JP7072213B2 - PET probe for imaging targeting hepatitis B antigen protein - Google Patents

Description

The present invention relates to a PET probe for imaging targeting a hepatitis B antigen protein.

Hepatitis B is caused by infection with hepatitis B virus (HBV) via blood and body fluids, and it is said that about 10% of infected people become chronic. Chronic hepatitis B progresses to cirrhosis, liver failure or hepatocellular carcinoma and is a life-threatening disease for patients, but there is no curative treatment at this time to prevent the production of new HBV and the spread of infection. However, there is only a palliative treatment that waits for infected cells to decrease or die due to immunity or metabolism, or for HBV to become inactivated.

Currently, interferon preparations that remove infected cells by immune action and nucleic acid analog preparations that inhibit the reverse transcription of HBV-DNA are used for the treatment of chronic hepatitis B. In particular, nucleic acid analog preparations exert a high therapeutic effect by suppressing HBV-DNA proliferation, but on the other hand, it is very difficult to determine when to stop medication. HBV is a DNA virus that exists in the nucleus of hepatocytes in the form of cccDNA (covalently closed circular DNA). When hepatocytes are infected, they invade the nucleus to produce pregenomic RNA, and by synthesizing HBV-DNA and various antigenic proteins from this RNA, this serves as a template to form new virus particles and proliferate (see scheme).

Therefore, even if the amount of HBV-DNA in the blood decreases due to medication, HBV in hepatocytes cannot be removed, and careless discontinuation of medication induces the emergence of drug-resistant strains, which may make treatment difficult. be. At present, there is no curative treatment that directly removes cccDNA, so treatment requires a long period of time, and as mentioned above, caution is required when deciding to stop medication. In addition, even if the hepatitis symptom recovers and the virus growth decreases or stops, it cannot be said that the virus is completely cured, and there is a problem that the latent virus is reactivated due to the appearance of a mutant strain or a decrease in immunity. Observation is required. The current diagnosis of hepatitis B pathology is mainly blood analysis, and invasive examination by liver biopsy is required for hepatitis B virus infection state in tissues and cells, and in the liver, which is a huge organ. It is not possible to know the overall infection distribution. Therefore, it is desired to develop a means for observing the intrahepatic HBV virus non-invasively.

Therefore, an object of the present invention is to develop a means for observing HBV virus in the liver non-invasively.

The present invention is a PET probe for imaging targeting a hepatitis B antigen protein containing a compound represented by the general formula (I) or a salt thereof. The compound represented by the general formula (I) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom.

[During the ceremony,
R ¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and carbon. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 3 and is
R ² is a phenyl group or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
R ³ is

(Here, R ⁴ is hydrogen or halogen.)
Is. ]

The PET probe for imaging of the present invention has an advantage that the intrahepatic HBV virus can be observed non-invasively.

A: It is a conceptual diagram which shows an example of the HBV replication activity evaluation nucleic acid. B: It is a conceptual diagram of a reporter pgRNA when the HBV replication activity evaluation vector in which the HBV replication activity evaluation nucleic acid shown by B: A is incorporated into an expression vector is introduced into a cell and expressed. In the reporter pgRNA, the introns in the reporter sequence have been removed by pre-mRNA splicing. C: It is a conceptual diagram of a reporter minus strand DNA (reporter (-) DNA) synthesized by the reverse transcription activity of HBV-Pol using the reporter pgRNA shown by B as a template. HBV replication activity evaluation Nucleic acid and reporter minus strand DNA can be distinguished by the presence or absence of an intron. FIG. 2 is a conceptual diagram showing an example of various expression vectors constituting the HBV replication activity evaluation system. A: It is a conceptual diagram of pBB-intron which is an example of an HBV replication activity evaluation vector. B: It is a conceptual diagram of pCI-HBV-Pol which is an expression vector of P gene which encodes HBV-Pol. C: It is a conceptual diagram of pCI-HBc which is an expression vector of C gene which encodes HBc. D: It is a conceptual diagram of pCI-HBx which is an expression vector of the X gene encoding HBx. FIG. 3A is the result of agarose gel electrophoresis. After gene transfer of the HBV replication activity evaluation vector into HeLa cells together with the combination of HBV protein expression vectors (C: HBc, P: HBV-Pol, X: HBx) shown in the figure, DNA extraction was performed to obtain a reporter minus strand DNA. The PCR product (131bp) when PCR was performed with the primer pair to be specifically detected is shown. FIG. 3B is a diagram showing a calibration curve of reporter minus strand DNA. DNA extracted from cells was serially diluted and prepared using real-time PCR. C: This is the result of real-time PCR using the DNA sample used in the experiment of A and quantitative analysis by the calibration curve of B. FIG. 3C shows the results of real-time PCR using the DNA sample used in the experiment of FIG. 3A and quantitative analysis by the calibration curve of FIG. 3B. FIG. 3D is a diagram showing the results of quantitative analysis of the correlation between the expression level of HBV protein (HBc, HBV-Pol, and HBx) and DNA replication by real-time PCR. FIG. 4 shows a liver tissue section HBc stained image. "Non-treatment" means that pCI-HBc was not administered, and "pCI-HBc Injected" means that pCI-HBc was administered. FIG. 5 shows a comparison of HBc and CRP concentrations in tissue lysates by pCI-HBc dose. FIG. 6 shows the PET imaging results using [ ¹¹ C] CBT-078 and [ ¹¹ C] CBT-039. It is a Maximum Industry Projection image 1 hour after administration. In the image, the white arrow indicates the liver, the black arrow indicates the bile excretion (duodenum / intestinal tract), Δ indicates the bladder, and ▲ indicates the position of the gallbladder. "078-HBc" is the PET imaging result of the HBc-expressing mouse using [ ¹¹ C] CBT-078, and "078-Vehicle" is the PET of the Vehicle (PBS) -administered mouse using [ ¹¹ C] CBT-078. The imaging result, "078-Blocking", is the PET imaging result of the competition inhibition test in which the unlabeled compound CBT-078 was administered to the HBc-expressing mice before the administration of [ ^11C ] CBT-078. [ ¹¹ C] PET imaging results of HBc-expressing mice using CBT-039, "039-Vehicle" indicates PET imaging results of Vehicle (PBS) -administered mice using [ ¹¹ C] CBT-039. FIG. 7 shows the time radioactivity curve in the liver. "078-HBc" is the PET imaging result of the HBc-expressing mouse using [ ¹¹ C] CBT-078, and "078-Vehicle" is the PET of the Vehicle (PBS) -administered mouse using [ ¹¹ C] CBT-078. The imaging result, "078-Blocking", is the PET imaging result of the competition inhibition test in which the unlabeled compound CBT-078 was administered to the HBc-expressing mice before the administration of [ ^11C ] CBT-078. [ ¹¹ C] PET imaging results of HBc-expressing mice using CBT-039, "039-Vehicle" indicates PET imaging results of Vehicle (PBS) -administered mice using [ ¹¹ C] CBT-039. FIG. 8 shows the tissue radioactivity distribution.

The present inventors aim at the development of a non-nucleic acid analog therapeutic agent having a different mechanism of action from existing therapeutic agents, and the core constituting the HB core-related molecule (HBc; HBV nucleocapsid) that forms virus particles produced by HBV infection. We have found a compound that targets (protein). HBc is a protein consisting of a total length of 183 amino acids, consisting of an N-terminal assembly domain (1-144 residues) and a C-terminal RNA / DNA binding domain (145-183 residues). HBc expressed in virus-infected cells forms a dimer by the assembly domain, and association occurs in this dimer as a unit to produce a nucleocapsid of HBV. Virus particles are produced by the uptake of pgRNA and polymerase during capsid formation and the production of HBV-DNA by reverse transcription reaction. The present inventors have found a compound having inhibitory activity by the HBV-DNA replication inhibition assay, which has separately developed an assay method for quantifying the dimer formation of HBc.

The present invention relates to a novel PET imaging probe targeting HBc, which comprises a compound having such inhibitory activity.

PET (positron emission tomography) is one of the molecular imaging techniques based on the detection of radiation emitted from positron emission tomography, and is a minimally invasive imaging of the pharmacokinetics of PET imaging probes in vivo. Can be done. The current diagnosis of hepatitis B pathology is mainly blood analysis, and invasive examination by liver biopsy is required for hepatitis B virus infection state in tissues and cells, and in the liver, which is a huge organ. It is not possible to know the overall infection distribution. If the HBV infection status can be directly confirmed by non-invasive diagnostic imaging using PET, it is expected to be useful information for treatment planning, decision of drug suspension, and the like. By quantitatively evaluating the HBc distribution in the liver tissue by diagnostic imaging, it is expected that it will be possible to make a rational decision to discontinue treatment based on an objective evaluation of the viral load in the substantial hepatocytes.

That is, the present invention
A PET probe for imaging targeting a hepatitis B antigen protein containing a compound represented by the general formula (I) or a salt thereof. The compound represented by the general formula (I) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom.

During the ceremony
R ¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and carbon. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 3 and is
R ² is a phenyl group or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
R ³ is

(Here, R ⁴ is hydrogen or halogen.)
Is.

In the PET probe for imaging targeting the hepatitis B antigen protein of the present invention,
The compound represented by the general formula (I) has R ³

Is preferable.

In the PET probe for imaging targeting the hepatitis B antigen protein of the present invention,
The compound represented by the general formula (I) is
R ³ is

And
R ¹ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, and a halogen.
R ² is a phenyl group or a phenyl group substituted with 1-3 selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkylthio having 1 to 3 carbon atoms, and a halogen. Is more preferable.

Further, in the PET probe for imaging targeting the hepatitis B antigen protein of the present invention,
The compound represented by the general formula (I) is a compound represented by the following general formula (XI), a compound represented by the general formula (XII), a compound represented by the general formula (XIII), or a general formula. It is preferably a compound represented by (XIV). The compound represented by the general formula (XI), the compound represented by the general formula (XII), the compound represented by the general formula (XIII), or the compound represented by the general formula (XIV) , ¹¹ C or ¹⁸ F if it contains a fluorine atom.

The compound represented by the general formula (XI) is shown below.

In formula (XI),
R ⁴ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 2 to 6 carbon atoms. Substituted by 1 to 3 selected from the group consisting of alkenyl 6 and alkynyl having 2 to 6 carbons, alkynyl having 2 to 6 carbons substituted with alkylsilyl having 1 to 6 carbons, phenyl, halogen, and dioxaboranyl. It is a phenyl group.

The compound represented by the general formula (XI) is
R ⁴ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, or an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, and a halogen. , And a phenyl group substituted with 1-3 selected from the group consisting of dioxaboranyl, preferably.
R ⁴ is more preferably a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, and a halogen.

The compound represented by the general formula (XII) is shown below.

In formula (XII),
R ⁵ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, and a halogen.

The compound represented by the general formula (XII) is
R ⁵ is preferably a phenyl group or a phenyl group substituted with 1-3 selected from the group consisting of alkyls having 1 to 6 carbon atoms and halogens.
R ⁵ is more preferably a phenyl group substituted with 1-3 halogens.

The compound represented by the general formula (XIII) is shown below.

In equation (XIII),
R ⁶ is a phenyl group or a phenyl group substituted with an alkyl having 1 to 3 carbon atoms and 1 to 6 carbon atoms, and R ⁷ is a phenyl group or a phenyl group substituted with 1 to 3 halogens.
R ⁸ is

Is.

The compound represented by the general formula (XIII) is
R ⁶ is preferably a phenyl group substituted with an alkyl having 1 to 3 carbon atoms of 1 to 6, and R ⁷ is preferably a phenyl group substituted with a halogen of 1 to 3.
R ⁶ is a phenyl group substituted with an alkyl having 1 to 3 carbon atoms of 1 to 6, and R ⁷ is a phenyl group substituted with a halogen of 1 to 3.
R ⁸ is

Is more preferable.

The compound represented by the general formula (XIV) is shown below.

In formula (XIV),
R ⁹ is a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.

The compound represented by the general formula (XIV) is
R ⁹ is a phenyl group, an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkylthio having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms substituted with halogen, and 1 to 3 carbon atoms. It is preferably a 1-3 substituted phenyl group selected from the group consisting of the alkyl-substituted amino, cyano, and halogens.

Further, the present invention
It is a compound represented by the general formula (XX) or a salt thereof, or a compound labeled with ¹¹ C or, if it contains a fluorine atom, labeled with ¹⁸ F.

The compound represented by the general formula (XX) is shown below.

In formula (XX),
R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and carbon. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 3 and is
R ¹² is a phenyl group or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
However, when R ¹² is a phenyl group substituted with halogen, R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, or an alkyl having 1 to 6 carbon atoms and an alkoxy having 1 to 6 carbon atoms. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, and dioxaboranyl. A phenyl group substituted with 3 or a phenyl group at which the o-position or p-position is substituted with halogen.
However, when R ¹² is a phenyl group, R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, and an alkenyl having 2 to 6 carbon atoms. , Select from the group consisting of 1-3 selected from the group consisting of alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 1 to 3 to be added.

The compound represented by the general formula (XX) is
R ¹¹ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, and a halogen.
R ¹² is a phenyl group or a phenyl group substituted with 1-3 selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkylthio having 1 to 3 carbon atoms, and a halogen. Is more preferable.

The compound represented by the general formula (XX) may be a compound represented by the general formula (XV).

In formula (XV),
R ^4'is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, and 2 to 6 carbon atoms. A phenyl group substituted with 1-3, selected from the group consisting of alkynyl, alkynyl having 2 to 6 carbons substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, and dioxaboranyl, or the o-position or p-position. Is a phenyl group substituted with halogen.

The compound represented by the general formula (XV) is
R ^4'is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, or an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkenyl having 2 to 6 carbon atoms, and an alkynyl having 2 to 6 carbon atoms. It is preferably a phenyl group substituted with 1-3 selected from the group consisting of halogen and dioxaboranyl.
R ^4'is more preferably a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, and a halogen.

The compound represented by the general formula (XX) may be a compound represented by the general formula (XII).

In formula (XII),
R ⁵ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, or a halogen.

The compound represented by the general formula (XII) is
R ⁵ is preferably a phenyl group or a phenyl group substituted with 1-3 selected from the group consisting of alkyls having 1 to 6 carbon atoms and halogens.
R ⁵ is more preferably a phenyl group substituted with 1-3 halogens.

The compound represented by the general formula (XX) may be a compound represented by the general formula (XVI).

In the formula (XVI),
R ⁹ is substituted with an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and an alkyl having 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of amino, cyano, and halogen.

The compound represented by the general formula (XVI) is
R ^9'is a phenyl group, an alkyl having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms, an alkylthio having 1 to 3 carbon atoms, an alkoxy having 1 to 3 carbon atoms substituted with halogen, and 1 to 3 carbon atoms. It is preferably a 1-3 substituted phenyl group selected from the group consisting of amino, cyano, and halogen substituted with 3 alkyls.

In the present invention, the "halogen" includes fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine, bromine, and more preferably fluorine.

In the present invention, the "alkyl having 1 to 6 carbon atoms" includes methyl, ethyl, n-propyl, isopropyl, 2-methylpropyl, n-butyl, t-butyl, pentyl, hexyl and the like. The "alkyl having 1 to 6 carbon atoms" is preferably an alkyl having 1 to 5 carbon atoms, more preferably an alkyl having 1 to 4 carbon atoms, and further preferably an alkyl having 1 to 3 carbon atoms.

In the present invention, the "alkylsilyl having 1 to 6 carbon atoms" means methylsilyl, trimethylsilyl, ethylsilyl, n-propylsilyl, isopropylsilyl, 2-methylpropylsilyl, n-butylsilyl, t-butylsilyl, pentylsilyl, hexylsilyl. And so on. The "alkylsilyl having 1 to 6 carbon atoms" is preferably an alkylsilyl having 1 to 5 carbon atoms, and more preferably an alkylsilyl having 1 to 4 carbon atoms.

In the present invention, "alkoxy having 1 to 6 carbon atoms" means alkyloxy having 1 to 6 carbon atoms, and specifically, methoxy, ethoxy, n-propyloxy, isopropyloxy, and 2-methylpropyloxy. , N-butyloxy, t-butyloxy, pentyloxy, hexyloxy and the like. The "alkoxy having 1 to 6 carbon atoms" is preferably an alkoxy having 1 to 5 carbon atoms (alkyloxy having 1 to 5 carbon atoms), and more preferably an alkoxy having 1 to 4 carbon atoms (alkyloxy having 1 to 4 carbon atoms). ), More preferably alkoxy having 1 to 3 carbon atoms (alkyloxy having 1 to 3 carbon atoms).

In the present invention, examples of the "alkoxy having 1 to 6 carbon atoms substituted with halogen" include methyloxy fluoride, ethyloxy fluoride, methyloxy iodide and the like. The "halogen-substituted alkoxy having 1 to 6 carbon atoms" is preferably a halogen-substituted alkoxy having 1 to 5 carbon atoms, more preferably a halogen-substituted alkoxy having 1 to 4 carbon atoms, and even more preferably. It is an alkoxy having 1 to 3 carbon atoms substituted with a halogen.

In the present invention, the "alkylthio having 1 to 6 carbon atoms" includes methylthio, ethylthio, n-propylthio, isopropylthio, 2-methylpropylthio, n-butylthio, t-butylthio, pentylthio, hexylthio and the like. The "alkylthio having 1 to 6 carbon atoms" is preferably an alkylthio having 1 to 5 carbon atoms, more preferably an alkylthio having 1 to 4 carbon atoms, and further preferably an alkylthio having 1 to 3 carbon atoms.

In the present invention, "cyclohexyl having 3 to 7 carbon atoms" includes cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. The "cyclohexyl group having 3 to 7 carbon atoms" is preferably a cyclohexyl having 4 to 7 carbon atoms, and more preferably a cyclohexyl having 4 to 6 carbon atoms.

In the present invention, the "alkenyl having 2 to 6 carbon atoms" includes vinyl, allyl, isopropenyl, butenyl, pentenyl, hexenyl and the like. The "alkenyl having 2 to 6 carbon atoms" is preferably an alkenyl having 2 to 5 carbon atoms, and more preferably an alkenyl having 2 to 4 carbon atoms.

In the present invention, "alkynyl having 2 to 6 carbon atoms" includes ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like. The "alkynyl having 2 to 6 carbon atoms" is preferably an alkynyl having 2 to 5 carbon atoms, and more preferably an alkynyl having 2 to 4 carbon atoms.

In the present invention, examples of the "alkynyl having 2 to 6 carbon atoms substituted with an alkylsilyl having 1 to 6 carbon atoms" include 1-trimethylsilylethynyl and 1-ethylsilylbutynyl.

In the present invention, the "amino substituted with an alkyl having 1 to 6 carbon atoms" means methylamino, ethylamino, n-propylamino, isopropylamino, 2-methylpropylamino, n-butylamino, t-butylamino. , Pentylamino, hexylamino and the like. The "amino acid substituted with an alkyl having 1 to 6 carbon atoms" is preferably an alkyl amino acid having 1 to 5 carbon atoms, more preferably an alkyl amino acid having 1 to 4 carbon atoms, and further preferably an alkyl amino acid having 1 to 3 carbon atoms. Is.

In the present invention, examples of the "phenyl substituted with an alkyl having 1 to 3 carbon atoms of 1 to 6" include 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 1-ethylphenyl and the like. The "phenyl substituted with an alkyl having 1 to 6 carbon atoms" is preferably a phenyl substituted with an alkyl having 1 to 5 carbon atoms, and more preferably a phenyl substituted with an alkyl having 1 to 4 carbon atoms.

In the present invention, "phenyl substituted with 1 to 3 halogens" means 1-fluorophenyl, 2-fluorophenyl, 3-fluorophenyl, 2,3,4-trifluorophenyl, 1-chlorophenyl, 2-. Examples thereof include chlorophenyl, 3-chlorophenyl, 1-bromophenyl, 2-bromophenyl, 3-bromophenyl, 1-iodophenyl, 2-iodophenyl and 3-iodophenyl.

In the present invention, the salt is a non-toxic and pharmaceutically acceptable conventional salt, for example, a salt with an alkali metal such as sodium and potassium, a salt with an alkaline earth metal such as calcium and magnesium, ammonium and the like. Salts with inorganic bases and salts with organic amines such as triethylamine, pyridine, picolin, ethanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethyleneamine, and hydrochloric acid, hydrobromic acid, sulfuric acid, Salts with inorganic acids such as phosphoric acid, salts with organic carboxylic acids such as formic acid, acetic acid, trifluoroacetic acid, maleic acid and tartaric acid, and sulfonic acids such as methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid. And salts with bases such as basic or acidic amino acids such as arginine, aspartic acid and glutamic acid or salts with acids.

In the present invention, "the compound represented by the general formula (I) is labeled with ¹¹ C or, if it contains a fluorine atom, it is labeled with ¹⁸ F".

(A) Any one of the carbon atoms in the general formula (I) is labeled with ¹¹ C.
(B) R ¹ in the general formula (I) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(C) R ² in the general formula (I) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(D) R ³ in the general formula (I) is

（ここで、Ｒ⁴は、ハロゲン（好ましくはフッ素）である。）
である場合、そのハロゲンが¹⁸Ｆで標識される、
のいずれかを意味する。

(Here, R ⁴ is a halogen (preferably fluorine).)
If, the halogen is labeled with ¹⁸ F,
Means one of.

(A) When any one of the carbon atoms in the general formula (I) is labeled with ¹¹ C, any one of the carbon atoms in R ¹ , R ² and R ³ is labeled with ¹¹ C. May be done. Specifically, R ¹ is substituted with 1-3 selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, and alkoxy having 1 to 6 carbon atoms substituted with halogen. In the case of a phenyl group, any one of the substituents, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, and an alkoxy having 1 to 6 carbon atoms substituted with halogen, is ¹¹ It may be labeled with C.

Examples of "the compound represented by the general formula (I) are labeled with ¹¹ C or ¹⁸ F when containing a fluorine atom" include the following compounds. ..

Similarly, in the present invention, "the compound represented by the general formula (XI) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom."

(A) Any one of the carbon atoms in the general formula (XI) is labeled with ¹¹ C.
(B) R ⁴ in the general formula (XI) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(C) In the general formula (XI)

Fluorine is labeled with ¹⁸ F,
Means one of.

Examples of "the compound represented by the general formula (XI) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

Similarly, in the present invention, "the compound represented by the general formula (XII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom."

(A) Any one of the carbon atoms in the general formula (XII) is labeled with ¹¹ C.
(B) When R ⁵ in the general formula (XII) is a phenyl group substituted with a halogen (preferably fluorine), the halogen is labeled with ¹⁸ F.
(C) In the general formula (XII)

Fluorine is labeled with ¹⁸ F,
Means one of.

Examples of "the compound represented by the general formula (XII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

Similarly, in the present invention, "the compound represented by the general formula (XIII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom."

(A) Any one of the carbon atoms in the general formula (XIII) is labeled with ¹¹ C.
(B) When R ⁷ in the general formula (XIII) is a phenyl group substituted with a halogen (preferably fluorine), the halogen is labeled with ¹⁸ F.
Means one of.

Examples of "the compound represented by the general formula (XIII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

Similarly, in the present invention, "the compound represented by the general formula (XIII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom."

(A) Any one of the carbon atoms in the general formula (XIV) is labeled with ¹¹ C.
(B) R ⁹ in the general formula (XIV) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(C) In the general formula (XIV)

Fluorine is labeled with ¹⁸ F,
Means one of.

In the present invention, "a compound represented by the general formula (XX) labeled with ¹¹ C or labeled with ¹⁸ F when it contains a fluorine atom" is defined as "a compound represented by the general formula (XX)".

(A) Any one of the carbon atoms in the general formula (XX) is labeled with ¹¹ C.
(B) R ¹¹ in the general formula (XX) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(C) R ¹² in the general formula (XX) is 1 to 3 selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a substituted phenyl group, its halogen is labeled with ¹⁸ F,
(D) In the general formula (XX)

Fluorine is labeled with ¹⁸ F,
Means one of.

Examples of "the compound represented by the general formula (XX) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

In the present invention, "a compound represented by the general formula (XV) labeled with ¹¹ C or labeled with ¹⁸ F when it contains a fluorine atom" is defined as "a compound represented by the general formula (XV)".

(A) Any one of the carbon atoms in the general formula (XV) is labeled with ¹¹ C.
(B) 1-3 selected from the group in which R ^4'in the general formula (XV) consists of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine). If it is a phenyl group substituted with, the halogen is labeled with ¹⁸ F,
(C) In the general formula (XV)

Fluorine is labeled with ¹⁸ F,
Means one of.

Examples of "the compound represented by the general formula (XV) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

In the present invention, "a compound represented by the general formula (XII) labeled with ¹¹ C or labeled with ¹⁸ F when it contains a fluorine atom" is defined as "a compound represented by the general formula (XII)".

(A) Any one of the carbon atoms in the general formula (XII) is labeled with ¹¹ C.
(B) When R ⁵ in the general formula (XII) is a phenyl group substituted with a halogen (preferably fluorine), the halogen is labeled with ¹⁸ F.
(C) In the general formula (XII)

Fluorine is labeled with ¹⁸ F,
Means one of.

Examples of "the compound represented by the general formula (XII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom" include, for example, the following compounds. ..

In the present invention, "a compound represented by the general formula (XVI) labeled with ¹¹ C or labeled with ¹⁸ F when it contains a fluorine atom" is defined as "a compound represented by the general formula (XVI)".

(A) Any one of the carbon atoms in the general formula (XVI) is labeled with ¹¹ C.
(B) R ^9'in the general formula (XVI) is selected from the group consisting of an alkoxy having 1 to 6 carbon atoms substituted with a halogen (preferably fluorine) and a halogen (preferably fluorine) 1-3. If it is a phenyl group substituted with, the halogen is labeled with ¹⁸ F,
(C) In the general formula (XVI)

Fluorine is labeled with ¹⁸ F,
Means one of.

The method for producing the compound represented by the general formula (I) of the present invention is as follows.

(In the formula, R ¹ , R ² , and R ³ are the same as the definitions in the formula (I).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (I) can be obtained, for example, by condensing a compound represented by the formula (XXXI) and an amine represented by the formula (XXXII). Alternatively, the compound represented by the general formula (I) can be obtained, for example, by condensing a compound represented by the formula (XXXIII) and an amine represented by the formula (XXXIV).

The condensation step can be performed using a conventionally known condensing agent. As the condensing agent, for example, carbodiimide-based, imidazole-based, triazine-based, phosphonium-based, uronium-based, halonium-based, and the like are known. Examples of the carbodiimide-based condensing agent include 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDCI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI ・ HCl), and N. , N'-dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIC) and the like. Examples of the imidazole-based condensing agent include N, N'-carbonyldiimidazole (CDI) and 1,1'-carbonyldi (1,2,4-triazole) (CDT). Examples of the triazine-based condensing agent include 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride n hydrate (DMT-MM). .. Phosphonium-based condensing agents include 1H-benzotriazole-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP) and 1H-benzotriazole-1-yloxytrypyrrolidinophosphonium hexafluorophosphate (BOP). PyBOP) and the like. Examples of the uronium-based condensing agent include {{[(1-cyano-2-ethoxy-2-oxoethylidene) amino] oxy} -4-morpholinomethylene} dimethylammonium hexanefluorophosphate (COMU). Examples of the halonium-based condensing agent include 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP).

The compound represented by the formula (XXXI) may be produced according to publicly known documents or may be commercially available, but it can also be produced as follows.

(In the formula, R ¹ and R ³ are the same as the definitions in the formula (I).
X is OH or alkoxy having 1 to 3 carbon atoms. )

The compound of the formula (XXXVI) and the amine represented by the formula (XXXIV) are condensed to obtain the compound of the formula (XXXV). This condensation can be carried out in the same manner as the condensation in the method for producing the compound represented by the general formula (I).

The compound of the formula (XXXV) is added to a mixture of palladium acetate and 1,3-bis (diphenylphosphino) propane under an inert gas atmosphere together with triethylamine, and the reaction vessel is replaced with carbon monoxide gas and heated. By stirring while stirring, the compound of the formula (XXXI) can be obtained.

The compound represented by the formula (XXXIII) may be produced according to publicly known documents or may be commercially available, but it can also be produced as follows.

(In the formula, R ² and R ³ are the same as the definitions in the formula (I).
X is OH or alkoxy having 1 to 3 carbon atoms. )

The compound of the formula (XXXVI) and the amine represented by the formula (XXXII) are condensed to obtain the compound of the formula (XXXVI). This condensation can be carried out in the same manner as the condensation in the method for producing the compound represented by the general formula (I).

The compound of the formula (XXXVI) is added to a mixture of palladium acetate and 1,3-bis (diphenylphosphino) propane under an inert gas atmosphere together with triethylamine, and the reaction vessel is replaced with carbon monoxide gas and heated. By stirring while stirring, the compound of the formula (XXXIII) can be obtained.

The compound represented by the general formula (XI) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ⁴ is the same as the definition in the formula (XI).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XII) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ⁵ is the same as the definition in the formula (XII).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XIII) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ⁶ , R ⁷ , and R ⁸ are the same as the definitions in the formula (XIII).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XIV) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(Same as the definition in the formula, R ⁹ , formula (XIV),
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XX) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ¹¹ and R ¹² are the same as the definitions in the formula (XX).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XX) can be obtained, for example, by condensing a compound represented by the formula (XXXX) and an amine represented by the formula (XXXXI). Alternatively, the compound represented by the general formula (XX) can be obtained, for example, by condensing a compound represented by the formula (XXXII) and an amine represented by the formula (XXXIII). This condensation can be carried out in the same manner as the condensation in the method for producing the compound represented by the general formula (I).

The compound represented by the formula (XXXX) may be produced according to a known document or may be commercially available, but it can also be produced as follows.

(In the equation, R ¹¹ is the same as the definition in the equation (XX).
X is OH or alkoxy having 1 to 3 carbon atoms. )

The compound of the formula (XXXIV) and the amine represented by the formula (XXXXIII) are condensed to obtain the compound of the formula (XXXV). This condensation can be carried out in the same manner as the condensation in the method for producing the compound represented by the general formula (I).

The compound of the formula (XXXV) is added to a mixture of palladium acetate and 1,3-bis (diphenylphosphino) propane under an inert gas atmosphere together with triethylamine, and the reaction vessel is replaced with carbon monoxide gas and heated. By stirring while stirring, the compound of the formula (XXXXX) can be obtained.

The compound represented by the formula (XXXXII) may be produced according to a known document or may be commercially available, but it can also be produced as follows.

(In the formula, R ¹² is the same as the definition in the formula (XX).
X is OH or alkoxy having 1 to 3 carbon atoms. )

The compound of the formula (XXXIV) and the amine represented by the formula (XXXXI) are condensed to obtain the compound of the formula (XXXXVI). This condensation can be carried out in the same manner as the condensation in the method for producing the compound represented by the general formula (I).

The compound of the formula (XXXXVI) is added to a mixture of palladium acetate and 1,3-bis (diphenylphosphino) propane under an inert gas atmosphere together with triethylamine, and the reaction vessel is replaced with carbon monoxide gas and heated. By stirring while stirring, the compound of the formula (XXXXII) can be obtained.

The compound represented by the general formula (XV) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ^4'is the same as the definition in the formula (XV).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The compound represented by the general formula (XVI) of the present invention can also be produced in the same manner. An example of the manufacturing scheme is shown below.

(In the formula, R ^9'is the same as the definition in the formula (XVI).
X is OH, a halogen atom or an alkoxy having 1 to 3 carbon atoms. )

The " ¹¹ C-labeled" compound of the present invention combines the method for producing the compound of the present invention with a conventionally known high-speed ¹¹ C-methylation reaction (Chem. Rec. 2014, 14, 516-541.). Can be manufactured. In addition, the compound "labeled with ¹⁸ F when it contains a fluorine atom" of the present invention is a conventionally known ¹⁸ F-labeling method (Org. Lett. 2015, 17, 17 in the method for producing the compound of the present invention. 5780-5783; Chem.Commun. 2016, 52, 8361-8364) can be produced in combination.

The PET probe for imaging of the present invention can be formulated into an appropriate dosage form such as an injection by a conventionally known method. As an injection, for example, an injection for intravenous administration, a medium, a pH adjuster, a buffer, a suspending agent, a solubilizer, an antioxidant, a preservative, etc. are represented by the general formula (I). It can be formulated by adding it to the compound or a salt thereof as needed and treating it by a conventional method.

[Abbreviation]
The following abbreviations may be used herein.
DMAP: 4-dimethylaminopyridine EDCl · HCl: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride DIPEA: diisopropylethylamine COMU: {{[(1-cyano-2-ethoxy-2-oxoethylidene)) Amino] Oxy} -4-morpholinomethylene} Dimethylammonium hexanefluorophosphate PBS: Phosphate buffered saline

<Manufacturing example 1>
Synthesis of intermediates Preparation of 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4)

Step 1: Preparation of 3-bromo-2-fluoro-N- (4-fluorophenyl) benzamide (2)

4-Fluoroaniline (2.20 g, 19.8 mmol, 2.0 eq) in a solution of 3-bromo-2-fluorobenzoic acid (1) (2.17 g, 9.91 mmol, 1 eq) in methylene chloride (100 mL). ), 4-Dimethylaminopyridine (DMAP) (121 mg, 0.991 mmol, 0.10 eq), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl · HCl) (2.85 g, 14.9 mmol (1.5 eq) was added at room temperature. After stirring at room temperature for 15 hours, water (about 100 mL) was added to the mixture. The resulting mixed solution was extracted 3 times with ethyl acetate (about 50 mL). The coalesced organic phase was washed with saturated brine (about 100 mL) and dried over anhydrous sodium sulfate. The organic phase was passed through silica gel of a short pad and filtered (eluted with ethyl acetate), and then the solvent was distilled off under reduced pressure. Pure 3-bromo-2-fluoro-N- (4-fluorophenyl) benzamide (2) (2.39 g, 7.66 mmol, 38.7%) was obtained as a colorless solid. It was used for the next reaction without further purification.

¹ H NMR (CDCl ₃ ) δ 7.06-7.12 (AA'BB'X, 2H), 7.22 (dd, J = 7.5, 7.5 Hz, 1H), 7.59-7.63 (AA'BB'X, 2H), 7.75 ( ddd, J = 7.5, 7.5, 1.5 Hz, 1H), 8.09 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H), 8.31 (br d, J = 14.3 Hz, 1H);
HRMS (ESI ⁺ ) m / z 333.9644 (calculated as C ₁₃ H ₈ BrF ₂ NNaO ⁺ , 333.9650, [M + Na] ⁺ ).

Step 2: Production of 2-fluoro-3-((4-fluorophenyl) carbamoyl) methyl benzoate (3)

1.3-Bis (diphenylphosphino) propane (947 mg, 2.) To a mixed solution of palladium acetate (516 mg, 2.30 mmol, 0.30 eq) in methanol (38 mL) and N, N-dimethylformamide (38 mL). 30 mmol, 0.30 eq) was added at room temperature under an argon atmosphere. After stirring at room temperature for 30 minutes, triethylamine and 3-bromo-2-fluoro-N- (4-fluorophenyl) benzamide (2) (2.39 g, 7.66 mmol, 1 eq) were added to the mixture. After replacing the reaction vessel containing the mixture with carbon monoxide, the mixture was heated and stirred at 75 ° C. for 20 hours. After returning to room temperature, the solution was filtered through silica gel on a short pad and eluted with diethyl ether. The solvent of the diethyl ether solution was distilled off under reduced pressure. After adding water to the residue, it was extracted 3 times with diethyl ether. The coalesced organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. By purifying the residue by silica gel column chromatography, 2-fluoro-3-((4-fluorophenyl) carbamoyl) methyl benzoate (3) (2.08 g, 7.14 mmol, 93.3%) is a colorless solid. Got as.

¹ H NMR (CDCl ₃ ) δ 3.99 (s, 3H), 7.05-7.12 (AA'BB'X, 2H), 7.39 (dd, J = 7.7, 7.7 Hz, 1H), 7.60-7.65 (AA'BB' X, 2H), 8.12 (ddd, J = 7.7, 7.7, 1.5 Hz, 1H), 8.36 (ddd, J = 7.7, 7.7, 1.5 Hz, 1H), 8.41 (br d, J = 14.5 Hz, 1H);
HRMS (ESI +) m / z 314.0599 (calculated as C ₁₅ H ₁₁ F ₂ NNaO ₃ ⁺ , 314.0651, [M + Na] +).

Step 3: Preparation of 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4)

2-Fluoro-3-((4-fluorophenyl) carbamoyl) Methyl benzoate (3) (1.98 g, 6.80 mmol, 1 eq) in methylene chloride (20 mL), methanol (10 mL) and water (10 mL) Sodium hydroxide (815 mg, 20.4 mmol, 3.0 eq) was added to the mixed solution at room temperature, and the mixture was stirred at room temperature for 4 hours. Hydrochloric acid (3M aqueous solution) was added to the solution after completion of the reaction until the pH became 1 or less. The solution was extracted 3 times with ethyl acetate (about 20 mL). The coalesced organic phase was washed with saturated brine (about 40 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent is distilled off from the organic phase under reduced pressure to obtain 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4) (1.40 g, 5.05 mmol, 75.4%). ) Was obtained as a white solid.

¹ H NMR (DMSO-d ₆ ) δ7.19-7.23 (AA'BB'X, 2H), 7.41 (dd, J = 7.7, 7.7 Hz, 1H), 7.72-7.75 (AA'BB'X, 2H) , 7.83-7.87 (m, 1H), 7.99 (ddd, J = 7.7, 7.7, 1.5 Hz, 1H), 10.59 (s, 1H);
HRMS (ESI +) m / z 300.0440 (calculated as C ₁₄ H ₉ F ₂ NNaO ₃ ⁺ , 300.0443, [M + Na] +).

<Manufacturing example 2>
Production of 2-Fluoro-3- (Phenylcarbamoyl) Benzoic Acid (7) Step 1: Production of 3-bromo-2-fluoro-N-phenylbenzamide (5)

Compound (5) was produced according to step 1 of Production Example 1 except that aniline was used instead of 4-fluoroaniline. Yield 32%.

¹ H NMR (CDCl ₃ ) δ 7.05-7.11 (AA'BB'X, 2H), 7.21 (dd, J = 7.9, 7.9 Hz, 1H), 7.60-7.63 (AA'BB'X, 2H), 7.75 ( ddd, J = 7.9, 7.5, 1.8 Hz, 1H), 8.08 (ddd, J = 7.9, 7.5, 1.8 Hz, 1H), 8.31 (br d, J = 13.4 Hz, 1H);
HRMS (ESI +) m / z 293.9922 (calculated as C ₁₃ H ₁₀ ON ⁷⁹ BrF ⁺ , 293.9924, [M + H] ⁺ ).

Step 2: Production of 2-fluoro-3- (phenylcarbamoyl) methyl benzoate (6)

Compound (6) used 3-bromo-2-fluoro-N-phenylbenzamide (5) instead of 3-bromo-2-fluoro-N- (4-fluorophenyl) benzamide (2), except that the compound (6) used 3-bromo-2-fluoro-N-phenylbenzamide (5). It was manufactured according to step 2 of Production Example 1. Yield 53%.

^{1 1} H NMR (CDCl ₃ ) δ 3.93 (s, 3H), 7.19 (dd, J = 7.5, 7.5 Hz, 1H), 7.37-7.41 (m, 3H), 7.67 (d, J = 7.5 Hz, 2H), 8.11 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H), 8.36 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H), 8.45 (br d, J = 14.3 Hz, 1H);
HRMS (ESI +) m / z 274.0870 (calculated as C ₁₅ H ₁₃ O ₃ NF ₊ , 274.0874, [M + H] ⁺ ).

Step 3: Production of 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7)

Compound 7 used methyl 2-fluoro-3- (phenylcarbamoyl) benzoate (6) instead of methyl 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoate (3). It was manufactured according to step 3 of Production Example 1. Yield 54%.

¹ H NMR (DMSO-d ₆ ) δ 7.10-7.14 (m, 1H), 7.34-7.43 (m, 3H), 7.72 (d, J = 8.4 Hz, 2H), 7.83-7.87 (m, 1H), 7.99 (ddd, J = 7.4, 7.4, 1.8 Hz, 1H), 10.54 (s, 1H);
HRMS (ESI +) m / z 260.0716 (calculated as C ₁₄ H ₁₁ O ₃ NF ⁺ , 260.0717, [M + H] ⁺ ).

<Manufacturing example 3>
Production of 2-Fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) Step 1: Production of 3-bromo-2-fluoro-3- (N- (o-tolyl) benzamide (8))

Compound 8 was produced according to step 1 of Production Example 1, except that 2-methylaniline was used instead of 4-fluoroaniline. Yield 80%.

^{1 1} H NMR (CDCl ₃ ) δ 2.37 (s, 3H), 7.14 (ddd, J = 7.5, 7.5, 1.1 Hz, 1H), 7.20-7.30 (m, 3H), 7.75 (ddd, J = 8.0, 6.8, 6.8, 1.8 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.12-8.16 (m, 1H), 8.29 (br d, J = 15.2 Hz, 1H);
HRMS (ESI +) m / z 308.0078 (calculated as C ₁₄ H ₁₂ ON ⁷⁹ BrF ⁺ , 308.0081, [M + H] ⁺ ).

Step 2: Production of methyl 2-fluoro-3- (o-tolylcarbamoyl) benzoate (9)

Compound (9) replaces 3-bromo-2-fluoro-N- (4-fluorophenyl) benzamide (2) with 3-bromo-2-fluoro-3- (N- (o-tolyl) benzamide (8). ) Was used, and the product was produced according to step 2 of Production Example 1. The yield was 79%.

¹ H NMR (DMSO-d ₆ ) δ 2.27 (s, 3H), 3.90 (s, 3H), 7.15-7.25 (m, 2H), 7.28 (d, J = 7.3 Hz, 1H), 7.43-7.47 (m) , 2H), 7.92-7.95 (m, 1H), 8.02 (ddd, J = 7.5, 7.5, 1.8 Hz, 1H), 10.01 (s, 1H);
HRMS (ESI +) m / z 288.1027 (calculated as C ₁₆ H ₁₅ O ₃ NF ⁺ , 288.1030, [M + H] ⁺ ).

Step 3: Production of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10)

Compound (10) uses methyl 2-fluoro-3- (o-tolylcarbamoyl) benzoate (9) instead of methyl 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoate (3). Except for the above, the product was manufactured according to step 3 of Production Example 1. Yield 63%.

¹ H NMR (DMSO-d ₆ ) δ 2.27 (s, 3H), 7.14-7.24 (m, 2H), 7.27 (d, J = 7.0 Hz, 1H), 7.41 (dd, J = 7.5, 7.5 Hz, 1H ), 7.45 (d, J = 7.5 Hz, 1H), 7.86-7.89 (m, 1H), 7.98 (dd, J = 7.0, 7.0 Hz, 1H), 9.95 (s, 1H);
HRMS (ESI +) m / z 274.0871 (calculated as C ₁₅ H ₁₃ O ₃ NF ⁺ , 274.0874, [M + H] ⁺ ).

<Example 1>
Preparation of compound CBT-078 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (o-tolyl) isophthalamide)

To a 1,2-dichloroethane solution (3.8 mL) of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (103 mg, 0.377 mmol, 1 equivalent) obtained in step 3 of Production Example 3. , 4-Fluoroaniline (46 μL, 0.49 mmol, 1.3 eq), 4-dimethylaminopyridine (9.1 mg, 75 μmol, 0.20 eq), and 1- (3-dimethylaminopropyl) -3-ethyl. Carbodiimide hydrochloride (109 mg, 0.565 mmol, 1.5 eq) was added and the mixture was heated and stirred at 50 ° C. overnight. After returning to room temperature, the reaction solution is adsorbed on silica gel and then purified by silica gel column chromatography (n-hexane / EtOAc = 4/1 to 2/1) to obtain CBT-078 (107 mg, 0.290 mmol, 76). 0.9%) was obtained as a colorless solid.

mp 167.6-169.1 ℃
¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 7.15-7.29 (m, 5H), 7.43-7.47 (m, 2H), 7.75-7.87 (m, 4H), 9.96 (s, 1H) , 10.61 (s, 1H);
HRMS (ESI +) m / z 389.1068 (calculated as C ₂₁ H ₁₆ F ₂ N ₂ NaO ₂ ⁺ , 389.1072, [M + Na] ⁺ ).

<Example 2>
Preparation of compound CBT-097 (2-fluoro-N ^1- (4-fluoro-2-methylphenyl) -N ^3- (o-tolyl) isophthalamide)

4-Fluoro-2-methylaniline in an N, N-dimethylformamide solution (1.2 mL) of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) obtained in step 3 of Production Example 3. (19.5 mg, 0.156 mmol, 1.3 eq), COMU (77.1 mg, 0.180 mmol, 1.5 eq) and diisopropylethylamine (31.0 mg, 0.240 mmol, 2.0 eq) at room temperature. Added. After stirring at room temperature overnight, water (about 1 mL) was added to the reaction solution. The solution was extracted 3 times with ethyl acetate (about 1 mL). The coalesced organic phase was washed with saturated brine (about 1 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / EtOAc = 2/1) to give CBT-097 (37.2 mg, 97.8 μmol, 81.4%) as a colorless solid.

mp 217.8-218.8 ℃
¹ H NMR (DMSO-d ₆ ) δ 2.29 (s, 3H + 3H, two signals overlapped), 7.07 (ddd, J = 8.5, 8.5, 2.8 Hz, 1H), 7.15-7.19 (m, 2H), 7.24 (dd) , J = 7.3, 7.3 Hz, 1H), 7.28 (d, J = 7.3 Hz, 1H), 7.43-7.48 (m, 3H), 7.84-7.87 (m, 2H), 9.97 (s, 1H), 9.99 ( s, 1H);
HRMS (ESI +) m / z 403.1226 (calculated as C ₂₂ H ₁₈ F ₂ N ₂ NaO ₂ ⁺ , 403.1229, [M + Na] ⁺ ).

<Example 3>
Preparation of compound CBT-042 (2-fluoro-N ¹ -phenyl-N ^3- (o-tolyl) isophthalamide)

Compound CBT-042 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (91.2 mg, 0.352 mmol) was prepared according to Example 1 except that 2-methylaniline was used instead of 4-fluoroaniline. Yield 50.6% (62.1 mg, 0.178 mmol).

mp 164.3-166.4 ℃
¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 7.11-1.19 (m, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.35-7.39 (AA'BB'X, 2H), 7.43-7.48 (m, 2H), 7.73-7.75 (AA'BB'X, 2H), 7.79-7.87 (m, 2H), 9.95 (s, 1H) , 10.54 (s, 1H);
HRMS (ESI +) m / z 371.1162 (calculated as C ₂₁ H ₁₇ FN ₂ NaO ₂ ⁺ , 371.1166, [M + Na] ⁺ ).

<Example 4>
Preparation of compound CBT-043 ( ² -fluoro-N1- ( ⁴ -methylphenyl) -N3-phenylisophthalamide)

Compound CBT-043 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (31.3 mg, 0.120 mmol) was prepared according to Example 1 except that 4-methylaniline was used instead of 4-fluoroaniline. Yield 29.1% (12.2 mg, 35.0 μmol).

<Example 5>
Preparation of compound CBT-044 ( ² -fluoro-N1- ( ⁴ -fluorophenyl) -N3-phenylisophthalamide)

Compound CBT-044 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (53.3 mg, 0.206 mmol) was prepared according to Example 1 except that 4-fluoroaniline was used instead of 4-fluoroaniline. Yield 68.0% (49.4 mg, 0.140 mmol).

<Example 6>
Preparation of compound CBT-053 (2-fluoro-N ^1- (2-fluorophenyl) -N ³ -phenylisophthalamide)

Compound CBT-053 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (47.9 mg, 0.185 mmol) was prepared according to Example 1 except that 4-fluoroaniline was used instead of 2-fluoroaniline. Yield 21.0% (13.6 mg, 38.6 μmol).

<Example 7>
Preparation of compound CBT-054 (2-fluoro-N ^1- (3-fluorophenyl) -N ³ -phenylisophthalamide)

Compound CBT-054 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (44.5 mg, 0.172 mmol) was prepared according to Example 1 except that 3-fluoroaniline was used instead of 4-fluoroaniline. Yield 46.0% (27.9 mg, 79.2 μmol).

mp 221.5-223.5 ℃
¹ H NMR (DMSO-d ₆ ) δ 6.97 (dddd, J = 8.4, 8.4, 2.5, 0.9 Hz, 1H), 7.10-7.15 (m, 1H), 7.34-7.48 (m, 5H), 7.69-7.74 ( m, 3H), 7.80-7.84 (m, 2H), 10.53 (s, 1H), 10.74 (s, 1H);
HRMS (ESI ⁺ ) m / z 375.0913 (calculated as C ₂₀ H ₁₄ F ₂ N ₂ NaO ₂ ⁺ , 375.0916, [M + Na] ⁺ ).

<Example 8>
Preparation of compound CBT-055 (2-fluoro-N ^1- (2-methoxyphenyl) -N ³ -phenylisophthalamide)

Compound CBT-055 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (30.4 mg, 0.117 mmol) was prepared according to Example 1 except that 2-methoxyaniline was used instead of 4-fluoroaniline. Yield 42.9% (18.3 mg, 50.2 μmol).

¹ H NMR (CDCl ₃ ) δ 3.93 (s, 3H), 6.94 (dd, J = 8.0, 1.4 Hz, 1H), 7.03 (ddd, J = 7.8, 7.8, 1.4 Hz, 1H), 7.13 (ddd, J) = 7.8, 7.8, 1.4 Hz, 1H), 7.18-7.23 (m, 1H), 7.39-7.44 (m, 3H), 7.70 (d, J = 7.8 Hz, 2H), 8.15-8.22 (m, 2H), 8.33 (br d, J = 9.7 Hz, 1H), 8.54 (dd, J = 8.0, 1.4 Hz, 1H), 8.93 (br d, J = 10.7 Hz, 1H);
HRMS (ESI +) m / z 387.1113 (calculated as C ₂₁ H ₁₇ FN ₂ NaO ₃ ⁺ , 387.1115, [M + Na] +).

<Example 9>
Preparation of compound CBT-056 (2-fluoro-N ^1- (2- (methylthio) phenyl) -N ³ -phenylisophthalamide)

Compound CBT-056 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (48.5 mg, 0.187 mmol) was prepared according to Example 1 except that 2-methylthioaniline was used instead of 4-fluoroaniline. Yield 17.1% (12.2 mg, 32.0 μmol).

¹ H NMR (DMSO-d ₆ ) δ 2.45 (s, 3H), 7.13 (dd, J = 7.5, 7.5 Hz, 1H), 7.23-7.32 (m, 2H), 7.35-7.47 (m, 4H), 7.56 (d, J = 7.5 Hz, 1H), 7.72-7.74 (m, 2H), 7.81 (dd, J = 7.5, 7.5 Hz, 1H), 7.90 (dd, J = 7.5, 7.5 Hz, 1H), 9.93 ( s, 1H), 10.56 (s, 1H);
HRMS (ESI +) m / z 403.0885 (calculated as C ₂₁ H ₁₇ FN ₂ NaO ₂ S ⁺ , 403.0887, [M + Na] ⁺ ).

<Example 10>
Preparation of compound CBT-057 (N ^1- (2-chlorophenyl) -2-phenyl-N ³ -phenylisophthalamide)

Compound CBT-057 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (200 mg, 0.772 mmol) was prepared according to Example 1 except that 2-chloroaniline was used instead of 4-fluoroaniline. Yield 12.0% (34.1 mg, 92.5 μmol).

¹ H NMR (DMSO-d ₆ ) δ 7.11-7.15 (m, 1H), 7.29 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H), 7.35-7.48 (m, 4H), 7.57 (dd, J = 7.5, 1.5 Hz, 1H), 7.70-7.84 (m, 4H), 7.89 (dd, J = 7.0, 7.0 Hz, 1H), 10.13 (s, 1H), 10.56 (s, 1H);
HRMS (ESI +) m / z 391.0619 (C ₂₀ H ₁₄ ³⁵ ClFN ₂ O ₂ Na ⁺ , calculated as 391.0620, [M + Na] ⁺ ).

<Example 11>
Preparation of compound CBT-058 (N ^1- (2-bromophenyl) -2-fluoro-N ³ -phenylisophthalamide)

Compound CBT-058 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (200 mg, 0.772 mmol) was prepared according to Example 1 except that 2-bromoaniline was used instead of 4-fluoroaniline. Yield 13.6% (43.5 mg, 0.105 mmol).

¹ H NMR (DMSO-d ₆ ) δ 7.11-7.15 (m, 1H), 7.23 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H), 7.35-7.39 (m, 2H), 7.43-7.48 (m, 2H), 7.72-7.75 (m, 4H), 7.81-7.85 (m, 1H), 7.91 (dd, J = 6.5, 6.5 Hz, 1H), 10.09 (s, 1H), 10.58 (s, 1H);
HRMS (ESI +) m / z 435.0116 (C ₂₀ H ₁₄ ⁷⁹ BrFN ₂ O ₂ Na ⁺ , calculated as 435.0115, [M + Na] ⁺ ).

<Example 12>
Preparation of compound CBT-059 (2-fluoro-N ¹ -phenyl-N ^3- (2-trifluoromethoxyphenyl) isophthalamide)

Compound CBT-059 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (45.1 mg, 0.174 mmol) was prepared according to Example 1 except that 2-trifluoromethoxyaniline was used instead of 4-fluoroaniline. Yield 4.1% (3.0 mg, 7.2 μmol).

<Example 13>
Preparation of compound CBT-060 (2-fluoro-N ^1- (4-methoxyphenyl) -N ³ -phenylisophthalamide)

Compound CBT-060 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (53.2 mg, 0.205 mmol) was prepared according to Example 1 except that 4-methoxyaniline was used instead of 4-fluoroaniline. Yield 57.6% (43.0 mg, 0.118 mmol).

<Example 14>
Preparation of compound CBT-064 (2-fluoro-N ^1- (4-dimethylaminophenyl) -N ³ -phenylisophthalamide)

Compound CBT-064 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (59.3 mg, 0.229 mmol) was prepared according to Example 1 except that 4-methoxyaniline was used instead of 4-fluoroaniline. Yield 38.4% (33.2 mg, 88.0 μmol).

<Example 15>
Preparation of compound CBT-065 (2-fluoro-N ^1- (4-fluoro-2-methylphenyl) -N ³ -phenylisophthalamide)

Compound CBT-065 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (29.4 mg, 0.113 mmol) was prepared according to Example 1 except that 4-fluoro-2-methylaniline was used instead of 4-fluoroaniline. Yield 37.0% (15.3 mg, 41.8 μmol).

¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 7.06 (dd, J = 8.5, 8.5, 3.0 Hz, 1H), 7.10-7.17 (m, 2H), 7.35-7.39 (m, 2H) , 7.42-7.46 (m, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.78-7.87 (m, 2H), 9.96 (s, 1H), 10.54 (s, 1H);
HRMS (ESI +) m / z 389.1070 (calculated as C ₂₁ H ₁₆ F ₂ N ₂ NaO ₂ ⁺ , 389.1072, [M + Na] ⁺ ).

<Example 16>
Preparation of compound CBT-073 (2-fluoro- (N ¹ , N ³ -bis (2-methylphenyl) isophthalamide))

To a 1,2-dichloroethane solution (2.0 mL) of 2-fluorophthalic acid (40.4 mg, 0.219 mmol, 1 eq), 2-methylaniline (56.5 mg, 0.527 mmol, 2.4 eq), 4-Dimethylaminopyridine (5.3 mg, 44 μmol, 0.20 eq) and EDCI HCl (105 mg, 0.548 mmol, 2.5 eq) were added at room temperature and stirred at room temperature for 1.5 hours. Hydrochloric acid (3M aqueous solution) was added to the reaction solution until the pH became 1 or less. The solution was extracted 3 times with ethyl acetate (about 20 mL). The combined organic phases were washed with saturated brine (about 40 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was washed with diethyl ether to give CBT-073 (36.7 mg, 0.101 mmol, 46.1%) as a white solid.

<Example 17>
Preparation of compound CBT-075 (2-fluoro-N ¹ -phenyl-N ^3- (m-tolyl) isophthalamide)

Compound CBT-075 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (68.1 mg, 0.263 mmol) was prepared according to Example 1 except that 3-methylaniline was used instead of 4-fluoroaniline. Yield 39.5% (36.2 mg, 0.104 mmol).

mp 181.9-184.1 ℃
¹ H NMR (DMSO-d ₆ ) δ 2.31 (s, 3H), 6.94 (d, J = 7.5 Hz, 1H), 7.10-7.15 (m, 1H), 7.24 (dd, J = 8.0, 8.0 Hz, 1H) ), 7.35-7.39 (m, 2H), 7.43 (dd, J = 7.5, 7.5 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.58 (br s, 1H), 7.72-7.74 (m) , 2H), 7.72-7.81 (m, 2H), 10.44 (s, 1H), 10.52 (s, 1H);
HRMS (ESI +) m / z 371.1166 (calculated as C ₂₁ H ₁₇ FN ₂ NaO ₂ ⁺ , 371.1166, [M + Na] +).

<Example 18>
Preparation of compound CBT-076 (2-fluoro-N ^1- (4-cyanophenyl) -N ³ -phenylisophthalamide)

Compound CBT-076 is a 2-fluoro-3- (phenylcarbamoyl) benzoic acid (7) obtained in step 3 of Production Example 2 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) (53.2 mg, 0.205 mmol) was prepared according to Example 1 except that 4-cyanoaniline was used instead of 4-fluoroaniline. Yield 19.5% (14.4 mg, 40.1 μmol).

<Example 19>
Preparation of compound CBT-088 (N ^1- (2-ethylphenyl) -2-fluoro-N ^3- (4-fluorophenyl) isophthalamide)

2-Fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4) (50.0 mg, 0.180 mmol, 1 equivalent) obtained in step 3 of Production Example 1 (2) To 0.0 mL), 2-ethylaniline (70.5 μL, 0.541 mmol, 3.0 eq) and COMU (154 mg, 0.361 mmol, 2.0 eq) were added at room temperature and stirred at 50 ° C. overnight. After returning to room temperature, hydrochloric acid (1 mol / L aqueous solution, about 2 mL) was added to the reaction solution. The solution was extracted 3 times with ethyl acetate (about 1 mL). The combined organic phase was saturated saline (about 1 mL). ), Then dried with anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / EtOAc = 2/1). Compound CBT-088 (56.0 mg, 0.154 mmol, 85.7%) was obtained as a colorless solid.

mp 169.1-169.8 ℃;
¹ H NMR (DMSO-d ₆ ) δ 1.16 (t, J = 7.5 Hz, 3H), 2.66 (q, J = 7.5 Hz, 2H), 7.19-7.26 (m, 4H), 7.29-7.32 (m, 1H) ), 7.41-7.47 (m, 2H), 7.74-7.85 (m, 4H), 9.96 (s, 1H), 10.61 (s, 1H);
HRMS (ESI +) m / z 403.1226 (calculated as C ₂₂ H ₁₈ F ₂ N ₂ NaO ₂ ⁺ , 403.1229, [M + Na] ⁺ ).

<Example 20>
Preparation of compound CBT-089 (2-fluoro-N ^1- (2-fluorophenyl) -N ^3- (4-fluorophenyl) isophthalamide)

A 1,2-dichloroethane solution of 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4) (50.0 mg, 0.180 mmol, 1 equivalent) obtained in step 3 of Production Example 1 ( To 2.0 mL), 2-fluoroaniline (52.2 μL, 0.541 mmol, 3.0 eq) and COMU (154 mg, 0.361 mmol, 2.0 eq) were added at room temperature and stirred at 50 ° C. overnight. .. After returning to room temperature, hydrochloric acid (1 mol / L aqueous solution, about 2 mL) was added to the reaction solution. The solution was extracted 3 times with ethyl acetate (about 1 mL). The combined organic phases were washed with saturated brine (about 1 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / EtOAc = 2/1) to give compound CBT-089 (17.6 mg, 50.0 μmol, 27.7%) as a colorless solid.

¹ H NMR (DMSO-d ₆ ) δ 7.18-7.34 (m, 5H), 7.44 (dd, J = 7.7, 7.7 Hz, 1H), 7.74-7.77 (AA'BB'X, 2H), 7.80-7.86 ( m, 3H), 10.29 (s, 1H), 10.59 (s, 1H);
HRMS (ESI +) m / z 393.0820 (calculated as C ₂₀ H ₁₃ F ₃ N ₂ NaO ₂ ⁺ , 393.0821, [M + Na] ⁺ ).

<Example 21>
Preparation of compound CBT-090 (N ^1- (2-chlorophenyl) -2-fluoro-N ^3- (4-fluorophenyl) isophthalamide)

A 1,2-dichloroethane solution of 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4) (50.0 mg, 0.180 mmol, 1 equivalent) obtained in step 3 of Production Example 1 ( 2-Chloroaniline (56.6 μL, 0.541 mmol, 3.0 eq) and COMU (154 mg, 0.361 mmol, 2.0 eq) were added to 2.0 mL) at room temperature and stirred at 50 ° C. overnight. .. After returning to room temperature, hydrochloric acid (1 mol / L aqueous solution, about 2 mL) was added to the reaction solution. The solution was extracted 3 times with ethyl acetate (about 1 mL). The coalesced organic phase was washed with saturated brine (about 1 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / EtOAc = 2/1) to give compound CBT-090 (trace amount) as a colorless solid.

¹ H NMR (CDCl ₃ ) δ 7.08-7.16 (m, 3H), 7.36 (dd, J = 7.7, 7.7 Hz, 1H), 7.44-7.51 (m, 2H), 7.62-7.66 (m, 2H), 8.18 -8.30 (m, 3H), 8.56 (d, J = 7.7 Hz, 1H), 8.86 (br d, J = 11.8 Hz, 1H);
HRMS (ESI +) m / z 409.0529 (C ₂₀ H ₁₃ O ₂ N ₂ ³⁵ ClF ₂ Calculated as Na ⁺ , 409.0526, [M + Na] ⁺ ).

<Example 22>
Preparation of compound CBT-091 (N ^1- (2-bromophenyl) -2-fluoro-N ^3- (4-fluorophenyl) isophthalamide)

Compound CBT-091 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). Examples of Examples except that benzoic acid (4) (44.3 mg, 0.160 mmol) was prepared by using 2-bromoaniline instead of 4-fluoro-2-methylaniline and setting the reaction temperature to 50 ° C. instead of room temperature. Manufactured according to 2. Yield 50.0% (34.5 mg, 80.0 μmol).

¹ H NMR (DMSO-d ₆ ) δ 7.19-7.25 (m, 3H), 7.43-7.49 (m, 2H), 7.71-7.79 (m, 4H), 7.82-7.85 (m, 1H), 7.91 (dd, J = 6.6, 6.6 Hz, 1H), 10.10 (s, 1H), 10.63 (s, 1H);
HRMS (ESI +) m / z 453.0018 (calculated as C ₂₀ H ₁₃ BrF ₂ N ₂ NaO ₂ ⁺ , 453.0021, [M + Na] ⁺ ).

<Example 23>
Preparation of compound CBT-092 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2-methoxyphenyl) isophthalamide)

A 1,2-dichloroethane solution of 2-fluoro-3-((4-fluorophenyl) carbamoyl) benzoic acid (4) (50.0 mg, 0.180 mmol, 1 equivalent) obtained in step 3 of Production Example 1 ( To 2.0 mL), 2-methoxyaniline (61.0 μL, 0.541 mmol, 3.0 eq) and COMU (154 mg, 0.361 mmol, 2.0 eq) were added at room temperature and stirred at 50 ° C. overnight. .. After returning to room temperature, hydrochloric acid (1 mol / L aqueous solution, about 2 mL) was added to the reaction solution. The solution was extracted 3 times with ethyl acetate (about 1 mL). The combined organic phases were washed with saturated brine (about 1 mL) and then dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off from the organic phase under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / EtOAc = 2/1) to give compound CBT-092 (23.7 mg, 65.0 μmol, 36.1%) as a colorless solid.

¹ H NMR (DMSO-d ₆ ) δ 3.86 (s, 3H), 6.99 (dd, J = 7.0, 7.0 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.15-7.25 (m, 3H) ), 7.46 (dd, J = 7.5, 7.5 Hz, 1H), 7.74-7.78 (m, 2H), 7.84 (dd, J = 7.0, 7.0 Hz, 1H), 7.94 (dd, J = 7.0, 7.0 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 9.58 (d, J = 5.7 Hz, 1H), 10.61 (s, 1H);
HRMS (ESI +) m / z 405.1021 (calculated as C ₂₁ H ₁₆ O ₃ N ₂ F ₂ Na ⁺ , 405.1021, [M + Na] ⁺ ).

<Example 24>
Preparation of compound CBT-093 (2-fluoro-N ^1- (o-tolyl) -N ^3- (3,4,5-trifluorophenyl) isophthalamide)

Compound CBT-093 was prepared by using 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (55.6 mg, 0.203 mmol) obtained in Step 3 of Production Example 3 and 4-fluoro-2. -It was produced according to Example 1 except that 3,4,5-trifluoroaniline was used instead of methylaniline. Yield 45.2% (36.9 mg, 91.7 μmol).

¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 7.17-7.25 (m, 2H), 7.28 (d, J = 7.5 Hz, 1H), 7.45-7.49 (m, 2H), 7.64-7.68 (m, 2H), 7.80-7.84 (m, 1H), 7.87-7.90 (m, 1H), 9.99 (s, 1H), 10.91 (s, 1H);
HRMS (ESI +) m / z 425.0883 (calculated as 425.0884, C ₂₁ H ₁₄ F ₄ N ₂ NaO ₂ ⁺ , [M + Na] +).

<Example 25>
Preparation of compound CBT-094 (N1- ( ⁴ -chlorophenyl) -2-fluoro- ^N3- (o-tolyl) isophthalamide)

Compound CBT-094 was prepared from 4-fluoroaniline using 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (46.6 mg, 0.171 mmol) obtained in step 3 of Production Example 3. It was produced according to Example 1 except that 4-chloroaniline was used instead. Yield 74.8% (49.0 mg, 0.127 mmol).

¹ H NMR (DMSO-d ₆ ) δ 2.29 (s, 3H), 7.17 (ddd, J = 7.5, 7.5, 1.1 Hz, 1H), 7.24 (dd, J = 7.5, 7.5 Hz, 1H), 7.28 (d) , J = 7.5 Hz, 1H), 7.42-7.48 (m, 4H), 7.78-7.89 (m, 4H), 9.97 (s, 1H), 10.71 (s, 1H);
HRMS (ESI +) m / z 409.0529 (calculated as C ₂₀ H ₁₃ F ₂ N ₂ NaO ₂ , 409.0526, [M + Na] ⁺ )

<Example 26>
Preparation of compound CBT-095 (2-fluoro-N ^1- (4-bromophenyl) -N ^3- (2-methylphenyl) isophthalamide)

Compound CBT-095 was prepared by using 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (44.9 mg, 0.164 mmol) obtained in step 3 of Production Example 3 of 4-fluoroaniline. It was produced according to Example 1 except that 4-bromoaniline was used instead. Yield 38.3% (26.9 mg, 62.9 μmol).

<Example 27>
Preparation of compound CBT-096 (2-fluoro-N ^1- (2-methylphenyl) -N ^3- (4-methoxyphenyl) isophthalamide)

Compound CBT-096 was prepared from 4-fluoroaniline using 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (56.3 mg, 0.206 mmol) obtained in step 3 of Production Example 3. It was produced according to Example 1 except that 4-methoxyaniline was used instead. Yield 64.0% (49.9 mg, 0.132 mmol).

<Example 28>
Compound CBT-098 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) ) Isophthalamide)

Compound CBT-098 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (175 mg, 0.631 mmol) instead of 4-fluoro-2-methylaniline 2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-) Il) Manufactured according to Example 2 except that aniline was used. Yield 26.5% (80.0 mg, 0.167 mmol).

<Example 29>
Preparation of compound CBT-099 (2-fluoro-N ^1- (2-biphenylyl) -N ^3- (4-fluorophenyl) isophthalamide)

Compound CBT-099 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (26.0 mg, 93.8 μmol) was produced according to Example 2 except that 2-phenylaniline was used instead of 4-fluoro-2-methylaniline. Yield 83.6% (33.6 mg, 78.4 μmol).

<Example 30>
Preparation of compound CBT-100 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (naphthalene-1-yl) isophthalamide)

Compound CBT-100 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). Example 2 except that benzoic acid (4) (31.3 mg, 0.113 mmol) was prepared by using 1-naphthylamine instead of 4-fluoro-2-methylaniline and the reaction temperature was 50 ° C. instead of room temperature. Manufactured according to. Yield 68.8% (31.3 mg, 77.8 μmol).

mp 209.1-209.9 ℃
¹ H NMR (DMSO-d ₆ ) δ 7.21-7.25 (m, 2H), 7.49 (dd, J = 7.5, 7.5 Hz, 1H), 7.55-7.62 (m, 3H), 7.75-7.80 (m, 3H) , 7.83-7.88 (m, 2H), 7.93-8.00 (m, 2H), 8.12 (d, J = 8.8 Hz, 1H), 10.58 (s, 1H), 10.64 (s, 1H);
HRMS (ESI +) m / z 425.1069 (calculated as C ₂₄ H ₁₆ F ₂ N ₂ NaO ₂ ⁺ , 425.1072, [M + Na] ⁺ )

<Example 31>
Preparation of compound CBT-101 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2-isopropylphenyl) isophthalamide)

Compound CBT-101 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). Examples of Examples except that benzoic acid (4) (34.7 mg, 0.125 mmol) was prepared by using 2-isopropylaniline instead of 4-fluoro-2-methylaniline and the reaction temperature was 50 ° C. instead of room temperature. Manufactured according to 2. Yield 51.5% (25.4 mg, 64.4 μmol).

¹ H NMR (DMSO-d ₆ ) δ 1.18 (d, J = 7.0 Hz, 6H), 3.26 (sept, J = 7.0 Hz, 1H), 7.19-7.25 (m, 3H), 7.28 (ddd, J = 7.5) , 7.5, 1.5 Hz, 1H), 7.34 (dd, J = 7.5, 1.5 Hz, 1H), 7.38 (dd, J = 7.5, 1.5 Hz, 1H), 7.45 (dd, J = 7.5, 7.5 Hz, 1H) , 7.74-7.85 (m, 4H), 10.01 (s, 1H), 10.62 (s, 1H);
HRMS (ESI +) m / z 417.1382 (calculated as C ₂₃ H ₂₀ O ₂ N ₂ F ₂ Na ⁺ 417.1391, [M + Na] ⁺ ).

<Example 32>
Preparation of compound CBT-102 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2- (1-propene-2-yl) phenyl) isophthalamide)

Compound CBT-102 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (34.3 mg, 0.124 mmol), 2- (1-propen-2-yl) aniline instead of 4-fluoro-2-methylaniline, reaction temperature instead of room temperature It was manufactured according to Example 2 except that the temperature was set to 50 ° C. Yield 12% (5.7 mg, 15 μmol).

<Example 33>
Preparation of compound CBT-103 (2-fluoro-N ^1- (2-tert-butylphenyl) -N ^3- (4-fluorophenyl) isophthalamide)

Compound CBT-103 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (22.9 mg, 82.6 μmol) was replaced with 2-tert-butylaniline instead of 4-fluoro-2-methylaniline, except that the reaction temperature was 60 ° C. instead of room temperature. Manufactured according to Example 2. Yield 61.6% (20.8 mg, 50.9 μmol).

<Example 34>
Preparation of compound CBT-104 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2- (trimethylsilyl) ethynylphenyl) isophthalamide)

Compound CBT-104 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (45.0 mg, 0.162 mmol) was replaced with 2- (trimethylsilyl) ethynylaniline instead of 4-fluoro-2-methylaniline, and the reaction temperature was set to 50 ° C. instead of room temperature. Was manufactured according to Example 2. Yield 53.8% (39.1 mg, 87.2 μmol).

<Example 35>
Preparation of compound CBT-105 (N ^1- (2-ethynylphenyl) -2-fluoro-N ^3- (4-fluorophenyl) isophthalamide)

Compound CBT-105 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). Examples of Examples except that benzoic acid (4) (36.4 mg, 0.131 mmol) was prepared by using 2-ethynylaniline instead of 4-fluoro-2-methylaniline and the reaction temperature was 50 ° C. instead of room temperature. Manufactured according to 2. Yield 52.7% (26.0 mg, 69.1 μmol).

¹ H NMR (DMSO-d ₆ ) δ 4.60 (s, 1H), 7.19-7.26 (m, 3H), 7.45-7.50 (m, 2H), 7.57 (dd, J = 7.7, 1.4 Hz, 1H), 7.74 -7.78 (AA'BB'X, 2H), 7.83-7.87 (m, 1H), 7.97 (br s, 1H), 7.99 (br s, 1H), 9.93 (d, J = 5.0 Hz, 1H), 10.64 (s, 1H);
HRMS (ESI +) m / z 377.1084 (calculated as C ₂₂ H ₁₅ N ₂ O ₂ F ₂ , 377.1096, [M + H] ⁺ ).

<Example 36>
Preparation of compound CBT-106 (N ¹ -cyclohexyl-2-fluoro-N ^3- (4-fluorophenyl) isophthalamide)

Compound CBT-106 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (37.6 mg, 0.136 mmol) according to Example 2 except that cyclohexylamine was used instead of 4-fluoro-2-methylaniline and the reaction temperature was 50 ° C. instead of room temperature. , Manufactured. Yield 83.0% (40.4 mg, 0.113 mmol).

¹ H NMR (DMSO-d ₆ ) δ 1.12-1.36 (m, 5H), 1.56-1.84 (m, 5H), 3.74-3.80 (m, 1H), 7.17-7.24 (AA'BB'X, 2H), 7.36 (dd, J = 7.5, 7.5 Hz, 1H), 7.64-7.68 (m, 1H), 7.70-7.77 (m, 3H), 8.29 (br d, J = 7.9 Hz, 1H), 10.53 (s, 1H) );
HRMS (ESI +) m / z 381.1382 (calculated as C ₂₀ H ₂₀ F ₂ N ₂ NaO ₂ ⁺ , 381.1385, [M + Na] ⁺ ).

<Example 37>
Preparation of compound CBT-107 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2-iodophenyl) isophthalamide)

Compound CBT-107 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). Examples of Examples except that benzoic acid (4) (33.2 mg, 0.120 mmol) was prepared by using 2-iodoaniline instead of 4-fluoro-2-methylaniline and the reaction temperature was 50 ° C. instead of room temperature. Manufactured according to 2. Yield 54.8% (31.4 mg, 65.7 μmol).

¹ H NMR (DMSO-d ₆ ) δ 7.07 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H), 7.19-7.25 (AA'BB'X, 2H), 7.44-7.49 (m, 2H), 7.57 ( d, J = 7.5 Hz, 1H), 7.74-7.79 (AA'BB'X, 2H), 7.83 (ddd, J = 7.0, 7.0, 1.5 Hz, 1H), 7.91-7.96 (m, 2H), 10.08 ( s, 1H), 10.65 (s, 1H);
HRMS (ESI +) m / z 500.9871 (C ₂₀ H ₁₃ F ₂ IN ₂ Calculated as NaO ₂ , 500.9882, [M + H] ⁺ ).

<Example 38>
Preparation of compound CBT-108 (2-fluoro-N ^1- (4-fluorophenyl) -N ^3- (2,6-dimethylphenyl) isophthalamide)

Compound CBT-108 is a 2-fluoro-3-((4-fluorophenyl) carbamoyl) obtained in step 3 of Production Example 1 instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10). ) Benzoic acid (4) (34.0 mg, 0.123 mmol) was replaced with 2,6-dimethylaniline instead of 4-fluoro-2-methylaniline, except that the reaction temperature was 50 ° C. instead of room temperature. Manufactured according to Example 2. Yield 61.8% (28.9 mg, 76.0 μmol).

<Example 39>
Preparation of compound CBT-114 (2-fluoro-N ^1- (3-fluorophenyl) -N ^3- (o-tolyl) isophthalamide)

Compound CBT-114 uses 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (28.5 mg, 0.104 mmol) and 3-fluoroaniline instead of 4-fluoro-2-methylaniline. Was produced according to Example 2 except that the reaction temperature was set to 50 ° C. instead of room temperature. Yield 78.2% (29.8 mg, 81.3 μmol).

¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 6.97 (dddd, J = 8.4, 8.4, 2.5, 0.9 Hz, 1H), 7.17 (dd, J = 7.0, 7.0 Hz, 1H), 7.23 (dd, J = 7.0, 7.0 Hz, 1H), 7.28 (d, J = 7.0 Hz, 1H), 7.38-7.49 (m, 4H), 7.71-7.73 (m, 1H), 7.82 (dd, J = 7.0) , 7.0 Hz, 1H), 7.87 (dd, J = 7.0, 7.0 Hz, 1H), 9.97 (s, 1H), 10.77 (s, 1H);
HRMS (ESI +) m / z 389.1068 (calculated as C ₂₁ H ₁₆ F ₂ N ₂ NaO ₂ ⁺ , 389.1072, [M + Na] ⁺ ).

<Example 40>
Preparation of compound CBT-115 (2-fluoro-N1- (2-fluorophenyl) -N3- (o-tolyl) isophthalamide)

Compound CBT-115 uses 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (31.9 mg, 0.117 mmol) and 2-fluoroaniline instead of 4-fluoro-2-methylaniline. Was produced according to Example 2 except that the reaction temperature was set to 50 ° C. instead of room temperature. Yield 68.4% (29.3 mg, 80.0 μmol).

¹ H NMR (DMSO-d ₆ ) δ 2.28 (s, 3H), 7.15-7.34 (m, 6H), 7.43-7.48 (m, 2H), 7.82-7.88 (m, 3H), 9.96 (s, 1H) , 10.31 (s, 1H);
HRMS (ESI +) m / z 389.1071 (calculated as C ₂₁ H ₁₆ F ₂ N ₂ NaO ₂ ⁺ , 389.1072, [M + Na] ⁺ ).

<Example 41>
¹¹ C-labeled compound CBT-078 (Compound [ ¹¹ C] CBT-078) Production Compound [ ¹¹ C] CBT-078 is produced in WO 2008/023780 and Chem. Eur. J. This was done with reference to 2009, 15, 4165-4171.

Reaction vessels 1 and 2 were prepared. The reaction was carried out using an oven-dried instrument, and the flow path was dried by flowing helium gas at a flow rate of 400 mL / min while heating the reaction vessels 1 and 2 at 250 ° C. before the start of the reaction. After the drying is completed, the reaction vessel 1 is cooled to −10 ° C. and the reaction vessel 2 is cooled to room temperature, and the compound CBT-098 (1.3 mg, 2.7 μmol) prepared in Example 28 and tris (dibenzylidene) are transferred to the reaction vessel 2. A tetrahydrofuran solution (300 μL) in which dipalladium (2.0 mg, 2.2 μmol), orthotrilphosphine (2.0 mg, 6.6 μmol), and potassium carbonate (1.2 mg, 8.7 μmol) were dissolved was introduced. .. [ ¹¹ C] About 2 minutes before the completion of carbon dioxide production, a tetrahydrofuran solution of lithium aluminum hydride hydride (0.1 mmol / L, 400 μL, ABXGmbH, 802.0001) was introduced into the reaction vessel 1.

By irradiating a nitrogen mixed gas containing a trace amount of oxygen (0.5%) with a proton laser (50 μA) using a cyclotron (CYPRIS HM-12S) manufactured by Sumitomo Heavy Industries, Ltd. for 52 minutes, [ ¹¹ C] ] Carbon dioxide (about 40 GBq) was synthesized. The synthesized [ ¹¹ C] carbon dioxide was transferred to an automatic synthesizer using oxygen-containing nitrogen gas (oxygen 0.5%, flow rate 400 mL / min) and bubbling into the solution of the reaction vessel 1. After confirming that the value of the RI detector adjacent to the reaction vessel 1 showed the maximum value, bubbling was stopped. Tetrahydrofuran in the reaction vessel 1 was distilled off by heating the reaction vessel 1 to 200 ° C. and flowing helium gas (flow rate 200 mL / min) for 90 seconds. After cooling the reaction vessel to room temperature, hydrogen iodide acid (55%, 500 μL) was added to the reaction vessel 1. This reaction vessel 1 was heated to 200 ° C. to synthesize [ ¹¹ C] methyl iodide. [ ¹¹ C] Methyl iodide was distilled and transferred to the reaction vessel 2 by flowing helium gas (flow rate 30 mL / min) while heating and bubbling into the solution of the already introduced reaction vessel 2. At this time, a column (inner diameter: 140 mm) filled with ascarite (1.6 g, Thomas Scientific, Inc, C049U90) and sikapent (2.0 g, Merck, 1.00543.0500) was installed between the reaction vessels. Unreacted [ ¹¹ C] carbon dioxide and the like, such as water other than ¹¹ C] methyl iodide and hydrogen iodide, were removed. After confirming that the value of the RI detector adjacent to the reaction vessel 2 showed the maximum value, bubbling was stopped and the reaction vessel 2 was heated at 65 ° C. for 5 minutes. After completion of the reaction, a mixed solvent of acetonitrile (500 μL) and water (500 μL) was introduced into the reaction vessel 2, and the obtained mixture was used as a membrane filter (PVDF, 0.2 μm, diameter 13 mm, GE Healthcare Life Science, 6779-1302). ), And then introduced into semi-preparative HPLC. The fraction containing compound [ ^11C ] CBT-078 was fractionated with reference to the UV sensor and RI detector attached to the HPLC. This fraction was transferred to a flask attached to an evaporator installed in a hot cell, heated under reduced pressure, and distilled off under reduced pressure for 3 minutes. After the inside of the flask is brought to normal temperature and pressure, a diluted solution for administration (physiological saline: propylene glycol: Tween80 = 100: 10: 1,1 mL) and physiological saline (1 mL) are introduced into the flask, and the mixture is rotated and stirred for 40 seconds. bottom. The obtained solution was transferred to a dosing vial while being permeated through a membrane filter (PVDF, 0.45 μm, diameter 33 mm, Merck, ^SLHV033NB ) to obtain a solution of compound [11C] CBT-078.

The total radioactivity of the obtained compound [ ¹¹ C] CBT-078 solution was measured with a dose calibrator (CAPINTEC, Inc., 5130-3235). The purity test was performed using HPLC equipped with a UV detector and an RI detector. Furthermore, assuming that the density of the obtained solution is 1 g / mL, the concentration is calculated from the UV absorption amount of the compound [ ¹¹ C] CBT-078 in HPLC, and the ratio with the total radioactivity amount is calculated to obtain non-radioactivity. Was calculated. Detailed analytical conditions for HPLC in semi-sorting and purity testing and examples of the results are shown below.

[Conditions for semi-prepared HPLC]
Column: COSMOSIL 5C18-MS-II (Nacalai Tesque) 10 × 20 mm and 10 × 250 mm are used in combination;
Flow rate: 5 mL / min; Mobile phase: acetonitrile / H ₂ O = 50: 50;
UV detection wavelength: 254 nm; retention time: 12.5 minutes.

[Analytical HPLC conditions]
column:
COSMOSIL 5C18-MS-II (Nacalai Tesque) 4.6 x 150 mm;
Flow rate: 1 mL / min; mobile phase: acetonitrile / H ₂ O = 55:45
UV detection wavelength: 254 nm; retention time: 6.1 minutes.

[[ ¹¹ C] Analysis result of CBT-078 solution ([ ¹¹ C] Data 35 minutes after carbon dioxide synthesis)]
Total radioactivity: 1501MBq; Specific activity: 108.5GBq / μmol; Solution volume: 1.9mL;
[ ¹¹ C] Weight concentration of CBT-078: 2.7 μg / mL; pH of solution: 7.0
Chemical purity: 93.0%; Radiochemical purity: 99.3%.

<Example 42>
¹¹ Production of C-labeled compound CBT-039 (Compound [ ¹¹ C] CBT-039) Using compound CBT-122 (1.2 mg, 2.7 μmol) in place of compound CBT-098 with Example 41. The same procedure was carried out to obtain compound [ ¹¹ C] CBT-039.

The compound CBT-122 contains 3- (phenylcarbamoyl) benzoic acid (51.3 mg, 0.213 mmol) instead of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10), 4-fluoro. Except for using 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline instead of -2-methylaniline and setting the reaction temperature to 50 ° C instead of room temperature. Was manufactured according to Example 2. Yield 40.1% (37.7 mg, 0.0852 mmol).

¹ H NMR (DMSO-d ₆ ) δ1.26 (s, 12H), 7.08-7.24 (m, 2H), 7.33-7.43 (m, 3H), 7.56 (d, J = 6.6 Hz, 1H), 7.62－ 7.87 (m, 4H), 8.15-8.26 (br s, 1H), 8.24 (d, J = 7.9 Hz, 1H), 8.62 (s, 1H), 10.49 (s, 1H), 11.56 (br s, 1H) ;
HRMS (ESI ⁺ ) m / z 443.2136 (calculated as C ₂₆ H ₂₈ O ₄ N ₂ B ⁺ , 443.2137, [M + H] ⁺ ).

[Conditions for semi-prepared HPLC]
Column: COSMOSIL 5C18-MS-II (Nacalai Tesque) 10 × 20 mm and 10 × 250 mm are used in combination;
Flow rate: 5 mL / min; Mobile phase: acetonitrile / H ₂ O = 50: 50;
UV detection wavelength: 254 nm; retention time: 9.8 minutes.

[Analytical HPLC conditions]
Column: COSMOSIL 5C18-MS-II (Nacalai Tesque) 4.6 x 150 mm;
Flow rate: 1 mL / min; mobile phase: acetonitrile / H ₂ O = 55:45
UV detection wavelength: 254 nm; retention time: 6.1 minutes.

Analysis results of compound [ ¹¹ C] CBT-039 solution ([ ¹¹ C] data 32 minutes after carbon dioxide synthesis)
Total radioactivity: 1049MBq; Specific activity: 50.0GBq / μmol; Solution volume: 1.7mL;
[ ¹¹ C] Weight concentration of CBT-039: 4.5 μg / mL; pH of solution: 7.0
Chemical purity: 96.1%; Radiochemical purity:> 99%.

<Example 43>
Production of Compound CBT-078 Labeled with ¹⁸ F (Compound [ ¹⁸ F] CBT-078) Production of Compound [ ¹⁸ F] CBT-078 was carried out by Chem. Commun. 2016, 52, 8361-8364 and Org. Let. This was done with reference to 2015, 17, 5780-5783.

A closed vial and a reaction V vial were prepared. The reaction was carried out using an oven-dried instrument, and the flow path was dried by repeating depressurization and pressurization with helium gas while heating the reaction vessel at 150 ° C. before the start of the reaction.

Compound CBT-140 (1.9 mg, 4.0 μmol) N, N-dimethylacetamide solution (300 μL) and copper trifluoromethanesulfonate (II) (7.2 mg, 20 μmol), pyridine (80.5 μL, 1.00 mmol). ) N, N-dimethylacetamide solution (300 μL) was prepared 10 minutes before the completion of production of [ ¹⁸ F] fluoride ion.

[ ¹⁸ O] H ₂ O (2 mL, ¹⁸ O:> 98 atom%, Taiyo Nippon Sanso Corp., F03-0027) using a cyclotron (CYPRIS HM-12S) manufactured by Sumitomo Heavy Industries, Ltd. with a proton laser (35 μA). ) Was irradiated for 40 minutes to synthesize [ ¹⁸ F] fluoride ion (about 40 GBq). The synthesized [ ¹⁸ F] fluoride ion was transferred into a closed vial installed inside the labeling synthesizer. After confirming that the value of the RI detector adjacent to the closed vial showed the maximum value, the closed vial was pressurized with nitrogen gas at a flow rate of 500 mL / min to contain [ ¹⁸ F] fluoride ions [ ¹⁸ O]. H ₂ O was transferred and passed through an ion exchange resin (Waters, 186004540) to capture [ ¹⁸ F] fluoride ions. After confirming that the value of the RI detector adjacent to the ion exchange resin showed the maximum value, the pressurization with nitrogen gas was stopped, and the [ ¹⁸ F] fluoride ion adsorbed on the ion exchange resin was trifluoromethane sulfone. The mixture was transferred into a V vial for reaction while being eluted with a mixed solvent of an aqueous solution (500 μL) containing potassium acid (5 mg) and potassium carbonate (50 μg) and acetonitrile (1000 μL). The eluted [ ¹⁸ F] potassium fluoride solution was depressurized at 130 ° C. for 5 minutes, and water was distilled off. Acetonitrile (300 μL) was added to the remaining small amount of solution, the pressure was reduced at 150 ° C. for 5 minutes, and the water was azeotropically boiled. This was repeated once more to prepare anhydrous [ ¹⁸ F] potassium fluoride in a reaction V vial. An N, N-dimethylacetamide solution (300 μL) of CBT-140 (1.9 mg, 4.0 μmol) was introduced into a reaction V vial and heated at 130 ° C. for 5 minutes. Subsequently, after cooling the V vial to room temperature, a solution of copper (II) trifluoromethanesulfonate (7.2 mg, 20 μmol) and pyridine (80.5 μL, 1.00 mmol) in N, N-dimethylacetamide (300 μL) was introduced. Then, it was heated at 130 ° C. for 20 minutes. After cooling the V-vial to room temperature, a mixed solvent of acetonitrile (500 μL) and water (500 μL) was introduced into the V-vial, and the obtained mixture was used as a membrane filter (PVDF, 0.2 μm, diameter 13 mm, GE Healthcare Life). After filtering with Science, 6779-1302), it was introduced into semi-preparative HPLC. The fraction containing the compound [ ^18F ] CBT-078 was fractionated with reference to the UV sensor and RI detector attached to the HPLC. This fraction was transferred to a flask attached to an evaporator installed in a hot cell, heated under reduced pressure, and distilled off under reduced pressure for 3 minutes. After the inside of the flask is brought to normal temperature and pressure, a diluted solution for administration (physiological saline: propylene glycol: Tween80 = 100: 10: 1,1 mL) and physiological saline (1 mL) are introduced into the flask, and the mixture is rotated and stirred for 40 seconds. bottom. The obtained solution was transferred to a dosing vial while being permeated through a membrane filter (PVDF, 0.45 μm, diameter 33 mm, Merck, SLHV033NB) to obtain a solution of compound [ ^18F ] CBT-078.

The total radioactivity of the solution of the obtained compound [ ^18F ] CBT-078 was measured with a dose calibrator (CAPINTEC, Inc., 5130-3235). The purity test was performed using HPLC equipped with a UV detector and an RI detector. Furthermore, assuming that the density of the obtained solution is 1 g / mL, the concentration is calculated from the UV absorption amount of the compound [ ¹⁸ F] CBT-078 in HPLC, and the ratio with the total radioactivity amount is calculated to obtain non-radioactivity. Was calculated. Detailed analytical conditions for HPLC in semi-sorting and purity testing and examples of the results are shown below.

[Conditions for semi-prepared HPLC]
Column: COSMOSIL 5C18-MS-II (Nacalai Tesque) 10 × 20 mm and 10 × 250 mm are used in combination;
Flow rate: 5 mL / min; Mobile phase: acetonitrile / H ₂ O = 48: 52;
UV detection wavelength: 254 nm; retention time: 18.1 minutes.

[Analytical HPLC conditions]
Column: COSMOSIL 5C18-MS-II (Nacalai Tesque) 4.6 x 150 mm;
Flow rate: 1 mL / min; mobile phase: acetonitrile / H ₂ O = 55:45
UV detection wavelength: 254 nm; retention time: 5.9 minutes.

[[ ¹⁸ F] Analysis result of CBT-078 solution ([ ¹⁸ F] Data after 81 minutes have passed since fluoride ion synthesis)]
Total radioactivity: 972MBq; Specific activity: 89.4GBq / μmol; Solution volume: 3.7mL;
[ ¹⁸ F] Weight concentration of CBT-078: 1.1 μg / mL;
Chemical purity: 94.5%; Radiochemical purity: 96.7%.

The compound CBT-140 was used in place of 2-fluoro-3- (o-tolylcarbamoyl) benzoic acid (10) (173 mg, 0.633 mmol) obtained in step 3 of Production Example 3 and 4-fluoroaniline. It was produced according to Example 1 except that 4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) aniline was used. Yield 59.9% (180 mg, 0.379 mmol).

¹ H NMR (DMSO-d ₆ ) δ1.30 (s, 12H), 2.28 (s, 3H), 7.14-7.31 (m, 3H), 7.41-7.50 (m, 2H), 7.64-7.70 (AA'BB) ′, 2H), 7.73-7.78 (AA ′ BB ′, 2H), 7.78-7.90 (m, 2H), 9.94 (s, 1H), 10.67 (s, 1H);
HRMS (ESI ⁺ ) m / z 475.2197 (calculated as C ₂₇ H ₂₉ O ₄ N ₂ BF ⁺ , 475.2199, [M + H] ⁺ ).

<Establishment of HBV replication activity evaluation method>
HBV replication activity evaluation method (method)
(1) Preparation of HBV replication activity evaluation vector (pBB-intron) With 99 bases at the 5'end (corresponding to positions 32 to 130 of HBV pgRNA) including the 1st DR1 sequence and the 1st ε sequence in HBV pgRNA of genotype C. , DR2 sequence, between 2'end 349 bases (corresponding to positions 3013 to 3215 and 1 to 146 of HBV pgRNA), including the 2nd DR1 and 2ε sequences, ie the 1st ε / DR2 sequence (of HBV pgRNA). (Corresponding to positions 131 to 3012) was replaced with a 320-base reporter sequence containing an intron to prepare the HBV replication activity evaluation nucleic acid represented by SEQ ID NO: 1 according to the seventh aspect of the present invention (FIG. 1A). This reporter sequence consists of 483 to 453 sites (31 base pairs) of the pCRE-MetLuc2 vector (Clontech), 857 to 989 sites (133 base pairs) of the pCI vector (Promega) and 452 sites of the pCRE-MetLuc2 vector. It is derived from the base sequence of ~ 337 sites (116 base pairs).

The CMV promoter (742 base pairs) and TATA-box (46 base pairs) were inserted upstream of the prepared HBV replication activity evaluation nucleic acid, and the synthetic polyA signal sequence (49 base pairs) was inserted downstream, and all these sequences were inserted into the pCI vector. The HBV replication activity evaluation vector according to the eighth aspect of the present invention, pBB-intron, was prepared by inserting into the backbone (containing only the phage f1 origin, the ampicillin resistance gene and the replication origin).

When this HBV replication activity evaluation vector is introduced into a host cell, mRNA is synthesized by RNA-pol II of the host cell. This mRNA is synthesized as pre-mRNA and then the intron in the reporter sequence is immediately removed by pre-mRNA splicing in the host cell. The mature mRNA with the intron spliced out corresponds to the reporter pgRNA (Fig. 1B). The reporter pgRNA is the original HBV replication activity evaluation vector by being reverse transcribed by the action of HBV-P expression vector, HBc expression vector, and HBV-Pol, HBc, and HBx expressed from the HBx expression vector described below. A reporter-minus-strand DNA containing a reporter sequence that is different from the reporter sequence of, ie, with the intron removed, is synthesized (FIG. 1C).

(2) Preparation of (pCI-HBV-P) HBV-P expression vector The sequence shown by SEQ ID NO: 5 artificially designed based on the P gene shown by SEQ ID NO: 4 of HBV is CMV of pCI vector (Promega). The HBV-P expression vector according to the eighth aspect of the present invention, pCI-HBV-P, was prepared by insertion under promoter control (FIG. 2B).

(3) Preparation of HBc expression vector (pCI-HBc) The sequence shown by SEQ ID NO: 3 artificially designed based on the C gene shown by SEQ ID NO: 2 of HBV is controlled by the CMV promoter of the pCI vector (Promega). Insertion was made to prepare pCI-HBc, which is the HBV-C expression vector according to the eighth aspect of the present invention (FIG. 2C).

(4) Preparation of HBx expression vector (pCI-HBx) The sequence shown by SEQ ID NO: 7 artificially designed based on the X gene shown by SEQ ID NO: 6 of HBV is controlled by the CMV promoter of the pCI vector (Promega). Insertion was made to prepare pCI-HBc, which is the HBx expression vector according to the eighth aspect of the present invention (FIG. 2D).

(5) HBV replication activity evaluation method An HBV replication activity evaluation vector, an HBV-P expression vector, an HBc expression vector, and an HBx expression vector were introduced into HeLa cells in various combinations (introduction step). The combination pattern is as described in FIG. 3A. The electroporation method was used for gene transfer into HeLa cells. An electroporator Nepa21 (Neppagene) introduced 10 μg of DNA into approximately 1 × 10 ⁶ HeLa cells under 125 V / 2.5 ms plus lenses conditions. The ratio of HBc expression vector: HBV-P expression vector: HBx expression vector is 9: 3: 1, and if there is an expression vector that is not introduced by combination, an empty vector (pCI) with the same ratio as the expression vector that is not introduced is used. Introduced. The ratio of the sum of the HBV replication activity evaluation vector and the other expression vectors was 1: 1 (5 μg: 5 μg), and for the expression vector not to be introduced, pCI corresponding to the ratio was introduced.

In addition, HBc / HBV-P / HBx expression to determine if the amount of reverse transcribed reporter-strand DNA reflects the function of HBc, HBV-Pol, and HBx in the cell. The ratio of the HBV replication activity evaluation vector: HBc / HBV-P / HBx expression vector was set to 1: 0 (5 μg: pCI 5 μg) without changing the vector ratio (HBc: HBV-P: HBx = 9: 3: 1). 1: 0.004 (5 μg: 0.02 μg), 1: 0.016 (5 μg: 0.08 μg), 1: 0.062 (5 μg: 0.31 μg), 1: 0.25 (5 μg: 1.25 μg) ) And 1: 1 (5 μg: 5 μg), the effect on the reverse transcription reaction was analyzed by the amount of reporter minus strand DNA.

After gene transfer, HeLa cells were cultured in DMEM supplemented with 2 mL of 10% FBS at 37 ° C. for 48 hours in the presence of 5% CO ₂ (culture step).

After culturing, DNA was extracted from HeLa cells using DNeasy Mini (Qiagen). As the primer, a primer pair of a forward primer consisting of the base sequence shown in SEQ ID NO: 8 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 9 was used. In this forward primer, the 2 bases at the 3'end match the 2 bases at the 5'end in the downstream exon, but not the 2 bases at the 5'end of the intron. Therefore, it functions as a primer only in the presence of a reporter-minus-stranded DNA having a reporter sequence from which the intron has been removed, and a 131-base DNA fragment is amplified using the reporter-minus-stranded DNA as a template.

In addition, for DNA derived from cells into which each expression vector was introduced in the combination shown in FIG. 3A, the PCR product after PCR was electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Further, for the preparation of the calibration line, the ratio of each expression vector of HBc: HBV-P: HBx is 9: 3: 1, and the ratio of the HBV replication activity evaluation vector: HBc / HBV-P / HBx expression vector is 1. The introduced HeLa cell-derived DNA was serially diluted to a ratio of 1, to prepare a template DNA, and real-time PCR was performed.

(result)
The results are shown in FIG.
FIG. 3A shows the results of staining the PCR product derived from the cells into which the expression vector was introduced in each combination with ethidium bromide after 2% agarose gel electrophoresis. C indicates an HBc expression vector containing the C gene, P indicates an HBV-P expression vector containing the P gene, and X indicates an HBx expression vector containing the X gene. + Indicates introduction into HeLa cells, and-indicates non-introduction. The 131 bp PCR product derived from the reporter-minus DNA was found only when the HBc expression vector and the HBV-P expression vector were introduced. Therefore, it was clarified that HBc and HVP-Pol are indispensable for reverse transcription of pgRNA. It was also confirmed that HBx significantly promotes reverse transcription by Hbc and HBV-Pol, although it is not essential for reverse transcription of pgRNA.

FIG. 3B is a calibration curve prepared from the results of real-time PCR of serially diluted DNA. As this figure shows, a precise calibration curve with R ² = 0.9996 was obtained, demonstrating that the amount of reverse transcribed reporter-strand DNA can be quantified by real-time PCR.

FIG. 3C shows the results of quantitative analysis using real-time PCR and the calibration curve of B. This is the result of quantitative analysis using the calibration curve shown in B after amplifying the DNA sample used in A by real-time PCR. A result similar to the analysis result by agarose gel electrophoresis of A was obtained.

FIG. 3D is a diagram showing the correlation between the amount of speech of HBV protein (HBc, HBV-P, HBx) and the amount of HBV DNA replication, that is, the amount of reporter minus chain DNA. Reverse transcription of pgRNA increased exponentially with the expression level of HBV protein. From this result, according to the HBV replication activity evaluation method using this HBV replication activity evaluation system, the activity of reverse transcription, that is, the replication activity is measured and quantified, reflecting the action of the HBV protein in the host cell. It became clear that it could be done.

<Evaluation of HBV replication activity>
The effect of the sample was verified using the HBV replication activity evaluation system.
(Method)
An HBV replication activity evaluation vector, an HBV-P expression vector, an HBc expression vector, and an HBx expression vector were introduced into HeLa cells according to the HBV replication activity evaluation method. The introduction ratio of each HBc: HBV-P: HBx expression vector was 9: 3: 1, and the introduction ratio of the HBV replication activity evaluation vector and the HBc + HBV-P + HBx expression vector was 1: 1 (5 μg: 5 μg). DMEM supplemented with 2 mL of 10% FBS was added to HeLa cells after the introduction step, and the mixture was dispensed into 96-well-plate at 0.1 mL / well, and then 0.04 μg / well, 0.08 μg / well, 0. Samples of 16 μg / well, 0.31 μg / well, 0.63 μg / well, 1.25 μg / well, 2.50 μg / well, and 5.00 μg / well were added and at 37 ° C. in the presence of 5% CO ₂ . It was cultured for 24 hours.

After culturing, 2% NP-40 / Proteinase K was added to each well to extract DNA from HeLa cells, and real-time PCR was performed according to the above-mentioned HBV replication activity evaluation method.

(result)
After amplification by real-time PCR, the reporter minus strand DNA was quantitatively analyzed by the calibration curve prepared by the above-mentioned HBV replication activity evaluation method, and EC ₅₀ was calculated. The obtained results are shown in Tables 2 to 5 below.

The results of EC ₅₀ obtained for the compounds obtained in Examples 1 to 40 as samples and HBV1106, Compound CBT-188, Compound CBT-039, and Compound CBT-083 by the above-mentioned method for measuring HBV replication activity are as follows. Shown in the table. The following known compounds were produced according to the known literature under the CAS Registry Number below.

As shown in Table 2, the compounds of formula (XI) have HBV replication inhibitory activity.

As shown in Table 3, the compound of formula (XII) has HBV replication inhibitory activity.

As shown in Table 4, the compound of formula (XIII) has HBV replication inhibitory activity.

As shown in Table 5, the compounds of formula (XIV) have HBV replication inhibitory activity.

<PET imaging with compound [ ^11C ] CBT-078 using HBc expression model mouse>
A PET imaging test using an HBc expression model mouse was carried out in order to examine the pharmacokinetics, excretion behavior, accumulation of HBc-expressing liver tissue in the liver tissue, and visualization ability by imaging of the compound [ ^11C ] CBT-078 produced in Example 41. bottom. All animal experiments were carried out in accordance with the guidelines for animal experiments of the National Institute of Physical and Chemical Research, with the approval of the Institute's Ethics Committee for research plans, and in compliance with laws and regulations.

-Creation of HBc expression model mouse The HBc expression model mouse was prepared by gene transfer of an expression vector pCI-HBc encoding only the HBc protein into BALB / c mice by the hydrodynamic injection method. The base sequence of pCI-HBc used is shown in SEQ ID NO: 10. [YK1] pCI-HBc used a PBS solution equivalent to 10% of body weight for 6-week-old male BALB / c mice. For example, if the body weight is 20 g, 2 ml of pCI-HBc / PBS solution is administered. At the time of administration, the mice were placed under isoflurane anesthesia on a hot pad to maintain body temperature, and the above solution was administered from the tail vein at a substantially constant velocity over 5 seconds using a Terumo 26G needle and a 2.5 ml disposable syringe. Dynamic injection was performed. After administration, the mice were immediately returned to their cages and recovered. pCI-HBc was dissolved in PBS solution of administration solution amount + 100 μl (dead volume), mixed with a mixer, and the whole amount was taken in a syringe and used for administration without any time. Mice were subjected to dissection about 24 hours after administration, and liver tissue was collected. Part of the liver tissue was used to prepare frozen tissue sections, and part was used to prepare tissue lysate solution. HBc expression was evaluated by immunohistochemical staining of frozen sections and ELISA analysis using plasma and liver lysate solutions.

Immunohistochemical staining is performed by M. O. Using the M Immunoduction kit (VECTOR), Anti-Hepatitis B Virus Antigen antibody (Abcam, ab8638) was used as the primary antibody, and M.I. O. Using M Biotinylated anti-mIgG, Vectorastein ABC-AP (VECTOR) was colored with the AP substrate after the reaction and nuclear-stained with hematoxylin. The stained image of the liver tissue section is shown in FIG. Expression of the HBc protein that was lightly stained over the entire section was confirmed by administration of pCI-HBc. In addition, a speckled dark-stained area was seen, which is thought to be due to the multimerization of HBc protein.

A commercially available HBc ELISA kit (QuickTiter® HBV Core Antigen ELISA Kit, CELL BIOLABS, INC) was used for the ELISA analysis. In order to compare the change in the HBc expression level depending on the amount of pCI-HBc administered, the HBc concentration in the liver lysate was compared at the pCI-HBc dose of 10, 20, 40, and 80 μg. In addition, in order to confirm the possibility of liver tissue damage by the hydrodynamic method, the CRP concentration in the liver lysate was also measured using the Mouse High-Sensitive CRP ELISA kit (KAMIYA Biomedical Company). As shown in FIG. 5, the highest HBc expression concentration in the 40 μg pCI-HBc administration group was found by comparing the HBc expression concentration for each dose calculated by ELISA. When the pCI-HBc dose was excessive, the HBc expression level decreased. On the other hand, the CRP concentration is not significantly different from that of the Vehicle group (administered only with PBS) without pCI-HBc administration, and it seems that there is no damage to the liver tissue.

From these results, it was decided to use the HBc expression model mouse used for the PET test 24 hours after administration of 40 μg of pCI-HBc having a high HBc concentration by the hydrodynamic injection method.

-PET imaging test PET imaging of compound [ ¹¹ C] CBT-078 was performed using the above HBc expression model mouse. Furthermore, in order to clarify the HBc specificity of the accumulation behavior of compound [ ^11C ] CBT-078, competitive inhibition was administered to the Vehicle group in which only PBS was hydrodynamically injected as a control group and the HBc-expressing mice to which the unlabeled compound CBT-078 was administered. It was decided to perform PET imaging for the (Blocking) group as well. Four HBc expression groups, three Vehicle groups, and three Blocking groups were used. Furthermore, imaging of 3 HBc expression groups and 3 Vehicle groups using [11C] CBT-039 as a negative control was also performed.

PET imaging was performed using an animal PET imaging device MicroPET Focus 220 (SIEMENS) under isoflurane anesthesia while maintaining body temperature with a rectal temperature-testing probe and a hot pad. Using a catheter placed in the tail vein in advance, PET imaging was started at the same time as the administration of compound [ ¹¹ C] CBT-078 or compound [ ¹¹ C] CBT-039, and data was collected for 90 minutes. The average body weight of the mice at the time of imaging was 19.0 ± 1.1 g, and the compound [ ¹¹ C] CBT-078 was about 47 MBq (≈267 ng) and the compound [ ¹¹ C] CBT-039 was about 39 MBq (≈356 ng) in each mouse. Was administered. In the Blocking group, the unlabeled compound CBT-078 was dissolved in dimethylsulfoxide at a concentration of 100 μg / μl, and then 1 μl was added to 1 ml of a mixed solution of 10% 2-hydroxypropyl-beta-cyclodextrin and 10% polyethylene glycol 300. 100 μl (= 10 μg) was administered 10 minutes prior to compound [ ¹¹ C] CBT-078 administration (approximately 37-fold equivalent of compound [ ¹¹ C] CBT-078). Mice were immediately dissected after PET imaging, blood and each organ were collected, weighed, and then radioactivity was measured with a gamma counter. After image reconstruction, the data collected by imaging was analyzed for liver accumulation using PET image analysis software ASIPro VM. At this time, by setting the region of interest VOI (Volume of Interest) only in a part of the right lobe of the liver, the influence of changes in the radioactivity distribution in the large blood vessels, gallbladder, and intestine is eliminated as much as possible for analysis. gone.

The result of the obtained PET image is shown in FIG. 6, and the time radioactivity curve in the liver by image analysis is shown in FIG. FIG. 8 shows the analysis results of the tissue radioactivity distribution by measuring the radioactivity of the anatomical sample. As can be seen from the PET image results, it was clarified that both compound [ ¹¹ C] CBT-078 and compound [ ¹¹ C] CBT-039 are excreted from the body by intestinal excretion and urine excretion via liver and bile. .. In the imaging results of compound [ ¹¹ C] CBT-078, a markedly high accumulation was observed in the liver of the HBc expression group at 1 hour after administration, the accumulation was not observed in the Vehicle group, and the accumulation was not observed in the Vehicle group than in the Vehicle group. Although it was slightly higher, it showed a significantly reduced accumulation compared with the HBc expression group. In the imaging results of compound [ ¹¹ C] CBT-039, the accumulation in the liver was lower than that of compound [ ¹¹ C] CBT-078, and no difference was observed between the HBc expression group and the Vehicle group. Compared to the compound [ ¹¹ C] CBT-078 administration group, the compound [ ¹¹ C] CBT-039 administration group had less systemic accumulation so that the outline of the mouse could not be seen, while a clear depiction of the gallbladder was observed. It is considered to be cleared quickly. From the results of the time radioactivity curve in the liver shown in FIG. 7, the difference in the clearance rate between the compound [ ¹¹ C] CBT-078 administration group and the compound [ ¹¹ C] CBT-039 administration group is clear. The hepatic accumulation of compound [ ¹¹ C] CBT-078 reached a maximum value immediately after administration and then gradually decreased until 90 minutes after administration, and the binding with HBc resulted in the hepatic accumulation of compound [ ¹¹ C] CBT-078. It seems that excretion is inhibited and the residence time in the liver is prolonged. In the Blocking group, the accumulation concentration at an early stage after administration is not different from that in the Vehicle group, but as time passes, it becomes higher than that in the Vehicle group. This is also thought to be due to the decrease in excretion rate due to the reaction with HBc. It is considered that the compound CBT-078 was sparingly soluble and the dose for Blocking was not sufficient, which caused a difference from the Vehicle group. The results of compound [ ¹¹ C] CBT-039 showed no difference between the HBc expression group and the Vehicle group, and they were excreted more rapidly than compound [ ¹¹ C] CBT-078 and almost leveled off 90 minutes after administration. There is. As a result of the tissue radioactivity distribution shown in FIG. 8, in compound [ ¹¹ C] CBT-078, the difference similar to the time radioactivity curve of the liver in all sites except urine, gallbladder, and intestinal tract, that is, HBc group> Blocking group> Vehicle There was a group difference in accumulation. On the other hand, in compound [ ¹¹ C] CBT-039, no significant difference was observed in the organ accumulation between the HBc group and the Vehicle group.

The results of compound [ ^11C ] CBT-078 showed remarkable accumulation in the liver in the HBc expression group, and the accumulation was decreased in the Vehicle group and the Blocking group, strongly suggesting that it was a specific accumulation. However, on the other hand, some hepatic accumulation was also observed in the Vehicle group, and the HBc expression manipulation by the hydrodynamic injection method affected the hepato-biliary excretion pathway, and the residence time of compound [ ^11C ] CBT-078 in the liver tissue was observed. There is concern that it may have contributed to the extension. However, in the results of the compound [ ^11C ] CBT-039 having a similar structure, there was no significant difference in the tissue radioactivity distribution between the HBc expression group and the Vehicle group, and the effect of the HBc expression manipulation on the hepatobiliary excretion pathway was almost nonexistent. It was suggested that there was none. This made it clearer that the accumulation of compound [ ^11C ] CBT-078 in the HBc-expressing liver was due to specific binding. From the above results, it was clarified that the compound [ ¹¹ C] CBT-078 specifically accumulated in the HBc-expressing liver, and the HBc-expressing liver was successfully visualized by PET imaging.

It is also applicable to the application of conducting a microdose clinical PET test for actual hepatitis B patients and providing useful information as diagnostic information from the correlation between blood HBc concentration in blood analysis and hepatic radioactivity accumulation in PET imaging. can.

Claims (6)

A PET probe for imaging targeting a hepatitis B antigen protein containing a compound represented by the general formula (I) or a salt thereof.

[During the ceremony,
R ¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and carbon. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 3 and is
R ² is a phenyl group or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
R ³ is

Is.
The compound represented by the general formula (I) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom. ] The compound represented by the general formula (I) is a compound represented by the following general formula (XI), a compound represented by the general formula (XII), a compound represented by the general formula (XIII), or a general formula ( The PET probe for imaging targeting the hepatitis B antigen protein according to claim 1, which is a compound represented by XIV).

[In formula (XI),
R ⁴ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 2 to 6 carbon atoms. Substituted by 1 to 3 selected from the group consisting of alkenyl 6 and alkynyl having 2 to 6 carbons, alkynyl having 2 to 6 carbons substituted with alkylsilyl having 1 to 6 carbons, phenyl, halogen, and dioxaboranyl. It is a phenyl group.
The compound represented by the general formula (XI) is labeled with ¹¹ C or ¹⁸ F if it contains a fluorine atom. ]

[In formula (XII),
R ⁵ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, and a halogen.
The compound represented by the general formula (XII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom. ]

[In equation (XIII),
R ⁶ is a phenyl group or a phenyl group substituted with an alkyl having 1 to 3 carbon atoms and 1 to 6 carbon atoms, and R ⁷ is a phenyl group or a phenyl group substituted with 1 to 3 halogens.
R ⁸ is

Is.
The compound represented by the general formula (XIII) is labeled with ¹¹ C or ¹⁸ F when it contains a fluorine atom. ]

[In formula (XIV),
R ⁹ is a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
It is labeled with ¹¹ C or with ¹⁸ F if it contains a fluorine atom.
The compound represented by the general formula (XIV) is labeled with ¹¹ C or ¹⁸ F if it contains a fluorine atom. ] A compound represented by the general formula (XX) or a salt thereof, or a compound labeled with ¹¹ C or labeled with ¹⁸ F if it contains a fluorine atom.

[During the ceremony,
R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and carbon. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 3 and is
R ¹² is a phenyl group or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of alkyl-substituted amino, cyano, and halogen.
However, when R ¹² is a phenyl group substituted with halogen, R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, or an alkyl having 1 to 6 carbon atoms and an alkoxy having 1 to 6 carbon atoms. 1 to selected from the group consisting of alkenyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, and dioxaboranyl. A phenyl group substituted with 3 or a phenyl group whose p-position is substituted with halogen.
However, when R ¹² is a phenyl group, R ¹¹ is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, or an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, and an alkenyl having 2 to 6 carbon atoms. , Select from the group consisting of 1-3 selected from the group consisting of alkynyl having 2 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, halogen, and dioxaboranyl. It is a phenyl group substituted with 1 to 3 to be added. ] The compound according to claim 3, wherein the compound represented by the general formula (XX) is a compound represented by the general formula (XV).

[During the ceremony,
R ^4'is a cyclohexyl group having 3 to 7 carbon atoms, a naphthyl group, a phenyl group, an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, and 2 to 6 carbon atoms. A phenyl group substituted with 1-3, selected from the group consisting of alkynyl, alkynyl having 2 to 6 carbons substituted with alkylsilyl having 1 to 6 carbon atoms, phenyl, and dioxaboranyl, or the o-position or p-position. Is a phenyl group substituted with halogen. ] The compound according to claim 3, wherein the compound represented by the general formula (XX) is a compound represented by the general formula (XII).

[During the ceremony,
R ⁵ is a phenyl group or a 1-3 substituted phenyl group selected from the group consisting of an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, or a halogen. ] The compound according to claim 3, wherein the compound represented by the general formula (XX) is a compound represented by the general formula (XVI).

[During the ceremony,
R ⁹ is substituted with an alkyl having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms, an alkylthio having 1 to 6 carbon atoms, an alkoxy having 1 to 6 carbon atoms substituted with halogen, and an alkyl having 1 to 6 carbon atoms. A 1-3 substituted phenyl group selected from the group consisting of amino, cyano, and halogen. ]

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