JP6187460B2 - Pyridopyrimidin-4-one derivatives - Google Patents

Description

The present invention relates to pyridopyrimidin-4-one derivatives that specifically bind to the V1b receptor of arginine-vasopressin (AVP). Further, it relates to a novel [ ³ H] -labeled ligand of a pyridopyrimidin-4-one derivative that specifically binds to the V1b receptor. The present invention also relates to a production method for efficient [ ³ H] -labeled ligand synthesis and a precursor useful for the method.

  AVP is a peptide consisting of 9 amino acids. AVP is biosynthesized by nerve cells in the brain, secreted from the posterior pituitary gland into the blood, and regulates blood pressure and fluid volume. AVP is also secreted into the anterior pituitary gland and stimulates the secretion of adrenocorticotropic hormone (ACTH).

The AVP receptor has so far been cloned into three subtypes of V1a, V1b and V2 receptors, all of which are G protein-coupled receptors. V1a and V1b receptors are coupled to Gq protein, and activation of the receptor enhances inositol phospholipid metabolism and increases intracellular Ca ²⁺ concentration (see Non-Patent Documents 1 and 2). . In addition, the V2 receptor is coupled to the Gs protein, and activation of the receptor increases the amount of intracellular cyclic AMP. V1a receptor is expressed in brain, liver, adrenal gland, vascular smooth muscle and the like. V2 receptor is expressed in the kidney and the like. V1b receptors are expressed in the brain, pituitary gland, etc., and in the brain, they are expressed in the hypothalamus, olfactory bulb, hippocampus, cerebral cortex, striatum, cerebellum, etc. (see Non-Patent Documents 3 and 4). ). In V1b receptor knockout mice, AVP-induced ACTH secretion is reduced (see Non-Patent Document 5). Therefore, it is suggested that the V1b receptor is involved in ACTH secretion from the anterior pituitary gland by AVP.

There are reports suggesting a relationship between AVP and AVP receptors and mental illness. For example, plasma AVP levels are elevated in depressed patients (see Non-Patent Document 6). Moreover, the relationship between the single-gene polymorphism of V1b receptor gene and the increase in the sensitivity of depression is suggested (refer nonpatent literature 7). On the other hand, there has been a report examining the effects of AVP receptor antagonists in animal models. So far, an orally administrable 1,3-dihydro-2H-indol-2-one compound has been created (see Patent Documents 1 to 3), and this compound is an animal model for evaluating antidepressant action (such as forced swimming test) ) And animal models (e.g., elevated plus maze test) that evaluate anxiolytic activity show antidepressant and anxiolytic activity (see Non-Patent Document 8 and Non-Patent Document 9). Furthermore, this compound suppresses blood ACTH increase induced by AVP and blood ACTH increase induced by restraint stress (see Non-Patent Document 8). Compounds disclosed in Patent Document 1, high affinity to V1b receptors ^{(1 × 10 -9 mol / L~4} × 10 -9 mol / L) and, V1b receptor compared to the other AVP receptors It is a compound that acts more selectively. From the above findings, there is a high possibility that the V1b receptor antagonist is useful as a therapeutic agent for mental illness.

A compound that selectively acts on the V1b receptor is not only applied to the development of a novel therapeutic agent targeting the V1b receptor, but also used as a radiolabeled substance such as [ ³ H], under physiological conditions. It is possible to examine the expression distribution of the V1b receptor or the change in the expression of the V1b receptor in a disease model animal, which is an effective tool for clarifying the physiological function of the V1b receptor. Expression of V1b receptor by autoradiography in pituitary slices using [ ³ H] -labeled 1,3-dihydro-2H-indol-2-one compound as an application example in in vitro binding test There is a report of distribution (Non-patent Document 10). In addition, there is a report of an autoradiography method using a pituitary slice in the case of a [ ³ H] labeled body having a quinazolin-4-one skeleton (Non-patent Document 11).

However, as in ^[3 H] label in the pyridopyrimidine-4-one derivatives disclosed in the present invention, administered intravenously to the rat at in vivo, ^[3 to pituitary by V1b receptor-specific binding To date there has been no report showing uptake of the H] label.

Moreover, although the pyridopyrimidin-4-one derivative couple | bonded specifically with V1b receptor is disclosed by patent document 4 and patent document 5, the pyridopyrimidin-4-one derivative of this invention, pyridopyrimidine There is no disclosure of [ ³ H] -labeled derivatives of -4-one derivatives and their production methods and precursors.

WO2001 / 0555130 WO2005 / 021534 WO2005 / 030755 WO2009 / 017236 JP 2010-173978 A

Sugimoto T, Kawashima H, J. Biol. Chem., 269, 27088-27092, 1994. Lolait S, Brownstein M, Proc. Natl. Acad. Sci. U S A, 92, 6783-6787, 1995. Vaccari C, Ostrowski N, Endocrinology, 139, 5015-5033, 1998. Hernando F, Burbach J, Endocrinology, 142, 1659-1668, 2001. Tanoue A, Tsujimoto G, J. Clin. Invest., 113, 302-309, 2004. van Londen L, Wied D, Neuropsychopharmacol., 17, 284-292, 1997. van West D, Claes S, Mol.Psychiatry, 9, 287-292, 2004. Gal CS, Fur GL, J. Pharmacol. Exp. Ther., 300, 1122-1130, 2002. Griebel G, Soubrie P, Proc. Natl. Acad. Sci. U S A, 99, 6370-6375, 2002. Gal CS, Ventura MA, Am J. Physiol. Regul. Integr. Comp. Physiol. 293, R938-R949, 2007. L.V.Unger, S.X.Vaidyanathan, 38th American Academy of Neuroscience, 2008, Program # / Poster # 845.21 / Z16

An object of the present invention is to provide an unlabeled body that specifically binds to the V1b receptor as well as a [ ³ H] -labeled body. Another object of the present invention is to provide a production method for efficient [ ³ H] -labeled ligand synthesis and a precursor useful for the method.

As a result of an sincere study on a compound that specifically binds to the V1b receptor, the present inventors have found a strong binding action to a certain pyridopyrimidin-4-one derivative, and an excellent [ ³ H] -labeled ligand. In addition, the inventors have found a production method for efficient [ ³ H] -labeled ligand synthesis, and a precursor useful for the method, and have completed the present invention.

Hereinafter, the present invention will be described in detail. Aspects of the present invention are as follows. The compounds (1) to (6) and (10) are hereinafter also referred to as “the present compound”. The production methods (11) to (14) are hereinafter also referred to as “the production method of the present invention”, and the compound (9) and the compound of the formula (III) are hereinafter also referred to as “precursors”.
(1) Formula (I)

(Where
X represents a nitrogen atom or the formula CH;
R ¹ represents C _1-5 alkyl (wherein the C _1-5 alkyl is the same or different 1 to 3 substituents selected from hydroxy, halogen atom, cyano, C _3-7 cycloalkyl, and C _1-5 alkoxy) May be substituted with a group),
R ² represents a hydrogen atom or C _1-5 alkyl (wherein the C _1-5 alkyl is at least one hydrogen atom is ³ H). )
Or a pharmaceutically acceptable salt thereof,
(2) In the above formula (I),
The compound or a pharmaceutically acceptable salt thereof according to (1), wherein R ² is C _1-5 alkyl (wherein the C _1-5 alkyl has at least one hydrogen atom is ³ H);
(3) In the above formula (I),
R ¹ is C _1-5 alkyl,
The compound or a pharmaceutically acceptable salt thereof according to (2), wherein R ² is C _1-5 alkyl (wherein the C _1-5 alkyl is at least one hydrogen atom is ³ H);
(4) In the above formula (I),
The compound or a pharmaceutically acceptable salt thereof according to either (2) or (3), wherein R ² is methyl (wherein the methyl has at least one hydrogen atom is ³ H),
(5) In the above formula (I),
The compound or pharmaceutically acceptable salt thereof according to any one of (2) to (4), wherein R ¹ is tert-butyl;
(6) In the above formula (I),
X is the formula CH;
The compound or a pharmaceutically acceptable salt thereof according to any one of (2) to (5), wherein R ¹ is tert-butyl;
(7) A V1b receptor labeling agent comprising the compound according to any one of (2) to (6) above or a pharmaceutically acceptable salt thereof,
(8) A pharmaceutical composition comprising the compound according to any one of (2) to (6) above or a pharmaceutically acceptable salt thereof,
(9) In the above formula (I),
R ¹ is C _1-5 alkyl,
The compound or a pharmaceutically acceptable salt thereof according to (1), wherein R ² is a hydrogen atom;
(10) Formula (II)

Or a pharmaceutically acceptable salt thereof,
(11) A method for producing the compound according to any one of (2) to (6) above or a pharmaceutically acceptable salt thereof, comprising the formula (III):

(Wherein R ¹ and X are as defined above) and the formula R ² -L ¹ (R ² is as defined above and L ¹ is a leaving group). And a compound represented by formula (I) in the presence of a base and a solvent,
(12) The method according to (11), wherein L ¹ is a 4-nitrobenzenesulfonyloxy group, a p-toluenesulfonyloxy group, a methanesulfonyloxy group, or a halogen atom.
(13) The method according to (11) or (12), wherein the base is potassium carbonate, cesium carbonate, triethylamine, or diisopropylethylamine.
(14) The method according to any one of (11) to (13), wherein the solvent is tetrahydrofuran, acetonitrile, N, N-dimethylformamide, or dimethyl sulfoxide.

The unlabeled product of the present invention showed binding inhibitory activity specific to the V1b receptor. Further, the [ ³ H] -labeled substance was stable without being metabolized after being administered in vivo, and a sufficient amount of transfer to the pituitary gland as a target organ was observed.

Furthermore, the production method of the present invention enables efficient supply of the [ ³ H] -labeled product. First, the process using a radiolabeled compound must be performed in a special facility for handling radioactive substances. In this respect, in the manufacturing method of the present invention, since such a process is performed in the final stage, the amount of work in a special facility can be minimized, and as a result, the complexity of the work is reduced and the work is accompanied. Cost can be reduced. Moreover, performing the said process in the last stage leads to preventing the loss of the labeling compound which will arise if there exists a subsequent additional process. It is extremely important to prevent such loss. This is because [ ³ H] -labeled raw materials are expensive. Precursor for ^[3 H] labeled ligand synthesis of the present invention has enabled the production of such efficient present invention ^[3 H] labeled ligands.

In the binding test using the rat anterior pituitary membrane, the relationship between the concentration of the compound A-3 of the present invention in the mixed reaction solution and the total binding, nonspecific binding and specific binding to the rat anterior pituitary membrane is shown. In the in vivo rat anterior pituitary binding test, the radioactivity concentration in the anterior pituitary gland and eyeball 30 minutes after administration of Compound A-3 of the present invention (no blocking) and the compound of Example A-250 described in WO2009 / 017236 Shows the radioactivity concentration (with blocking) in the anterior pituitary gland and eyeball when pretreated.

The terms used in the present specification have the following meanings.
The “halogen atom” is a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
The “C _1-5 alkyl group” means a linear or branched alkyl group having 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec -Butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl group and the like can be mentioned.
The “C _3-7 cycloalkyl group” is a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl group.
The “C _1-5 alkoxy group” means a linear or branched alkoxy group having 1 to 5 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, Examples include sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, tert-pentyloxy groups, and the like.
As the “leaving group”, an arylsulfonyloxy group (the aryl of the arylsulfonyloxy group may be substituted with a C _1-5 alkyl group, a nitro group, a halogen atom, or the like), C _1-5 alkylsulfonyloxy A group (the C _1-5 alkyl group of the C _1-5 alkylsulfonyloxy group may be substituted with a halogen atom), a halogen atom, and the like. The aryl is a monocyclic to bicyclic aromatic carbocycle, and examples thereof include phenyl, 1-naphthyl, and 2-naphthyl.
Examples of the arylsulfonyloxy group (the aryl of the arylsulfonyloxy group may be substituted with a C _1-5 alkyl group, a nitro group, a halogen atom, or the like) include a 4-nitrobenzenesulfonyloxy group, p-toluenesulfonyloxy Groups and the like.
As the C _1-5 alkylsulfonyloxy group (the C _1-5 alkyl group of the C _1-5 alkylsulfonyloxy group may be substituted with a halogen atom), a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group Etc.
Examples of the “base” include inorganic bases such as potassium carbonate and cesium carbonate, and organic bases such as triethylamine and diisopropylethylamine.
“ ³ H” is a radioactive isotope of a hydrogen atom and is tritium. It is also written as tritium and may be abbreviated as T.

  As used herein, “pharmaceutically acceptable salt” means a pharmaceutically acceptable acid addition salt, and the acid used includes sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid. Salts with inorganic acids such as acetic acid, benzoic acid, oxalic acid, lactic acid, malic acid, tartaric acid, fumaric acid, maleic acid, citric acid, malonic acid, mandelic acid, gluconic acid, galactaric acid, glucoheptonic acid, glycol Salts with organic acids such as acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, naphthalene-2-sulfonic acid are included. Conversion from the educt to the salt can be performed by conventional methods.

  Preferred embodiments of the compound of the present invention are listed below.

  X is preferably a compound of formula CH.

R ¹ is preferably a compound which is C _1-5 alkyl, more preferably a compound which is isopropyl or tert-butyl, and even more preferably a compound which is tert-butyl.

R ² is preferably a compound which is labeled C _1-5 alkyl with 1-3 ³ H, more preferably a compound which is labeled methyl with 1-3 ³ H, 3 pieces of ³ H Further preferred is a compound which is methyl labeled with.

In another preferred embodiment of R ^2, R ² is a compound which is a hydrogen atom.

Preferred profiles of compounds acting on V1b receptors and their [ ³ H] -labeled compounds show potent and specific binding inhibitory activity against V1b receptors, and other receptors (eg, V1a receptor, V2 receptor) And the like are preferably compounds that do not exhibit binding inhibitory activity.

  In addition, when this invention compound forms a hydrate or a solvate, they are also contained in the scope of the present invention. Similarly, pharmaceutically acceptable salts of hydrates or solvates of the compounds of the invention are also included within the scope of the invention. Further, the compound of the present invention includes all enantiomers, diastereomers, equilibrium compounds, mixtures of these in arbitrary proportions, racemates and the like.

  The compound of the present invention can be produced, for example, according to the method shown below.

  The compounds (I), (II), (III) and their pharmaceutically acceptable salts of the present invention can be synthesized using various organic synthesis methods known to those skilled in the art. For example, the production method is shown below, but is not limited to this synthesis method. In the following examples of production methods, the compound may form a salt that does not hinder the reaction.

  The compound represented by the formula (I) can be produced by the synthesis method shown in Scheme 1.

Wherein R ¹ and X are as defined above, R ² is C _1-5 alkyl (wherein the C _1-5 alkyl is at least one hydrogen atom is ³ H) L ¹ is A leaving group means a 4-nitrobenzenesulfonyloxy group, a p-toluenesulfonyloxy group, a methanesulfonyloxy group, a halogen atom, etc.)

  The compound represented by formula (I) can be obtained by reacting the compound represented by formula (III) with the compound represented by formula (1-a) (step 1-1). The reaction in Step 1-1 is carried out in a solvent such as tetrahydrofuran, acetonitrile, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixed solvent thereof at room temperature to a temperature near the boiling point of the solvent. The reaction proceeds due to the presence of an inorganic base such as potassium carbonate or cesium carbonate, or an organic base such as triethylamine or diisopropylethylamine. In particularly preferred reaction conditions, the base is cesium carbonate and the solvent is in DMF.

The compound (III) of the present invention in which R ² is a hydrogen atom can be produced by the synthesis method shown in Scheme 2.

(In the formula, R ¹ and X are as defined above. Hal represents a halogen atom. L ² represents a protecting group for a phenolic hydroxyl group [Protective Groups in Organic Synthesis, 4th edition] See John Wiley & Sons, Inc.] L ³ represents a leaving group, which means a p-toluenesulfonyloxy group, a methanesulfonyloxy group, a halogen atom, or the like. .)

The compound represented by the formula (2-b) can be obtained by converting the compound represented by the formula (2-a) into a boronic acid derivative and then hydroxylating with a peracid (Step 2-1). ). This step can be carried out according to the method described in WO2006 / 021886. The compound represented by the formula (2-d) can be produced by three different steps of Step 2-2, Step 2-3, and Step 2-4. That is, step 2-2 can be obtained by reacting the compound represented by formula (2-b) with the compound represented by formula (2-c). The compound represented by the formula (2-c) may form a salt. The reaction in step 2-2 can be carried out in the same manner as in step 1-1. The compound represented by the formula (2-d) can be obtained by reacting the compound represented by the formula (2-b) with the compound represented by the formula (2-e) under the conditions of Mitsunobu reaction. Yes (step 2-3). A comprehensive overview of the Mitsunobu reaction can be found in Synthesis. 1981, 1-28; Chem. Asian J. 2007, 2, 1340-1355 .; Chem. Pharm. Bull. 2003, 51 (4), 474-476. Furthermore, the compound represented by the formula (2-d) is obtained by subjecting the compound represented by the formula (2-a) and the compound represented by the formula (2-e) to the conditions for the etherification reaction using a palladium catalyst. It can be obtained by reacting (step 2-4). A comprehensive overview of palladium-catalyzed etherification reactions can be found in M. Paulucki, JP Wolfe, SL Buchwald, J. Am. Chem. Soc., 1996, 118, 10333 .; G. Mann, JF Hartwig, J. Am Chem. Soc. 1996, 118, 13109 .; M. Watanabe, M. Nishiyama, Y. Koie, Tetrahedron Lett. 1999, 40, 8837 .; Q. Shelby, N. Kataoka, G. Mann, JF Hartwig, J Am. Chem. Soc. 2000, 122, 10718 .; KE Torraca, S. Kuwabe, SL Buchwald, J. Am. Chem. Soc. 2000, 122, 12907 .; CA Parrish, SL Buchwald, J. Org. Chem 2001, 66, 2498 .; PM Karen, E. Torraca, X. Huang, CA Parrish, and SL Buchwald, J. Am. Chem. Soc, 2001, 10770-10771 .; Andrei V. Vorogushin, Xiaohua Huang, and Found in Stephen L. Buchwald J. Am. Chem. Soc., 2005, 8146 -8149. The compound represented by the formula (III) deprotects the L ² group of the compound represented by the formula (2-d) by a general method [Protective Groups in Organic Synthesis] 4th edition, see John Wiley & Sons, INC.] (Step 2-5).

  Among the compounds represented by the formula (2-a), the compound represented by the formula (3-d) can be produced by the synthesis method shown in Scheme 3.

(In the formula, R ¹ , L ² , X and Hal are the same as above. L ⁴ represents C _1-5 alkyl.)

  The compound represented by the formula (3-d) is obtained by subjecting the compound represented by the formula (3-a) and the aldehyde represented by the formula (3-b) to dehydration condensation, and 1,2-dihydropyrido After obtaining the pyrimidin-4-one derivative (3-c) (step 3-1), it can be obtained by subsequent oxidation (step 3-2). The condensation reaction in step 3-1 proceeds in the presence of an organic acid such as acetic acid in a solvent such as ethanol or 2-propanol under temperature conditions near the boiling point of the reaction solvent, and is a by-product using a dehydrating agent such as molecular sieves. By removing the water, the condensation reaction proceeds more smoothly. The oxidation reaction in Step 3-2 uses an oxidizing agent such as active manganese dioxide, and is in a solvent such as chloroform, N, N-dimethylformamide, tetrahydrofuran, or a mixed solvent thereof, from room temperature to a temperature near the boiling point of the reaction solvent. Progress under conditions. In addition, the compound represented by the formula (3-d) is obtained by mixing the compound represented by the formula (3-a) and the compound represented by the formula (3-e) in a solvent such as ethanol, 2-propanol, and tetrahydrofuran. , And can be condensed under temperature conditions near the boiling point of the reaction solvent (step 3-3).

  The compound represented by the formula (II) can be produced by the synthesis method shown in Scheme 4.

(In the formula, L ³ is the same as above.)

  The compound represented by formula (II) can be obtained by reacting the compound represented by formula (4-a) with the compound represented by formula (2-c) (step 4-1). The compound represented by the formula (2-c) may form a salt. The reaction in Step 4-1 can be performed in the same manner as in Step 1-1.

  Hereinafter, the present invention will be described in more detail with reference examples, examples, and test examples. However, the present invention is not limited to these examples, and may be changed without departing from the scope of the present invention.

In the following reference examples and examples, “Phase Separator” at the time of post-processing is ISOLUTE (registered trademark) Phase Separator of Biotage. "SNAP Cartridge KP-NH" when purified using column chromatography is Biotage's SNAP Cartridge KP-NH, "SNAP Cartridge KP-Sil" is Biotage's SNAP Cartridge KP-Sil, "SNAP Cartridge HP “Sil” has Biotage SNAP Cartridge HP-Sil, “SNAP Cartridge HP-Sphere” has Biotage SNAP Cartridge Ultra, “Chromatorex NH” has Chloramex (registered trademark) NH, TLC plate NH ( Thin-layer chromatography (TLC) using Fuji Silysia), silica gel 60, silica gel The 0N, using silica gel, commercially available from Kanto Chemical Corporation. When purifying using preparative thin layer chromatography (PTLC), Merck silica gel 60F ₂₅₄ , 20 cm × 20 cm was used. Waters Sunfire prep C18 OBD, 5.0 μm, φ30 × 50 mm or YMC-Actus Trial C18, 5.0 μm, φ30 × 50 mm was used for “reverse phase column chromatography” at the time of purification.

Each instrument data described in the following reference examples and examples was measured with the following measuring instruments.
NMR spectrum: JEOL JNM-ECA600 (600 MHz), JEOL NM-ECA500 (500 MHz), Varian UNITYNOVA300 (300 MHz), Varian GEMINI2000 / 200 (200 MHz)
MS spectrum: Shimadzu LCMS-2010EV or micromass Platform LC

In the following Reference Examples and Examples, high performance liquid chromatography mass spectrum (LCMS) was measured under the following conditions.
Condition 1
Measuring instrument: micromass Platform LC and Agilent Agilent 1100
Column: Waters SunFire C18, 2.5 μm, φ4.6 × 50 mm
Solvent: Solution A; 0.1% trifluoroacetic acid-containing water, Solution B; 0.1% trifluoroacetic acid-containing acetonitrile gradient: 0 minutes (A solution / B solution = 90/10), 0.5 minutes (A solution / B liquid = 90/10), 5.5 minutes (A liquid / B liquid = 20/80), 6.0 minutes (A liquid / B liquid = 1/99), 6.3 minutes (A liquid / B Liquid = 1/99)
Flow rate: 1 mL / min, detection method: 254 nm
Ionization method: Electron impact ionization (Electron Spray Ionization: ESI)
Condition 2-1
Measuring machine: Agilent 2900 and Agilent 6150
Column: Waters Acquity CSH C18, 1.7 μm, φ2.1 × 50 mm
Solvent: solution A; 0.1% formic acid-containing water, solution B; 0.1% formic acid-containing acetonitrile gradient: 0 minutes (solution A / solution B = 80/20), 1.2-1.4 minutes (solution A) / B liquid = 1/99)
Flow rate: 0.8 mL / min, detection method: 254 nm
Condition 2-2
Measuring equipment, column, and solvent are the same as those in Condition 2-1.
Gradient, flow rate: 0.8 mL / min, 0 min (A liquid / B liquid = 95/5), 1.20 min (A liquid / B liquid = 50/50), 1.0 mL / min, 1.38 min (A liquid) / B liquid = 3/97)
Detection method: 254 nm

In the following examples, radio HPLC was measured under the following conditions.
Measuring machines: LC-20AD (Shimadzu Corporation), SCL-10Avp (Shimadzu Corporation), SPD-20A (Shimadzu Corporation), 525TR (PerkinElmer)
Column: Atlantis T3, 3 μm, φ4.6 × 150 mm (Waters)

The mobile phase was measured by changing the solvent composition for each example.
Mobile phase when analyzing the compound of Example A-3: 10 mmol / L ammonium acetate (pH 7.0) / acetonitrile (65/35, v / v)
Mobile phase when analyzing compound of Example B-2: 10 mmol / L ammonium acetate (pH 7.0) / acetonitrile (70/30, v / v)
Mobile phase when the compound of Example C-2 was analyzed: 10 mmol / L ammonium acetate (pH 7.0) / acetonitrile (75/25, v / v)
Flow rate: 1.0 mL / min
Detection wavelength: 254 nm

In the following examples, mass spectrometry of [ ³ H] -labeled product was measured under the following conditions.
Measuring machine: LTQ XL (Thermo Fisher Scientific)
Mode: ESI (+)
In the following Reference Examples and Examples, the compound name was named by ACD / Name (ACD / Labs 12.01, Advanced Chemistry Development Inc.).

In the examples, the following terms and reagents are expressed as follows.
AcOK (potassium acetate), BBr ₃ (boron tribromide), CHCl ₃ (chloroform), DMF (N, N-dimethylformamide), EtOAc (ethyl acetate), EtOH (ethanol), MeOH (methanol), Na ₂ SO ₄ (anhydrous sodium sulfate), NaHCO ₃ (sodium bicarbonate), PdCl ₂ (dppf) · CH ₂ Cl ₂ {[1,1′-bis (diphenylphosphino) ferrocene] palladium (II) dichloride dichloromethane complex (1: 1)}, TFA (trifluoroacetic acid), THF (tetrahydrofuran).

  Example A-1: N-tert-butyl-2- [2- (3-methoxyphenyl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] Synthesis of pyrimidine-3 (4H) -yl] acetamide (A-1)

(1) 2- [6-Bromo-2- (3-methoxyphenyl) -4-oxo-1,4-dihydropyrido [2,3-d] pyrimidin-3 (2H) -yl] -N-tert-butyl Synthesis of acetamide (A-1-1)

Under a nitrogen stream, 2-amino-5-bromo-N- [2- (tert-butylamino) -2-oxoethyl] nicotinamide (25.0 g), 3-methoxybenzaldehyde (31.0 g), acetic acid (22. A suspension of 8 g) of EtOH (500 mL) was heated to reflux for 5 hours while dewatering with a Dean-Stark apparatus. After allowing to cool, the reaction solvent was evaporated under reduced pressure, EtOAc was added to the resulting residue, and the resulting solid was collected by filtration and dried to give the title compound (33.4 g, colorless solid).
MS (ESI neg.) M / z: 445 ([MH] ^- ).

(2) 2- [6-Bromo-2- (3-methoxyphenyl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] -N-tert-butylacetamide (A-1- 2) Synthesis

Under a nitrogen stream, a suspension of compound A-1-1 (33.0 g) obtained in Example A-1 (1), MnO ₂ (32.1 g) in THF (512 mL) and CHCl ₃ (130 mL) was added. The mixture was heated to reflux for 5 hours. After filtration using hot Celite (registered trademark), the filtrate was washed with THF (600 mL), and the filtrate was concentrated under reduced pressure. EtOAc was added to the resulting residue, and the resulting solid was collected by filtration and dried to give the title compound (28.7 g, colorless solid).
MS (ESI pos.) M / z: 445 ([M + H] ⁺ ).

(3) N-tert-butyl-2- [6-hydroxy-2- (3-methoxyphenyl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] acetamide (A-1- 3) Synthesis

Under a nitrogen stream, compound A-1-2 (4.45 g) obtained in Example A-1 (2), 4,4,4 ′, 4 ′, 5,5,5 ′, 5′-octamethyl- A solution of 2,2′-bi-1,3,2-dioxaborolane (5.08 g), PdCl ₂ (dppf) .CH ₂ Cl ₂ (408 mg) and AcOK (2.94 g) in DMSO (45 mL) was brought to 100 ° C. And stirred for 2 hours. After allowing to cool, water (200 mL) was added, and the resulting solid was collected by filtration and dried to obtain a solid (9.91 g, brown solid). An aqueous solution of NaHCO ₃ (1.68 g / 15 mL of water) was added to a solution of the obtained solid (9.91 g) in THF (25 mL) and EtOH (25 mL), and then ice-cooled, while maintaining the reaction solution temperature at 8 ° C. or lower, After adding 30% aqueous hydrogen peroxide (3.40 mL), the mixture was stirred for 2 hours. An aqueous sodium sulfite solution (3.78 g / 50 mL of water) was added to the reaction solution, and the mixture was stirred for 15 minutes. CHCl ₃ (100 mL) and saturated brine (100 mL) were added for liquid separation, and the aqueous layer was extracted twice with CHCl ₃ (50 mL). The organic layer was dried over Na ₂ SO ₄ , the desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (SNAP Cartridge HP-Sphere, mobile phase: CHCl ₃ / MeOH = 100/0 to 90/10; v / v) to give the title compound (3.69 g, gray solid) Got.
MS (ESI pos.) M / z: 383 ([M + H] ⁺ ).

(4) N-tert-butyl-2- [2- (3-methoxyphenyl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] pyrimidine-3 ( 4H) -yl] acetamide (A-1)

Compound A-1-3 (3.65 g), 4- (3-chloropropyl) morpholine hydrochloride (2.29 g), cesium carbonate (15.6 g) and iodine obtained in Example A-1 (3) A suspension of potassium fluoride (0.792 g) in DMF (36 mL) was stirred at an external temperature of 85 ° C. for 4 hours. The reaction mixture was allowed to cool, saturated aqueous NaHCO ₃ solution (120 mL), EtOAc (120 mL) and toluene (20 mL) were added and separated, and the organic layer was washed with saturated brine (120 mL). The aqueous layer was extracted twice with EtOAc (120 mL) and toluene (20 mL), and the combined organic layers were dried over Na ₂ SO ₄ . After the desiccant was filtered off, the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (SNAP Cartridge HP-Sphere, mobile phase: CHCl ₃ / MeOH = 99/1 to 90/10; v / v) to give the title compound (4.55 g, brown amorphous) Got.
MS (ESI pos.) M / z: 510 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.35 (9 H, s), 2.01-2.08 (2 H, m), 2.48 (4 H, br.s.), 2.55 (2 H, t, J = 7.0 Hz), 3.73 (4 H, t, J = 4.5 Hz), 3.83 (3 H, s), 4.19 (2 H, t, J = 6.4 Hz), 4.52 (2 H, s), 5.45 (1 H, s), 7.02-7.05 (1 H, m), 7.23 (1 H, d, J = 7.4 Hz), 7.25-7.26 (1 H, m), 7.37 (1 H, t, J = 7.8 Hz), 7.95 (1 H, d, J = 3.3 Hz), 8.72 (1 H, d, J = 3.3 Hz).

  Example A-2: N-tert-butyl-2- [2- (3-hydroxyphenyl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] Synthesis of pyrimidine-3 (4H) -yl] acetamide

Compound A-1 (1.00 g) obtained in Example A-1 (4) was dissolved in CHCl ₃ (20 mL), and BBr ₃ (1 mmol / L dichloromethane solution, 9.81 mL) was added dropwise under ice cooling. And stirred at room temperature for 24 hours. MeOH was added to the reaction solution under ice-cooling, CHCl ₃ (80 mL) and saturated aqueous NaHCO ₃ solution (100 mL) were added, and the mixture was separated, and washed with saturated brine (100 mL). The aqueous layer was extracted twice with CHCl ₃ (100 mL), and the organic layers were combined and dried over Na ₂ SO ₄ . After the desiccant was filtered off, the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (SNAP Cartridge KP-NH, mobile phase: CHCl ₃ / MeOH = 100/0 to 90/10; v / v). EtOAc (1.5 mL) and n-hexane (1.5 mL) were added to the obtained crude product, and the mixture was heated to reflux for 10 minutes, cooled to room temperature, and stirred for 12 hours. The solid was collected by filtration and dried to give the title compound (400 mg, light brown solid).
MS (ESI pos.) M / z: 496 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.32 (9 H, s), 2.01-2.07 (2 H, m), 2.42-2.51 (4 H, m), 2.54 (2 H, t , J = 6.8 Hz), 3.73 (4 H, t, J = 4.3 Hz), 4.15-4.20 (2 H, m), 4.51 (2 H, s), 5.48 (1 H, s), 6.89-6.93 ( 1 H, m), 7.06 (1 H, d, J = 7.4 Hz), 7.12-7.15 (1 H, m), 7.24 (1 H, t, J = 7.8 Hz), 7.93-7.97 (1 H, m ), 8.39 (1 H, br.s.), 8.66 (1 H, d, J = 3.3 Hz).

  Example Aa-1: N-tert-butyl-2- [2- (3-methoxyphenyl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] Synthesis of pyrimidine-3 (4H) -yl] acetamide (A-1)

A suspension of compound A-2 (30 mg) obtained in Example A-2, methyl 4-nitrobenzenesulfonate (13.1 mg) and cesium carbonate (30 mg) in DMF (600 μL) was stirred at room temperature for 16 hours. The insoluble material was filtered off and purified by reverse phase silica gel column chromatography (mobile phase: 0.1% TFA MeCN / H ₂ O = 10/90 to 90/10; v / v). The fraction was neutralized with saturated aqueous NaHCO ₃ solution, extracted with CHCl ₃ , dried over Na ₂ SO ₄ , the desiccant was filtered off, and the solvent was evaporated under reduced pressure to give the title compound (6.4 mg, pale brown Amorphous) was obtained.
MS (ESI pos.) M / z: 510 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.35 (9 H, s), 2.02-2.08 (2 H, m), 2.48 (4 H, br.s.), 2.53-2.58 (2 H, m), 3.72-3.76 (4 H, m), 3.83 (3 H, s), 4.19 (2 H, t, J = 6.4 Hz), 4.52 (2 H, s), 5.43 (1 H, s ), 7.03-7.06 (1 H, m), 7.22-7.24 (1 H, m), 7.25-7.26 (1 H, m), 7.37 (1 H, t, J = 8.1 Hz), 7.95 (1 H, d, J = 3.3 Hz), 8.72 (1 H, d, J = 3.3 Hz).

Example A-3: N-tert-butyl-2- [2- {3-[( ³ H ₃ ) methyloxy] phenyl} -6- [3- (morpholin-4-yl) propoxy] -4- Synthesis of Oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] acetamide (A-3)

In the same manner as in Example Aa-1, the title compound was synthesized from compound A-2 using 4-nitrobenzenesulfonic acid ( ³ H ₃ ) methyl (synthesis quantity 347 MBq, specific activity 3.17 TBq / mmol). 37MBq / mL ethanol solution was prepared.
Radio-HPLC Retention time: 9.36min
MS (ESI pos.) M / z: 516 ([M + H] ⁺ ).

  Example B-1 N-tert-butyl-2- [2- (6-hydroxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2, Synthesis of 3-d] pyrimidin-3 (4H) -yl] acetamide (B-1)

(1) 2- [6-Bromo-2- (6-methoxypyridin-2-yl) -4-oxo-1,4-dihydropyrido [2,3-d] pyrimidin-3 (2H) -yl] -N Synthesis of tert-butylacetamide (B-1-1)

  In the same manner as in Example A-1 (1), the title compound (8.28 g) was obtained from 6-methoxypyridine-2-carbaldehyde (5.0 g).

(2) 2- [6-Bromo-2- (6-methoxypyridin-2-yl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] -N-tert-butylacetamide ( Synthesis of B-1-2)

In the same manner as in Example A-1 (2), the title compound (6.86 g, colorless solid) was obtained from compound B-1-1 (8.28 g) obtained in Example B-1 (1). Obtained.
MS (ESI pos.) M / z: 446 ([M + H] ⁺ ).

(3) N-tert-butyl-2- [6-hydroxy-2- (6-methoxypyridin-2-yl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] acetamide ( Synthesis of B-1-3)

In the same manner as in Example A-1 (3), the title compound (3.85 g, gray solid) was obtained from compound B-1-2 (6.22 g) obtained in Example B-1 (2). Obtained.
MS (ESI pos.) M / z: 384 ([M + H] ⁺ ).

(4) N-tert-butyl-2- [2- (6-methoxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] Synthesis of pyrimidine-3 (4H) -yl] acetamide (B-1-4)

In the same manner as in Example A-1 (4), the title compound (1.31 g, yellow amorphous) was obtained from compound B-1-3 (1.80 g) obtained in Example B-1 (3). Obtained.
MS (ESI pos.) M / z: 511 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.24 (9 H, s), 2.01-2.09 (2 H, m), 2.48 (4 H, br.s.), 2.55 (2 H, t, J = 7.0 Hz), 3.70-3.77 (4 H, m), 3.94 (3 H, s), 4.20 (2 H, t, J = 6.2 Hz), 5.08 (2 H, s), 5.39 (1 H, s), 6.87 (1 H, d, J = 8.3 Hz), 7.67-7.71 (1 H, m), 7.72-7.77 (1 H, m), 7.96 (1 H, d, J = 3.3 Hz) , 8.73 (1 H, d, J = 3.3 Hz).

(5) N-tert-butyl-2- [2- (6-hydroxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] Synthesis of pyrimidine-3 (4H) -yl] acetamide (B-1)

Compound B-1-4 (700 mg) obtained in Example B-1 (4) and sodium iodide (1.44 g) were suspended in acetonitrile (20 mL), and chlorotrimethylsilane (1.21 mL) was added. . After stirring at room temperature for 15 minutes, the mixture was stirred at an external temperature of 85 ° C. for 1 hour. The reaction mixture was allowed to cool, water (20 mL) was added under ice cooling, saturated aqueous NaHCO ₃ solution (100 mL) and CHCl ₃ (100 mL) were added, and the mixture was separated, and washed with saturated brine (100 mL). The aqueous layer was extracted three times with CHCl ₃ (100 mL), the organic layers were combined and dried over Na ₂ SO ₄ , the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (SNAP Cartridge HP-Sphere, mobile phase: CHCl ₃ / MeOH = 98/2 to 85/15; v / v). EtOAc (6 mL) and n-hexane (3 mL) were added to the resulting crude product, and the precipitate was collected by filtration and dried to give the title compound (290 mg, light brown amorphous).
MS (ESI pos.) M / z: 497 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.41 (9 H, s), 1.99-2.09 (2 H, m), 2.48 (4 H, br.s.), 2.55 (2 H, t, J = 6.8 Hz), 3.69-3.76 (4 H, m), 4.20 (2 H, t, J = 6.2 Hz), 4.61 (2 H, s), 6.09 (1 H, s), 6.73 (1 H, d, J = 8.3 Hz), 6.96 (1 H, d, J = 6.6 Hz), 7.48-7.54 (1 H, m), 7.87-7.93 (1 H, m), 8.74 (1 H, d, J = 3.3 Hz), 10.54-11.09 (1 H, m).

  Reference Example Ba-1: N-tert-butyl-2- [2- (6-methoxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2, Synthesis of 3-d] pyrimidin-3 (4H) -yl] acetamide (B-1-4)

A suspension of compound B-1 obtained in Example B-1 (30 mg), methyl 4-nitrobenzenesulfonate (13.1 mg) and cesium carbonate (30 mg) in DMF (600 μL) was stirred at room temperature for 16 hours. The insoluble material was filtered off and purified by reverse phase silica gel column chromatography (mobile phase: 0.1% TFA MeCN / H ₂ O = 10/90 to 90/10; v / v). The fraction was neutralized with saturated aqueous NaHCO ₃ solution, extracted with CHCl ₃ , dried over Na ₂ SO ₄ , the desiccant was filtered off, and the solvent was evaporated under reduced pressure to give the title compound (11 mg, pale brown amorphous). Got.
MS (ESI pos.) M / z: 511 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.24 (9 H, s), 2.02-2.09 (2 H, m), 2.48 (4 H, br.s.), 2.53-2.58 (2 H, m), 3.71-3.76 (4 H, m), 3.94 (3 H, s), 4.20 (2 H, t, J = 6.4 Hz), 5.08 (2 H, s), 5.39 (1 H, s ), 6.88 (1 H, dd, J = 8.3, 0.8 Hz), 7.69 (1 H, dd, J = 7.4, 0.8 Hz), 7.75 (1 H, dd, J = 8.3, 7.4 Hz), 7.96 (1 H, d, J = 2.9 Hz), 8.73 (1 H, d, J = 3.3 Hz).

Example B-2: N-tert-butyl-2- [2- {6-[( ³ H ₃ ) methyloxy] pyridin-2-yl} -6- [3- (morpholin-4-yl) propoxy ] -4-Oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] acetamide (B-2)

In the same manner as in Reference Example Ba-1, the title compound was synthesized from compound B-1 using methyl 4-nitrobenzenesulfonate ( ³ H ₃ ) (synthesis amount 235 MBq, specific activity 3.15 TBq / mmol). ) A 11.5 MBq / mL ethanol solution was prepared.
Radio-HPLC Retention time: 15.81min
MS (ESI pos.) M / z: 517 ([M + H] ⁺ ).

  Example C-1: 2- [2- (6-Hydroxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] pyrimidine- Synthesis of 3 (4H) -yl] -N-isopropylacetamide (C-1)

(1) 2- [6-Bromo-2- (6-methoxypyridin-2-yl) -4-oxo-1,4-dihydropyrido [2,3-d] pyrimidin-3 (2H) -yl] -N -Synthesis of isopropylacetamide (C-1-1)

  In the same manner as in Example A-1 (1), 2-amino-5-bromo-N- {2-[(1-methylethyl) amino] -2-oxoethyl} pyridine-3-carboxamide (31. 0g) and 6-methoxypyridine-2-carbaldehyde (40.5g) gave the title compound (39.4g, light brown solid).

(2) 2- [6-Bromo-2- (6-methoxypyridin-2-yl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] -N-isopropylacetamide (C- 1-2) Synthesis

In the same manner as in Example A-1 (2), the title compound (31.4 g, colorless solid) was obtained from compound C-1-1 (39.0 g) obtained in Example C-1 (1). Obtained.
MS (ESI pos.) M / z: 432 ([M + H] ⁺ ).

(3) 2- [6-Hydroxy-2- (6-methoxypyridin-2-yl) -4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] -N-isopropylacetamide (C- Synthesis of 1-3)

In the same manner as in Example A-1 (3), the title compound (31.3 g, brown solid) was obtained from compound C-1-2 (30.0 g) obtained in Example C-1 (2). Obtained.
MS (ESI pos.) M / z: 370 ([M + H] ⁺ ).

(4) N-isopropyl-2- [2- (6-methoxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] pyrimidine- Synthesis of 3 (4H) -yl] acetamide (C-1-4)

In the same manner as in Example A-1 (4), the title compound (1.73 g, pale brown solid) was obtained from compound C-1-3 (3.70 g) obtained in Example C-1 (3). Got.
MS (ESI pos.) M / z: 497 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.09 (6 H, d, J = 6.6 Hz), 2.00-2.10 (2 H, m), 2.43-2.51 (4 H, m), 2.55 (2 H, t, J = 7.2 Hz), 3.70-3.77 (4 H, m), 3.93 (3 H, s), 3.95-4.04 (1 H, m), 4.20 (2 H, t, J = 6.4 Hz), 5.06 (2 H, s), 5.45 (1 H, d, J = 7.8 Hz), 6.88 (1 H, d, J = 8.3 Hz), 7.70 (1 H, d, J = 7.4 Hz), 7.73-7.77 (1 H, m), 7.97 (1 H, d, J = 3.3 Hz), 8.74 (1 H, d, J = 2.9 Hz).

(4) 2- [2- (6-Hydroxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3-d] pyrimidine-3 (4H) -Ill] -N-isopropylacetamide (C-1) synthesis

The title compound (600 mg, pale yellow solid) was obtained using the compound C-1-4 (700 mg) obtained in Example C-1 (4) in the same manner as in Example B-1 (5). It was.
MS (ESI pos.) M / z: 483 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.23 (6 H, d, J = 6.2 Hz), 2.03-2.11 (2 H, m), 2.44-2.61 (6 H, m), 3.74 (4 H, br. S.), 4.16-4.23 (3 H, m), 4.63 (2 H, s), 6.08-6.13 (1 H, m), 6.74 (1 H, d, J = 8.7 Hz) , 6.99 (1 H, d, J = 6.6 Hz), 7.52 (1 H, dd, J = 9.1, 7.0 Hz), 7.86-7.94 (1 H, m), 8.72-8.81 (1 H, m), 10.69 -11.12 (1 H, m).

  Reference Example Ca-1: N-isopropyl-2- [2- (6-methoxypyridin-2-yl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2,3- d] Synthesis of pyrimidine-3 (4H) -yl] acetamide (C-1-4)

A suspension of compound C-1 (30 mg), methyl 4-nitrobenzenesulfonate (13.5 mg) and cesium carbonate (30 mg) obtained in Example C-1 in DMF (600 μL) was stirred at room temperature for 16 hours. The insoluble material was filtered off and purified by reverse phase silica gel column chromatography (mobile phase: 0.1% TFA MeCN / H ₂ O = 10/90 to 90/10; v / v). The fraction was neutralized with saturated aqueous NaHCO ₃ solution, extracted with CHCl ₃ , dried over Na ₂ SO ₄ , the desiccant was filtered off, and the solvent was evaporated under reduced pressure to give the title compound (11 mg, light brown solid) Got.
MS (ESI pos.) M / z: 497 ([M + H] ⁺ ).
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm); 1.09 (6 H, d, J = 6.6 Hz), 2.00-2.11 (2 H, m), 2.48 (4 H, br.s.), 2.55 (2 H, t, J = 7.0 Hz), 3.71-3.76 (4 H, m), 3.93 (3 H, s), 3.94-4.04 (1 H, m), 4.20 (2 H, t, J = 6.2 Hz), 5.06 (2 H, s), 5.49 (1 H, d, J = 7.4 Hz), 6.88 (1 H, dd, J = 8.3, 0.8 Hz), 7.70 (1 H, d, J = 7.0 Hz), 7.73-7.78 (1 H, m), 7.93-7.99 (1 H, m), 8.74 (1 H, d, J = 2.9 Hz).

Example C-2: N-isopropyl-2- [2- {6-[( ³ H ₃ ) methyloxy] pyridin-2-yl} -6- [3- (morpholin-4-yl) propoxy]- Synthesis of 4-oxopyrido [2,3-d] pyrimidin-3 (4H) -yl] acetamide (C-2)

In the same manner as in Reference Example Ca-1, the title compound was synthesized from Compound C-1 using 4-nitrobenzenesulfonic acid ( ³ H ₃ ) methyl (synthesis quantity 357 MBq, specific activity 3.16 TBq / mmol). ), 37.0 MBq / mL ethanol solution was prepared.
Radio-HPLC Retention time: 15.07min
MS (ESI pos.) M / z: 503 ([M + H] ⁺ ).

Test example 1
-V1b receptor binding test 293FT cells in which human V1b receptor was transiently expressed were collected, and 15 mmol / L Tris-HCl buffer (pH 7.4, 2 mmol / L magnesium chloride, 0.3 mmol / L ethylenediaminetetraacetic acid). In 1 mmol / L glycol ether diamine tetraacetic acid). The obtained homogenate was centrifuged at 50,000 × g for 20 minutes at 4 ° C., and the precipitate was added to 75 mmol / L Tris-HCl buffer (pH 7.4, 12.5 mmol / L magnesium chloride, 0.3 mmol / L ethylenediamine tetrachloride). Acetic acid, 1 mmol / L glycol ether diamine tetraacetic acid, 250 mmol / L sucrose included) were resuspended to prepare a crude film sample, and stored at −80 ° C. until the binding test was performed. In the binding test, the crude membrane preparation was diluted with 50 mmol / L Tris-HCl buffer (pH 7.4, 10 mmol / L magnesium chloride, containing 0.1% bovine serum albumin), and each test compound, And [ ³ H] AVP (final concentration 0.4-1 nmol / L) and incubated for 60 minutes at room temperature. The test compound is diluted stepwise with DMSO, and the final concentration of the test compound at the time of mixing is 0.01 nmol / L to 1 μmol / L. After the incubation, the mixed solution was suction filtered through a GF / C filter infiltrated with 0.3% polyethyleneimine. After drying this GF / C filter and adding a scintillator, the radioactivity remaining on the filter was measured using a top count (Perkin Elmer). The radioactivity in the presence of 10 μmol / L unlabeled AVP was 0%, and the radioactivity in the absence of unlabeled AVP was 100%. A dose response curve was prepared from the radioactivity in the presence of each concentration of the test compound, and the 50% inhibitory concentration (IC ₅₀ value) of the test compound was calculated. The IC ₅₀ values of compounds A-1, B-1-4 and C-1-4 are shown in Table 1.

Test example 2
Rat anterior pituitary membrane binding test (Compound A-3 binding test)
The rat anterior pituitary gland was excised and 10 mmol / L HEPES buffer (pH 7.4, 1 mmol / L ethylenediaminetetraacetic acid, 250 mmol / L sucrose, 0.1 mmol / L fluorinated phenylmethane) in a wet weight. (Including sulfonyl and protease inhibitors). The obtained homogenate was centrifuged at 1,000 × g and 4 ° C. for 10 minutes, and the obtained supernatant was further centrifuged at 50,000 × g and 4 ° C. for 20 minutes. The obtained precipitate was suspended in a 10 mmol / L HEPES buffer and stored at −80 ° C. as a crude membrane preparation until the binding test was performed. In the binding test, this crude membrane preparation was diluted with 50 mmol / L Tris-HCl buffer (pH 7.4, 10 mmol / L magnesium chloride, 0.1% bovine serum albumin contained), and compound A-3 ( (Final concentration 0.17 to 9.6 nmol / L) and incubated for 60 minutes at room temperature. After incubation, the mixed solution was suction filtered into a GF / C filter infiltrated with 0.3% polyethyleneimine and washed with refrigerated 50 mmol / L Tris-HCl buffer (pH 7.4, containing 10 mmol / L magnesium chloride). did. After drying the GF / C filter and adding a scintillator, the radioactivity remaining on the filter was measured using a top count. Radioactivity in the presence of 10 μmol / L AVP was defined as non-specific binding, and the difference from radioactivity in the absence of AVP (total binding) was defined as specific binding. The binding dissociation constant was calculated from the binding saturation curve in which the relationship between the final radioactivity concentration and the specific binding during the binding test of Compound A-3 was plotted. The results are shown in FIG.

  As shown in FIG. 1, specific binding increased in a concentration-dependent manner on the low concentration side of Compound A-3, and specific binding was saturated on the high concentration side. The bond dissociation constant was 0.41 nmol / L.

Test example 3
In vivo rat anterior pituitary binding test (Compound A-3 binding test)
Rats were administered Compound A-3 (0.9 nmol / kg, 3.0 MBq / kg) via the tail vein, and 30 minutes after administration, the anterior pituitary gland and eyeballs were removed. The extracted tissue is homogenized in 1 mL of ice-cold Tris-HCl buffer (pH 7.4, containing 10 mmol / L magnesium chloride), and rapidly sucked into a GF / B filter infiltrated with 0.3% polyethyleneimine. Filter and wash with ice-cold Tris-HCl buffer. After drying this GF / B filter and adding a scintillator, the radioactivity was measured using a liquid scintillation counter. The radioactivity concentration was corrected by the protein concentration in the filtered tissue homogenate. The results are shown in FIG.

  As a blocking test, 2- [2- (3-chloro-4-fluorophenyl) -6- [3- (morpholin-4-yl) propoxy] -4-oxopyrido [2, which has binding affinity for the V1b receptor 3-d] pyrimidin-3 (4H) -yl] -N-isopropylacetamide (Example A-250 described in WO2009 / 017236) was administered into the tail vein at 1 mg / kg, and after 5 minutes compound A-3 Was administered via the tail vein and the same operation as described above was performed. The results are shown in FIG.

  As shown in FIG. 2, an increase in the radioactivity concentration was observed in the anterior pituitary gland in which V1b receptor was highly expressed, and no radioactivity concentration was observed in the eyeball without V1b receptor expression (no blocking). . In the blocking test, the increase in radioactivity concentration in the anterior pituitary gland was completely suppressed.

The non-labeled substance of the present invention has a binding inhibitory activity specific to the V1b receptor, and is useful as a tool for pharmacological, pharmacokinetic and safety studies. In addition, the [ ³ H] -labeled substance is stable without being metabolized after being administered in vivo, and a sufficient amount of transfer to the pituitary gland, which is the target organ, has been observed. Pharmacology, pharmacokinetics and safety Useful as a tracer for research. Further, the production method of the present invention enables efficient supply of the ^[3 H] label, precursors for the ^[3 H] label synthesis may enable such efficient production method Is useful.

Claims (13)

Formula (I)

(Where
X represents a nitrogen atom or the formula CH;
R ¹ represents C _1-5 alkyl (wherein the C _1-5 alkyl is the same or different 1 to 3 substituents selected from hydroxy, halogen atom, cyano, C _3-7 cycloalkyl, and C _1-5 alkoxy) May be substituted with a group),
R ² represents C _1-5 alkyl (wherein the C _1-5 alkyl is at least one hydrogen atom is ³ H). )
Or a pharmaceutically acceptable salt thereof. In the above formula (I),
R ¹ is A compound or a pharmaceutically acceptable salt thereof according to claim 1 is a C _1-5 alkyl. In the above formula (I),
The compound according to claim 1 or 2 , or a pharmaceutically acceptable salt thereof, wherein R ² is methyl (wherein the methyl is at least one hydrogen atom is ³ H). In the above formula (I),
R ¹ is A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 is tert- butyl. In the above formula (I),
X is the formula CH;
R ¹ is A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 is tert- butyl. A V1b receptor labeling agent comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof. Formula (I)

(Where
X represents a nitrogen atom or the formula CH;
R ¹ Is C _1-5 Alkyl (the C _1-5 Alkyl is hydroxy, halogen atom, cyano, C _3-7 Cycloalkyl, and C _1-5 May be substituted with the same or different 1 to 3 substituents selected from alkoxy),
R ² Represents a hydrogen atom. )
Or a pharmaceutically acceptable salt thereof. In the above formula (I),
The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein R ¹ is C _1-5 alkyl . Formula (I)

(Where
X represents a nitrogen atom or the formula CH;
R ¹ represents C _1-5 alkyl (wherein the C _1-5 alkyl is the same or different 1 to 3 substituents selected from hydroxy, halogen atom, cyano, C _3-7 cycloalkyl, and C _1-5 alkoxy) May be substituted with a group),
R ² represents C _1-5 alkyl (wherein the C _1-5 alkyl is at least one hydrogen atom is ³ H). )
A compound represented by or a method for producing a pharmaceutically acceptable salt thereof, Formula (III):

(Wherein R ¹ and X are as defined above) and the formula R ² -L ¹ (R ² is as defined above and L ¹ is a leaving group). And a compound represented by formula (1) in the presence of a base and a solvent. The method according to claim 10 , wherein L ¹ is a 4-nitrobenzenesulfonyloxy group, a p-toluenesulfonyloxy group, a methanesulfonyloxy group, or a halogen atom. The method according to claim 10 or 11 , wherein the base is potassium carbonate, cesium carbonate, triethylamine, or diisopropylethylamine. The method according to any one of claims 10 to 12 , wherein the solvent is tetrahydrofuran, acetonitrile, N, N-dimethylformamide, or dimethyl sulfoxide.