Nitrogen-containing cyclic compound having no asymmetric carbon or salt thereof

Abstract

The present invention provides a novel nitrogen-containing cyclic compound which can be synthesized at low cost and has high safety, or a salt of the nitrogen-containing cyclic compound. The nitrogen-containing cyclic compound is represented by general formula (I), general formula (II), or general formula (III). R1 represents an alkyl group having 1 to 10 carbon atoms, R2 represents a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, or a monocyclic, fused cyclic, or polycyclic aryl group having one or two 5-membered or 6-membered aromatic rings, and each R3 independently represents an alkyl group having 1 to 10 carbon atoms.

Description

Nitrogen-containing cyclic compounds or salts thereof that lack an asymmetric carbon atom

[0001] This invention relates to novel nitrogen-containing cyclic compounds or salts thereof, and more particularly to nitrogen-containing cyclic compounds or salts thereof that can be used as research reagents and pharmaceuticals.

[0002] Nitrogen-containing cyclic compounds are considered important compounds in fields such as pharmaceuticals and agrochemicals, and are widely used in various applications such as pesticides and plant disease control agents.

[0003] Examples of nitrogen-containing cyclic compounds include pyrazoline derivatives. Because pyrazoline derivatives have high utility in a wide range of applications, many researchers are working on their novel synthesis. For example, in the pharmaceutical field, certain pyrazoline derivatives have been shown to have anticonvulsant effects (see Non-Patent Literature 1).

[0004] Furthermore, for example, 2,3-diaryl-pyrazoline derivatives, one type of pyrazoline derivative, have been shown to have inhibitory activity against enzymes that degrade the neuropeptide neurotensin (see Patent Document 1).

[0005] Furthermore, in the field of medicine, hypoxia-inducible factor (HIF) activators (hereinafter referred to as HIF activators) were discovered by Dr. William Kellin, who received the 2019 Nobel Prize in Physiology or Medicine, and are attracting attention due to their useful properties.

[0006] Hypoxia-inducible factor (HIF) is a protein that is expressed when cells and tissues are in a hypoxic state, and it promotes angiogenesis to alleviate the hypoxic condition. Furthermore, HIF is a "master transcription factor" that promotes the transcription of many genes, including erythrocyte production hormone and vascular endothelial growth factor, and plays an important role in resolving hypoxia.

[0007] However, hypoxia-inducible factor (HIF) is hydroxylated by the iron atom in prolyl hydroxylase (PHD), followed by ubiquitination by the von Hippel-Lindau (VHL) protein, and then degraded by the proteasome. In other words, under normal conditions, hypoxia-inducible factor (HIF) is constantly degraded and kept at a low level, thus being negatively regulated.

[0008] Therefore, it is believed that inhibiting prolyl hydroxylase (PHD) with small molecule reagents can suppress the proteasomal degradation of hypoxia-inducible factor (HIF), thereby increasing the tissue's resistance to hypoxic conditions. Long-term suffering from lifestyle-related diseases such as diabetes, hypertension, and arteriosclerosis can lead to diseases associated with hypoxia in peripheral tissues, i.e., ischemia.

[0009] The use of HIF activators is being considered as a treatment for such ischemia-related diseases. In particular, while blood transfusions and intravenous injections of erythropoietin protein are indicated for the treatment of chronic kidney disease, these have an impact on patients' financial burden and a decline in quality of life due to increased hospital visits, so there is a need for less burdensome oral medications.

[0010] In fact, several HIF activators have been marketed as oral treatments for chronic kidney disease. In September 2019, roxadustat (FG4592), developed by Astellas Pharma / FibroGen (see Non-Patent Document 2), received manufacturing and marketing approval in Japan. Subsequently, vadadustat (MT-6548), developed by Tanabe Mitsubishi Pharma / Akebia (see Patent Document 2), enarodustat (JTZ-951), developed by Japan Tobacco / Torii Pharmaceutical (see Non-Patent Document 3), and daprodustat (GSK-1278863), developed by GlaxoSmithKline (see Patent Document 3) were also marketed.

[0011] Thus, conventional nitrogen-containing cyclic compounds, as shown in, for example, the above-mentioned Patent Documents 2 and 3 and Non-Patent Documents 2 and 3, are used as HIF activators for the treatment of chronic kidney disease. Their main component is a compound having a 2-oxoglutarate (2-OG) skeleton (2-OG analog).

[0012] This 2-OG analog compound acts as a cofactor by coordinating to the active site of prolyl hydroxylase (PHD), and its competitive inhibition inactivates prolyl hydroxylase (PHD), thereby preventing the degradation of hypoxia-inducible factor (HIF).

[0013] However, because the synthesis of 2-OG analogs is complex, conventional HIF activators that primarily contain such 2-OG analogs are expensive drugs.

[0014] Thus, while pyrazoline derivatives, which are nitrogen-containing cyclic compounds, are useful in a wide range of fields, including pharmaceuticals and agrochemicals, their complex synthesis reactions make them expensive compounds, and they cannot be said to be fully utilized in various applications, such as pharmaceuticals in the pharmaceutical field.

[0015] Furthermore, since 2-oxoglutaric acid (2-OG) is a cofactor for more than 60 types of proteins in the body, conventional HIF activators that primarily contain 2-OG-like compounds may activate or inhibit proteins other than prolyl hydroxylase (PHD), potentially leading to side effects.

[0016] In this context, the present inventors have invented a novel nitrogen-containing cyclic compound that, unlike the prior art described above, does not have a 2-oxoglutaric acid (2-OG) skeleton, can be synthesized at low cost, and can also be used as an HIF activator (see Patent Document 4).

[0017] International Publication WO2003 / 078400, U.S. Patent Publication No. 20040254215, International Publication WO2007 / 150011, Japanese Patent Publication No. 2022-066061

[0018] Ogoshi, Y. et al. ACS Med. Chem. Lett. 2017, 8 (12), 1320

[0019] However, the conventional nitrogen-containing cyclic compounds described in Patent Document 4 are asymmetric carbon-containing compounds, in which a structure with an asymmetric carbon is an essential component. Asymmetric carbon-containing compounds generally exhibit completely different properties in their optical isomers (R-form and S-form), and it is extremely difficult to distinguish, separate, and purify each of them.

[0020] For example, regarding the safety risks associated with optical isomers, thalidomide, a drug used to treat multiple myeloma and severe erythema nodosum leprosy, is well-known. The R-isomer of thalidomide does not cause limb deformities, while the S-isomer does. Because the optical isomers of thalidomide had different efficacy (Blaschke, G. et al. Arzneimitt. Forsch. 1979, 29, 1640-1642), the cause of limb deformities became a problem in the 1950s.

[0021] For these reasons, safety is a particularly important issue in pharmaceuticals. However, conventional nitrogen-containing cyclic compounds, such as those described in Patent Document 4, pose safety risks due to optical isomerism. Furthermore, nitrogen-containing cyclic compounds that simply lack optical isomers do not necessarily exhibit superior medicinal effects.

[0022] The present invention was made to solve the above problems and aims to provide a novel nitrogen-containing cyclic compound or a salt thereof that can be synthesized at low cost and has high safety. One of its applications is to provide an inexpensive and safe composition that exhibits HIF activation ability by inhibiting prolyl hydroxylase (PHD). For example, it aims to provide a safe composition that can be used as an oral treatment for chronic kidney disease.

[0023] The inventors of this invention diligently conducted research on novel compounds with high safety that could be applied to pharmaceuticals, and discovered a novel nitrogen-containing cyclic compound that does not contain optical isomers. Furthermore, they found that this compound can be prepared through an extremely simple reaction process and exhibits excellent medical effects, leading to the completion of the present invention.

[0024] Thus, according to the present invention, a nitrogen-containing cyclic compound without an asymmetric carbon, represented by the following general formula (I), general formula (II), or general formula (III), or a salt thereof, is provided.

[0025]

[0026] In the above general formulas (I), (II), and (III), R １ R is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms. ２ R is an aryl group having 1 to 2 aromatic rings, consisting of a hydrogen atom, a substituted or unsubstituted C1 to C10 alkyl group, or a substituted or unsubstituted 5-membered or 6-membered ring, in a monocyclic, fused, or polycyclic structure. ３ Each of these is independently a substituted or unsubstituted linear or cyclic alkyl group having 1 to 10 carbon atoms. １ They may be joined to each other to form a ring.

[0027] Furthermore, compositions containing nitrogen-containing cyclic compounds or salts thereof that do not have this chiral carbon are also provided.

[0028] The results of measuring the HIF transcriptional activity of compound 5a according to Example 1 of the present invention are shown.

[0029] A nitrogen cyclic compound or salt thereof without an asymmetric carbon according to an embodiment of the present invention is represented by the following general formula (I), general formula (II), or general formula (III).

[0030]

[0031] Here, in the above general formulas (I), (II), and (III), R １ R is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms. ２is a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, or an aryl group having a monocyclic structure, a condensed ring structure, or a polycyclic structure consisting of 1 to 2 aromatic rings of a substituted or unsubstituted 5-membered or 6-membered ring, R ３ are each independently a substituted or unsubstituted linear or cyclic alkyl group having 1 to 10 carbon atoms. Two Rs １ may be bonded to each other to form a ring.

[0032] R １ is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, preferably a lower alkyl group having 1 to 5 carbon atoms, more preferably a lower alkyl group having 1 to 3 carbon atoms, and for example, it can be a methyl group, an ethyl group or a propyl group. Two Rs １ may be bonded to each other's carbon atoms to form a ring, or may be bonded through other carbon atoms or the like to form a ring.

[0033] R ２ is a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, or an aryl group having a monocyclic structure, a condensed ring structure, or a polycyclic structure consisting of 1 to 2 aromatic rings of a substituted or unsubstituted 5-membered or 6-membered ring, preferably one or two phenyl groups optionally substituted with a lower alkyl group having 1 to 9 carbon atoms, or a lower alkyl group having 1 to 9 carbon atoms, and examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a dodecyl group, a propyl group, a butyl group, a pentyl group, a neopentyl group, an octyl group, a phenyl group, a phenylethyl group, etc. This lower alkyl group having 1 to 9 carbon atoms may be linear or branched.

[0034] R 3 are each independently a substituted or unsubstituted linear or cyclic alkyl group having 1 to 10 carbon atoms, more preferably a linear or cyclic alkyl group having 1 to 6 carbon atoms, and for example, it can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a cyclohexyl group.

[0035] The present inventors have succeeded in synthesizing the nitrogen-containing cyclic compounds or salts thereof according to the present invention, specifically pyrazolin derivatives or pyrazolidine derivatives as distinct nitrogen-containing cyclic compounds or salts thereof represented by the above general formulas (I), (II), or (III), respectively, thereby arriving at the present invention.

[0036] In other words, each of the above general formulas (I), (II), or (III) does not represent, for convenience, a tautomutation of the same compound (keto-enol tautomutation, enamine-imine tautomutation), but rather represents a nitrogen-containing cyclic compound or a salt thereof that has been synthesized separately as an independent pyrazoline derivative or pyrazolidine derivative.

[0037] The term "the salt" above refers to a salt that retains the biological and pharmaceutical effects of the free acid or free base of the nitrogen-containing cyclic compound represented by the above general formula (I), general formula (II), or general formula (III). For example, as a salt derived from a base, salts containing alkali metal or alkaline earth metal cations can be included, such as sodium salts, potassium salts, lithium salts, cesium salts, calcium salts, magnesium salts, etc. Due to ease of handling, the nitrogen-containing cyclic compound according to the present invention can be in salt form, more preferably salts containing alkaline earth metals, such as sodium salts, potassium salts, lithium salts, and cesium salts, and due to ease of handling, the sodium salt form is preferred.

[0038] The pyrazolidine derivative or salt thereof, which is a nitrogen-containing cyclic compound of the present invention represented by general formula (I), can be produced using a three-step reaction represented by the following reaction formula (A).

[0039]

[0040] The reaction steps (1) to (3) in these three stages will be explained in more detail below. The reaction steps for general formulas (II) and (III) are the same as described below.

[0041] (First stage) As shown in the following reaction equation (A-1), first, R is added to acrylic anhydride. 1 Compound 1, to which two R groups are attached, 3Compound 2, which has a pyrimidine skeleton containing the compound, is subjected to an alkanoylation (acylation) reaction by heating under a sulfuric acid catalyst to obtain compound 3. Known methods can be used for this alkanoylation (e.g., Holt-Martyn,JP et al. Chem. Med. Chem. 2020, 15 (3), 270-273).

[0042]

[0043] (Second step) As shown in the following reaction equation (A-2), hydrazine monohydrate is added to the obtained compound 3 and refluxed under ethanol to obtain compound 4 having a pyrazolidine skeleton. The reflux time is not particularly limited, but can be 1 hour to several hours, for example, 1 hour. Known methods can be used for this pyrazolidine formation reaction (e.g., Abdel Latif, N. et al. Bioorg. Chem. 2016, 67, 116.).

[0044]

[0045] (Third step) Next, as shown in the following reaction equation (A-3), R is added to the obtained compound 4. 2 Carbonate esters (acid anhydrides) containing R 2 The pyrazolidine derivative 5 according to the present invention can be obtained by heating in the presence of a carboxylic acid containing the specified compound to carry out an alkanoylation (acylation) reaction, and its production by this reaction has actually been confirmed (see the examples described below). The heating temperature is not particularly limited, but can be 100°C to 200°C, for example, 125°C. The heating time is not particularly limited, but can be 1 to 10 hours, for example, 2 hours. For this alkanoylation (acylation) reaction, known methods can be used.

[0046]

[0047] The nitrogen-containing cyclic compounds or salts thereof of the present invention can be obtained as pyrazoline derivatives or pyrazolidine derivatives or salts thereof, with respect to any of the general formulas (I), (II), and (III), by the extremely simple three-step reaction (1) to (3) described above.

[0048] The nitrogen-containing cyclic compounds obtained in this manner, represented by general formula (I), general formula (II), or general formula (III), can form salt forms that retain the biological and pharmaceutical effects of their free acid or free base. One example of such a salt can be represented by the following general formula (IV).

[0049]

[0050] In the above general formula (IV), M + This represents a cation of an alkali metal or alkaline earth metal.

[0051] Thus, the salts derived from the base can be, for example, sodium salts, potassium salts, lithium salts, cesium salts, calcium salts, magnesium salts, and so on. The nitrogen-containing cyclic compound according to the present invention can be in salt form, more preferably salts containing alkaline earth metals, such as sodium salts, potassium salts, lithium salts, and cesium salts, due to their ease of handling, and the sodium salt form is preferred due to its ease of handling.

[0052] The composition according to this embodiment contains the novel nitrogen-containing cyclic compound or a salt thereof described above, and can be used for a variety of applications.

[0053] For example, in the pharmaceutical field, if there is a safe composition that can be manufactured inexpensively and exhibits prolyl hydroxylase (PHD) inhibitory activity, it is thought that it would be possible to provide cheaper and safer HIF activators and reagents than conventional ones due to their hypoxia-inducible factor (HIF) activating effect. This has the potential to realize an excellent, inexpensive, and easy-to-use treatment for chronic kidney disease, but conventionally, no such excellent composition exists that ensures high safety.

[0054] In this regard, the present inventors have discovered that the above-mentioned composition according to the present invention not only ensures high safety, particularly in the pharmaceutical field, by eliminating the possibility of optical isomers, but also, surprisingly, exhibits excellent proline hydroxylase (PHD) activity inhibitory ability. This proline hydroxylase (PHD) activity inhibitory ability makes it possible to activate hypoxia-inducible factor (HIF), resulting in the realization of a new HIF activator with a highly safe basic structure.

[0055] In fact, the composition according to the present invention not only exhibits high safety due to the absence of optical isomers, but has also been confirmed to exhibit superior activity compared to conventional HIF activators in terms of hypoxia-inducible factor (HIF) activity (see the examples described below). In other words, the composition according to the present invention achieves both high safety and superior HIF activity, which were not possible with conventional HIF activators.

[0056] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0057] The analysis was performed using the following equipment. 1 H-NMR, 13 1C-NMR: Varian NMR 400 MHz system; Gel Permeation Chromatography (GPC): LaboACE LC-5060 manufactured by Nippon Analytical Engineering Co., Ltd.

[0058] (Example 1) Preparation of pyrazoline derivatives Preparation of 5-(1-acetyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (compound 5a) (General formula (A): R 1 =CH3, R 2 =CH3, R 3 =CH3)

[0059]

[0060] The following synthesis reaction was carried out according to the general formula (A) above.

[0061]

[0062] A 200 mL two-necked round-bottom flask was flame-dried under an argon atmosphere, then 50 mmol of DCC (manufactured by Tokyo Chemical Industry Co., Ltd.) and 100 mL of dichloromethane were added and the mixture was stirred. 100 mmol of 3,3-dimethylacrylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered three times with dichloromethane (the solvent was removed by distillation after each filtration), and the filtrate was collected and vacuum-dried to obtain acrylic anhydride in 76% yield.

[0063] A 10 mL two-necked round-bottom flask fitted with a reflux condenser was flame-dried under an argon atmosphere. Then, 3 mL of the previously synthesized acrylic anhydride, 10 mmol of 1,3-dimethylbarbituric acid (manufactured by Tokyo Chemical Industry Co., Ltd.), and 10 drops of concentrated sulfuric acid were added, and the mixture was stirred at 150°C for 1 hour. Compound 3a was obtained in 57% yield by suction filtration with an acetone:water = 1:1 solution, followed by washing with hot water, and finally vacuum drying of the filtrate.

[0064] A 200 mL two-necked round-bottom flask, fitted with a reflux tubing, was flame-dried under an argon atmosphere. Then, 4 mmol of the previously synthesized compound 3a, 2 equivalents of hydrazine hydrate (Nacalai Tesque), and ethanol were added, and the mixture was refluxed for 1 hour. After the reaction was complete, the mixture was filtered with ethanol, and the filtrate was vacuum-dried to obtain compound 4a in 71% yield.

[0065] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.3 mmol of the previously synthesized compound 4a, 0.6 mL of acetic acid, and 0.6 mL of acetic anhydride were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was filtered with ethanol and vacuum-dried to obtain pure compound 5a, 5-(1-acetyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5a), as a white solid (yield 85%). The structure is 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(1-acetyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5a): white solid; 1H NMR (400 MHz, CDCl3) δ 1.63 (s, 6H), 2.23 (s, 3H), 3.33 (s, 6H), 3.71 (s, 2H); 13 C NMR (101 MHz, CDCl3) δ 21.4, 26.5, 27.6, 51.0, 61.6, 85.7, 151.2, 156.9, 161.9, 164.4.

[0066]

[0067] (Example 2) Preparation of 5-(1-isobutyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ab) (R 1 =CH3, R 2 = i Pr, R 3 =CH3)

[0068]

[0069] A 10 mL two-necked flask was flame-dried under an argon atmosphere. Then, 0.3 mmol of compound 4a, 0.6 mL of isobutyric acid (Tokyo Chemical Industries), and 0.6 mL of isobutyric anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added, and the mixture was concentrated using an evaporator and vacuum-dried. The mixture was then filtered with ethanol and vacuum-dried to obtain pure compound 5ab, 5-(1-isobutyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ab), as a white solid (99% yield). The structure is... 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(1-isobutyryl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5ab): white solid; 1H NMR (400 MHz, CDCl3) δ 1.24 (d, J = 6.7 Hz, 6H), 1.66 (s, 6H), 2.77 (q, J = 6.4 Hz, 1H), 3.34 (d, J = 22.6 Hz, 6H), 3.71 (d, J = 70.4 Hz, 2H), 14.45 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 18.8, 26.7, 27.5, 27.7, 27.7, 32.3, 50.7, 61.8, 85.5, 151.2, 156.9, 161.8, 164.3, 171.4

[0070]

[0071] (Example 3) Preparation of 5-(1-hexanoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ac) (R 1 =CH3, R 2 = n Pen, R 3 =CH3)

[0072]

[0073] A 10 mL two-necked flask was flame-dried under an argon atmosphere. Then, 0.3 mmol of compound 4a, 0.6 mL of hexanoic acid (Tokyo Chemical Industries), and 0.6 mL of hexanoic anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added, and the mixture was concentrated using an evaporator and vacuum-dried. The mixture was then filtered over hexane and vacuum-dried to obtain pure compound 5ac, 5-(1-hexanoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ac), as a white solid (92% yield). The structure is... 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(1-hexanoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5ac): white solid; 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 6.7 Hz, 3H), 1.37 (t, J = 3.5 Hz, 4H), 1.64 (s, 6H), 1.73 (s, 2H), 2.44 (d, J = 7.5 Hz, 2H), 3.29 (s, 3H), 3.35 (s, 3H), 3.69 (s, 2H); 13 C NMR (101 MHz, CDCl3) δ 13.6, 22.2, 24.1, 26.5, 27.5, 27.7, 31.3, 33.9, 50.7, 62.0, 85.6, 151.2, 156.7, 161.8, 164.3, 167.0

[0074]

[0075] (Example 4) 5-(1-decanoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ad (R) 1 =CH3, R 2 = n Non 3 =CH3)

[0076]

[0077] A 10 mL two-necked flask was flame-dried under an argon atmosphere. Then, 0.3 mmol of compound 4a, 0.6 mL of decanoic acid (Tokyo Chemical Industries), and 0.6 mL of decanoic acid anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added, and the mixture was concentrated using an evaporator and vacuum-dried. Separation was then performed by gel permeation chromatography (GPC), and vacuum-drying yielded pure compound 5ad, specifically 5-(1-decanoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ad), as a white solid (yield 21%). The structure is... 1 1H NMR and 13TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(1-decanoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5ad): white solid; 1 H NMR (400 MHz, CDCl3) δ 0.86-0.89 (m, 3H), 1.28-1.40 (m, 12H), 1.63 (s, 6H), 1.68-1.75 (m, 2H), 2.42 (t, J = 7.4 Hz, 2H), 3.33 (d, J = 22.8 Hz, 6H), 3.68 (s, 2H); 13 C NMR (101 MHz, CDCl3) δ 13.8, 22.5, 24.6, 26.5, 27.5, 27.7, 29.1, 29.1, 29.2, 29.2, 31.7, 33.9, 50.8, 61.8, 85.6, 151.2, 156.9, 161.9, 164.4, 167.1.

[0078]

[0079] (Example 5) Preparation of 5-(1-benzoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ae) (R 1 =CH3, R 2 = Ph, R 3 =CH3)

[0080]

[0081] A 10 mL two-necked flask was flame-dried under an argon atmosphere. Then, 0.3 mmol of compound 4a, 4 mmol of benzoic acid (Tokyo Chemical Industries), and 2 mmol of benzoic anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added, and the mixture was concentrated using an evaporator and vacuum-dried. The mixture was then filtered with ethanol and vacuum-dried to obtain pure compound 5ae, 5-(1-benzoyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5ae), as a white solid (yield 87%). The structure is... 11H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(1-benzoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5ae): white solid; 1 H NMR (400 MHz, CDCl3) δ 1.59 (s, 6H), 3.30 (s, 6H), 3.69 (s, 2H), 7.45-7.56 (m, 5H), 13.51 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 26.3, 27.5, 27.7, 50.3, 63.1, 85.8, 127.5, 128.6, 131.2, 134.3, 151.2, 157.7, 161.9, 164.3, 165.5

[0082]

[0083] (Example 6) Preparation of 5-(5,5-dimethyl-1-(3-phenylpropanoyl)pyrazolidine-3-ylidene)-1,3-dimethylbarbituric acid (compound 5af) (R 1 =CH3, R 2 = EtPh, R 3 =CH3)

[0084]

[0085] A 200 mL two-necked round-bottom flask was flame-dried under an argon atmosphere. Then, 100 mmol of 3-phenylpropionic acid, 50 mmol of DCC, and 100 mL of dichloromethane were added, and the mixture was stirred at room temperature. After the reaction was complete, the mixture was filtered three times with dichloromethane (the solvent was removed by distillation after each filtration), and the filtrate was collected and vacuum-dried to obtain 3-phenylpropionic anhydride in 99% yield as a compound containing 3-phenylpropionic acid.

[0086] A 10 mL two-necked round-bottom flask was flame-dried under an argon atmosphere. Then, 0.3 mmol of compound 4a, 4 mmol of 3-phenylpropionic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.6 mL of previously synthesized 3-phenylpropionic anhydride were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added, and the mixture was concentrated using an evaporator and vacuum-dried. Subsequently, the mixture was filtered with ethanol and vacuum-dried to obtain pure compound 5af, 5-(5,5-dimethyl-1-(3-phenylpropanoyl)pyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5af), as a white solid (yield 74%). The structure is 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 5-(5,5-dimethyl-1-(3-phenylpropanoyl)pyrazolidin-3-ylidene)-1,3-dimethylbarbituric acid (5af): white solid; 1 H NMR (400 MHz, CDCl3)δ 1.57 (d, J = 30.4 Hz, 6H), 2.74 (s, 2H), 3.05 (s, 2H), 3.32 (d, J = 19.3 Hz, 6H), 3.53 (s, 1H), 3.78 (s, 1H), 7.25 (dt, J = 33.3, 7.3 Hz, 5H), 13.06 (s, 1H), 14.30 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 25.9, 27.7, 30.4, 31.1, 35.0, 36.6, 50.1, 51.5, 61.1, 62.7, 85.6, 85.7, 126.5, 128.5, 128.6, 140.2, 151.7, 156.3, 157.5, 162.4, 164.2, 165.4, 166.8

[0087]

[0088] (Example 7) Preparation of 5-(1-acetyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dicyclohexylbarbituric acid (compound 5b) (R 1=CH3, R 2 =CH3, R 3 = c Hex)

[0089]

[0090] A 50 mL two-necked round-bottom flask fitted with a reflux condenser was flame-dried under an argon atmosphere. Then, 9 mL of the previously synthesized acrylic anhydride, 6 mmol of 1,3-dimethylbarbituric acid (manufactured by Tokyo Chemical Industry Co., Ltd.), and 3 drops of concentrated sulfuric acid were added, and the mixture was stirred at 150°C for 1 hour. Compound 3b was obtained in 78% yield by suction filtration with an acetone:water = 1:1 solution, followed by washing with hot water, and vacuum drying of the filtrate.

[0091] A 200 mL two-necked round-bottom flask, fitted with a reflux tubing, was flame-dried under an argon atmosphere. Then, 4 mmol of the previously synthesized compound 3b, 2 equivalents of hydrazine hydrate (Nacalai Tesque), and ethanol were added, and the mixture was refluxed for 1 hour. After the reaction was complete, the solvent was removed by distillation, and the mixture was vacuum-dried. The mixture was then filtered with water, and the filtrate was vacuum-dried to obtain compound 4b in 71% yield.

[0092] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.13 mmol of the previously synthesized compound 4b, 0.26 mL of acetic acid, and 0.26 mL of acetic anhydride were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was separated by gel permeation chromatography (GPC). By vacuum drying, pure compound 5b, 5-(1-acetyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-dicyclohexylbarbituric acid (compound 5b), was obtained as a white solid (80% yield). The structure is 1 TMS was determined by 1H NMR, with each component set to 0 ppm. 5-(1-acetyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dicyclohexylbarbituric acid (5b): white solid; 1H NMR (400 MHz, CDCl3) δ 1.23-1.41 (m, 6H), 1.65 (d, J = 14.8 Hz, 12H), 1.83 (s, 4H), 2.22 (s, 3H), 2.38 (q, J = 12.3 Hz, 4H), 3.66 (s, 2H), 4.74 (td, J = 12.0, 3.4 Hz, 2H)

[0093]

[0094] (Example 8) Preparation of 1,3-dicyclohexyl-5-(5,5-dimethyl-1-propanoylpyrazolidine-3-ylidene)barbituric acid (compound 5ba) (R 1 =CH3, R 2 = Et, R 3 = c Hex)

[0095]

[0096] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.2 mmol of the previously synthesized compound 4b, 0.4 mL of propanoic acid (Tokyo Chemical Industries), and 0.4 mL of propanoic anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was filtered with hot water and vacuum-dried to obtain 1,3-dicyclohexyl-5-(5,5-dimethyl-1-propanoylpyrazolidine-3-ylidene)barbituric acid (compound 5ba) as a pure brown solid (yield 43%). The structure is 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 1,3-dicyclohexyl-5-(5,5-dimethyl-1-propionylpyrazolidin-3-ylidene)barbituric acid (5 ba): brown solid; 1H NMR (400 MHz, CDCl3) δ 1.14-1.36 (m, 9H), 1.64 (d, J = 23.4 Hz, 12H), 1.83 (s, 4H), 2.34-2.49 (m, 6H), 3.64 (s, 2H), 4.74 (t, J = 12.0 Hz, 2H); 13 C NMR (101 MHz, CDCl3) δ 8.5, 25.2, 25.3, 26.5, 29.1, 29.3, 51.2, 54.2, 55.0, 61.7, 86.2, 150.3, 157.2, 162.1, 164.6, 168.1.

[0097]

[0098] (Example 9) Preparation of 1,3-dicyclohexyl-5-(1-isobutyl-5,5-dimethylpyrazolidine-3-ylidene)barbituric acid (compound 5bb) (R 1 =CH3, R 2 = i Pr, R 3 = c Hex)

[0099]

[0100] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.2 mmol of the previously synthesized compound 4b, 0.4 mL of isobutyric acid (Tokyo Chemical Industries), and 0.4 mL of isobutyric anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was filtered with hot water and vacuum-dried to obtain 1,3-dicyclohexyl-5-(1-isobutyl-5,5-dimethylpyrazolidine-3-ylidene)barbituric acid (compound 5bb) as a pure brown solid (yield 37%). The structure is 1 1H NMR and 13 TMS was determined by 13C NMR, with each component set to 0 ppm. 1,3-dicyclohexyl-5-(1-isobutyryl-5,5-dimethylpyrazolidin-3-ylidene)barbituric acid (5bb): brown solid;1 1H NMR (400 MHz, CDCl3) δ 1.20 - 1.36 (m, 12H), 1.62 (s, 12H), 1.83 (s, 4H), 2.38 (q, J = 12.3 Hz, 4H), 2.76 - 2.83 (m, 1H), 3.64 (s, 2H), 4.71 - 4.77 (m, 2H), 14.89 - 13.34 (1H), 13 13C NMR (101 MHz, CDCl3) δ 18.6, 18.7, 25.2, 25.3, 26.5, 29.1, 29.3, 32.4, 33.3, 51.2, 54.2, 55.0, 61.7, 86.2, 150.3, 157.1, 162.1, 164.6, 171.7.

[0101]

[0102] (Example 10) Preparation of 5-(1-benzoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dicyclohexylbarbituric acid (5bc) (R 1 =CH3, R 2 =Ph, R 3 = c Hex)

[0103]

[0104] A 10 mL two-necked eggplant flask attached with a reflux condenser was flame-dried under an argon atmosphere, then 0.3 mmol of the previously synthesized compound 4b, 4 mmol of benzoic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), and 2 mmol of benzoic anhydride (manufactured by Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was stirred at 125 °C for 2 hours. After completion of the reaction, toluene was added and concentrated, and separated by gel permeation chromatography (GPC), and dried under vacuum to obtain 5-(1-benzoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dicyclohexylbarbituric acid (Compound 5bc) as a yellow oily substance (yield 48%). The structure was 1 1H NMR and 1313C NMR was used to determine TMS with reference to 0 ppm respectively. 5-(1-benzoyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-dicyclohexylbarbituric acid (5bc): yellow oil; 1 1H NMR (400 MHz, CDCl3) δ 1.15 - 1.36 (m, 6H), 1.60 (d, J = 17.7 Hz, 12H), 1.81 (d, J = 11.6 Hz, 4H), 2.34 (dt, J = 33.7, 11.6 Hz, 4H), 3.67 (s, 2H), 4.63 - 4.76 (m, 2H), 7.44 - 7.56 (m, 5H), 13.76 (s, 1H); 13 13C NMR (101 MHz, CDCl3) δ 25.3, 26.3, 26.4, 26.5, 29.2, 29.3, 50.9, 54.1, 54.2, 54.7, 54.8, 62.9, 86.4, 127.6, 128.5, 131.0, 134.7, 150.4, 157.8, 162.2, 164.5, 165.5.

[0105]

[0106] (Example 11) Preparation of 1,3-dicyclohexyl-5-(1-hexanoyl-5,5-dimethylpyrazolidin-3-ylidene)barbituric acid (5bd) (R 1 =CH3, R 2 = n Pen, R 3 = c Hex)

[0107]

[0108] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.2 mmol of the previously synthesized compound 4b, 0.4 mL of hexanoic acid (Tokyo Chemical Industries), and 0.4 mL of hexanoic anhydride (Tokyo Chemical Industries) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was separated by gel permeation chromatography (GPC). Vacuum drying was then performed to obtain 1,3-dicyclohexyl-5-(1-hexanoyl-5,5-dimethylpyrazolidine-3-ylidene)barbituric acid (5bd) as a pure compound 5bd in a yellow oily substance (yield 64%). The structure is 1 TMS was determined by 1H NMR, with each component set to 0 ppm. 1,3-dicyclohexyl-5-(1-hexanoyl-5,5-dimethylpyrazolidin-3-ylidene)barbituric acid (5bd): yellow oil; 1 H NMR (400 MHz, CDCl3) δ 0.91-0.94 (m, 4H), 1.22-1.38 (m, 12H), 1.62-1.72 (m, 14H), 1.83 (s, 4H), 2.34-2.44 (m, 6H), 3.64 (s, 2H), 4.74 (td, J = 12.0, 3.4 Hz, 2H)

[0109]

[0110] (Example 12) Preparation of 1,3-dicyclohexyl-5-(5,5-dimethyl-1-(3-phenylpropanoyl)pyrazolidine-3-ylidene) barbituric acid (5be) 1 =CH3, R 2 = EtPh, R 3 = c Hex)

[0111]

[0112] A 10 mL two-necked round-bottom flask attached to a reflux condenser was flame-dried under an argon atmosphere. Then, 0.3 mmol of the previously synthesized compound 4b, 4 mmol of 3-phenylpropionic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.6 mL of the previously synthesized 3-phenylpropionic anhydride were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was separated by gel permeation chromatography (GPC). Vacuum drying was performed to obtain 1,3-dicyclohexyl-5-(1-hexanoyl-5,5-dimethylpyrazolidine-3-ylidene)barbituric acid (5bd) as a pure compound 5bd in a yellow oily substance (yield 64%). The structure is 1 TMS was determined by 1H NMR, with each component set to 0 ppm. 1,3-dicyclohexyl-5-(5,5-dimethyl-1-(3-phenylpropanoyl)pyrazolidin-3-ylidene)barbituric acid (5bd): yellow oil; 1 H NMR (400 MHz, CDCl3) δ 1.27-1.39 (m, 6H), 1.61 (dd, J = 26.2, 12.0 Hz, 12H), 1.83 (s, 4H), 2.37 (d, J = 11.8 Hz, 4H), 2.74 (t, J = 7.4 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H), 3.59 (s, 2H), 4.71-4.76 (m, 2H), 7.17-7.29 (m, 5H), 14.47 (s, 1H);

[0113]

[0114] (Example 13) Preparation of 5-(1-acetyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-diisopropylbarbituric acid (compound 5c) (General formula (A): R 1 =CH3, R 2 =CH3, R 3 = i Pr)

[0115]

[0116] A 10 mL two-necked round-bottom flask, fitted to a reflux condenser, was flame-dried under an argon atmosphere. Then, 0.4 mmol of the previously synthesized compound 4c, 0.8 mL of acetic acid (Nacalai Tesque), and 0.8 mL of acetic anhydride (Nacalai Tesque) were added, and the mixture was stirred at 125°C for 2 hours. After the reaction was complete, toluene was added to concentrate the mixture, and it was separated by gel permeation chromatography (GPC). Vacuum drying was then performed to obtain pure compound 5c, 5-(1-acetyl-5,5-dimethylpyrazolidine-3-ylidene)-1,3-diisopropylbarbituric acid (5c), as a clear solid (91% yield). The structure is... 1 TMS was determined by 1H NMR, with each component set to 0 ppm. 5-(1-acetyl-5,5-dimethylpyrazolidin-3-ylidene)-1,3-diisopropylbarbituric acid (5c): transparent solid; 1 H NMR (400 MHz, CDCl3) δ 5.208-5.156 (m, 2H), 3.828 (brs, 1H), 3.561 (brs, 1H), 2.253 (s, 3H), 1.637 (s, 6H), 1.455-1.438 (d, J = 6.8 Hz, 12H);

[0117]

[0118] (Yield for each reaction step) The yield for each reaction step in each of the above examples is summarized below.

[0119] (1) First step (Alkanoylation (acylation) reaction)

[0120]

[0121]

[0122] (2) Second stage (pyrazolidine formation reaction)

[0123]

[0124]

[0125] (3) Third step (Alkanoylation (acylation) reaction)

[0126]

[0127]

[0128] (Example 14) Using the HRE luciferase assay system previously developed by the inventors (Tsujita, T.; Kawaguchi, Si.; Dan, T.; Baird, L.; Miyata, T.; Yamamoto, M., Hypoxia-Sensitive Reporter System for High-Throughput Screening. Tohoku J. Exp. Med. 2015, 235 (2), 151-159), the HIF activation ability (HIF transcriptional activity) of the compounds in each of the above examples was measured to determine whether they showed a signal indicating HIF activation.

[0129] The RE luciferase assay system used SK-N-BE(2)-C cells (SKN:HRE-NLuc) that stably expressed nano-Luc (Promega) under modified HIF transcriptional regulatory regions. SKN:HRE-NLuc cells were placed in 384-well plates (Corning) in quantities of 7.0 × 10⁶. 3 Cells were seeded in cell / wells and pre-cultured for 16 hours at 37°C and 5% CO2. The following day, the culture medium was replaced with serially diluted media containing various drugs starting from 100 μM, and cultured for 24 hours. After stimulation, 1 / 3 volume of luciferase assay (NanoGlo Luciferase Assay, Promega) was added to the medium, and the luminescence intensity was measured using a multimode microplate reader (SpectraMax iD3, Molecular Devices). Based on HIF transcriptional activity, the relative activity of each drug was determined with 1% DMSO stimulation set as 1.

[0130] SKN:HRE-NLuc is loaded into a 384-well plate (Corning) in 7.0 x 10 3Cells were seeded in cell / wells and pre-cultured for 16 hours at 37°C and 5% CO2. The following day, the culture medium was changed to one containing various drugs serially diluted from 100 μM, and the cells were cultured for 24 hours. After stimulation, a cell counting kit (Cell Counting Kit-8, Dojin Chemical Laboratories) was added to 1 / 10 of the culture medium, and the color reaction was allowed to develop in an incubator for 1 hour. Subsequently, the absorbance at 450 nm was measured using a multimode microplate reader (SpectraMax iD3, Molecular Devices). Finally, the viability of cells treated with 1% DMSO was set to 100%, and the viability of each stimulated cell was determined.

[0131] Figure 1 shows the measurement results for the HIF transcriptional activity of compound 5a. The results obtained, including those for the compounds in each of the other examples, are summarized in the table below. The nitrogen-containing cyclic compound in Patent Document 4 is denoted as PyrzA as a comparative example and used as the standard for comparison. In the table, the 5x activity ratio indicates the ratio of (5x activity concentration of commercially available roxadustat (FG-4592, manufactured by Astellas Pharma / FibroGen)) / (5x activity concentration of each compound). LC 50 This indicates a concentration that reduces cell viability by 50%. Furthermore, the HIF transcriptional activity in the table indicates the following: + PyrzA (reference) or higher ++ 5x activity ratio 1 or higher

[0132]

[0133] The results confirmed that all compounds in each example exhibited high HIF activation activity. All other compounds in each example showed significantly higher HIF activation activity than the nitrogen-containing cyclic compound (PyrzA) in the comparative example, Patent Document 4. Furthermore, extremely high HIF activation activity was confirmed for compounds 5ac, 5b, and 5bc. Compound 5bb, in particular, showed extremely high HIF activation activity, and no cytotoxicity was detected.

[0134]

[0135] Regarding cytotoxicity, compounds 5b and 5ba showed only slight cytotoxicity. However, the cytotoxicity level of compound 5b was measured in LC for the activity level of 0.36 μM or higher. 50Since the concentration is around 24 μM, which is nearly 100 times lower, it indicates very weak cytotoxicity and a certain level of safety has been confirmed. For compound 5ba, LC was performed against the active action region of 1.3 μM or higher. 50 The difference between this and the 84 μM level is nearly 100 times, indicating very weak cytotoxicity. No cytotoxicity was detected in any of the other compounds used in the examples, confirming their high level of safety.

Claims

1. A nitrogen-containing cyclic compound without an asymmetric carbon, represented by the following general formula (I), general formula (II), or general formula (III). (In the above general formulas (I), (II), and (III), R １ R is a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms. ２ R is an aryl group having 1 to 2 aromatic rings, consisting of a hydrogen atom, a substituted or unsubstituted C1 to C10 alkyl group, or a substituted or unsubstituted 5-membered or 6-membered ring, in a monocyclic, fused, or polycyclic structure. ３ Each of these is independently a substituted or unsubstituted linear or cyclic alkyl group having 1 to 10 carbon atoms. １ They may be joined to each other to form a ring.

2. R 1 A nitrogen-containing cyclic compound without an asymmetric carbon, as described in claim 1, wherein the asymmetric carbon is composed of a lower alkyl group having 1 to 5 carbon atoms.

3. R 2 A nitrogen-containing cyclic compound without an asymmetric carbon, as described in claim 1, comprising one or two phenyl groups or a lower alkyl group having 1 to 9 carbon atoms, which may be substituted with a lower alkyl group having 1 to 9 carbon atoms.

4. R 3 A nitrogen-containing cyclic compound without an asymmetric carbon, as described in claim 1, wherein the asymmetric carbon is composed of a linear or cyclic alkyl group having 1 to 6 carbon atoms.

5. A composition containing a nitrogen-containing cyclic compound without an asymmetric carbon as described in claim 1.

6. The composition according to claim 5, for use in inhibiting the activity of proline hydroxylase.

7. The composition according to claim 5 for use in a disease treatable by inhibition of proline hydroxylase activity.

8. The composition according to claim 5, wherein the disease is selected from the group consisting of kidney disease, chronic kidney disease, ischemic disease, hypertension, diabetes, and arteriosclerosis.

9. The composition according to claim 5, wherein the embodiment is a pharmaceutical product, a quasi-drug, or a reagent.

10. The composition according to claim 9, for oral administration.

11. An orally administered hypoxia-inducible factor activator containing the composition described in claim 10.

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