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WO2011087051A1 - Agent thérapeutique pour l'athérosclérose, comprenant un antagoniste du récepteur s1p2 - Google Patents

Agent thérapeutique pour l'athérosclérose, comprenant un antagoniste du récepteur s1p2 Download PDF

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WO2011087051A1
WO2011087051A1 PCT/JP2011/050429 JP2011050429W WO2011087051A1 WO 2011087051 A1 WO2011087051 A1 WO 2011087051A1 JP 2011050429 W JP2011050429 W JP 2011050429W WO 2011087051 A1 WO2011087051 A1 WO 2011087051A1
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group
optionally substituted
alkyl
hydrogen atom
apoe
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陽 多久和
安雄 岡本
飛 王
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Kanazawa University NUC
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Kanazawa University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to a therapeutic agent for atherosclerosis.
  • This application claims priority from Japanese Patent Application No. 2010-006300, which is incorporated herein by reference.
  • Sphingosine 1-phosphate is attracting attention as a lipid growth factor that exerts a variety of effects on various cell types including vascular endothelial cells, such as cell proliferation, cell motility / morphological regulation, and cell differentiation. ing. Initially, it was suggested that these biological activities of S1P may be due to intracellular actions, but in 1998, a specific cell surface receptor for S1P was identified, and many of these actions are S1P receptors. It has been clarified that the process is performed (see Non-Patent Document 1).
  • Biosynthesis and degradation of S1P The biosynthesis of S1P starts from ceramide. Ceramide is hydrolyzed to sphingosine by ceramidase. Sphingosine is phosphorylated by sphingosine kinase (SphK) to generate S1P.
  • SphK sphingosine kinase
  • S1P degrading enzymes S1P phosphohydrolase (SPP) and S1P lyase (SPL).
  • SPP S1P phosphohydrolase
  • SPL S1P lyase
  • S1P is present in plasma at concentrations on the order of about 10 ⁇ 7 M, and serum S1P concentrations are somewhat higher.
  • Platelets are an important source of S1P. This is due to the unique feature that platelets have high SphK activity but lack SPL activity that degrades S1P. S1P, which is abundantly stored in platelets, is released in large amounts outside of cells upon stimulation of platelet aggregation. For this reason, it is considered that serum, which is a blood supernatant after platelet aggregation, has a higher S1P concentration than plasma. In addition to platelets, red blood cells and vascular endothelium are also considered important sources of plasma S1P.
  • S1P vascular cell action lipoproteins
  • HDL high-density lipoprotein
  • LDL low-density lipoprotein
  • S1P mainly promotes cell proliferation and migration, but ceramide and sphingosine, which are precursors, are known to promote cell apoptosis. This suggests that the S1P synthesis / degradation system is an important system that determines cell viability.
  • S1P receptor S1P-specific G protein-coupled receptors, identified five subtypes (S1P 1 / Edg (Endothelialdifferentiation gene ) 1, S1P 2 / Edg5 / AGR16, S1P 3 / Edg3, S1P 4 / Edg6, S1P 5 / Edg8) Many, if not all, of S1P actions are thought to be transmitted into cells via these Edg family S1P receptors.
  • the tissue expression distribution of S1P receptors varies from subtype to subtype. In rats and mice, S1P 1 , S1P 2 and S1P 3 are widely expressed in almost all organs and tissues throughout the body.
  • SlP 4 in lymphoid tissue and lung, SlP 5 nervous system, expressed in the spleen is localized.
  • Many organs express multiple S1P receptor subtypes.
  • S1P 1 and S1P 3 are strongly expressed in vascular endothelial cells.
  • the expression of S1P 2 is detected in the vascular endothelium of some organs, but it is low in human umbilical vein endothelial cells (HUVEC), which are often used in experiments.
  • UUVEC human umbilical vein endothelial cells
  • S1P 1 activates Ras-ERK (extracellular signal-regulated kinase), activation of phospholipase C (PLC), adenylate via Gi. It is coupled to the inhibition of cyclase.
  • Ras-ERK extracellular signal-regulated kinase
  • PLC phospholipase C
  • S1P 3 is coupled to inhibition of Ras-ERK and adenylate cyclase via Gi, as well as S1P 1 , coupled to PLC activation via Gq, and Rho activation via G12 / 13. is doing.
  • S1P 2 is the signal pathway most predominantly coupled to Rho via G12 / 13.
  • S1P is essential for angiogenesis in the fetal period, especially the process of accumulation of vascular medial smooth muscle cells and pericytes into the vascular wall (vascular maturation or remodeling), and development of the nervous system.
  • NO nitric oxide
  • Atherosclerosis and S1P Atherosclerosis is considered to be a chronic inflammatory change due to injury of the blood vessel wall, and many inflammatory cells and platelets are actually involved in the formation of arteriosclerotic lesions, and various cytokines secreted from these cells .
  • Chemokines, growth factors, bioactive lipids and the like activate vascular endothelial cells, vascular smooth muscle cells, monocytes, and macrophages involved in arteriosclerosis. That is, vascular endothelial cells bring about expression of adhesion molecules such as VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intracellular adhesion molecule-1) and production of cell migration substances on the cell surface.
  • S1P may be an anti-arteriosclerosis factor because S1P promotes differentiation into anti-inflammatory macrophages via S1P 1 receptor from S1P 1 receptor agonist / blocker experiments was suggested.
  • S1P 1 and S1P 3 receptor gene knockdown experiments show that S1P is essential for endothelial adhesion of monocytes via S1P 1 and S1P 3 receptors (enhanced expression of VCAM-1 and ICAM-1). It has also been suggested that may be an atherosclerotic factor.
  • S1P at the individual animal level is an atherosclerotic factor or an anti-arteriosclerotic factor.
  • vascular endothelial cells, vascular smooth muscle cells, monocytes, macrophages, and foam cells involved in arteriosclerosis each express several types of S1P receptors. There are also many unclear points regarding whether the body subtype is involved in the development of atherosclerosis.
  • the present invention aims to provide a therapeutic agent for atherosclerosis.
  • SlP 1 and S1P 3 receptors function suppression is reported to reduce atherosclerotic lesion size of the aorta in mice.
  • S1P 2 receptor plays in atherosclerosis.
  • mice S1P 2 deficient (gene knockout (KO) mice
  • ApoE KO a component protein of lipoprotein.
  • ApoE ⁇ / ⁇ mice were mated to produce S1P 2 ⁇ / ⁇ , ApoE ⁇ / ⁇ double KO mice, and a high cholesterol diet was loaded to induce atherosclerosis.
  • ApoE-/-mice were treated with 1- [1,3-Dimethyl-4- (2-methylethyl) -1H-pyrazolo [3,4-b] pyridine-6-yl, a S1P 2 receptor blocker for 3 months
  • administration of] -4- (2,6-dichloro-4-pyridinyl) -semicarbazide (JTE-K1) was performed, the formation of atherosclerotic lesions in the aorta was suppressed compared to the non-administered group.
  • S1P 2 receptor promotes the formation of atherosclerotic lesions, and at least a part of the mechanism involves the promotion of macrophage oxidized LDL accumulation by S1P 2 receptor. This suggests that the present inventors have found that blockade of the S1P 2 receptor by a drug suppresses atherosclerosis and completed the present invention.
  • a therapeutic agent for atherosclerosis comprising an S1P 2 receptor antagonist as an active ingredient.
  • R 1 represents a hydrogen atom, a C 1-6 alkyl group, a haloalkyl group, an optionally substituted aryl group, an optionally substituted aralkyl group or —COR 7 (where R 7 represents C 1-6 An alkyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, a C 1-6 alkoxy group, an optionally substituted aryloxy group or an optionally substituted aralkyloxy group) Is;
  • R 2 is a hydrogen atom, an optionally substituted C 1-6 alkyl group or an optionally substituted aryl group;
  • R 3 is a hydrogen atom, an optionally substituted C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-6 alkoxycarbonyl group, a haloalkyl group, a C 3-7 cycloalkyl group, or a substituted A good aryl group;
  • R 4 is a hydrogen
  • R 14 represents a hydrogen atom, a C 1-6 alkyl group, an optionally substituted aralkyloxycarbonyl group, an optionally substituted aryloxycarbonyl group, or A heteroaryl C 1-6 alkyl group), —O—, —CO—, —CONH— (wherein the nitrogen atom is bonded to ring A), —CH 2 —, —NHCH 2 — (wherein carbon atom) Is bonded to ring A) or a single bond; --------- is a double bond or a single bond; Ring A is an aryl group, a heterocyclic residue, or a C 3-7 cycloalkyl group]
  • R 1 is a hydrogen atom, a C 1-6 alkyl group or —COR 7 (where R 7 is a C 1-6 alkyl group, an aryl group which may be substituted, or an optionally substituted group).
  • R 2 is a C 1-6 alkyl group or a substituted
  • R 3 is a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-6 alkoxycarbonyl group, a haloalkyl group, a C 3-7 cycloalkyl group or a substituted aryl group.
  • R 4 is a hydrogen atom or a C 1-6 alkyl group
  • R 5 is an optionally substituted aryl group, an optionally substituted heterocyclic residue, or C 2.
  • R 6 is preferably a hydrogen atom, C 1-6 alkyl group, a halogen atom, an optionally substituted aryl group, optionally substituted heterocyclic residue, or C 2-12 alkoxy
  • the therapeutic agent for atherosclerosis according to [2].
  • R 1 is a C 1-6 alkyl group
  • R 2 is a C 1-6 alkyl group
  • R 3 is a C 1-6 alkyl group
  • R 4 is a hydrogen atom.
  • R 5 and R 6 may be the same or different and are a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, or a halogen atom
  • X is —NH—, —N ⁇ , —CH 2 — or —O—
  • Y is —NH— or ⁇ N—
  • Z is —CO—
  • W is —NH—
  • ring A is an aryl group, preferably a phenyl group or heteroaryl.
  • R 1 and R 2 are methyl groups, R 3 is isopropyl groups, R 4 is hydrogen, X is —NH—, Y is —NH—, X is —CO—, W is —NH—, and ring A is The therapeutic agent for atherosclerosis according to [2], wherein the pyridyl group, R 5 and R 6 are independently hydrogen, chlorine, fluorine, bromine, methyl group or methoxy group.
  • the medicament containing the S1P 2 receptor antagonist of the present invention as an active ingredient blocks the S1P 2 receptor, decreases the ability of macrophages infiltrated into the endovascular subcutaneous tissue to take up oxidized LDL, and is oxidized. LDL pumping ability is promoted, and foam cell formation of macrophages is suppressed. As a result, formation of atherosclerotic lesions can be suppressed.
  • FIG. 1A shows the results of ApoE ⁇ / ⁇ , S1P 2 + / + mice
  • FIG. 1B shows the results of ApoE ⁇ / ⁇ , S1P 2 +/ ⁇ mice
  • FIG. 1C shows the results of ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ mice.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency
  • S1P 2 +/ ⁇ indicates S1P 2 receptor hetero deficiency.
  • the arteriosclerotic lesion is stained red. It is a figure which shows the atherosclerotic lesion area of each gene modification mouse
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency. It is a figure which shows the fixed_quantity
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • b and d are enlarged images of a and c, respectively.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency. It is a figure which shows the Azan staining image of the aortic valve annulus cross section of each gene modification mouse.
  • b and d are enlarged images of a and c, respectively.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency.
  • ApoE ⁇ / ⁇ indicates ApoE deficiency
  • S1P 2 ⁇ / ⁇ indicates S1P 2 receptor deficiency.
  • FIGA shows OXLDL-untreated S1P 2 WT
  • FIG. 9B shows oxLDL-untreated S1P 2 KO
  • FIG. 9C shows oxLDL-treated S1P 2 WT
  • FIG. 9D shows oxLDL-treated S1P 2 KO.
  • S1P 2 WT represents a wild-type mouse
  • S1P 2 KO represents an S1P 2 receptor-deficient mouse. It is a figure which shows the fixed_quantity
  • S1P 2 WT represents a wild-type mouse
  • S1P 2 KO represents an S1P 2 receptor-deficient mouse. It is a figure which shows the analysis result of the gene expression of the arteriosclerosis related molecule
  • S1P 2 WT represents a wild-type mouse
  • S1P 2 KO represents an S1P 2 receptor-deficient mouse. It is a figure which shows the quantification result of the gene expression change of the arteriosclerosis related molecule
  • S1P 2 WT represents a wild-type mouse, and S1P 2 KO represents an S1P 2 receptor-deficient mouse. It is a figure which shows the fixed_quantity
  • Control group is a diagram showing an aortic Oilred-O staining images (FIG. 14A) and SlP 2 receptor blocker treated group mice ( Figure 14B). Is a diagram illustrating an aortic atherosclerotic lesion area of the control group and SlP 2 receptor blocker treated group mice. *: P ⁇ 0.05.
  • FIG. 16A shows an X-gal stained image of the aortic sinus
  • FIG. 16B shows X-gal staining of the aortic atherosclerotic lesion.
  • a and SlP 2 expressing cells in B are stained blue.
  • FIG. 16C shows the result of double staining of X-gal staining and immunostaining of macrophage marker Mac-3. Mac-3 positive (brown) macrophages are also positive for X-gal staining.
  • FIG. 16A shows an X-gal stained image of the aortic sinus
  • FIG. 16B shows X-gal staining of the aortic atherosclerotic lesion.
  • a and SlP 2 expressing cells in B are stained blue.
  • FIG. 16C shows the result of double staining of X-gal staining and immunostaining of macrophage marker Mac-3. Mac-3 positive (brown) macrophages are also positive for X-gal staining.
  • 16D shows the result of double staining of Xgal staining and immunostaining of vascular endothelial marker CD31.
  • Part of the CD-31 positive (brown) endothelium is X-gal staining positive. It is a figure which shows the result of suppression of atherosclerosis by transplantation of S1P 2 ⁇ / ⁇ mouse bone marrow.
  • mice High cholesterol diet in mice (BMT mice) transplanted with ApoE-/-, S1P 2 + / + mice and ApoE-/-, S1P 2 + / + mouse bone marrow or ApoE-/-, S1P 2 -/-mice bone marrow And atherosclerotic lesions of the isolated aorta were evaluated by Oiled O staining. It is a figure which shows the fixed_quantity
  • Is a diagram showing changes in the intracellular signaling of SlP 2 deficient mice macrophages activated Rho shows changes in. Macrophages isolated from ApoE ⁇ / ⁇ , S1P 2 + / + mice and ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ mice after pretreatment with ROCK inhibitor Y-27632 or S1P 2 blocker JTE-K1, or Stimulated with S1P without pretreatment.
  • It is a diagram showing changes in the intracellular signaling of SlP 2 deficient mice macrophages activated Rho shows a quantitative value of the change in.
  • Macrophages isolated from ApoE-/-, S1P 2 + / + mice and ApoE-/-, S1P 2 -/-mice, or after pretreatment with ROCK inhibitor Y-27632 or S1P 2 antagonist JTE-K1 Stimulated with S1P without treatment Is a diagram showing changes in the intracellular signaling of SlP 2 deficient mice macrophages, showing the mRNA expression of ABCA1. Is a diagram showing changes in the intracellular signaling of SlP 2 deficient mice macrophages, showing the mRNA expression of LXR-alpha. It shows cell migration reaction of SlP 2 deficient mice macrophages. Macrophages were added to the upper layer of the Boyden chamber shown in FIG.
  • FIG. 20 shows a specific example of a compound represented by the general formula (1) of the present invention (continuation of FIG. 20-1).
  • FIG. 20 shows a specific example of a compound represented by the general formula (1) of the present invention (continuation of FIG. 20-2).
  • FIG. 20 shows a specific example of the compound represented by the general formula (1) of the present invention (continuation of FIG. 20-3).
  • FIG. 4 shows a specific example of a compound represented by the general formula (1) of the present invention (continuation of FIG. 20-4).
  • FIG. 5 shows a specific example of a compound represented by the general formula (1) of the present invention (continuation of FIG. 20-5).
  • FIG. 6 shows a specific example of a compound represented by the general formula (1) of the present invention (continuation of FIG. 20-6).
  • FIG. 18 is a view showing specific examples of the compound represented by the general formula (1) of the present invention (continuation of FIG. 20-7).
  • the present invention is a therapeutic agent for atherosclerosis comprising an S1P 2 receptor antagonist as an active ingredient.
  • An antagonist refers to a compound that acts on a receptor molecule in a living body and blocks signal transduction or the like, and an S1P 2 receptor antagonist has an S1P 2 receptor antagonistic action.
  • Arteriosclerosis is classified into atherosclerosis (atherosclerosis), arteriosclerosis, and medial sclerosis.
  • the S1P 2 receptor antagonist of the present invention is effective against atherosclerosis.
  • a therapeutic agent for atherosclerosis comprising the S1P 2 receptor antagonist of the present invention as an active ingredient suppresses the accumulation of macrophages (foam cells) at the site of atherosclerotic lesions, and reduces the ability of macrophages to take up oxidized LDL, It also promotes the ability to pump oxidized LDL. As a result, the formation of atherosclerotic lesions is suppressed.
  • S1P 2 receptor antagonist of the present invention examples include a pyrazolopyridine derivative represented by the following general formula (1), or a pharmaceutically acceptable salt thereof.
  • R 1 represents a hydrogen atom, a C 1-6 alkyl group, a haloalkyl group, an optionally substituted aryl group, an optionally substituted aralkyl group or —COR 7 (where R 7 represents C 1-6 An alkyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, a C 1-6 alkoxy group, an optionally substituted aryloxy group or an optionally substituted aralkyloxy group) Is;
  • R 2 is a hydrogen atom, an optionally substituted C 1-6 alkyl group or an optionally substituted aryl group;
  • R 3 is a hydrogen atom, an optionally substituted C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-6 alkoxycarbonyl group, a haloalkyl group, a C 3-7 cycloalkyl group, or a substituted A good aryl group;
  • R 4 is a hydrogen
  • R 14 represents a hydrogen atom, a C 1-6 alkyl group, an optionally substituted aralkyloxycarbonyl group, an optionally substituted aryloxycarbonyl group, or A heteroaryl C 1-6 alkyl group), —O—, —CO—, —CONH— (wherein the nitrogen atom is bonded to ring A), —CH 2 —, —NHCH 2 — (wherein carbon atom) Is bonded to ring A) or a single bond; --------- is a double bond or a single bond; Ring A is an aryl group, a heterocyclic residue, or a C 3-7 cycloalkyl group]
  • R 1 is a C 1-6 alkyl group, a hydrogen atom or —COR 7 (where R 7 is a C 1-6 alkyl group, an aryl group, an aralkyl group, a C 1-6 alkoxy group, an aryloxy group, Group or an aralkyloxy group);
  • R 2 is a C 1-6 alkyl group or an aryl group;
  • R 3 is a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkoxycarbonyl group, a haloalkyl group, a C 3-7 cycloalkyl group or an aryl group;
  • R 4 is a hydrogen atom or a C 1-6 alkyl group;
  • R 5 and R 6 are the same or different and each represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 1-6 alkoxycarbonyl group, a carboxy
  • examples of the “halogen atom” include a chlorine atom, a bromine atom, a fluorine atom and an iodine atom, and R 5 and R 6 in the above formula are preferably chlorine atoms.
  • C 1-6 alkyl group examples include linear or branched alkyl groups having 1 to 6 carbon atoms. Examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, and a hexyl group.
  • a linear or branched alkyl group having 1 to 4 carbon atoms may be mentioned.
  • R 1 in the above formula is preferably a methyl group or an ethyl group, more preferably a methyl group
  • R 5 and R 6 are preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, or an isobutyl group.
  • R 7 is preferably a methyl group or an ethyl group.
  • R 9 is preferably a C 1-4 alkyl group (more preferably a methyl group), R 11 is preferably a C 1-4 alkyl group (more preferably a methyl group), and R 12 is preferably methyl, R 14 is preferably methyl, R 15 is preferably methyl or isobutyl, R 16 is preferably Is a methyl group, R 17 is preferably a methyl group, R 18 is preferably a methyl group, R 19 is preferably a methyl group.
  • the C 1-6 alkyl group in R 2 , R 3 and R 4 may be substituted with the following substituents, and the substituents may be the same or different, and The number and position of substituents are not limited.
  • substituents include an aralkyloxy group (preferably having a total carbon number of 7 to 10, specifically benzyloxy group, phenethyloxy group, etc.), hydroxyl group, carboxyl group, halogen atom, carbamoyl group, amino group and the like. Is mentioned.
  • Specific examples of the “ optionally substituted C 1-6 alkyl group” are as follows.
  • R 2 in the above formula is preferably a methyl group, a tert-butyl group or a benzyloxymethyl group, more preferably a methyl group or a tert group.
  • -Butyl group, particularly preferably methyl group, and R 3 is preferably methyl group, ethyl group, tert-butyl group, isobutyl group, isopropyl group, 1-benzyloxypropan-2-yl group or 1- A hydroxypropan-2-yl group, more preferably a methyl group, an ethyl group, a tert-butyl group, an isobutyl group or an isopropyl group, particularly preferably a methyl group or an isopropyl group, and R 4 is preferably a methyl group Group, isopropyl group, 1-benzyloxypropan-2-yl group or 1-hydroxypropan-2-yl group. Particularly preferred is a methyl group.
  • C 1-6 alkoxy group examples include linear or branched alkoxy groups having 1 to 6 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group. Tert-butoxy group, pentyloxy group, tert-pentyloxy group or hexyloxy group.
  • a straight chain or branched alkoxy group having 1 to 4 carbon atoms (for example, methoxy group, ethoxy group, isopropoxy group, butoxy group, tert-butoxy group) is preferable. More preferably, it is a methoxy group or an ethoxy group.
  • R 3 in the above formula is preferably a methoxy group
  • R 5 and R 6 are preferably a methoxy group or an ethoxy group
  • R 7 is preferably a methoxy group.
  • C 2-12 alkoxyalkyl group means an alkoxyalkyl in which the alkoxy part has the same meaning as the above alkoxy group and the alkyl part has the same meaning as the above alkyl group (that is, the total carbon number is 2 to 12).
  • Examples thereof include a methoxymethyl group, an ethoxymethyl group, a propoxymethyl group, a butoxymethyl group, a pentyloxymethyl group, a hexyloxymethyl group, and an ethoxyethyl group.
  • R 5 and R 6 in the above formula are preferably a methoxymethyl group, an ethoxymethyl group, or a tert-butoxymethyl group, and more preferably a methoxymethyl group or a tert-butoxymethyl group.
  • an alkyl moiety such as a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, an isopropoxycarbonyl group, a butoxycarbonyl group, an isobutoxycarbonyl group or a tert-butoxycarbonyl group has a carbon number Examples thereof include 1 to 5 alkoxycarbonyl groups.
  • a methoxycarbonyl group, an ethoxycarbonyl group or a tert-butoxycarbonyl group is preferred.
  • R 3 , R 5 and R 6 in the above formula are preferably a methoxycarbonyl group
  • R 10 is preferably a methoxycarbonyl group or an ethoxycarbonyl group
  • R 12 is preferably a methoxycarbonyl group or tert-butoxy It is a carbonyl group.
  • haloalkyl group examples include those in which the above C 1-6 alkyl group is substituted with the above halogen atom.
  • chloromethyl group, bromomethyl group, fluoromethyl group, difluoromethyl group, trifluoromethyl group, trichloromethyl Group, tribromomethyl group, trichloroethyl group, trifluoroethyl group, pentafluoropropyl group or chlorobutyl group, etc. preferably chloromethyl group, bromomethyl group, fluoromethyl group, trifluoromethyl group or trichloromethyl group Can be mentioned. More preferably, it is a trifluoromethyl group.
  • R 1 in the above formula is preferably a 2,2,2-trifluoroethyl group
  • R 3 , R 5 and R 6 are preferably a trifluoromethyl group or a difluoromethyl group.
  • C 2-6 alkynyl group examples include linear or branched alkynyl groups having 2 to 6 carbon atoms, such as ethynyl group, propynyl group, butynyl group, 2-pentynyl group, 3- Examples include a pentynyl group, a 2-hexynyl group, a 3-hexynyl group, and a straight chain or branched alkynyl group having 2 to 4 carbon atoms is preferable.
  • R 5 and R 6 in the above formula are preferably an ethynyl group.
  • C 1-6 alkylamino group examples include amino groups monosubstituted by 1 to 6 linear or branched alkyl groups, such as methylamino group, ethylamino group, propylamino group. Group, butylamino group and the like. Preferably, a methylamino group or an ethylamino group is used.
  • R 5 and R 6 in the above formula are preferably a methylamino group or an ethylamino group.
  • di (C 1-6 alkyl) amino group examples include amino groups disubstituted by 1 to 6 linear or branched alkyl groups such as a dimethylamino group and a diethylamino group. , Dipropylamino group, dibutylamino group and the like, preferably dimethylamino group and diethylamino group.
  • R 5 and R 6 in the above formula are preferably a dimethylamino group or a diethylamino group.
  • acyl group includes a formyl group having 1 carbon atom; an alkanoyl group having 2 to 6 carbon atoms (for example, an acetyl group, a propionyl group, a butyryl group, or a pivaloyl group); or 1 to 3 carbon atoms on an aryl group
  • An aroyl group for example, benzoyl group, etc .; the substituent here is synonymous with the substituent of the following “optionally substituted aryl group” may be mentioned.
  • a formyl group, an acetyl group, a pivaloyl group or a benzoyl group is preferred.
  • R 5 and R 6 in the above formula are preferably acetyl groups.
  • aryl group for example, a C 6-12 aryl group such as a phenyl group, a naphthyl group, a biphenyl group and the like, which may be substituted with at least one substituent, preferably a phenyl group Is mentioned.
  • the substituents may be the same or different, and the number and position of the substituents are not particularly limited.
  • an aralkyl group such as phenylethyl group
  • an amide group (-CONH 2); - NHCOR 16 group
  • R 16 is, C 1-6 alkyl group, optionally substituted An aryl group or an optionally substituted aralkyl group
  • -CONHR 17 group (wherein R 17 represents a C 1-6 alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl); Haloalkyl group (eg, trifluoromethyl group, etc.); C 1-6 alkylsulfonyl group (eg, methylsulfonyl group, ethylsulfonyl group, etc.); sulfamoyl group; —SO 2 NHR 18 group (wherein R 18 represents a C 1-6 alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group); —SO 2 N (R 19 ) 2
  • R 15 , R 16 , R 17 , R 18 , R 19 in the above formula each independently represent a C 1-6 alkyl group, an optionally substituted aryl group or an optionally substituted aralkyl group.
  • C 1-6 alkyl group is synonymous with “C 1-6 alkyl group” represented by such R 1 mentioned above
  • optionally substituted aryl group such as R 5 mentioned above
  • the “optionally substituted aryl group” has the same meaning as the “optionally substituted aralkyl group” such as R 7 described later.
  • a phenyl group substituted with a group or a halogen atom (eg, bromophenyl group), and R 13 is a phenyl group or a phenyl group substituted with a halogen atom (eg, bromophenyl group, chlorophenyl group, dichlorophenyl group, etc.)
  • R 5 and R 6 are phenyl group, chlorophenyl group, dichlorophenyl group, chloro-fluorophenyl group, chloro-methoxyphenyl group, fluorophenyl group, difluorophenyl group, trifluorophenyl group, fluoro-methylphenyl group , Trifluoro-dimethoxyphenyl group, bromophenyl group, trif Fluoromethylphenyl group, hydroxyphenyl group, methylphenyl group, dimethylphenyl group, ethylphenyl group, tert-butylphenyl group
  • the aryl moiety is a phenyl group (wherein the phenyl group may be substituted with the above-mentioned substituents which may be substituted, and the number and position of the substituents are particularly limited)
  • aryloxycarbonyl group examples include C 7-13 aryloxycarbonyl groups in which the aryl portion has the same meaning as the above aryl group. It may be substituted with a group.
  • the number and position of substituents are not particularly limited. Examples thereof include a phenoxycarbonyl group and a naphthyloxycarbonyl group, and a phenoxycarbonyl group is preferable.
  • R 12 and R 14 in the above formula are preferably a phenoxycarbonyl group.
  • C 3-7 cycloalkyl group refers to a cycloalkyl group having 3 to 7 carbon atoms, specifically, cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, 1-methylcyclohexyl group, cycloheptyl group. Etc.
  • Preferable examples include cycloalkyl groups having 3 to 6 carbon atoms, and specific examples include cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group. More preferably, a cyclopropyl group or a cyclohexyl group is mentioned.
  • R 3 in the above formula is preferably a cyclopropyl group.
  • Ring A is preferably a cyclohexyl group.
  • R 5 and R 6 are preferably a cycloalkyl group having 4 to 7 carbon atoms (eg, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl).
  • Heterocyclic residue means a 5- to 6-membered aromatic heterocyclic ring containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur atoms in addition to carbon atoms as atoms constituting the ring, saturated A heterocyclic ring, an unsaturated heterocyclic ring, or a condensed heterocyclic ring obtained by condensing these heterocyclic rings with a benzene ring.
  • Specific examples include a thiophen-2-yl group, a thiophen-3-yl group, and a furan-2-yl group.
  • Ring A in the above formula is preferably a pyridyl group (eg, pyridin-2-yl group, pyridin-3-yl group, pyridin-4-yl group, etc.), benzoxazol-2-yl group, morpholin-4-yl Group, benzimidazol-2-yl group, pyrimidin-2-yl group, pyrimidin-4-yl group, thienyl group (for example, thiophen-2-yl group, thiophen-3-yl group), furan-2-yl Group, furan-3-yl group, pyrrol-2-yl group, pyrrol-3-yl group, pyrazol-3-yl group, thiazolyl group (for example, thiazol-4-yl group, thiazol-5-yl group, etc.) Benzoimidazol-2-yl group, benzothiophen-2-yl group, benzofuran-2-yl group, etc., more preferably
  • the “heterocyclic residue” of R 5 and R 6 in the above formula is the same as the above heterocyclic residue in the heterocyclic moiety and is substituted with the substituent described in the above “optionally substituted aryl group”.
  • the number and position of the substituents are not particularly limited.
  • the “heterocyclic residue” is preferably a pyridyl group (eg, pyridin-2-yl group, pyridin-3-yl group, pyridin-4-yl group), a thiazolyl group (eg thiazol-4-yl group).
  • thienyl group thiophen-2-yl group, thiophen-3-yl group
  • pyridyl group pyridin-4-yl group
  • thienyl group examples include a chloropyridyl group, a chloro-methoxypyridyl group, a dichloropyridyl group, and the like.
  • Heteroaryl C 1-6 alkyl group means a heteroaryl part in which the heteroaryl part is the above-mentioned heterocyclic residue, and the alkyl part is a straight chain or branched alkyl group having 1 to 6 alkyl parts.
  • Arylalkyl groups for example, pyridylmethyl groups (eg 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group etc.), quinolinylmethyl groups (eg 2-quinolinylmethyl group etc.), indolylmethyl Groups (for example, 2-indolylmethyl group, 3-indolylmethyl group, etc.), thiophen-2-ylmethyl group, thiophen-3-ylmethyl group, 2-furanylmethyl group, 3-furanylmethyl group, 1H-benzimidazole-2 -Ylmethyl group, benzothiazol-2-ylmethyl group, 2- (thiophen-2-yl) ethyl group, 2- (furan- 2-yl) ethyl group and the like.
  • pyridylmethyl groups eg 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group etc.
  • quinolinylmethyl groups
  • R 14 in the general formula (1) is preferably a 3-pyridylethyl group (eg, 2- (pyridin-3-yl) ethyl group).
  • R 1 is a hydrogen atom, a C 1-6 alkyl group or —COR 7 (where R 7 is a C 1-6 alkyl group, an optionally substituted aryl group) , aralkyl group which may be substituted, a C 1-6 alkoxy group, a substituted aryloxy group, or an optionally substituted aralkyloxy group),
  • R 2 is, C 1-6 An alkyl group or an optionally substituted aryl group
  • R 3 is a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, a C 2-6 alkoxycarbonyl group, a haloalkyl group, a C 3-7
  • R 4 is a hydrogen atom or a C 1-6 alkyl group
  • R 5 is, for example, an optionally substituted aryl group, an optionally substituted heterocyclic residue, or
  • R 6 is preferably a hydrogen atom, a C 1-6 alkyl group, a halogen atom, an optionally substituted aryl group, an optionally substituted heterocyclic residue or a C 2-12 alkoxyalkyl group, More preferably, an aryl group which may be substituted with at least one substituent selected from the group consisting of a halogen atom, a haloalkyl group, a hydroxyl group, a C 1-6 alkyl group, a C 1-6 alkoxy group and a nitro group, It is a heterocyclic residue, or is a hydrogen atom, a C 1-6 alkyl group, a halogen atom or a C 2-12 alkoxyalkyl group.
  • Ring A is a pyridyl group, a thiazolyl group, or a thienyl group.
  • R 1 is a C 1-6 alkyl group
  • R 2 is a C 1-6 alkyl group
  • R 3 is a C 1-6 alkyl group
  • R 4 is a hydrogen atom
  • R 5 and R 6 may be the same or different and are a hydrogen atom, a C 1-6 alkyl group, a C 1-6 alkoxy group, or a halogen atom
  • X is —NH—, —N ⁇ , — CH 2 — or —O—
  • Y is —NH— or ⁇ N—
  • Z is —CO—
  • W is —NH—
  • ring A is an aryl group, preferably a phenyl group or hetero An aryl group, more preferably a pyridyl group.
  • R 1 is selected from the group consisting of a methyl group, an ethyl group, a benzyl group, hydrogen, a phenyl group, a phenyl group substituted with a methyl group, and —C—CF 3 —, and particularly preferably a methyl group It is.
  • R 2 is selected from the group consisting of a methyl group, hydrogen, —C—O—Bn— (Bn is a benzyl group), a phenyl group, and a tert-butyl group, and particularly preferably a methyl group.
  • R 3 is a methyl group, an isopropyl group, —C (Me) —C—O—Bn— (Me is a methyl group, Bn is a benzyl group), —C (Me) -C—OH— (Me is a methyl group) , Phenyl group, tert-butyl group, ethyl group, isobutyl group, methoxy group, and CF 3 , particularly preferably isopropyl group, and R 4 is hydrogen or methyl group, particularly preferably Hydrogen.
  • X represents —NH—, —N ⁇ , —N—Me—, —CH 2 —, —CH ⁇ , —CH—Me—, —O—, —N (—NH—CO—O—CH— Me) —, and —CH (R 11 ) — (where R 11 is a hydrogen atom or a C 1-6 alkyl group), and Y is —NH—, —CH 2 —.
  • -CO -, - O - CH -, - N-CO 2 -Me -, - N-CO 2 -Ph -, - N-CO 2 -tBu -, - N-CO 2 -Bn -, - N—CO 2 —Ph—, —N—Bn—, —N—Me—, —N (CONH—Ph—Cl 2 ) —, —N (CO—NH—Ph—Br) —, —N (CO— NH—Ph—Cl 2 ) —, —N (CO—NH—Ph—Cl) —, —N (CO—NH—Ph—Cl) —, —N (R 12 ) — (where R 12 is a hydrogen atom), and a single bond , Z is CO -, - CS -, - CH 2 -, and is selected from the group consisting of a single bond, W is, -NH -
  • Ring A is selected from the group consisting of a pyridyl group, a thiazolyl group, a thienyl group, a phenyl group, and a morpholine group, and particularly preferably a pyridyl group.
  • R 5 is, for example, an optionally substituted aryl group, an optionally substituted heterocyclic residue, or a C 2-12 alkoxyalkyl group, and more preferably an optionally substituted aryl group It is.
  • R 6 is preferably a hydrogen atom, a C 1-6 alkyl group, a halogen atom, an optionally substituted aryl group, an optionally substituted heterocyclic residue or a C 2-12 alkoxyalkyl group.
  • a halogen atom preferably a haloalkyl group, a hydroxyl group, a C 1-6 alkyl group, a C 1-6 alkoxy group, and a nitro group.
  • It is an aryl group or a heterocyclic residue, or is a hydrogen atom, a C 1-6 alkyl group, a halogen atom or a C 2-12 alkoxyalkyl group.
  • R 5 and R 6 are preferably independently halogen or hydrogen, and preferred halogen is fluorine or chlorine. Particularly preferably, R 5 and R 6 are both chlorine.
  • Ring A is preferably a pyridyl group.
  • FIGS. 20-1 to 20-8 show combinations of R 1 , R 2 , R 3 , R 4 , X, Y, Z, W, A, R 5 , and R 6 .
  • R 1 and R 2 are a methyl group
  • R 3 is an isopropyl group
  • R 4 is hydrogen
  • X is —NH -
  • Y is -NH-
  • X is -CO-
  • W is -NH-
  • ring A is a pyridyl group
  • R 5 and R 6 are independently hydrogen, chlorine, fluorine, bromine, methyl group, or methoxy group
  • both R 5 and R 6 are chlorine, and in this case, the bonding positions of chlorine are the 2 and 6 positions, the 3 and 5 positions, the 2 and 3 positions, the 2 and 5 positions, and the 3 and 4 positions.
  • Specific compounds of the S1P 2 receptor antagonist contained as an active ingredient in the therapeutic agent of the present invention include the following compounds.
  • pyrazolopyridine compounds and derivatives described in WO2003 / 051876 pamphlet and JP2004-123539A can be used as the S1P 2 receptor antagonist of the present invention.
  • the above compound can be synthesized by a known method.
  • the S1P 2 receptor antagonist of the present invention also includes an antagonist antibody against the S1P 2 receptor.
  • the antibody blocks the receptor by binding to the S1P 2 receptor, prevents the ligand from binding to the receptor, and inhibits signal transduction.
  • the antibody includes both a monoclonal antibody and a polyclonal antibody, and is preferably a monoclonal antibody. Also included are humanized antibodies, human antibodies, and chimeric antibodies. The antibody can be prepared by a known technique.
  • the “pharmaceutically acceptable salt” means, for example, various inorganic acid addition salts such as hydrochloride, hydrobromide, sulfate, phosphate or nitrate; acetate, propionate, succinic acid Salt, glycolate, lactate, malate, oxalate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, tosyl
  • Various organic acid addition salts such as acid salts or ascorbates; salts with various amino acids such as aspartate or glutamate are included, but are not limited thereto. In some cases, it may be a hydrate, hydrate or solvate.
  • the therapeutic agent of the present invention includes carriers, excipients, thickeners, preservatives, stabilizers, antioxidants, binders, disintegrants, wetting agents, lubricants, colorants, Ingredients such as fragrances, flavoring agents, suspending agents, emulsifiers, solubilizing agents, buffering agents, tonicity agents, surfactants, soothing agents, sulfur-containing reducing agents, absorption improvements, solid stabilization, etc.
  • Various other functional components can be appropriately blended for the purpose.
  • carriers and excipients are water-soluble or sparingly soluble, such as sugars, polysaccharides, dextrins, celluloses, synthetic or semi-synthetic polymers, amino acids, polyamino acids, proteins, phosphorus Examples thereof include lipids.
  • the administration route of the therapeutic agent of the present invention includes transdermal, intravenous, intramuscular, subcutaneous and oral.
  • the dosage form of the therapeutic agent can be appropriately set depending on the administration method. Specific examples include liquid solutions such as aqueous solutions, emulsions and suspensions, and tablets. Usually, since oral administration is preferable, an aqueous solution or a tablet is preferable.
  • the dose can be appropriately set depending on the administration method, the age, weight, and medical condition of the patient to be applied, but is preferably 0.005 to 5 mg / kg body weight, more preferably 0.005 to 1 mg / kg body weight at a time. It can be administered once a day or divided into several times every day.
  • S1P 2 receptor antagonists are [N- (1H-4-isopropyl-1,3-dimethylpyrazolo [3,4-b] pyrid-6-yl) amino-N ′-(2 , 6-Dichloropyridin-4-yl) urea was synthesized and used.
  • the compound is referred to as JTE-K1.
  • Mouse bone marrow-derived macrophages were obtained by collecting bone marrow cells from mouse femur and tibia and culturing them for 1 week in the presence of macrophage colony stimulating factor (M-CSF, Sigma). Differentiation into macrophages was confirmed by immunostaining using rat monoclonal anti-Mac-3 (macrophage marker) antibody (BD Pharmingen) and Alexa488-labeled anti-rat Ig antibody.
  • M-CSF macrophage colony stimulating factor
  • Mouse peritoneal macrophages were collected by injecting 4% thioglycolic acid into the peritoneal cavity of mice for 3-4 days, and then the peritoneal cavity was washed with cold phosphate buffered saline and collected.
  • phosphate buffered saline and 4% neutral buffered paraformaldehyde aqueous solution were injected from the mouse heart and fixed by reflux, and then the aorta from the origin of the aorta Collected to the left and right common iliac artery bifurcation.
  • the aortic atherosclerotic lesion area is determined by incising and expanding the aortic root to the abdominal aorta, staining with lipid red-O, and then calculating the area of the sclerotic lesion using image analysis software. Expressed as a percentage (% value).
  • Real-time PCR analysis was performed by synthesizing cDNA using TaqMan Reverse Transcriptase Reagents (Applied Biosystems), performing PCR using TaqMan probe (Applied Biosystems), and detecting and analyzing with an Applied Biosystems 7300 real-time PCR system.
  • Rho GTP-linked Rho
  • the amount of active Rho was quantified by the pull-down method using a glutathione-s-transferase fusion recombinant protein (binding to Sepharose beads) at the Rho binding site of Rhothekin, a protein that binds Rho (non-patent literature). 1).
  • bands were detected using anti-phosphorylated Thr850MYPT1 antibody, anti-non-phosphorylated MYPT1, anti-phosphorylated p65NF- ⁇ B antibody and anti-non-phosphorylated p65NF- ⁇ B antibody, and the density was quantified .
  • Band density was quantified with a densitometer (PDI, San Francisco, USA).
  • N3 mice and N7 mice showed no difference in weight gain between ApoE ⁇ / ⁇ S1P 2 + / + and ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ mice throughout the study period.
  • blood total cholesterol 1492 ⁇ 284 (mean ⁇ standard error) vs. 1749 ⁇ 291 mg / dl
  • neutral fat 174 ⁇ 23 vs. 97 ⁇ 22 mg / dl
  • HDL cholesterol 10 ⁇ 2 vs. 15 ⁇ 3.2 mg / dl
  • the oil red-O stained images of the aorta are shown in FIGS. 1A to 1C, and the quantitative results of the area of oil red-O-positive atherosclerotic lesions are shown in FIG.
  • the ratio of the atherosclerotic lesion area to the total lumen area of the entire aorta from the aortic root (thoracic aorta) to the abdominal aorta is ApoE-/-, S1P 2 + / + (N7), ApoE-/ It was 28.8 ⁇ 6.7, 20.7 ⁇ 6.5, and 8.0 ⁇ 0.6%, respectively, in the-, S1P 2 +/- (N7) and ApoE-/-, S1P 2 -/-(N7) mice.
  • the amount of collagen fibers in the atherosclerotic lesion was evaluated by Azan staining. There was no difference in collagen fiber mass among ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ mice, and ApoE ⁇ / ⁇ , S1P 2 + / + mice (FIGS. 5A and B and FIGS. 6A and B).
  • Macrophage infiltration in atherosclerotic lesions was evaluated by immunostaining with an anti-Mac3 antibody that specifically recognizes macrophages.
  • the Mac3 positive area of the atherosclerotic lesion was lower in ApoE ⁇ / ⁇ and S1P 2 ⁇ / ⁇ mice than in ApoE ⁇ / ⁇ and S1P 2 + / + mice (FIGS. 7A and B).
  • ⁇ -smooth muscle actin ( ⁇ -SMA) positive smooth muscle cells were determined by anti- ⁇ -SMA antibody, a vascular smooth muscle marker. Immunostained. In ApoE-/-, S1P 2 -/-mice, the ⁇ -SMA positive region of the intima was larger than in ApoE-/-, S1P 2 + / + mice, and smooth muscle cell infiltration was stronger ( 8A and B).
  • ⁇ -SMA smooth muscle actin
  • Atherosclerosis-related gene expression The effect of the presence or absence of S1P 2 receptor on the gene expression of other arteriosclerosis-related molecules in blood vessels was examined. 6-week-old male ApoE-/-, S1P 2 + / + and ApoE-/-, S1P 2 -/-were fed a high cholesterol diet, reared for 3-4 months, and the aorta removed and RT-PCR Was used to evaluate mRNA expression. In the aorta of ApoE-/-, S1P 2 -/-mice, mRNA expression of TNF- ⁇ , IFN- ⁇ , IL-6 was decreased compared to ApoE-/-, S1P 2 + / + mice ( FIG. 13A).
  • VCAM-1 an adhesion molecule that is expressed in endothelial cells and mediates infiltration of leukocytes in blood into the subendothelium, is ApoE-/-, S1P 2 -/- It was reduced compared to 2 + / + mice (FIGS. 13B and 13C).
  • the atherosclerotic lesion area in the entire aorta was 20.5 ⁇ 8.93 and 10.0 ⁇ 5.80% in the control group and the test group, respectively, and the test group showed a significantly lower value than the control group (P ⁇ 0.05).
  • SlP 2 locus SlP 2 receptor expressed in bone marrow-derived cells including the monocytes / macrophages (LacZ) mice knocked gene (SlP 2 LacZ / LacZ mice (detailed in our recently published laboratory paper Wa Du et al. Cancer Res), X-gal staining was performed using sections of aortic tissue and combined with immunostaining in arteriosclerotic vessels S1P 2 expressing cells were identified.
  • vascular endothelium, smooth muscle, and annulus mesenchymal vesicles were positive for X-gal staining (stained in blue), that is, expressed S1P 2 (FIG. 16A).
  • X-gal staining stained in blue
  • FIG. 16B Double staining of X-gal staining and Mac-3 immunostaining, which is a macrophage marker, indicates that Mac-3 positive (brown) macrophages are also positive for X-gal staining and macrophages express S1P 2 (FIG. 16C).
  • Monocytes / macrophages play an important role in the development of atherosclerosis.
  • chimeric mice were prepared using bone marrow transplantation (BMT). ApoE ⁇ / ⁇ and S1P 2 ⁇ / ⁇ mice bone marrow were transplanted into ApoE ⁇ / ⁇ and S1P 2 + / + mice that had received a lethal dose of radiation (ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ BMT mice).
  • the average atherosclerotic lesion area for the entire vascular wall area from the thoracic aorta to the abdominal aorta is about 20% on average in ApoE ⁇ / ⁇ , S1P 2 + / + BMT mice and ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ BMT mice.
  • ApoE ⁇ / ⁇ , S1P 2 ⁇ / ⁇ BMT mice showed significantly lower values than ApoE ⁇ / ⁇ , S1P 2 + / + BMT mice. From these results, it was shown that the S1P 2 receptor expressed in bone marrow-derived cells is important for the development of atherosclerosis.
  • the medicament containing the S1P 2 receptor antagonist of the present invention as an active ingredient is useful for the treatment of atherosclerosis.

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Abstract

L'invention porte sur un agent thérapeutique pour l'athérosclérose. L'agent thérapeutique pour l'athérosclérose comprend un antagoniste du récepteur S1P2 comme ingrédient actif.
PCT/JP2011/050429 2010-01-14 2011-01-13 Agent thérapeutique pour l'athérosclérose, comprenant un antagoniste du récepteur s1p2 Ceased WO2011087051A1 (fr)

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US12161625B2 (en) 2016-08-26 2024-12-10 Gilead Sciences, Inc. Substituted pyrrolizine compounds and uses thereof
US10836769B2 (en) 2018-02-26 2020-11-17 Gilead Sciences, Inc. Substituted pyrrolizine compounds and uses thereof
US11420974B2 (en) 2018-02-26 2022-08-23 Gilead Sciences, Inc. Substituted pyrrolizine compounds and uses thereof

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