaction solution in which free oxamyl was not added. The concentration of oxamyl in the sample can be calculated using a calibration curve prepared in advance based on the inhibition rate of the reaction solution to which a known concentration of oxamyl has been added.

According to the above-mentioned indirect competitive inhibition ELISA method, the monoclonal antibodies OXM H3 and OXM of the present invention are
2-3, OXM3-2 and OXM6-8 each gave about 1.00 oxamyl by the indirect competitive inhibition ELISA method.
0-10,000 ng / ml, about 100-1,000 ng
/ Ml, about 100-1,000 ng / ml and about 10
It can be measured in the range of -100 ng / ml (Example 18).

Alternatively, the measurement of oxamyl can be carried out, for example, by the direct competitive inhibition ELISA method using the monoclonal antibody of the present invention as described below. (A) First, the monoclonal antibody of the present invention is immobilized on a carrier. (B) The surface of the carrier on which the antibody has not been immobilized is blocked with an antigen-independent, for example, protein. (C) Separately from the above step, a mixture is prepared by adding an enzyme-linked hapten obtained by binding an oxamyl derivative and an enzyme to a sample containing various concentrations of oxamyl. (D) The mixture is reacted with the antibody-immobilized carrier. (E) The amount of oxamyl in the sample is determined from the calibration curve prepared in advance by measuring the amount of the immobilized antibody-enzyme-bound hapten complex.

There is no particular limitation on the carrier for immobilizing the monoclonal antibody in the step (a), and the ELISA is used.
What is routinely used in the method can be used, and examples thereof include a 96-well microtiter plate.
The immobilization of the monoclonal antibody can be carried out, for example, by placing a buffer solution containing the monoclonal antibody on the carrier and incubating. The composition and concentration of the buffer solution may be the same as those used in the indirect competitive inhibition ELISA method described above. Alternatively, an amino group-bonded microtiter plate to which an antibody has been bound by a chemical binding method can also be used.

The blocking in the step (b) may occur because, in the carrier on which the antibody is immobilized, there is a portion where the oxamyl and the enzyme-bound hapten in the sample to be added later are adsorbed regardless of the antigen-antibody reaction. , To prevent it. The blocking agent and the method therefor can be the same as those used in the indirect competitive inhibition ELISA method described above.

The enzyme-bound hapten used in step (c) may be prepared by any method without particular limitation as long as it is a method for binding an oxamyl derivative to an enzyme. For example, the above-mentioned activated ester method can be adopted. The prepared enzyme-linked hapten is mixed with a sample containing oxamyl.

As the hapten to be bound to a labeling substance such as an enzyme, not only the oxamyl derivative itself used for antibody preparation but also the formula (1) or Other derivatives represented by (4), and other oxamyl-analogous compounds not included in formulas (1) to (4) can also be used.

In the step (d), the mixture is brought into contact with the antibody-immobilized carrier, and a complex inhibitory reaction between oxamyl and the enzyme-bound hapten in the mixture produces a complex between these and the immobilized antibody. The sample containing oxamyl should be diluted with an appropriate buffer before use. But not limited to
The reaction is performed, for example, at room temperature for about 1 hour. After completion of the reaction, the carrier is washed with a buffer solution to remove unreacted enzyme-bound hapten. As the washing solution, for example, a borate buffer solution containing 60 mM NaCl can be used.

Further, in the step (e), a chromogenic substrate solution that reacts with the enzyme of the enzyme-bound hapten is added in the same manner as in the indirect competitive inhibition ELISA method described above, and the absorbance is measured to calculate the amount of oxamyl from the calibration curve. You can

Monoclonal antibody OXM6-8 of the present invention
Is about 0.5-500 ng / ml, preferably 8-250 ng of oxamyl by direct competitive inhibition ELISA.
/ Ml can be measured (Examples 23 and 24, Figures 2 and 3).

Methanol Tolerance of the Antibody of the Present Invention Monoclonal Antibody OXM6-8 which is an Embodiment of the Present Invention
Furthermore, according to the above direct competitive inhibition ELISA method, oxamyl can be recognized in a concentration-dependent manner in the presence of methanol at a concentration of about 0-20% (Example 25). Considering that oxamyl is easily soluble in an organic solvent and the analysis is generally carried out in an organic solvent such as methanol, such characteristics of the monoclonal antibody of the present invention are very effective.

Cross-Reactivity of the Antibody of the Present Invention The cross-reactivity of the monoclonal antibody of the present invention can be examined by the above-mentioned direct competitive inhibition ELISA method or indirect competitive inhibition method. OXM6-8 is, for example, mesomil, which is a related compound in the direct competitive inhibition ELISA method,
Cross-reacts with butocarboxime and alanicalb at 0.3% or less. (Example 26, Table 1).

Hereinafter, the present invention will be described in detail with reference to Examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily make modifications and changes to the present invention based on the description of the present specification, and those are included in the technical scope of the present invention.

[0103]

EXAMPLES Production Example Synthesis of Oxamyl Derivative-C

[Chemical 20]

4- (benzyloxycarbonylamino)
Synthesis of butyric acid (2) 28 g (270 mmol) of 4-aminobutyric acid (1) was added to 2
It was dissolved in 140 ml of a specified aqueous sodium hydroxide solution and ice-cooled. While vigorously stirring the solution, benzyl chloroformate (70 g, 410 mmol) and 200 ml of a 2N aqueous sodium hydroxide solution were alternately added dropwise in three times. After the completion of the dropping, the mixture was stirred for 30 minutes at ice-cooling temperature, and further stirred overnight at room temperature. After unreacted benzyl chloroformate was extracted with ice-cooled diethyl ether (150 ml × 3 times), concentrated hydrochloric acid was added under ice-cooling to adjust the pH to about 2. The mixture was extracted with dichloromethane (150 ml × 4 times), the collected dichloromethane extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. Dissolve the concentrate in a small amount of ethyl acetate,
White crystals precipitated by adding n-hexane thereto were collected by suction filtration to obtain a target product (2) (60 g, 93% yield).

4- (benzyloxycarbonylamino)
Synthesis of tert-butyl butyrate (3) 28 g (118 mmol) of 4- (benzyloxycarbonylamino) butyric acid (2) in dichloromethane (30
0 ml) was cooled to −16 ° C. with sodium chloride-ice, and a stream of isobutene was slowly poured into the solution for 5 minutes. Next, phosphoric acid (0.92 g, 9.4 mmol) and boron trifluoride diethyl ether complex (2.5 g, 17.7).
(mmol) was slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was allowed to stir to room temperature and continued stirring overnight.
After adding a saturated aqueous sodium hydrogen carbonate solution and stirring for 30 minutes, the mixture was extracted with dichloromethane (150 ml × 4 times). The collected dichloromethane extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was purified by silica gel column chromatography (n-hexane-ethyl acetate = 4: 1) to obtain the desired product (3) as a colorless transparent liquid (yield 31
g, yield 90%).

Of tert-butyl 4-aminobutyrate (4)
Synthesis 11.7 g (40 mmol) of 4- (benzyloxycarbonylaminobutyric acid) tert-butyl (3) in methanol (300 ml) was added with 1.0 g of palladium carbon until hydrogen absorption was completed in the presence of hydrogen. It was stirred. Next, the reaction solution was filtered and concentrated under reduced pressure, and the solvent was completely removed by a vacuum pump to obtain the target product (4) as a yellow liquid (yield 6.1 g, yield 100%).

4- (phenoxycarbonylamino) butyric acid
Synthesis of tert-butyl (5) In a dichloromethane solution (50 ml) of phenyl chlorocarbonate (8.0 g, 5 mmol), 6.0 g (3.
8 mmol) tert-butyl 4-aminobutyrate (4)
Was added dropwise to a dichloromethane solution (50 ml), and then a solution of triethylamine (5.0 g, 5 mmol) in dichloromethane (50 ml) was added dropwise. After the completion of dropping, the solution was stirred as it was until it reached room temperature, and was further stirred overnight. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction to remove sodium sulfate, and then concentrated under reduced pressure. The concentrate was dissolved in a small amount of ethyl acetate, and n-hexane was added to the concentrate to collect the precipitated white crystals by suction filtration to obtain the target product (5) (yield 7.4 g, yield 70%). ).

Synthesis of (6) 3.2 g (20 mmol) of oxamyl oxime and 4-
Tert-Butyl (benzyloxycarbonylamino) butyrate (5) (5.6 g, 20 mmol) was dissolved in 50 ml of dichloromethane, and 3.0 g (20 mmol) under ice cooling.
1,8-diazabicyclo [5.4.0] undec-7
20 ml of a dichloromethane solution of ene (DBU) was added dropwise. After stirring for 1 hour under ice cooling, the mixture was further stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 2) to obtain 3.6 g (yield 52%) of (6) as a colorless transparent liquid.

Synthesis of (7) 5.6 g (16 mmol) of ester compound (6) was added to 1
Dissolve in benzene (00 ml), trifluoroacetic acid 10 m
After adding l, the mixture was stirred at room temperature for 1 day. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (ethyl acetate) to obtain 3.9 g as a colorless transparent liquid (yield 84%).
Oxamyl derivative-C (7) of This liquid became white crystals when left in a freezer at -20 ° C for a long time. ¹ H-NMR (CDCl ₃ ) 1.86 to 2.02 (m, 2H), 2.33 (s, 3H), 2.24 to 2.58 (t, 2H), 3.10 (s, 3H). ), 3.12 (s, 3H), 3.18 to 3.49 (m, 2H), 6.04 to 6.32 (t, 1H), 7.35 to 7.72 (br, 1H).

Example 1 Synthesis of Oxamyl Derivative-1

[Chemical 21]

6- (benzyloxycarbonylamino)
Synthesis of caproic acid (2) 13.1 g (100 mmol) of 6-aminocaproic acid (1) was dissolved in 50 ml of 2N aqueous sodium hydroxide solution and cooled with ice. While vigorously stirring this solution, benzyl chloroformate (20.5 g, 120 mmol) and 60 ml of a 2N aqueous sodium hydroxide solution were alternately added dropwise twice. After the completion of the dropping, the mixture was stirred for 30 minutes at ice-cooling temperature, and further stirred overnight at room temperature. After unreacted benzyl chloroformate was extracted with ice-cooled diethyl ether (150 ml x 3 times), concentrated hydrochloric acid was added dropwise at ice-cooling temperature to adjust the pH.
Was adjusted to about 2. Dichloromethane (150 ml x 4
The collected dichloromethane extracts were dried over anhydrous sodium sulfate, the sodium sulfate was removed by suction filtration, and the filtrate was concentrated under reduced pressure. The concentrate was dissolved in a small amount of ethyl acetate, and n-hexane was added thereto. The precipitated white crystals were isolated by suction filtration to obtain the target product (2) (yield 22 g, yield 83%).

6- (benzyloxycarbonylamino)
Synthesis of tert-butyl caproate (3) A solution of 5.3 g (20 mmol) of 6- (benzyloxycarbonylamino) caproic acid (2) in dichloromethane (100 ml) was cooled to −16 ° C. with salt-ice, and then cooled. A stream of isobutene was slowly poured into the solution for 5 minutes. Then phosphoric acid (0.1 g, 1.8 mmol)
And boron trifluoride diethyl ether complex (0.37m
(1, 3 mmol) was slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was allowed to stir to room temperature and continued stirring overnight. After adding a saturated aqueous sodium hydrogen carbonate solution and stirring for 30 minutes, the mixture was extracted with dichloromethane (150 ml × 4 times). The collected dichloromethane extracts were dried over anhydrous sodium sulfate and the sodium sulfate was removed by suction filtration.
It was concentrated under reduced pressure. This concentrate was purified by silica gel column chromatography (n-hexane: ethyl acetate = 4: 1) to obtain the target product (3) as a colorless transparent liquid (amount 4.0 g, yield 63%).

[0113]Tert-Butyl 6-aminocaproate
Synthesis of (4) 4.0 g (12.5 mmol) of 6- (benzyloxy
Carbonylaminocaproic acid) tert-butyl (3)
Methanol solution (100 ml) with 0.3 g of palladium
Absorption of hydrogen is completed in the presence of hydrogen by adding mucarbon
Stirred until. Next, the reaction solution is filtered and then concentrated under reduced pressure.
The target product as a yellow liquid when the solvent is completely removed with an empty pump
(4) was obtained (yield 2.3 g, yield 100%).

Synthesis of (5) 1.4 g (7.5 mmol) of 6-aminocaproic acid t
To 50 ml of a solution of ert-butyl (4) and 2.0 (7.1 mmol) of phenyl carbonate derivative of oxamyl oxime in acetonitrile, under ice-cooling, 1.1 g (7.2).
mmol) 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in acetonitrile 10
ml was added dropwise. After stirring for 1 hour under ice-cooling, further 1 hour at room temperature
Stir for hours. The reaction solution was concentrated under reduced pressure and subjected to silica gel chromatography (n-hexane: ethyl acetate = 1: 1).
Purified in 1), 2.0 g (yield 76
%) (5) was obtained.

Synthesis of (6) 2.0 g (5.3 mmol) of the ester compound (5) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (ethyl acetate) to obtain 1.2 g (yield 71%) of oxamyl derivative-1 (6) as a transparent liquid. ¹ H-NMR (CDCl ₃ ) 1.18 to 1.82 (m, 6H), 2.20 to 2.49
(Overlap, 5H), 2.98 to 3.40 (overlap, 8
H), 5.89 to 6.21 (t, 1H), 7.50 to
7.92 (br, 1H)

Example 2 Synthesis of Oxamyl Derivative-2

[Chemical formula 22]

7- (benzyloxycarbonylamino)
Synthesis of heptanoic acid (2) 2.2 g (15.2 mmol) of 7-aminoheptanoic acid (1) was dissolved in 7.6 ml of a 2N aqueous sodium hydroxide solution and ice-cooled. While vigorously stirring this solution, benzyl chloroformate (3.1 g, 18.2 mmol) and 9.1 ml of a 2N aqueous sodium hydroxide solution were added dropwise. After the dropwise addition was completed, the mixture was stirred for 30 minutes at ice-cooling temperature and then at room temperature for 5 hours. Ice-cold diethyl ether (50 ml x
(3 times), unreacted benzyl chloroformate was extracted, and then concentrated hydrochloric acid was added under ice cooling to adjust the pH to about 2. The mixture was extracted with dichloromethane (50 ml × 4 times), the collected dichloromethane extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was dissolved in a small amount of ethyl acetate, and n-hexane was added to the concentrate to collect the precipitated white crystals by suction filtration to obtain the target product (2) (yield 3.0 g, yield 71%). .

7- (benzyloxycarbonylamino)
Synthesis of tert-butyl heptanoate (3) 3.0 g (10.8 mmol) of 7- (benzyloxycarbonylamino) heptanoic acid (2) in dichloromethane (150 ml) was cooled to −16 ° C. with salt-ice. Then, a stream of isobutene was slowly poured into the solution for 5 minutes. Next, 0.1 ml of concentrated sulfuric acid was slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was continuously stirred under ice cooling for 3 hours and further at room temperature overnight. The reaction solution was directly concentrated under reduced pressure and purified by silica gel column chromatography (n-hexane-ethyl acetate = 4: 1) to obtain the target product (3) as a colorless transparent liquid (yield 2.2 g, yield 61). %).

[0119]Tert-Butyl 7-aminoheptanoate
Synthesis of (4) 2.2 g (6.6 mmol) of 7- (benzyloxyca
Rubonylamino) tert-butyl heptanoate (3)
1.0 g of palladium in methanol solution (200 ml)
Add carbon until hydrogen absorption is complete in the presence of hydrogen.
It was stirred at. Next, the reaction solution is filtered and then concentrated under reduced pressure.
The target product as a yellow liquid when the solvent is completely removed with an empty pump.
(4) was obtained (amount 1.25 g, yield 95%).

[0120]7- (phenoxycarbonylamino) hep
Synthesis of tert-butyl titanate (5) Diphenyl chlorocarbonate (1.6 g, 10 mmol)
1.25g at ice-cold temperature in loromethane solution (50ml)
(6.2 mmol) 7-aminoheptanoic acid tert-
Add butyl (4) in dichloromethane (50 ml) dropwise.
Then, triethylamine (1.0 g, 10 mmo
A solution of l) in dichloromethane (50 ml) was added dropwise. drop
After completion of the above, the solution was cooled with ice for 1 hour and then at room temperature overnight.
Continued to stir. Wash the reaction solution with saturated saline and
Dehydrate with sodium acidate and filter with sodium sulfate by suction filtration.
After removing, it was concentrated under reduced pressure. This concentrate is purified by silica gel chromatography.
Matography (n-hexane: ethyl acetate = 19: 1)
→ 9: 1 → 4: 1) to obtain a transparent liquid 1.
7 g (yield 85%) of (5) was obtained.

Synthesis of (6) 0.9 g (20 mmol) of oxamyl oxime and 7-
(Phenyloxycarbonylamino) heptanoic acid ter
1.7 g (20 mmol) of t-butyl was dissolved in 50 ml of acetonitrile, and 0.9 g (5.9 mmo) under ice cooling.
10 ml of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in 1) in acetonitrile was added dropwise. After stirring under ice cooling for 3 hours, the reaction solution was directly concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 1 → ethyl acetate).
1.7 g (yield 83%) of (6) was obtained as a transparent liquid.

Synthesis of (7) 1.7 g (4.4 mmol) of the ester compound (6) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 2: 1 → ethyl acetate) to give 1.2 g (yield 82%) of oxamyl derivative-2 (7) as white crystals. Got ¹ H-NMR (CDCl ₃ ) 1.21 to 1.43 (m, 4H), 1.43 to 1.71 (m, 4H), 2.21 to 2.40 (overlap, 5H), 3.06 (S, 3H), 3.10 (s, 3H), 3.17 to 3.31 (m, 2H), 6.10 to 6.26 (t, 1H), 10.30 to 10.55 (br) , 1H)

Example 3 Synthesis of Oxamyl Derivative-3

[Chemical formula 23]

8- (benzyloxycarbonylamino)
Synthesis of octanoic acid (2) 5.0 g (31.4 mmol) of 8-aminooctanoic acid (1) was dissolved in 16 ml of 2N aqueous sodium hydroxide solution, and ice-cooled. While vigorously stirring this solution, benzyl chloroformate (7.0 g, 41 mmol) and 20 ml of a 2N aqueous sodium hydroxide solution were alternately added dropwise in two batches. After completion of the dropping, 100 ml of THF was added, and stirring was continued at room temperature for 7 hours. After unreacted benzyl chloroformate was extracted with ice-cooled diethyl ether (50 ml × 3 times), concentrated hydrochloric acid was added under ice-cooling to adjust the pH to about 2. The mixture was extracted with dichloromethane (50 ml × 4 times), the collected dichloromethane extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. Dissolve the concentrate in a small amount of ethyl acetate,
White crystals precipitated by adding n-hexane thereto were collected by suction filtration to obtain the target product (2). (Yield 7.6
g, yield 83%).

8- (benzyloxycarbonylamino)
Synthesis of tert-butyl octanoate (3) 7.6 g (26 mmol) of 8- (benzyloxycarbonylamino) octanoic acid (2) in dichloromethane (250 ml) salt-cooling to minus 16 ° C with ice,
A stream of isobutene was slowly poured into the solution for 5 minutes. Next, 0.3 ml of concentrated sulfuric acid was slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was allowed to stir for 1 hour and then overnight. The reaction solution was directly concentrated under reduced pressure and subjected to silica gel column chromatography (n-hexane-ethyl acetate = 4:
The target product (3) was obtained as a colorless transparent liquid by purification in 1) (yield 5.4 g, yield 60%).

[0126]Tert-butyl 8-aminooctanoate
Synthesis of (4) 5.4 g (15.5 mmol) of 8- (benzyloxy)
Carbonylamino) tert-butyl octoate (3)
0.5 g of paradium in methanol solution (100 ml) of
Absorption of hydrogen is completed in the presence of hydrogen by adding mucarbon
Stirred until. Next, the reaction solution is filtered and then concentrated under reduced pressure,
The target substance is obtained as a yellow liquid when the solvent is completely removed with a vacuum pump.
(4) was obtained. (Yield 3.5 g, yield 106%).

[0127]8- (phenoxycarbonylamino) octane
Synthesis of tert-butyl titanate (5) Diphenyl chlorocarbonate (3.8 g, 24 mmol)
3.5 g of loromethane solution (100 ml) at ice cooling temperature
(16 mmol) 8-aminooctanoic acid tert-bu
A solution of chill (4) in dichloromethane (10 ml) was added dropwise.
And then triethylamine (2.4g, 24mmol)
Dichloromethane solution (10 ml) was added dropwise. End of dripping
After completion, stir the solution as it is until it reaches room temperature, and
Continued to stir overnight. After washing the reaction solution with saturated saline solution
Dehydrate with sodium sulfate and filter with suction to remove sodium sulfate.
After removing the bubbles, the solution was concentrated under reduced pressure. The concentrate is silica gel
Column chromatography (n-hexane-ethyl acetate
= 19: 1 → 9: 1 → 4: 1) and a colorless transparent liquid
The target product (5) was obtained as a body (yield 4.9 g, yield 90).
%).

Synthesis of (6) 1.8 g (11 mmol) of oxamyl oxime and 3.
1 g (9.2 mmol) of tert-butyl 8- (phenoxycarbonylamino) octanoate (5) was dissolved in 150 ml of acetonitrile, and 1.7 g (11 m) under ice cooling.
20 m of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in acetonitrile)
1 was added dropwise. After stirring under ice cooling for 3 hours, the reaction solution was directly concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 1 → ethyl acetate) to give 3.2 g of a colorless transparent liquid (yield 86 %) (6) was obtained.

Synthesis of (7) 3.2 g (7.9 mmol) of the ester compound (6) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solution was concentrated under reduced pressure and subjected to silica gel chromatography (n
Purification with -hexane: ethyl acetate = 1: 1 → ethyl acetate) gave 3.9 g (yield 84%) of oxamyl derivative-3 (7) as a transparent liquid. This solution is minus 2
When left in a freezer at 0 ° C for a long time, white crystals were formed. ¹ H-NMR (CDCl ₃ ) 1.20 to 1.44 (s, 6H), 1.45 to 1.71 (m, 4H), 2.25 to 2.42 (overlap, 5H), 3.08 (S, 3H), 3.10 (s, 3H), 3.15 to 3.32 (m, 2H), 5.87 to 6.02 (br, 1H)

Example 4 Synthesis of Oxamyl Derivative-4

[Chemical formula 24]

11-Bromoundecanoic acid tert-butyl
Synthesis of (2) 13.3 g (50 mmol) of 11-bromoundecanoic acid (1) in dichloromethane (200 ml) was added to sodium chloride-
After cooling to −16 ° C. with ice, a stream of isobutene was slowly poured into the solution for 5 minutes. Next, 0.5 ml of concentrated sulfuric acid was slowly added dropwise with a syringe, and isobutene was slowly poured again for 5 minutes. The solution was continuously stirred under ice cooling for 2 hours and further at room temperature for 3 hours.
The reaction solution was directly concentrated under reduced pressure and subjected to silica gel column chromatography (n-hexane-ethyl acetate = 19: 1).
The desired product (2) was obtained as a colorless transparent liquid by purification with (10.8 g, 67% yield).

11- (phthalimidyl) undecanoic acid t
Synthesis of ert-butyl (3) 25.5 g (79 mmol) of tert-butyl 11-bromoundecanoate (2) was dissolved in DMF (500 ml) and 15 g (81 mmol) potassium phthalimide was added at room temperature. The solution was kept stirring at room temperature for 2 nights. The reaction solution was partitioned with ether-water (150 ml x 4).
Times), the collected ether layer was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (n-hexane-ethyl acetate = 9: 1) to obtain the target product (3) as a liquid. (Yield 19 g, Yield 62%).

11-aminoundecanoic acid tert-butyl
(4) Synthesis of 10 g (26 mmol) of tert-butyl 11- (phthalimidyl) undecanoate (3) in ethanol (150 ml), 1.0 g (31 mmol) of anhydrous hydrazine in 50 ml of ethanol under ice cooling. Dropped. 2 hours under ice-cooling, 3 hours at room temperature, and 30 at 50 ℃
Stir for minutes. The reaction solution was concentrated under reduced pressure, dissolved in ethyl acetate, hexane was added, and the precipitate was removed by filtration. The filtrate was concentrated, and the concentrate was subjected to silica gel chromatography (n-hexane: ethyl acetate = 1: 1 → chloroform:
Purification with methanol = 9: 1) gave 2.0 g (30% yield) of (5) as a yellow liquid.

11- (phenoxycarbonylamino) w
Synthesis of tert-butyl ndecanoate (5) 4.0 g of phenyl chloroformate (3.5 g, 22.3 mmol) in dichloromethane (50 ml) at ice-cooling temperature.
(15.6 mmol) 11-aminoundecanoic acid te
Dichloromethane solution of rt-butyl (4) (50 ml)
Was added dropwise, and then triethylamine (2.2 g, 22 m
A dichloromethane solution (50 ml) of (mol) was added dropwise. After the completion of dropping, the solution was continuously stirred under ice cooling for 1 hour and further at room temperature for 3 hours. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1) to obtain 4.5 g (yield 77%) of (5) as a transparent liquid.

Synthesis of (6) 3.0 g (18.5 mmol) of oxamyl oxime and 4.5 g (12 mmol) of tert-butyl 11- (phenyloxycarbonylamino) undecanoate were dissolved in 100 ml of acetonitrile and cooled under ice-cooling. 2.0 g (13
mmol) 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in acetonitrile 20
ml was added dropwise. After stirring under ice cooling for 3 hours, the reaction solution was directly concentrated under reduced pressure and subjected to silica gel chromatography (n-
Purification with hexane: ethyl acetate = 1: 1) gave 3.8 g (72% yield) of (6) as a transparent liquid.

Synthesis of (7) 3.8 g (8.5 mmol) of the ester compound (2) was dissolved in 80 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 1 → ethyl acetate) to give 2.5 g (yield 76%) of oxamyl derivative-4 (7) as white crystals. Got ¹ H-NMR (CDCl ₃ ) 1.12 to 1.40 (s, 12H), 1.40 to 1.71 (m, 4H), 2.20 to 2.39 (overlap, 5H), 3.07. (S, 3H), 3.09 (s, 3H), 3.16 to 3.32 (m, 2H), 5.93 to 6.08 (t, 1H), 10.02 to 10.73 (br , 1H)

Example 5 Synthesis of Oxamyl Derivative-5

[Chemical 25]

4- (benzyloxycarbonylamino)
Synthesis of cyclohexanecarboxylic acid (2) 5.0 g (35 mmol) of 4-aminocyclohexanecarboxylic acid (1) was added to 2 N aqueous sodium hydroxide solution 1
It was dissolved in 7.5 ml and ice-cooled. While vigorously stirring this solution, benzyl chloroformate (7.8 g, 46 mmo
1) and 23 ml of 2N aqueous sodium hydroxide solution were added dropwise. After completion of dropping, the mixture was returned to room temperature and stirred overnight. After unreacted benzyl chloroformate was extracted with ice-cooled diethyl ether (50 ml × 3 times), concentrated hydrochloric acid was added under ice-cooling to adjust the pH to about 2. Diethyl ether (50 ml
The extracted diethyl ether extracts were dried over anhydrous sodium sulfate, the sodium sulfate was removed by suction filtration, and the filtrate was concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (n-hexane-ethyl acetate = 1: 1) to obtain the desired product (2) as white crystals (yield 7.2 g, 91% yield).

4- (benzyloxycarbonylamino)
Combined with tert-butyl cyclohexanecarboxylate (3)
A solution of 7.2 g (26 mmol) of 4- (benzyloxycarbonylamino) cyclohexanecarboxylic acid (2) in dichloromethane (150 ml) was added with sodium chloride-ice to obtain a minus 1
After cooling to 6 ° C, a stream of isobutene was slowly poured into the solution for 5 minutes. Next, 0.25 ml of concentrated sulfuric acid was slowly added dropwise with a syringe, and isobutene was slowly poured again for 10 minutes. The solution was left stirring overnight and allowed to come to room temperature. The reaction solution was directly concentrated under reduced pressure and purified by silica gel column chromatography (n-hexane-ethyl acetate = 4: 1) to obtain the target product (3) as white crystals (yield 5.2 g, 66% yield). .

4-aminocyclohexanecarboxylic acid te
Synthesis of rt-butyl (4) 0.5 g in 5.0 g (15 mmol) of tert-butyl 4- (benzyloxycarbonylamino) cyclohexanecarboxylate (3) in methanol (150 ml).
Palladium carbon was added and stirred in the presence of hydrogen until the absorption of hydrogen was completed. Next, the reaction solution was filtered and concentrated under reduced pressure, and the solvent was completely removed by a vacuum pump to obtain the target product (4) as a yellow liquid (amount 3.0 g, yield 100).
%).

4- (phenoxycarbonylamino) shik
Synthesis of tert-butyl rohexanecarboxylate (5) Phenyl chlorocarbonate (2.8 g, 2.8 g, was added to a dichloromethane solution (100 ml) of tert-butyl 4-aminocyclohexanecarboxylate (3.0 g, 15 mmol) (4) at ice cooling temperature. 18 mmol) in dichloromethane (1
0 ml) and then triethylamine (1.5 ml).
g, 15 mmol) in dichloromethane (10 ml)
Was dripped. After completion of dropping, the solution was cooled with ice for 3 hours,
Further, stirring was continued at room temperature for 1 hour. The reaction solution was washed with saturated brine, dried over anhydrous sodium sulfate, filtered with suction to remove sodium sulfate, and then concentrated under reduced pressure. The concentrate was purified by silica gel chromatography (n-hexane: ethyl acetate = 4: 1) to obtain 2.9 g (yield 60
%) (5) was obtained.

Synthesis of (6) 1.4 g (8.6 mmol) of oxamyl oxime and 4
2.7 g (8.5 mmo) of tert-butyl- (phenyloxycarbonylamino) cyclohexanecarboxylate
l) was dissolved in 100 ml of acetonitrile, and under ice cooling, 1.3 g (8.6 mmol) of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) in 15 ml of acetonitrile was added dropwise. After stirring under ice-cooling for 5 hours, the reaction mixture was directly concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 2) to give 2.8 g (yield 85%) of (6). Obtained.

Synthesis of (7) 2.8 g (7.2 mmol) of hapten ester (6) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (ethyl acetate) to give 1.9 g (yield 79%) of oxamyl derivative-5 (7) as white crystals.
Got ¹ H-NMR (CDCl ₃ ) 1.50 to 2.04 (overlap, 8H), 2.31 (s, 3H), 2.48 to 2.62 (m, 1H), 3.06 (s, 3H) ), 3.09 (s, 3H), 3.63 to 3.81 (m, 1H), 5.92 to 6.09 (d, 1H).

Example 6 Synthesis of oxamyl derivative-6

[Chemical formula 26]

4- (benzyloxycarbonylamino )
Synthesis of (tyl ) cyclohexanecarboxylic acid (2) 25 g (160 mmol) of 4- (aminomethyl) cyclohexanecarboxylic acid (1) was dissolved in 80 ml of a 2N aqueous sodium hydroxide solution and ice-cooled. Benzyl chloroformate (36 g, 210 m
mol) and 2N sodium hydroxide aqueous solution 104 ml
Was divided into two portions and dropped alternately. After the dropping is completed,
It returned to room temperature and stirred overnight. After unreacted benzyl chloroformate was extracted and removed with ice-cooled diethyl ether (50 ml × 3 times), concentrated hydrochloric acid was added under ice-cooling to adjust the pH to about 2. It was extracted with diethyl ether (50 ml × 4 times), the collected diethyl ether extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. The concentrate was dissolved in ethyl acetate and hexane was added to obtain (2) as white crystals (yield 3
0.6 g, yield 66%).

[0146]4- (benzyloxycarbonylamino)
Tyl) tert-butyl cyclohexanecarboxylate
Synthesis of (3) 10.6 g (36 mmol) of 4- (benzyloxyca
Rubonylaminomethyl) cyclohexanecarboxylic acid
A dichloromethane solution (150 ml) of (2) was added to sodium chloride-ice.
And cool it to minus 16 ° C with isobutene.
The airflow was slowly infused for 5 minutes. Next, concentrated hydrochloric acid 0.4
Slowly add ml by syringe and re-isobutene.
Was slowly poured for 10 minutes. Leave this solution as is
Stirring was continued for 1 day. The reaction mixture is concentrated under reduced pressure as it is.
Kagel column chromatography (n-hexane-acetic acid
The target compound was obtained as white crystals after purification with ethyl = 4: 1).
(3) was obtained (yield 7.5 g, yield 59%).

[0147]4- (aminomethyl) cyclohexanecal
Synthesis of tert-butyl borate (4) 7.2 g (20.1 mmol) of 4- (benzyloxy)
Carbonylamino) tert-butyl heptanoate (3)
1.0 g of palladium in methanol solution (200 ml) of
Absorption of hydrogen is completed in the presence of hydrogen by adding mucarbon
Stirred until. Next, the reaction solution is filtered and then concentrated under reduced pressure,
The target substance is obtained as a white solid when the solvent is completely removed with a vacuum pump.
(4) was obtained (yield 4.4 g, 100% yield).

[0148]4- (phenoxycarbonylaminomethyi
) Tert-Butyl cyclohexanecarboxylate (5)
Synthesis of 4.4 g (21 mmol) of 4- (aminomethyl) sik
Dichloromethane of tert-butyl rohexanecarboxylate (4)
Chlorocarbonic acid was added to a dichloromethane solution (100 ml) at ice-cooling temperature.
Phenyl (4.2 g, 26.8 mmol) dichlorometh
Tan solution (20 ml) was added dropwise, followed by triethylamine.
Dichloromethane solution (2.5g, 25mmol)
(10 ml) was added dropwise. After completion of dropping, add this solution to
The mixture was stirred as it was overnight. After washing the reaction solution with saturated saline solution
Dehydrate with sodium sulfate and filter with suction to remove sodium sulfate.
After removing the bubbles, the solution was concentrated under reduced pressure. The concentrate is silica gel
Chromatography (n-hexane: ethyl acetate = 4:
Purified in 1), 4.8 g (69% yield) as a transparent liquid.
%) (5) was obtained.

Synthesis of (6) 1.67 g (10 mmol) of oxamyl oxime and 4
3.33 g (10 mmo) tert-butyl- (phenyloxycarbonylaminomethyl) hexanecarboxylate
l) was dissolved in 150 ml of acetonitrile, and 152 ml of an acetonitrile solution of 1.52 g (10 mmol) of 1,8-azabicyclo [5.4.0] undec-7-ene (DBU) was added dropwise under ice cooling. After stirring under ice-cooling for 3 hours, the reaction solution was concentrated under reduced pressure as it was and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 2 → ethyl acetate) to obtain 3.3 g (yield 82%) ( 6) was obtained.

Synthesis of (7) 3.3 g (8.2 mmol) of the ester compound (6) was dissolved in 50 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (ethyl acetate → chloroform: methanol = 4: 1) to obtain 2.2 g (yield 100%) of oxamyl derivative-6 (7) as white crystals. . ¹ H-NMR (CDCl ₃ ) 0.88 to 1.08 (t, 2H), 1.31 to 1.6
2 (overlap, 3H), 1.73 to 1.90 (m, 2
H), 1.93 to 2.08 (m, 2H), 2.14 to
2.32 (overlap, 4H), 2.94-3.20 (over
lap, 8H), 5.89 to 6.07 (t, 1H)

Example 7 Synthesis of Oxamyl Derivative-7

[Chemical 27]

4- (benzyloxycarbonylamino )
Synthesis of (tyl ) benzoic acid (2) 4.5 g (30 mmol) of 4-aminomethylbenzoic acid (1) was dissolved in 15 ml of 2N aqueous sodium hydroxide solution and cooled with ice. While vigorously stirring this solution, benzyl chloroformate (6.6 g, 39 mmol) and 20 ml of a 2N aqueous sodium hydroxide solution were added dropwise. After completion of dropping, the mixture was returned to room temperature and stirred overnight. After unreacted benzyl chloroformate was extracted and removed with ice-cooled diethyl ether (50 ml × 3 times), concentrated hydrochloric acid was added under ice-cooling to adjust the pH to about 2. It was extracted with diethyl ether (50 ml × 4 times), the collected diethyl ether extracts were dried over anhydrous sodium sulfate, and sodium sulfate was removed by suction filtration, followed by concentration under reduced pressure. Dissolve the concentrate in ethyl acetate,
Hexane was added to obtain (2) as white crystals (yield 6.0 g, yield 71%).

4- (benzyloxycarbonylamino )
Synthesis of tert) tert-butyl benzoate (3) 5.8 g (20 mmol) of 4- (benzyloxycarbonylaminomethyl) benzoic acid (2) was suspended in dichloromethane (250 ml) and salt-ice was added to minus 16 ° C.
The mixture was cooled to 0 ° C. and a stream of isobutene was slowly poured into the suspension for 5 minutes. Next, 0.7 ml of concentrated hydrochloric acid was slowly added dropwise with a syringe, and isobutene was slowly poured again for 20 minutes. The solution was left stirring for 2 days. The reaction solution was directly concentrated under reduced pressure and subjected to silica gel column chromatography (n-hexane-ethyl acetate = 4:
Purification in 1) gave the desired product (3) (yield 3.78 g,
Yield 54%).

4- (aminomethyl) benzoic acid tert-
Synthesis of butyl (4) 3.78 g (11 mmol) of tert-butyl 4- (benzyloxycarbonylaminomethyl) benzoate (3) in methanol (200 ml) was added with 0.5 g of palladium carbon in the presence of hydrogen. Stir until hydrogen uptake is complete. Next, the reaction solution was filtered and then concentrated under reduced pressure, and the solvent was completely removed by a vacuum pump to obtain the target product (4) (amount 2.2 g, yield 96%).

[0155]4- (phenoxycarbonylaminomethyi
Le) Synthesis of tert-butyl benzoate (5) 2.2 g (10.6 mmol) of 4- (aminomethyl)
A solution of tert-butyl benzoate (4) in dichloromethane
(100 ml), phenyl chlorocarbonate at ice-cold temperature
(2.1 g, 13.4 mmol) in dichloromethane
(10 ml) dropwise and then triethylamine (1.
1 g, 11 mmol) in dichloromethane (10 m
l) was added dropwise. Stir for 2 hours under ice cooling, then for 3 hours at room temperature.
After stirring, the reaction solution was washed with saturated saline and then dried over anhydrous sodium sulfate.
After dehydration with um and removing sodium sulfate by suction filtration
It was concentrated under reduced pressure. This concentrate is purified by silica gel chromatography.
Purify with phy (n-hexane: ethyl acetate = 9: 1)
Then, 1.93 g (yield 55%) of white crystals was obtained.
(5) was obtained.

Synthesis of (6) 0.96 g (5.9 mmol) of oxamyl oxime and tert-butyl 4- (phenyloxycarbonylaminomethyl) benzoate (1.9 g, 5.8 mmol) were dissolved in 100 ml of acetonitrile, and iced. 0.88g under cold conditions
(5.8 mmol) of 1,8-diazabicyclo [5.
4.0] 15 ml of an acetonitrile solution of undec-7-ene (DBU) was added dropwise. After stirring under ice-cooling for 4.5 hours, the reaction solution was directly concentrated under reduced pressure and purified by silica gel chromatography (n-hexane: ethyl acetate = 1: 2 → ethyl acetate) to give 1.53 g (yield 67%). (6)
Got

Synthesis of (7) 1.5 g (3.8 mmol) of the ester compound (6) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (hexane: ethyl acetate = 1: 1 → ethyl acetate).
As a white crystal, 0.8 g (yield 62%) of oxamyl derivative-7 (7) was obtained. ¹ H-NMR (CDCl ₃ ) 2.33 (s, 3H), 3.07 (s, 3H), 3.09 (s, 3H), 4.45 to 4.61 (d, 2H), 6. 29-6.35 (br, 1H), 7.38-7.48 (d, 2H), 7.99-8.12 (d, 2H).

Example 8 Oxamyl Derivatives-8 and 9
Synthesis of

[Chemical 28]

Synthesis of (2) 4.0 g (25 mmol) of oxamyl oxime (1)
And tert-butyl bromoacetate 5.0 g (25 mmo
l), and 1 g of potassium carbonate 12 g (87 mmol)
In addition to 00 ml of acetonitrile, the mixture was stirred at 80 ° C. under reflux for 3 hours. The reaction solution was filtered through Celite, the filtrate was concentrated, and then purified by silica gel chromatography (hexane-ethyl acetate = 1: 1) to obtain 6.0 g (yield 87%) of (2) as white crystals.

Synthesis of (3) 5.8 g (21 mmol) of (2) was added to dichloromethane 1
After dissolving in 00 ml and adding 10 ml of trifluoroacetic acid, the mixture was stirred at room temperature for 3 hours. The reaction solution is concentrated under reduced pressure,
Purification by silica gel chromatography (hexane-ethyl acetate = 1: 1) gave 3.9 g (yield 84%) of oxamyl derivative-8 (3) as white crystals. ¹ H-NMR (CDCl ₃ ) 2.25 (s, 3H), 3.03 (s, 3H), 3.0
6 (s, 3H), 4.76 (s, 2H), 8.10
(S, 1H).

Synthesis of (4) 2.2 g (10 mmol) of (3), 1.6 g (10 m
mol) tert-butyl 4-aminobutyrate, and N-
Hydroxysuccinimide (HONSu) 1.5 g (1
(3 mmol) in a THF solution (30 ml) was ice-cooled to dicyclohexylcarbodiimide (DCC) (2.5 g, 12 m).
50 ml of THF solution of (mol) was added dropwise. After the dropping was completed, the mixture was stirred at room temperature overnight. After removing the precipitated dicyclohexylurea by filtration, the filtrate was concentrated and purified by silica gel chromatography (hexane-ethyl acetate = 1: 1) to give 2.4 g (yield 66%) of an ester compound (as white crystals). 4) was obtained.

Synthesis of (5) 2.3 g (6.4 mmol) of the ester compound (4) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated and purified by silica gel chromatography (ethyl acetate) to give 1.3 g of white crystals (yield 67
%) Of oxamyl derivative-9 (5) was obtained. ¹ H-NMR (CDCl ₃ ) 1.78 to 2.18 (m, 2H), 2.09 to 2.50 (overlap, 5H), 3.04 (s, 3H), 3.06 (s, 3H ), 3.41 (q, 2H), 4.62 (s, 2H), 6.48 to 6.85 (br, 1H), 7.80 to 8.50 (br, 1H).

Example 9 Synthesis of Oxamyl Derivative-10

[Chemical 29]

Synthesis of (2) 0.8 g (5 mmol) of oxamyl oxime (1) and 0.7 g (7 mmol) of succinic anhydride were dissolved in 30 ml of dichloromethane and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (hexane: ethyl acetate = 1: 1 → ethyl acetate) to obtain 1.0 g (yield 78%) of (2) as white crystals. ¹ H-NMR (CDCl ₃ ) 2.33 (s, 3H), 2.68 to 2.88 (m, 4H), 3.06 (s, 3H), 3.11 (s, 3H), 9. 10 to 9.50 (br, 1H)

Example 10 Combination of oxamyl derivative-11
Success

[Chemical 30]

Synthesis of (2) 1.9 g (10.4 mmol) of adipic acid chloride was dissolved in 100 ml of dichloromethane and cooled with ice to give 1.7 g.
10 ml of a dichloromethane solution of oxamyl oxime (10.5 mmol) and 10 ml of a dichloromethane solution of 0.8 g (10 mmol) of pyridine were added dropwise, and the mixture was stirred for 1 hour under ice cooling. 2.0 g (12 mmol) of veratryl alcohol in dichloromethane 10 ml, 0.8 g
(10 mmol) of pyridine in dichloromethane 10
ml was added dropwise, and the mixture was stirred under ice cooling for 30 minutes and further at room temperature for 1.5 hours. The reaction solution was washed with water, dehydrated over anhydrous magnesium sulfate, filtered, and concentrated, followed by silica gel chromatography (n
-Hexane: ethyl acetate = 1: 1 → ethyl acetate) for crude purification. This crude product was directly used in the next reaction.

2.0 g of the crude product containing the synthesis (2) of (3) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure and subjected to silica gel chromatography (hexane: ethyl acetate =
Purification with 1: 1) gave 1.3 g (43% overall yield from (1)) of (3). ¹ H-NMR (CDCl ₃ ) 1.64-1.83 (m, 4H), 2.33 (s, 3H), 2.36-2.45 (t, 2H), 2.45-2.56. (T, 2H), 3.05 (s, 3H), 3.11 (s, 3H)

Example 11 Combination of Oxamyl Derivative-12
Success

[Chemical 31]

Synthesis of (2) 3.5 g (50 mmol) of sodium thiomethoxide was dissolved in 150 ml of ethanol, and 9.0 g under ice cooling.
(50 mmol) of 1-bromo-3,3-dimethyl-2
20 ml of an ethanol solution of butanone (1) was added dropwise. After stirring at room temperature for 2.5 hours, the reaction solution was filtered and then concentrated. This concentrate was subjected to silica gel chromatography (n-
Purification with hexane: ethyl acetate = 1: 9 → ethyl acetate) yielded 4.4 g (60% yield) of (2) as a transparent liquid.
Got

Synthesis of (3) 4.4 g (30 mmol) of (2) was added to ethanol 200.
After dissolving in ml, 2.5 g (36 mmol) of hydroxylamine hydrochloride and 3.5 g (36 mmol) of potassium acetate were added, and the mixture was heated under reflux at 110 ° C. for 5 hours. The reaction solution was filtered, and the filtrate was concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1 → 4: 1) to give 4.0 g (yield 82%) of (3) as a transparent liquid. Got

Synthesis of (4) 2.6 g (8.4 mmol) of tert-butyl 6- (phenyloxycarbonylamino) hexanoate was dissolved in 200 ml of acetonitrile, and then 1.5 g under ice cooling.
20 ml of a solution of (9.3 mmol) 3,3-dimethyl-1- (methylthio) -2-butanone oxime (thiophanoxoxime) (3) in acetonitrile, then 1.3 g (8.5 mmol) of 1,8- 20 ml of an acetonitrile solution of diazabicyclo [5.4.0] undec-7-ene (DBU) was added dropwise. 5 under ice cooling
After stirring for an hour, the reaction solution was directly concentrated and subjected to silica gel chromatography (n-hexane: ethyl acetate = 9:
Purified by 1 → 1: 1), 2.3g as a transparent liquid
(4) of (yield 73%) was obtained.

Synthesis of (5) 2.3 g (6.1 mmol) of (4) was dissolved in 200 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was directly concentrated under reduced pressure and purified by silica gel chromatography (hexane: ethyl acetate = 4: 1 → 2: 1 → 1: 1) to give 1.35 g (yield 69%) of (5) as a transparent liquid. Got ¹ H-NMR (CDCl ₃ ) 1.24 (s, 9H), 1.31 to 1.48 (m, 2H), 1.54 to 1.78 (m, 4H), 2.25 (s, 3H). ), 2.32 to 2.43 (t, 2H), 3.23 to 3.36 (m, 2H), 3.44 (s, 2H), 6.24 to 6.38 (br, 1H).

Example 12 Combination of Oxamyl Derivative-13
Success

[Chemical 32]

Synthesis of (2) 2.3 g (57.5 mmol) of sodium hydroxide was dissolved in 50 ml of ethanol, and 4.5 g (7
10 ml of an ethanol solution of 2.6 mmol) ethyl mercaptan was added dropwise. 10 g (55.9 mmol) of 1
20 ml of an ethanol solution of -bromo-3,3-dimethyl-2-butanone (1) was added dropwise and stirred overnight. The reaction solution was filtered, concentrated, and purified by silica gel chromatography (n-hexane: ethyl acetate = 49: 1 → 9: 1) to obtain 7.4 g (yield 83%) of (2) as a transparent liquid. Obtained.

Synthesis of (3) 7.4 g (46.3 mmol) of (2) was added to ethanol 2
Dissolved in 00 ml, 3.86 g (55.5 mmol)
Hydroxylamine hydrochloride and 5.44g (55.5mm
Ol) potassium acetate was added and the mixture was heated under reflux at 110 ° C. for 5 hours. The reaction solution is filtered, and the filtrate is concentrated and then subjected to silica gel chromatography (n-hexane: ethyl acetate =
Purification by 9: 1) gave 7.12 g (88% yield) of (3) as a clear liquid.

Synthesis of (4) 1.75 g (10 mmol) of oxime (3) was dissolved in 100 ml of acetonitrile to give 3.07 g (10 mmo).
20 ml of an acetonitrile solution of tert-butyl 6- (phenyloxycarbonylamino) hexanoate of 1) was added all at once under ice cooling. Then 1.52g (10mmo
20 ml of an acetonitrile solution of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) of 1) was added dropwise under ice cooling, and the mixture was stirred for 3 hours as it was. The reaction solution was concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1 → 1: 1) to obtain 2.86 g (yield 74%) of (4) as a transparent liquid.

Synthesis of (5) 2.8 g (7.2 mmol) of (4) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solution was concentrated under reduced pressure and purified by silica gel chromatography (hexane: ethyl acetate = 4: 1 → 1: 1 → ethyl acetate) to obtain 1.67 g (yield 70%) of (5) as white crystals. It was ¹ H-NMR (CDCl ₃ ) 1.17 to 1.32 (overlap, 12H), 1.32 to 1.50 (m, 2H), 1.51 to 1.76 (m, 4H), 2.30. -2.41 (m, 2H), 2.65-2.76 (q, 2H), 3.21-3.32 (m, 2H), 3.44 (s, 2H), 6.22-6 .38 (br, 1H)

Example 13 Combination of Oxamyl Derivative-14
Success

[Chemical 33]

Synthesis of (2) 1.12 g (28 mmol) of sodium hydroxide was dissolved in 50 ml of ethanol, and 2.5 g (33 mm) was added at room temperature.
ol) of isopropyl mercaptan in ethanol 1
5 ml was added dropwise. 15 g of an ethanol solution of 5.0 g (28 mmol) of 1-bromo-3,3-dimethyl-2-butanone (1) was added dropwise at room temperature and stirred for 2 days. The reaction solution was partitioned with aqueous potassium carbonate solution-dichloromethane, and the dichloromethane layer was dehydrated with anhydrous magnesium sulfate,
After filtration, concentration, and purification by silica gel chromatography (n-hexane: ethyl acetate = 49: 1), 4.3 g (yield 88%) of (2) was obtained as a transparent liquid.

Synthesis of (3) 4.3 g (24 mmol) of (2), 2.0 g (28.
8 mmol) of hydroxylamine hydrochloride and 2.8 g (2
8.6 mmol) potassium acetate to 200 m ethanol
In addition to 1, the mixture was heated under reflux at 110 ° C. for 5 hours. The reaction solution was filtered, and the filtrate was concentrated and purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1) to give 4.49 g (yield 96%) of (3) as white crystals.
Got

Synthesis of (4) 1.9 g (10 mmol) of oxime (3) was dissolved in 50 ml of acetonitrile to give 3.07 g (10 mmol).
10-ml of a solution of 6- (phenyloxycarbonylamino) hexanoate tert-butyl in acetonitrile at room temperature was added. Then 1.52 g (10 mmol) of 1,8
10 ml of a solution of diazabicyclo [5.4.0] undec-7-ene (DBU) in acetonitrile was added dropwise under ice cooling, and the mixture was stirred as it was for 2 hours and further at room temperature for 2 hours. The reaction solution was concentrated and subjected to silica gel chromatography (n-hexane: ethyl acetate = 9: 1 → 4: 1 →
Purification by 1: 1) gave 3.23 g (80% yield) of (4) as a transparent liquid.

Synthesis of (5) 3.2 g (8.0 mmol) of (4) was dissolved in 150 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution is concentrated under reduced pressure,
Purification by silica gel chromatography (hexane: ethyl acetate = 4: 1 → 1: 1) gave 1.51 g (yield 5
(5%) of (5) was obtained. ¹ H-NMR (CDCl ₃ ) 1.23 (s, 9H), 1.24 to 1.31 (d, 6H), 1.32 to 1.48 (m, 2H), 1.51 to 1.76. (M, 4H), 2.29 to 2.41 (m, 2H), 3.04 to 3.19 (m, 1H), 3.21 to 3.35 (m, 2H), 3.42 (s). , 2H), 6.22 to 6.38 (br, 1H)

Example 14 Combination of oxamyl derivative-15
Success

[Chemical 34]

Synthesis of (2) 1.12 g (28 mmol) of sodium hydroxide was dissolved in 50 ml of ethanol, and 2.5 g (33 mm) was added at room temperature.
ol) n-propyl mercaptan ethanol 15 m
1 was added dropwise. 15 g of an ethanol solution of 5.0 g (28 mmol) of 1-bromo-3,3-dimethyl-2-butanone (1) was added dropwise at room temperature and stirred for 2 days. The reaction solution was partitioned with aqueous potassium carbonate solution-dichloromethane, and the dichloromethane layer was dried over anhydrous magnesium sulfate, filtered,
After concentration and purification by silica gel chromatography (n-hexane: ethyl acetate = 49: 1), 4.28 g (yield 88%) of (2) was obtained as a transparent liquid.

Synthesis of (3) 6.16 g (35.4 mmol) of (2), 3.0 g
(42.5 mmol) hydroxylamine hydrochloride and 4.
16 g (42.5 mmol) of potassium acetate was added to 200 ml of ethanol, and the mixture was heated under reflux at 80 ° C. overnight. The reaction solution was filtered, and the filtrate was concentrated and then purified by silica gel chromatography (n-hexane: ethyl acetate = 9: 1) to obtain 6.25 g (yield 93%) of (3) as a transparent liquid.

Synthesis of (4) 2.0 g (16 mmol) of oxime (3) and 3.1 g
(10 mmol) tert-butyl 6- (phenyloxycarbonylamino) hexanoate was added to acetonitrile 5
It was dissolved in 0 ml. Then 1.52 g (10 mmol) of 1,8-diazabicyclo [5.4.0] undec-7-
20 ml of an acetonitrile solution of ene (DBU) was added dropwise under ice cooling, and the mixture was stirred as it was for 2 hours and further at room temperature for 2 hours. The reaction solution was concentrated and subjected to silica gel chromatography (n-hexane: ethyl acetate = 9: 1 → 4:
When purified in 1), it was 2.83 g as a transparent liquid (yield 7
0%) of (4) was obtained.

Synthesis of (5) 2.8 g (7.0 mmol) of (4) was dissolved in 100 ml of dichloromethane, 10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution is concentrated under reduced pressure,
Silica gel chromatography (hexane → hexane:
Purified with ethyl acetate = 9: 1 → 4: 1), 1.2 g
(5) with a yield of 50% was obtained. ¹ H-NMR (CDCl ₃ ) 0.91 to 1.03 (t, 3H), 1.23 (s, 9)
H), 1.32 to 1.48 (m, 2H), 1.53 to
1.77 (m, 6H), 2.31 to 2.41 (t, 2)
H), 2.61 to 2.70 (t, 2H), 3.23 to
3.34 (q, 2H), 3.42 (s, 2H), 6.2
3 to 6.38 (br, 1H).

Example 15 Immunogen and Screening
Preparation of Antigens for Use Carrier proteins were bound to the oxamyl derivatives-1 to -15 and oxamyl derivative-C prepared in Examples 1-14 and Production Examples above by the activated ester method to prepare immunogens and screening antigens. . First, using oxamyl derivative as a hapten,
mol was dissolved in 50 μl of DMSO. Next, D-N-hydroxysuccinimide (5 μmol) was added to these solutions.
After dissolving in 10 μl of MSO and adding, 1-ethyl-3
-(3-Dimethylaminopropyl) carbodiimide (4
μmol) was dissolved in 20 μl of DMSO and added. After reacting for 1.5 hours at room temperature, 85 mM was added to this reaction solution.
BS dissolved in 500 μl of borate buffer (pH 8.0)
10 mg each of A or KLH was further added, and the mixture was reacted again at room temperature for 1.5 hours. After completion of the reaction, the mixture was dialyzed against a Dulbecco's phosphate buffer solution (hereinafter referred to as "PBS (-)") to prepare an oxamyl derivative-KLH conjugate and an oxamyl derivative-BSA conjugate, respectively.

Example 16 Immunization 100 μg of the conjugate of the oxamyl derivative and KLH prepared in Example 15 was dissolved in 50 μl of PBS (−), emulsified and mixed with an equal volume of Freund's complete adjuvant, and then Bal.
b / c mice were inoculated intraperitoneally. One month later, booster immunization with 1/4 of each initial immunization amount
Four days later, the same amount as the booster dose was used for final immunization.

Example 17 Collection of antiserum and antibody activity
Measurement Blood was collected from the tail vein of the mouse after the booster immunization to prepare an antiserum. The activity of the obtained antiserum was measured by ELI using the conjugate of the oxamyl derivative prepared in Example 15 and BSA.
It was measured by the SA method.

BSA conjugates (4
μg / ml) was dissolved in PBS (−), added to a 96-well microtiter plate at 100 μl / well, and left standing at 4 ° C. overnight to immobilize. Then 300 μl / well blocking buffer {1% BSA
Was replaced with 85 mM borate buffer (pH 8.0) containing 60 mM NaCl, and blocking was performed at room temperature for 1 hour. The wells were washed with a washing solution (borate buffer containing 60 mM NaCl), and the antiserum was added to 100%.
μl / well was added, and the mixture was reacted at room temperature for 1 hour. After washing three times with the washing solution, the second antibody dilution solution {0.3% BSA and 6
85 mM borate buffer with 0 mM NaCl (p
(H8.0)} 1000-fold diluted with peroxidase-conjugated anti-mouse IgG antibody (manufactured by Capel) was added at 100 μl / well, and reacted at room temperature for 1 hour. After washing three times with a washing solution, a substrate solution of peroxidase {3, 3 ', 5,
5′-tetramethylbenzidine (100 μg / ml),
Color was developed with 0.1 M sodium acetate buffer (pH 5.5) containing 0.006% hydrogen peroxide for 10 minutes, the reaction was stopped with 1N sulfuric acid, and the absorbance at 450 nm was measured.

Example 18 Indirect competitive inhibition ELISA method
Reactivity of Antiserum with Oxamyl The antiserum whose activity was confirmed in Example 3 was evaluated for its reactivity with oxamyl by the indirect competitive inhibition ELISA method.

First, in the same manner as in Example 17, the diluted solution {1
Oxamyl solution diluted to an appropriate concentration with 85 mM borate buffer (pH 8.0) containing 50 mM NaCl was added at 50 μl / well, and immediately thereafter, 50 μl of an antiserum solution diluted to an appropriate concentration with the same diluent was added. / Well was added and mixed, and the mixture was reacted at room temperature for 1 hour. After washing three times, it was reacted with the second antibody by the same method as the ELISA method shown in Example 17, and after the color development, the absorbance at 450 nm was measured.

As shown in FIG. 1, the antiserum prepared from the oxamyl derivative-1 had the highest reactivity with oxamyl.

Example 19 Preparation of Monoclonal Antibody Cell fusion was carried out by using spleen cells in the mouse 3 days after the final immunization of Example 16. After removing large solids with a stainless mesh, the spleen cells taken out in the Dulbecco's medium were washed 3 times with the Dulbecco's medium, and then the ratio of the number of mouse myeloma cells P3-X63-Ag8.653 to 5: 1 was obtained. (Spleen cells: Myeloma cells) were mixed and centrifuged (1,200 rpm, 5 minutes) to collect cell precipitates. 50% polyethylene glycol (molecular weight 1,500) preheated to 37 ° C on this cell pellet
1 ml was added to fuse the cells. For cell fusion, 10 ml of Dulbecco's medium was gradually added, and fetal bovine serum (hereinafter referred to as “FB
It was stopped by the addition of an additional 1 ml of "S"). Fused cells were hypoxanthine (100 μM) and aminopterin (0. 0%) in Dulbecco's medium (hereinafter referred to as “Dulbeco / 10% FBS medium”) supplemented with 10% FBS.
4 μM), and HA supplemented with thymidine (16 μM)
After suspending in T medium, it was dispensed at 2 × 10 ⁵ cells / well on a 96-well polystyrene plate and cultured at 37 ° C. in the presence of 5% carbon dioxide for 10 days to 14 days. After culturing, each well was screened for the presence of antibody activity.

The reactivity of the antibody was measured by the same method as in Example 17 using the oxamyl derivative-BSA conjugate prepared in Example 15.

The hybridoma in the well showing the reactivity of the derivative-BSA conjugate was subjected to cell cloning by the limiting dilution method to obtain a monoclonal antibody-producing cell.
Of these, OXM6-8 obtained by using the oxamyl derivative 1-BSA conjugate as an antigen was deposited on July 3, 1997 with the deposit number FERM P-16301, Institute of Biotechnology, Institute of Industrial Science and Technology (Tsukuba, Ibaraki 305, Japan). 1-3, Ichihigashi
No.).

Example 20 Indirect competitive inhibition ELISA method
According to the reactivity of the monoclonal antibody with oxamyl , among the hybridomas obtained in Example 19, four hybridomas (OXMH3, OXM2-3, OXM3-) were used.
2, OXM6-8) produced monoclonal antibodies (hereinafter the monoclonal antibody uses the same name as the hybridoma producing them) for reactivity with oxamyl by the indirect competitive inhibition ELISA method as in Example 18. .

From the absorbance at each concentration obtained for each monoclonal antibody, the following formula:

[Chemical 35] Was used to calculate the inhibition rate. As a result, OXM H3 was about 1,000 to 10,000 ng / ml, OXM2-3 and OXM3-2 were about 100 to 10,000 ng / ml, and
XM6-8 was able to measure oxamyl in the range of about 10-100 ng / ml. Therefore, the antibody OXM6 prepared by using the oxamyl derivative 1 synthesized in Example 1
It was revealed that -8 shows the highest reactivity with oxamyl.

Example 21 Purification of Monoclonal Antibody First, hybridomas were cultured in a Dulbecco / 10% FBS medium. Ammonium sulfate was added to the culture supernatant to 50% saturation, and the mixture was stirred at 4 ° C. overnight. Distilled water was added to the resulting precipitate to solubilize it, which was then dialyzed against PBS (-) to give A.
It was purified by vid AL gel (manufactured by Bioprobe International) column chromatography.

Example 22 Oxamyl derivative and HRP
Preparation of 1.25 μmol of each oxamyl derivative shown in Table 1
It was dissolved in 50 μl of MSO. To this solution was added N-hydroxysuccinimide (5.5 μmo) dissolved in DMSO.
l) 3 μl, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (3.5 μmol)
7 μl was added and reacted at room temperature for 1 hour. To this reaction solution, add 40 μl of 1M sodium hydrogen carbonate, and add HRP.
250 μl of the solution (20 mg / ml) was added and mixed, and the mixture was reacted at room temperature for 3 hours. The low molecular weight compound was removed from the obtained reaction solution by a gel filtration column (G-25) to obtain an oxamyl derivative-HRP conjugate.

Example 23 Direct competitive inhibition ELISA
Between OXM6-8 monoclonal antibody and oxamyl
The monoclonal antibody OXM6-8 purified in Example 21
And the oxamyl derivative-HRP conjugate prepared in Example 22 and used to directly competitively inhibit the reactivity with oxamyl.
The comparison was made by the ISA method. The direct competitive inhibition ELISA method was performed according to the following procedure.

First, OXM6-8 purified in Example 21.
Was dissolved in 50 mM sodium carbonate buffer (pH 8.3) at a concentration of 2 μg / ml, added to a 96-well microtiter plate at 100 μl / well, and then allowed to stand at 4 ° C. overnight for immobilization. . Then 300 μl /
The wells were replaced with blocking buffer and blocked for 1 hour at room temperature. Separately from this plate, an oxamyl solution and an oxamyl derivative-HRP conjugate were added to a final concentration of 0.15% BSA and 60 mM NaCl in an 85 mM borate buffer solution (pH 8.0).
100 μl / for the blocked plate, mixed in
After adding in a well, the mixture was reacted at room temperature for 1 hour. After washing 5 times with a washing solution, color was developed with a substrate solution of peroxidase, and the absorbance at 450 nm was measured.

As shown in FIG. 2, OXM6-8 was about 4-500 ng / m by direct competitive inhibition ELISA.
It was possible to measure oxamyl in a measuring range of 1.

[0205]Example 24 Direct competitive inhibition ELISA method
Effect of Different Oxamyl Derivatives Oxamyl having a different structure from that used for antibody production
Use the conjugate of the derivative and HRP as a labeling competitive compound
Then, the direct competitive inhibition ELISA method was performed in the same manner as in Example 23.
went.

As a result, as shown in FIG. 3, with respect to the monoclonal antibody OXM6-8 prepared by using oxamyl derivative-1 as an immunogen, oxamyl derivative-5 (Example 5) in which a cyclohexane ring was introduced into the linker, or Oxamyl derivative with the amino group near the linker removed-
Oxamyl derivative-1 was obtained when 10 (Example 9) or -11 (Example 10) was used as a competitor.
It was revealed that oxamyl can be measured with higher sensitivity than when it is used by itself. That is, for example, when the oxamyl derivative-11 is used, it is about 0.5-20 ng.
It became possible to measure oxamyl in the range of / ml.

Example 25 Direct competitive inhibition ELISA
OXM6-8 in Methanol Tolerance In the direct competitive inhibition ELISA method shown in Example 23, the effect of methanol on the measurement system was examined using oxamyl derivative-4 as the labeled competitive compound. As a result, 2
When the methanol concentration was up to about 0%, it reacted with oxamyl with almost the same inhibition rate.

Example 26 Oxime analogs of OXM6-8
Cross-Reactivity with Compounds The cross-reactivity of OXM6-8 with oxamyl analogs was investigated by a direct competitive inhibition ELISA method using oxamyl derivative-4 as a labeled competing compound. Result is,
The IC ₅₀ values are shown in Table 1 as the concentrations at which the absorbance when these compounds were not added were inhibited by 50%.

[0209]

[Table 1] It was found that OXM6-8 hardly cross-reacted with the related compounds shown in Table 1.

[Brief description of drawings]

FIG. 1 shows the reactivity of antisera obtained by using the oxamyl derivative of the present invention with oxamyl by an indirect competitive inhibition ELISA method.

FIG. 2 shows the monoclonal antibody OXM6 of the present invention.
8 shows the reactivity of -8 with oxamyl by a direct competitive inhibition ELISA method.

FIG. 3 shows the monoclonal antibody OXM6 of the present invention.
8 shows the reactivity with oxamyl when the labeled competitive compound was changed in the direct competitive inhibition ELISA method of -8.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N 33/531 A01N 37/10 33/577 C12N 5/00 B // A01N 37/10 15/00 C (72) Inventor Miyake Shiro 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental and Immunological Technology (72) Inventor Yuki Yamaguchi 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside the Institute for Environmental and Immunological Technology (72) Invention Kabe Beppu 1-27-14 Hamamatsucho, Minato-ku, Tokyo Inside Institute for Environmental Immunotechnology (56) Reference JP-A-8-231591 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 16/16-16/44 A01N 37/10-37/14 C12P 21/08 CA (STN) REGISTRY (STN)

Claims (10)

(57) [Claims] 1. The following formulas (1)-(4): [In the formulas (1)-(4), R ₁ is the following formula (R _1a )-, which may be optionally substituted with 1 to 2 halogen atoms or an alkyl group having 1 to 3 carbon atoms; (R _1d ): [Chemical _formula 2] R ₂ is (CH ₂ ) _nc , CH ₂ CONH (CH ₂ ) _nd , or CO (CH ₂ ) _ne , wherein nc is 1
-3 is an integer, nd is an integer of 1-5, and ne
Is an integer of 1-5; R ₃ is an alkyl group having 1-6 carbon atoms; na is an integer of 5-15; and nb is 1-1.
It is an integer of 0], and an antigen is produced by binding a compound having a structure represented by any one of the following formulas to a polymer compound, and by using the antigen, the following formula (5): A method for producing an antibody reactive with a compound of formula (5) or a fragment thereof capable of binding to an antigen, comprising producing an antibody reactive with a compound having a structure represented by: 2. A method manufactured by the method according to claim 1.
An antibody or a fragment thereof capable of binding to an antigen, which is reactive with the compound of formula (5). 3. An antibody or fragment thereof capable of binding to an antibody or an antigen, which is reactive with the compound of formula (5) according to claim 2, which is prepared using a compound of formula (1) in which na = 5. 4. The antibody according to claim 2 or 3, which is a monoclonal antibody, or a fragment thereof capable of binding to an antigen. 5. The antibody or the fragment thereof capable of binding to the antibody or the antigen according to any one of claims 2 to 4, which is OXM6-8. 6. The method according to any one of claims 2 to 5.
A hybridoma that produces an antibody . 7. The hybridoma according to claim 6, which has been deposited under deposit number FERM P-16301. 8. An immunological assay method for a compound represented by the formula (5), which comprises using the antibody or the fragment thereof capable of binding to the antigen according to any one of claims 2 to 5. . 9. The immunological method according to claim 8, further comprising using a conjugate of the compound represented by any one of formulas (1) to (4) with a polymer compound or a labeled compound. Measuring method. (10) A compound represented by any of formulas (1)-(4) for producing the antibody or the fragment thereof capable of binding to the antibody according to any one of (2) to (5). The immunological assay method according to claim 9, which is different from the compound used.