Myofibroblast deactivation agent and therapeutic or prophylactic agent

Abstract

Provided is a therapeutic or prophylactic agent for a disease, a disorder, or a condition selected from the group consisting of fibrosis, organ failure accompanied by fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, the therapeutic or prophylactic agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. Also provided is a myofibroblast deactivation agent containing a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R1, R2, and R3 are each independently selected from the group consisting of hydrogen, halogen, C1-4 alkyl, C1-4 alkoxy, C1-4 alkylthio, and morpholin-4-yl, wherein the C1-4 alkyl, the C1-4 alkoxy, and the C1-4 alkylthio may be substituted with a substituent selected from hydroxy and -NR5R6; R4 is selected from the group consisting of C1-4 alkyl and C1-4 alkoxy; and R5 and R6 are each independently selected from the group consisting of hydrogen and C1-4 alkyl.]

Description

Myofibroblast deactivators and therapeutic or prophylactic agents

[0001] The present invention relates to a therapeutic or prophylactic agent for a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure associated with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway.

[0002] Fibrosis is a condition characterized by excessive deposition of extracellular matrix, such as collagen, leading to organ dysfunction. Fibrosis, which causes fibrosis, is a part of the wound healing process and occurs in a variety of organs and tissues, including the heart, brain, digestive tract, skin, lungs, kidneys, liver, pancreas, hematopoietic organs, retroperitoneum, mediastinum, and joints. Tissue wound healing is achieved through the differentiation of fibroblasts and mesenchymal cells into myofibroblasts, which then close the wound and synthesize and deposit extracellular matrix such as collagen. After wound healing, myofibroblasts disappear through apoptosis, but due to persistent damage such as inflammation, myofibroblasts may remain without undergoing apoptosis. In this case, the extracellular matrix increases abnormally, causing excessive fibrosis. Excessive fibrosis causes organ dysfunction and progresses the pathogenesis, making the treatment and prevention of fibrosis a crucial issue.

[0003] For example, hepatic stellate cells (HSCs) play a major role in the fibrosis process in the liver. The liver consists of hepatocytes (hepatocytes) and non-parenchymal cells (hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, Pit cells, etc.), and the liver's connective tissue is composed of extracellular matrix (collagen, etc.) and cells localized therein. When hepatic stellate cells are activated and transformed, the proliferation and accumulation of connective tissue are promoted. Hepatic stellate cells in a normal liver (also called "quiescent hepatic stellate cells") produce little extracellular matrix, but when activated, they change their properties and morphology to myofibroblasts (also called "activated hepatic stellate cells"), and are known to synthesize and produce large amounts of extracellular matrix as the number of cells increases. The proliferation and accumulation of connective tissue in the liver that occurs in this way is thought to cause liver dysfunction, further damage to hepatocytes, and further proliferation and accumulation of connective tissue, leading to cirrhosis and liver cancer.

[0004] To recover from fibrosis, it is thought that targeting myofibroblasts such as activated hepatic stellate cells and deactivating or suppressing their activation is effective (Non-Patent Document 1). In previous research, the present inventors succeeded in stably preparing activated hepatic stellate cells by inducing quiescent hepatic stellate cells from iPS cells, with the aim of developing a drug discovery screening system that reflects the activation process of hepatic stellate cells in a culture system (Patent Documents 1, 2, Non-Patent Document 2). However, effective treatments and therapeutic drugs for fibrosis have not yet been established.

[0005] International Publication No. 2016 / 148216, International Publication No. 2020 / 166726

[0006] Lv,Ke et al.,Front.Immunol.,2022;13:1042983Koui Y,et al.Stem Cell Reports.2021

[0007] There is a need for new medicines that are effective in treating or preventing conditions such as fibrosis.

[0008] As a result of diligent research, the inventors have discovered that certain pyridopyrimidine compounds are effective in treating or preventing diseases, disorders, or conditions selected from the group consisting of fibrosis, organ failure associated with fibrosis, and organ failure related to downregulation of the AMPK signaling pathway, and have completed the present invention. In particular, the present invention is based on the discovery that certain pyridopyrimidine compounds have deactivating effects on activated myofibroblasts, such as activated hepatic stellate cells, and activating effects on AMPK. The present invention is, for example, as follows:

[0009] [1] A therapeutic or prophylactic agent for a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C１～４ Selected from the group consisting of alkylthio and morpholin-4-yl, said C １～４ alkyl, C １～４ alkoxy, and C １～４ alkylthio may be substituted with a substituent selected from hydroxy and -NR ５ R ６ , and R ４ is selected from the group consisting of C １～４ alkyl and C １～４ alkoxy, and R ５ and R ６ are each independently selected from the group consisting of a hydrogen atom and C １～４ alkyl. [2] At least one of R １ , R ２ , and R ３ is not a hydrogen atom, and R ４ is C １～２ alkyl. The therapeutic or prophylactic agent according to [1]. [3] The therapeutic or prophylactic agent according to [1] or [2], wherein the compound is selected from the group consisting of and pharmaceutically acceptable salts thereof. [4] R １ , R[[ID=​​​​​​​​​​​​​​​​​​A therapeutic or prophylactic agent according to any one of [1] to [3], which may be substituted with substituents selected from [1] to [3]. [5] A therapeutic or prophylactic agent according to any one of [1] to [4], wherein the disease, disorder or condition is fibrosis. [6] A therapeutic or prophylactic agent according to claim [5], wherein the disease, disorder or condition is fibrosis occurring in the heart, brain, gastrointestinal tract, skin, lungs, kidneys, liver, pancreas, hematopoietic organs, retroperitoneum, mediastinum, joints, muscles, blood vessels, eyes, breasts, or genitals. [7] A therapeutic or prophylactic agent according to any one of [1] to [5], wherein the fibrosis is fibrosis occurring in the liver, kidneys, or heart. [8] A therapeutic or prophylactic agent according to any one of [1] to [7], wherein the organ failure is at least one selected from hepatic failure, renal failure, and heart failure. [9] A therapeutic or prophylactic agent according to any one of [1] to [8] that promotes the expression of TCF21.

[10] A therapeutic or prophylactic agent according to any one of [1] to [9], which (i) reduces the expression level of at least one fibrosis marker, (ii) increases the expression level of at least one quiescent hepatic stellate cell marker, (iii) increases the expression level of at least one liver regeneration induction marker, (iv) reduces the accumulation of F-actin, and / or (v) transitions activated myofibroblasts into a quiescent state. [10a] The therapeutic or prophylactic agent according to

[10] , wherein the fibrosis marker comprises at least one selected from extracellular matrix selected from collagen, laminin, and fibronectin; actin; transforming growth factor (TGFB); PAI1; and WNT, the quiescent hepatic stellate cell marker comprises at least one selected from LHX2, LRAT, NES, and NGFR, and the liver regeneration induction marker comprises at least one selected from MDK and PTN. [10b] The therapeutic or prophylactic agent according to

[10] , wherein the fibrosis marker comprises at least one selected from COL1A1, COL1A2, COL3A1, ACTA2, TGFB1, and PAI1, the quiescent hepatic stellate cell marker comprises at least one selected from LHX2 and LRAT, and the liver regeneration induction marker comprises at least one selected from MDK and PTN.

[0010]

[11] The therapeutic or prophylactic agent according to any one of [1] to [4], wherein the disease, disorder or condition is heart failure. [11a] The therapeutic or prophylactic agent according to

[11] , wherein the disease, disorder or condition is heart failure with fibrosis.

[12] The therapeutic or prophylactic agent according to

[11] , wherein (i) increases mitochondrial metabolism in cardiomyocytes, (ii) induces activation of AMPK in cardiomyocytes, and / or (iii) induces at least one of inhibiting nuclear translocation of MRTF-A, decreasing SRF transcriptional activity, or inhibiting phosphorylation of Src in cardiac fibroblasts.

[13] A therapeutic or prophylactic agent according to

[11] or

[12] , which (i) reduces the expression level of at least one fibrosis marker gene selected from COL1A1, COL1A2, POSTN, CCN2, MEOX1, and ACTA2, (ii) increases the expression level of the metabolism-related marker gene PPARGC1A, and / or (iii) reduces the expression level of at least one heart failure marker selected from NPPA (ANP) and NPPB (BNP).

[14] A therapeutic or prophylactic agent according to any one of [1] to

[13] , which inhibits the Rho-GTPase signaling pathway in myofibroblasts.

[0011]

[15] A myofibroblast deactivator comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl.

[16] The deactivator according to

[15] , wherein the myofibroblasts are activated hepatic stellate cells, activated cardiac fibroblasts, or activated renal fibroblasts. [16a] The deactivator according to any one of

[15] to

[16] , wherein the myofibroblasts are activated hepatic stellate cells.

[17] An AMPK activator comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyls. ]

[18] R １ , R ２ , and R ３ At least one of them is not a hydrogen atom, but R ４ C １～２ A deactivating agent according to any one of

[15] to

[17] , wherein the compound is A deactivating agent according to any one of

[15] to

[18] , selected from the group consisting of pharmaceutically acceptable salts thereof. [18b] R １ , R ２ , and R ３is independently selected from the group consisting of a hydrogen atom, a halogen atom, C １～４ alkyl, C １～４ alkoxy, C １～４ alkylthio, and morpholin-4-yl, wherein the C １～４ alkyl, C １～４ alkoxy, and C １～４ alkylthio may be substituted with a substituent selected from -NR ５ R ６ The therapeutic or prophylactic agent according to any one of

[15] to

[18] .

[0012]

[19] Use of a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof for the production of a medicament for the treatment or prevention of a disease, disorder or condition selected from the group consisting of fibrosis, organ failure accompanied by fibrosis, and organ failure associated with down-regulation of the AMPK signaling pathway. [In the formula, R １ , R ２ , and R ３ are each independently selected from the group consisting of a hydrogen atom, a halogen atom, C １～４ alkyl, C １～４ alkoxy, C １～４ alkylthio, and morpholin-4-yl, wherein the C １～４ alkyl, C １～４ alkoxy, and C １～４ alkylthio may be substituted with a substituent selected from hydroxy and -NR ５ R ６ ; R ４ is selected from the group consisting of C １～４ alkyl and C １～４ alkoxy; R ５ and R ６ are each independently selected from the group consisting of a hydrogen atom and C １～４ alkyl. ]

[0013]

[20] A method for treating or preventing a disease, disorder or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, comprising administering a therapeutically effective amount of a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof to a subject in need thereof. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl groups.

[0014]

[21] A compound represented by formula (I) below or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease, disorder or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl groups.

[0015]

[22] Use of a compound represented by formula (I) below or a pharmaceutically acceptable salt thereof for the treatment or prevention of a disease, disorder or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl groups.

[0016]

[23] R １ , R ２ , and R ３ At least one of them is not a hydrogen atom, but R ４ C １～２A compound or salt for use according to the use described in

[19] or

[22] , the method described in

[20] , or the use described in

[21] , which is alkyl.

[24] The compound is A compound or salt for use as described in any of

[19] ,

[22] to

[23] , the method described in any of

[20] ,

[23] , or any of

[21] ,

[23] , selected from the group consisting of pharmaceutically acceptable salts thereof.

[25] R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is -NR ５ R ６The compound or salt for use according to any of

[19] ,

[22] to

[24] , the method according to any of

[20] ,

[23] to

[24] , or any of

[21] ,

[23] to

[24] , which may be substituted with substituents selected from.

[26] The compound or salt for use according to any of

[19] ,

[22] to

[25] , the method according to any of

[20] ,

[23] to

[25] , or any of

[21] ,

[23] to

[25] , which is the disease, disorder or condition, which is fibrosis.

[27] The compound or salt for use according to any of

[19] ,

[22] to

[26] , the method according to any of

[20] ,

[23] to

[26] , or any of

[21] ,

[23] to

[26] , which is the disease, disorder or condition, which is fibrosis.

[28] The use according to any one of

[19] ,

[22] to

[27] , the method according to any one of

[20] ,

[23] to

[27] , or the compound or salt for use according to any one of

[21] ,

[23] to

[27] , wherein the fibrosis is fibrosis occurring in the liver, kidney, or heart.

[29] The use according to any one of

[19] ,

[22] to

[28] , the method according to any one of

[20] ,

[23] to

[28] , or the compound or salt for use according to any one of

[21] ,

[23] to

[28] , which promotes the expression of TCF21.

[30] A compound or salt for use according to any one of

[19] ,

[22] to

[29] , the method according to any one of

[20] ,

[23] to

[29] , or any one of

[21] ,

[23] to

[29] , which (i) reduces the expression level of at least one fibrosis marker, (ii) increases the expression level of at least one quiescent hepatic stellate cell marker, (iii) increases the expression level of at least one hepatic regeneration induction marker, (iv) reduces the accumulation of F-actin, and / or (v) transitions activated myofibroblasts into a quiescent state.[30a] The use, method, or compound or salt according to

[30] , wherein the fibrosis marker comprises at least one selected from extracellular matrix selected from collagen, laminin, and fibronectin; actin; transforming growth factor (TGFB); PAI1; and WNT; the quiescent hepatic stellate cell marker comprises at least one selected from LHX2, LRAT, NES, and NGFR; and the liver regeneration induction marker comprises at least one selected from MDK and PTN. [30b] The use, method, or compound or salt according to

[30] , wherein the fibrosis marker comprises at least one selected from COL1A1, COL1A2, COL3A1, ACTA2, TGFB1, and PAI1; the quiescent hepatic stellate cell marker comprises at least one selected from LHX2 and LRAT; and the liver regeneration induction marker comprises at least one selected from MDK and PTN.

[0017]

[31] The use described in

[19] or

[22] , the method described in

[20] , or the compound or salt for use described in

[21] , wherein the disease, disorder or condition is heart failure. [31a] The use, method, or compound or salt described in

[31] , wherein the disease, disorder or condition is heart failure with fibrosis.

[32] The use, method, or compound or salt described in

[31] , wherein (i) increases mitochondrial metabolism in cardiomyocytes, (ii) induces activation of AMPK in cardiomyocytes, and / or (iii) induces at least one of the following in cardiac fibroblasts: inhibition of nuclear translocation of MRTF-A, reduction of SRF transcriptional activity, or inhibition of Src phosphorylation.

[33] Uses, methods, or compounds or salts according to

[31] or

[32] that (i) reduce the expression level of at least one fibrosis marker gene selected from COL1A1, COL1A2, POSTN, CCN2, MEOX1, and ACTA2; (ii) increase the expression level of the metabolism-related marker gene PPARGC1A; and / or (iii) reduce the expression level of at least one heart failure marker selected from NPPA (ANP) and NPPB (BNP).

[34] Uses according to any one of

[19] ,

[22] to

[33] , methods according to any one of

[20] ,

[23] to

[33] , or compounds or salts for use according to any one of

[21] ,

[23] to

[33] that inhibit the Rho-GTPase signaling pathway in myofibroblasts.

[0018] According to one embodiment of the present invention, a novel therapeutic or prophylactic agent is provided for a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure associated with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway. In particular, according to one embodiment of the present invention, the pyridopyrimidine compound of the present invention has a deactivating effect on myofibroblasts such as activated hepatic stellate cells and can function as a deactivator of myofibroblasts. According to this embodiment, it is possible to deactivate myofibroblasts, which are activated fibroblasts, and restore them to a quiescent cell morphology and cellular characteristics, thereby improving fibrosis and / or normalizing or restoring tissue function, and the compound of this embodiment can be used for the treatment or prevention of fibrosis. Furthermore, according to one embodiment of the present invention, the pyridopyrimidine compound of the present invention also has a deactivating effect on senescent hepatic stellate cells. In addition, according to one embodiment of the present invention, the compound described herein can suppress the activation of myofibroblasts and can function as a myofibroblast activation inhibitor (anti-fibrotic agent). Therefore, according to this embodiment, it is possible to suppress collagen expression and / or suppress fibrosis, and the compound of this embodiment can be used for the treatment or prevention of fibrosis. Furthermore, according to this embodiment, the compound can be used to treat or prevent organ failure accompanied by fibrosis. Moreover, according to one embodiment of the present invention, the compound can induce the activity of AMPK. According to a specific embodiment of the present invention, the compound has the effect of improving mitochondrial metabolism in cardiomyocytes. Therefore, it can function as a therapeutic agent that restores the function of the organ itself not only in diseases accompanied by fibrosis, but also in organ failure related to downregulation of the AMPK signaling pathway (for example, heart failure, especially heart failure accompanied by mitochondrial dysfunction), and the compound can be used to treat or prevent organ failure such as heart failure.

[0019] Figure 1 shows the expression levels of various marker genes during the deactivation induction of activated hepatic stellate cells by the pyridopyrimidine compound PD1. ACTA2 and COL1A1 were used as fibrosis marker genes, TCF21 as a deactivation marker gene, LHX2 and LRAT as quiescent hepatic stellate cell markers, and MDK and PTN as liver regeneration induction markers. The expression of fibrosis markers decreased in a PD1 concentration-dependent manner, while the expression of deactivation markers, quiescent hepatic stellate cell markers, and liver regeneration induction markers increased. Each sample size was 5. Figure 2 shows the results of cell morphology observation by fluorescence microscopy during the deactivation induction of activated hepatic stellate cells. Figures 3(A) and 3(B) show the evaluation results of the ability of pyridopyrimidine compound PD1 to suppress the activation of hepatic stellate cells during the activation process. Day 5 represents activated hepatic stellate cells cultured for 5 days from quiescent hepatic stellate cells, Day 7 (DMSO) represents activated hepatic stellate cells cultured for 5 days from quiescent hepatic stellate cells with the addition of DMSO and cultured for 2 days, and Day 7 (PD1) represents activated hepatic stellate cells cultured for 5 days from quiescent hepatic stellate cells with the addition of PD1 and cultured for 2 days. Figure 3(A) shows the results of principal component analysis. Figure 3(B) shows the expression levels of various marker genes in each cell. COL1A1, COL1A2, COL3A1, COL4A1 were used as fibrosis marker genes, TGFB1, TGFB2, TGFB3 as fibrosis-promoting factors, CXCL12 as a chemokine family gene, MMP7, MMP13 as matrix metalloproteinase family genes, and TCF21 as a deactivation marker gene. Day 7 (DMSO) showed increased expression of fibrosis markers compared to day 5, suggesting that day 5 was during the activation process of hepatic stellate cells. When PD1 was added during the activation process of hepatic stellate cells (day 5), it decreased the expression of fibrosis markers and increased the expression of deactivation markers compared to the negative control (DMSO). The sample size for each group was 3. Figures 4(A) and 4(B) show the evaluation results of the deactivation-inducing ability of the pyridopyrimidine compound PD1 to activate hepatic stellate cells undergoing cellular senescence.Xray day 8 represents senescent hepatic stellate cells (13 days total culture) obtained by irradiating activated hepatic stellate cells (5 days cultured) with radiation (X-rays) and culturing them for an additional 8 days; Xray day 10 (DMSO) represents senescent hepatic stellate cells (13 days cultured) with DMSO added and cultured for 2 days (15 days total culture); and Xray day 10 (PD1) represents senescent hepatic stellate cells (13 days cultured) with PD1 added and cultured for 2 days (15 days total culture). Figure 4(A) shows the results of principal component analysis. Figure 4(B) shows the expression levels of various marker genes in each cell. The fibrosis marker gene, quiescent hepatic stellate cell marker gene, and deactivation marker gene used in Figure 4(B) are the same as in Figure 3. When PD1 was added (Xray day 10 (PD1)), the expression of fibrosis markers decreased and the expression of deactivation markers increased compared to the negative controls, Xray day 10 (DMSO) and Xray day 8 (before PD1 addition). The sample size for each group was 3. Figure 4(C) shows the results of Wester blot analysis of each cell group. qHSC (day 0) represents quiescent hepatic stellate cells, aHSC days 1, 3, and 5 represent activated hepatic stellate cells cultured for 1, 3, and 5 days respectively from quiescent hepatic stellate cells, and Xray d1, d3, d5, d7, d10, and d12 represent cells that were irradiated with X-rays after 5 days of culture and then cultured for 1, 3, 5, 7, 10, and 12 days respectively. p21 and p16 are cellular senescence markers, and β-actin is an actin protein and a loading control. An increase in p16 protein expression was observed from X-ray d5 (5 days after X-ray irradiation), confirming the induction of cellular senescence. Figure 5 shows the expression levels of various marker genes in the deactivation induction of activated hepatic stellate cells by the pyridopyrimidine compound PD1 and the existing pulmonary fibrosis treatments Pirespa and Ofev. COL1A1, COL1A2, and COL3A1 were used as fibrosis markers, TGFB1 as a fibrosis-promoting factor, TCF21 as a deactivation marker, and PTN as a liver regeneration induction marker. Compared to Pirespa and Ofev, PD1 suppressed the expression of fibrosis markers and fibrosis-promoting factors, and promoted the expression of deactivation markers and liver regeneration induction markers. The sample size for each study was 3 or more.Figure 6 shows the expression levels of the fibrosis marker (ACTA2) gene during the deactivation induction of activated hepatic stellate cells by various pyridopyrimidine compounds (PD1, PD2, PD3, PD4, or PD5). Each sample size is 3 or more. Figure 7 shows the expression levels of the fibrosis marker (COL1A1) gene during the deactivation induction of activated hepatic stellate cells by various pyridopyrimidine compounds (PD1, PD2, PD3, PD4, or PD5). Each sample size is 3 or more. Figure 8 shows the expression levels of the deactivation marker (TCF21) gene during the deactivation induction of activated hepatic stellate cells by various pyridopyrimidine compounds (PD1, PD2, PD3, PD4, or PD5). Each sample size is 3 or more. Figure 9 shows the results of microscopic observation of cell morphology during the deactivation induction of activated hepatic stellate cells by various pyridopyrimidine compounds (PD1, PD2, PD3, PD4, or PD5). Figure 10 shows a conceptual diagram of animal experiments using a hepatic fibrosis model mouse. Figure 11 shows the evaluation results of liver tissue sections from a hepatic fibrosis model mouse using Sirius Red staining. Collagen fibers were observed in the Control (DMSO administration group), but disappeared in the PD1 administration group. Figure 12 shows the expression levels of marker genes in the liver tissue of a hepatic fibrosis model mouse. Col1a1, Col1a2, and Col3a1, which are fibrosis markers, were used as marker genes. In the PD1 administration group, the expression levels of fibrosis markers were reduced compared to the Control (DMSO administration group). Figure 13 shows the evaluation results of hepatomegaly (liver / body weight ratio) and liver function (serum total protein, albumin, and ALT levels) after PD1 administration in a hepatic fibrosis model mouse. The liver / body weight ratio was significantly reduced with PD1 administration. Serum total protein, albumin, and ALT levels were significantly improved with PD1 administration. Figure 14 shows a conceptual diagram of animal experiments using a mouse model of renal fibrosis induced by unilateral ureteral ligation. Figure 15 shows the results of the drug efficacy evaluation of PD1 in a mouse model of renal fibrosis induced by unilateral ureteral ligation. Administration of PD1 significantly reduced the expression of fibrosis marker genes (Col1a1, Col1a2, Col3a1, Tgfb1). Figure 16 shows a conceptual diagram of animal experiments using a mouse model of heart failure induced by transverse aortic coarctation. Figure 17 shows the results of cardiac ultrasound examination in a mouse model of heart failure induced by transverse aortic coarctation.The graph shows the temporal changes in ventricular septal thickness (IVS (mm)), left ventricular posterior wall thickness (LVPW (mm)), left ventricular end-diastolic diameter (LVDd (mm)), left ventricular end-systolic diameter (LVDs (mm)), and left ventricular diameter shortening percentage (FS (%)). preTAC indicates the preoperative period before transverse aortic coarctation (TAC), and pTAC1W-5W indicates 1-5 weeks post-TAC, respectively. In the PD1 administration group, the increase in LVDd and LVDs after 2 weeks post-surgery, which was observed in the negative control group, was significantly suppressed (suppression of cardiac hypertrophy), and the decrease in FS was suppressed (improvement of systolic function). Figure 18 shows FAST green and picrosilius red stained images of a mouse model of heart failure induced by transverse aortic coarctation. The fibrotic area was reduced in the PD1 administration group compared to Control (DMSO administration group). Figure 19 shows the expression levels of heart failure marker genes in a mouse model of heart failure induced by transverse aortic coarctation. In the figure, Sham represents sham surgery, TAC5W represents the negative control group (DMSO administration group) 5 weeks post-TAC surgery, and PD1 represents the PD1 administration group 5 weeks post-TAC surgery. The sample size is n=2 for each group. In the PD1 administration group, the expression levels of heart failure markers (ANP and BNP) were significantly reduced (improved cardiac function). Figure 20 is a schematic diagram showing the mechanism of action of pyridopyrimidine compounds (PD) (e.g., PD1) according to the present invention on cardiomyocytes and fibroblasts. The left figure shows the effect on cardiomyocytes (improvement of mitochondrial metabolism via the AMPK-PGC1α pathway), and the right figure shows the effect on activated fibroblasts (deactivation via inhibition of the Rho-GTPase / MRTF-A pathway). Figure 21 shows the therapeutic effect of PD1 on pressure overload heart failure model (TAC) mice. Figure 21A shows the experimental protocol, Figure 21B shows the temporal changes in cardiac function (LVDd, LVDs, FS) by transthoracic echocardiography, Figure 21C shows picrosilius red staining of cardiac tissue and quantification of fibrotic area (scale bar in the figure is 100 μm), and Figure 21D shows DAPI / WGA fluorescence staining of myocardial tissue sections (DAPI: blue, WGA: green, scale bar in the figure is 20 μm). Figure 22 shows an overview of single-nuclear RNA-seq (snRNA-seq) analysis in a pressure-overload heart failure model mouse. Figure 22A shows the experimental protocol, and Figure 22B shows the UMAP plot and cell composition ratio of all cells. Figure 23 shows the results of subcluster analysis of cardiac fibroblasts in snRNA-seq.Figure 23A shows the UMAP plot, Figure 23B shows the cluster composition ratio for each group, and Figure 23C shows the violin plot of representative fibrosis marker gene expression. Figure 24A shows the results of co-expression gene network analysis and gene ontology analysis for the gene group upexpressed in cluster 0 of cardiac fibroblasts, and Figure 24B shows a schematic diagram of the Rho-GTPase signaling pathway. Figure 25 shows the results of RNA velocity analysis of cardiac fibroblasts. It shows the direction (vector) of cell state transitions in the PD administration group (pTAC + PD). Figure 26 is a conceptual diagram of cell state transitions showing plasticity in cardiac fibroblasts. Figure 27 shows the results of subcluster analysis of cardiomyocytes in snRNA-seq. Figure 27A shows the UMAP plot, Figure 27B shows the cluster composition ratio for each group, and Figure 27C shows the violin plot of expression of mitochondrial metabolism-related genes, etc. Figure 28 shows the effect of therapeutic administration of PD1 to a mouse model of advanced heart failure (administration started 6 weeks post-TAC surgery). Figure 28A shows the experimental protocol, Figure 28B shows the temporal changes in cardiac function (IVS, LVPW, LVDd, LVDs, FS) by transthoracic echocardiography, and Figure 28C shows the picrosilius red staining image of cardiac tissue and the quantitative results of fibrosis area (scale bar in the figure is 100 μm). Figure 29 shows the results of in vitro analysis using mouse cardiac fibroblasts. Figure 29A shows the experimental protocol, Figure 28B shows the immunohistochemical staining images of MRTF-A and actin (phaloidin) (DAPI: blue, Phalloidin: green, Mrtf-A: red), and Figure 29C shows the quantitative results of fluorescence intensity of the immunohistochemical staining images (MRTF-A and actin). Figure 30 shows the results of in vitro analysis using mouse cardiac fibroblasts. Figure 30A shows the expression level of the Col1a1 gene, Figure 30B shows the phosphorylation analysis of Src and Smad2 by Western blotting, and Figure 30C shows the quantitative results of the phosphorylation levels of Src and Smad2 (pSrc / Src, pSmad2 / Smad2). Figure 31 shows the analysis results using cardiac fibroblasts derived from human heart failure patients.Figure 31A shows the experimental protocol, Figure 31B shows immunohistochemical staining images of MRTF-A and actin (phalloidin) (DAPI: blue, Phalloidin: green, Mrtf-A: red) (scale bar in the figure indicates 50 μm), and Figure 31C shows the expression levels of representative fibrosis marker genes (COL1A1, POSTN, ACTA2) (normalized by RPS28 expression level, n=3 for each group). The fibrosis marker genes (COL1A1, POSTN, ACTA2) are representative fibroblast activation and extracellular matrix (collagen fiber) related genes. Figure 32 shows the results of gene expression analysis using human iPS cell-derived cardiomyocytes (iPSC-CM). Figure 32A shows the pathway analysis by RNA-seq (bubble plot) when PD1 was administered under normal culture conditions, and Figure 32B shows the results of GSEA analysis. Figure 33 shows oxidative stress (H. ２ O ２ Figure 33A shows the results of metabolic analysis of iPSC-CM under oxidative stress. Figure 33A is a Western blot image showing the phosphorylation level of AMPK, Figure 33B shows the change in mitochondrial respiratory capacity (oxygen consumption rate: OCR) over time, and Figure 33C shows the quantitative results of respiratory reserve and maximal respiratory capacity. Each group has n=10. Figure 34 shows the results of contractile function analysis of iPSC-CM under oxidative stress. Figure 34A shows the effect of PD1 on calcium transients (waveform, amplitude), and Figure 34B shows the effect of PD1 on contractile behavior (beat rate, contraction velocity, deformation distance). Figure 35 shows the evaluation results of mitochondrial ROS (mtROS) in iPSC-CM. The left image is a fluorescence microscope image (Hoechst, mtROS), and the right image shows the quantitative results of fluorescence intensity. Figure 36 shows the evaluation results of mitochondrial membrane potential (MT-1) in iPSC-CM. The image on the left is a fluorescence microscope image (Hoechst, MT-1), and the image on the right shows the quantitative results of the fluorescence intensity.

[0020] Embodiments of the present invention will be described below. The scope of the present invention is not limited to these descriptions, and other examples may be modified and implemented as appropriate, as long as they do not impair the spirit of the invention. The upper and lower limits of the numerical ranges described herein can be combined arbitrarily. For example, if "A to B" and "C to D" are described, the ranges "A to D" and "C to B" are also included in the scope of the present invention as numerical ranges. Ｘ～Ｙ In the quotation marks, X and Y represent the number of carbon atoms. For example, "C １～４ " indicates that the number of carbon atoms is between 1 and 4. The term "approximately" means that, when referring to a numerical value or range of numerical values, the referred numerical value or range of numerical values ​​is an approximation within experimental variability (or within statistical experimental error), and that the numerical value or range of numerical values ​​varies by 1% to 15% (e.g., 1% to 10%, especially 1% to 5%) of the stated numerical value or range of numerical values. In some embodiments, approximately means within a standard deviation using measurements that are generally accepted in the art. In some embodiments, "approximately" means a range of + / - 10% of a particular value. In some embodiments, approximately means a range of + / - 10% of a particular value.

[0021] 1. Definitions In this specification, "alkyl group" means a saturated aliphatic hydrocarbon group consisting of a linear, branched, cyclic, or combination thereof. A cyclic alkyl group is also called a cycloalkyl group. The number of carbon atoms in an alkyl group is not particularly limited, but for example, it may have 1 to 4 carbon atoms (C １～４ ) For example, C １～ C ８ Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, and cyclobutyl. Alkyl groups may have one or more substituents. In this specification, "alkoxy group" refers to a group in which an oxygen atom (O) is bonded to the terminus of the alkyl group. Examples include saturated alkoxy groups that are linear, branched, cyclic, or combinations thereof. The number of carbon atoms in an alkoxy group is not particularly limited, but for example, it may have 1 to 4 carbon atoms (C １～４) Specifically, examples include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, etc. The alkoxy may have one or more substituents. In this specification, "alkylthio group" refers to a structure in which the alkyl group is bonded to a sulfur atom, and examples include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof. The number of carbon atoms in the alkylthio group is not particularly limited, but for example, it may have 1 to 4 carbon atoms (C １～４ ) The alkylthio may have one or more substituents. In this specification, "halogen atom" is a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), or an iodine atom (I). For example, the halogen atom is a fluorine atom (F) or a chlorine atom (Cl). In this specification, substituents on alkyl, alkoxy, and alkylthio are not particularly limited, but examples include alkyl groups, alkylene groups, alkynyl groups, hydroxyl groups (OH), alkoxy groups, halogen atoms (which may be any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), cyano groups, amino groups, mono- or disubstituted amino groups, substituted silyl groups, nitro groups, azide groups, aryl groups, heteroaryl groups, carbocyclic groups, heterocyclic groups, and acyl groups.

[0022] In this specification, "pharmaceutically acceptable salt" means that it does not cause any decrease in safety or concern about toxicity. Pharmaceutically acceptable salts are not particularly limited, but include, for example, salts with acids or salts with bases. Examples of salts with acids include inorganic salts such as hydrochloride, hydrobromide, sulfate, and phosphate, and organic salts such as formic acid, acetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, tartaric acid, stearic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and trifluoroacetic acid. Examples of salts with bases include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, organic base salts such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, arginine, and lysine, and ammonium salts.

[0023] In this specification, "pharmaceutically acceptable carrier" means a solvent and / or additive that can be commonly used in the pharmaceutical technology field and does not cause any safety concerns or toxicity concerns. Examples of pharmaceutically acceptable solvents include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. These are preferably sterilized and, if necessary, adjusted to be isotonic with blood. Examples of pharmaceutically acceptable additives include excipients, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring and deodorizing agents, analgesics, stabilizers, and isotonic agents.

[0024] The terms "effective amount" or "therapeutic effective amount" of the compound in this invention refer to the amount of the active compound that elicits a biological or medical response to a target, or that relieves symptoms, alleviates a condition, slows or delays disease progression, or prevents a disease.

[0025] In this specification, “subject” refers to an animal. Examples of subjects include, but are not limited to, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, birds, and similar animals. In preferred embodiments, the subject is a mammal, most preferably a human.

[0026] In this specification, "myofibroblasts" refers to fibroblasts characterized by the expression of α-SMA, and is also called activated fibroblasts. Myofibroblasts appear in various organs. Myofibroblasts are thought to differentiate from mesenchymal cells such as fibroblasts, epithelial cells, and vascular pericytes present in various organs and the circulating blood, and are involved in various organ fibroses and the regeneration of various organs (e.g., Pakshir, P et al., Journal of Cell Science, 2020, 133(13), jcs227900; Niu, C et al., Front. Cell Dev. Biol., 24 October 2024, 12:1490315, etc.). In this invention, myofibroblasts include activated stellate cells (activated stellate cells) that exhibit a myofibroblast-like phenotype characterized by the expression of α-smooth muscle actin (α-SMA). Myofibroblasts can be identified by immunostaining using detectably labeled anti-α-SMA antibodies. Furthermore, myofibroblasts can be identified by the expression of the Acta2 gene in RNA-seq analysis, etc. It is primarily ACTA2-positive myofibroblasts that induce fibrosis in many organs and tissues. Examples of such myofibroblasts include activated hepatic stellate cells, activated cardiac fibroblasts, activated renal fibroblasts, activated pulmonary fibroblasts, activated cutaneous fibroblasts, activated gastrointestinal fibroblasts, activated ocular fibroblasts, activated uterine fibroblasts, and activated bone marrow fibroblasts.

[0027] In this specification, "astrocytosis" typically refers to astrocyte-like cells that have vitamin A (VA) storage capacity. In this invention, astrocytosis encompasses all states: quiescent, activated, and deactivated. Astrocytosis is normally present in various organs such as the liver and pancreas, and in this invention, astrocytosis encompasses these cells. For example, astrocytosis in the pancreas and liver are called pancreatic astrocytosis and hepatic astrocytosis, respectively. When astrocytosis is activated by stimuli such as inflammation, it is known to exhibit a myofibroblast-like phenotype characterized by the expression of α-smooth muscle actin (α-SMA), proliferate, and produce large amounts of collagen, which causes fibrosis. In some embodiments, astrocytosis refers to hepatic astrocytosis. In this specification, hepatic astrocytosis in a quiescent state is described as "quiescent hepatic astrocytosis," hepatic astrocytosis in an activated state is described as "activated hepatic astrocytosis," and hepatic astrocytosis in a deactivated state is described as "deactivated hepatic astrocytosis" or "quiescent-like hepatic astrocytosis." In some embodiments, the stellate cells may be senescent stellate cells (e.g., senescent hepatic stellate cells).

[0028] In this specification, "activated hepatic stellate cells" are not particularly limited as long as they are hepatic stellate cells in an activated state, and may be, for example, induced from quiescent hepatic stellate cells or spontaneously occurring. Activated hepatic stellate cells obtained by conventionally known methods may be used as activated hepatic stellate cells. Even dormant (quiescent) hepatic stellate cells are known to produce extracellular matrix such as collagen to form connective tissue, which is important for the normal organization of organ structure. On the other hand, when hepatic stellate cells are activated, they begin to produce large amounts of excess collagen and inflammatory molecules. Activated hepatic stellate cells can be prepared, for example, using quiescent hepatic stellate cells derived from pluripotent stem cells, and specifically, they can be prepared using quiescent hepatic stellate cells obtained by methods described in Patent Document 2 (International Publication No. 2020 / 166726) and Non-Patent Document 2 (Koui Y, et al., Stem Cell Reports, 2021). In this specification, "activated hepatic stellate cells" include activated stellate cells that have undergone cellular senescence (activated senescent stellate cells).

[0029] In this specification, "deactivating myofibroblasts" refers to the transformation of an activated myofibroblast into a quiescent-like cell morphology. This transformation to a "quiescent-like state" includes not only a complete return to the quiescent state, but also a transition from an "activated state" with high extracellular matrix (collagen, etc.) production capacity to a "quiescent-like state" with low extracellular matrix production capacity (for example, a state in which α-SMA is expressed but collagen expression is reduced) (see Figures 23C and 26). For example, in the case of activated stellate cells, "deactivation" refers to the transformation of an activated stellate cell (for example, an activated hepatic stellate cell) into a quiescent-like (deactivated) stellate cell (for example, a deactivated hepatic stellate cell (quiescent-like hepatic stellate cell)). Similarly, in the case of activated cardiac fibroblasts and activated renal fibroblasts, "deactivation" refers to the conversion of the cell state to a quiescent state (e.g., deactivated cardiac fibroblasts, deactivated renal fibroblasts). Deactivated cells possess properties similar to those of quiescent cells before activation. The conversion of the cell state to a quiescent state involves altering the properties, morphology, shape, and cell cycle of myofibroblasts. The properties, morphology, shape, and cell cycle of myofibroblasts can be determined by the cytoskeleton (CSK) and extracellular matrix (ECM). The cytoskeleton includes actin filaments (F-actin), intermediate filaments, microtubules, and talin, etc. The extracellular matrix includes collagen, hyaluronic acid, and proteoglycans, etc. The return of the cell morphology to a quiescent state can also be determined by a decrease in connective tissue and / or a decrease in its accumulation. The decrease in connective tissue and / or the decrease in its accumulation can be determined by the amount of extracellular matrix such as collagen.

[0030] In this specification, the cell morphology of a quiescent-like state obtained by deactivating and converting myofibroblasts from an "activated state" is referred to as the "deactivated state." In this specification, the cell morphology of the "quiescent state" or "quiescent-like state" is, for example, a fibroblast-like cell or fibroblast-like cell that produces the minimum amount of collagen necessary to maintain the structure of an organ and forms connective tissue.

[0031] The state of a cell (i.e., whether it is quiescent, activated, or deactivated) can be confirmed, for example, by measuring the production of proteins and gene expression mainly expressed in quiescent, activated, and deactivated cells. When examining or comparing the presence or absence of gene expression in cells, a method can be used in which the mRNA expression of each protein or gene is measured using the respective protein or gene as a marker. The quantification and analysis of gene expression levels are performed by commonly used gene expression quantification methods, such as quantitative RT-PCR. The primers used in quantitative RT-PCR may be those known in the industry as appropriate.

[0032] Deactivation markers can be used as one indicator of myofibroblast deactivation. While there are no particular limitations on the deactivation markers, deactivation of activated hepatic stellate cells can be confirmed by using TCF21 expression, F-actin accumulation, or suppression of fibrosis marker expression. "TCF21" is an abbreviation for Transcription factor 21. Increased expression of a deactivation marker gene (e.g., TCF21) means that activated myofibroblasts have been deactivated. Reduced accumulation of F-actin indicates that activated myofibroblasts have been deactivated.

[0033] Furthermore, quiescent markers can be used as one of the indicators of myofibroblast deactivation. Quiescent markers are not particularly limited, but for example, NGFR and / or genes expressed at the same time as NGFR (e.g., LRAT, NES, LHX2, etc.) can be used. Quiescent markers are genes that are mainly expressed in cells in a quiescent (or quiescent) state, and can be used as indicators of gene expression in deactivated hepatic stellate cells when activated hepatic stellate cells are deactivated and acquire a quiescent-like cell morphology. An increase in the expression of quiescent marker genes means that activated cells in fibroblast-like cells (myofibroblasts) have been deactivated. "NGFR" is an abbreviation for Nerve Growth Factor Receptor. "NES" is an abbreviation for Nestin. "LRAT" is an abbreviation for Lecthin Retinol Acyltransferase, a retinol esterification enzyme. "LHX2" is an abbreviation for LIM Homeobox 2.

[0034] Furthermore, liver regeneration induction markers can be used as one indicator of myofibroblast deactivation. While there are no particular limitations on the liver regeneration induction markers, examples include MDK and PTN. "MDK" is an abbreviation for Midkine. "PTN" is an abbreviation for Pleiotrophin. Liver regeneration induction markers can be used as indicators of improved liver function. An increase in the expression of liver regeneration induction marker genes means that activated cells in fibroblast-like cells (myofibroblasts) have been deactivated.

[0035] Fibrosis markers can be used as indicators of suppressed myofibroblast activation. Examples of fibrosis markers, though not particularly limited, include extracellular matrix (ECM) such as collagen, laminin, and fibronectin; actin; transforming growth factor (TGFB); PAI1; and WNT. Extracellular matrix components such as collagen, laminin, and fibronectin are cell adhesion proteins and can be used as indicators of fibrosis. Examples of WNT, though not particularly limited, include Wnt1, Wnt3a, Wnt7b, Wnt5a, and Wnt4. For example, known fibrosis markers such as COL1A1, COL1A2, COL3A1, ACTA2, and TGFB1 can be used. "COL1A1" is an abbreviation for type I collagen α1. "COL1A2" is an abbreviation for type I collagen α2. "COL3A1" is an abbreviation for type III collagen α1. "TGFB1" is an abbreviation for transforming growth factor-b1. "ACTA2 (αSMA)" is α-smooth muscle actin 2. "PAI1" is plasminogen activator inhibitor 1. A decrease in the expression of fibrosis marker genes means that the activation of myofibroblasts has been suppressed, or that activated myofibroblasts have been deactivated.

[0036] In addition to those mentioned above, Postn, Ccn2, and Meox1, which are specifically highly expressed in activated fibroblasts, can also be used as fibrosis markers. "Postn" is periostin, "Ccn2" is CTGF (Connective Tissue Growth Factor), and "Meox1" is mesenchyme homeobox 1. These can reflect the activation state of fibroblasts.

[0037] Heart failure markers can be used as indicators of treatment effectiveness. While not limited to specific markers, examples include natriuretic peptides (ANP, BNP). "ANP" stands for Atrial Natriuretic Peptide, and "NPPA" is its gene. "BNP" stands for Brain Natriuretic Peptide, and "NPPB" is its gene. A decrease in the expression of these markers indicates a reduction in cardiac load and improvement in cardiac function.

[0038] Furthermore, metabolic markers can be used as indicators of the effects (metabolic improvement) of the compounds of the present invention on cardiomyocytes. Examples of metabolic markers include genes involved in mitochondrial biosynthesis and fatty acid metabolism, such as PGC-1α (Ppargc1a). "PGC-1α" is Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha. Increased expression of these markers indicates improvement in mitochondrial function or energy metabolism in cardiomyocytes.

[0039] Deactivation of myofibroblasts can also be achieved by observing their cell morphology. The observation of cell morphology is not particularly limited and can be carried out by those skilled in the art using any known method. For example, it can be done using a fluorescence microscope. When using a fluorescence microscope, cytoskeletal molecules such as F-Actin may be stained.

[0040] In some forms, "deactivation" refers to the cessation or suppression of fiber production. In some forms, "deactivation" refers to the presence of fewer cytoskeletal fibers, such as actin (e.g., F-actin), in the deactivated state compared to the activated state. For example, myofibroblasts (e.g., activated stellate cells) produce a large amount of cytoskeletal fibers, while deactivated cell morphology (quiescent-like stellate cells) shows a decrease in cytoskeletal fibers. In some forms, "deactivation" refers to the improvement of fibrosis and / or the normalization of organ function. In some forms, "deactivation" refers to the promotion of the expression of TCF21, a deactivation marker. In some forms, "deactivation" refers to the expression of liver regeneration induction markers (e.g., PTN and MDK), resulting in improved fibrosis and normalization of liver function. In some forms, "deactivation" indicates an increase in the expression of quiescent markers (e.g., NGFR, LRAT, NES, LHX2, etc.). In some forms, "deactivation" refers to the phenomenon that occurs when the damage caused by fibrosis is removed or reduced from an organ.

[0041] In some forms, "inhibition of activation" indicates a decrease in the expression of fibrosis markers (e.g., COL1A1, COL1A2, COL3A1, ACTA2, TGFB1, PAI1, POSTN, CCN2, and / or MEOX1).

[0042] In this specification, "fibrosis" refers to the process by which extracellular matrix such as collagen accumulates in organs and tissues. In this specification, "improvement of fibrosis" refers to the process by which extracellular matrix such as collagen decreases or disappears from organs and tissues.

[0043] In this specification, "fibrosis" refers to the accumulation of extracellular matrix (ECM) following trauma, inflammation, tissue repair, immune response, cell hyperplasia, and abnormal proliferation. Fibrosis is a disease that occurs in various organs and tissues and is known to be an underlying disease that progresses to cirrhosis, renal failure, heart failure, pancreatic cancer, etc., and includes fibrosis in organs and tissues. Depending on the organ to which fibrosis has progressed and the degree of progression, symptoms associated with fibrosis, such as inflammation and atrophy, may be observed. The "treatment" of the present invention includes not only the prevention and treatment of the above-mentioned fibrosis, but also the suppression of the progression of fibrosis in tissues, the reduction of inflammation, the reduction of symptoms associated with fibrosis, and maintenance to prevent recurrence. Treatment of symptoms associated with fibrosis is also included in the present invention. In some embodiments, "treatment or prevention of fibrosis" and "improvement of fibrosis" are accompanied by improvement of organ and tissue function.

[0044] In this specification, "organ failure" refers to a state in which the function of an organ is significantly impaired, making it impossible to maintain homeostasis in the body. Organ failure in this invention also includes multi-organ failure, in which multiple organs fail simultaneously or in a chain reaction. Furthermore, organ failure in this invention includes those that occur as fibrosis progresses (e.g., heart failure, liver failure, kidney failure, respiratory failure). In this specification, "heart failure" refers to a state in which the pumping function of the heart is impaired, making it impossible to supply the necessary blood to the tissues of the body. Heart failure in this invention includes not only heart failure accompanied by myocardial fibrosis, but also heart failure accompanied by impaired energy metabolism of cardiomyocytes and mitochondrial dysfunction. In this specification, "AMPK" is an abbreviation for AMP (Adenosine monophosphate-activated protein) kinase, a type of protein kinase that regulates intracellular energy metabolism and is a major regulator in many biological processes. The AMPK signaling pathway includes glucose and lipid metabolism and affects the expression of related genes and proteins. When AMPK is phosphorylated, its activity increases, further regulating downstream proteins in the AMPK signaling pathway, thereby regulating metabolism in the liver, skeletal muscle, heart, lipid tissue, and pancreas. Therefore, the medicinal effects of AMPK activation may be potentially effective in treating many diseases. In this specification, "AMPK activation" refers to increased activity of the AMPK signaling pathway, indicated by an increase in the phosphorylation level of the AMPK protein or altered expression of downstream target factors of AMPK (e.g., PGC-1α). In this specification, “organ failure associated with downregulation of the AMPK signaling pathway” means dysfunction of an organ or tissue in which a decrease in the activity or expression level of AMPK in the tissue or cell contributes to the development, progression, or maintenance of the pathological condition. Such organ failure includes diseases or conditions resulting from decreased AMPK activity that involve impaired intracellular energy metabolism (e.g., mitochondrial dysfunction), impaired maintenance of cellular homeostasis (e.g., autophagy dysfunction), and / or increased oxidative stress.

[0045] 2. Pyridopyrimidine Compounds The present invention is based on the discovery that certain compounds described herein exhibit a deactivating effect on activated hepatic stellate cells, which is beneficial in the treatment of fibrosis. The compounds used in the present invention are compounds represented by the following formula (I) or pharmaceutically acceptable salts thereof (hereinafter also referred to as "pyridopyrimidine compounds"), and these compounds have a deactivating effect on myofibroblasts.

[0046] In equation (I), R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl. １ , R ２ , and R ３ In the case of C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ It may be substituted with a substituent selected from. In some embodiments, R １ , R ２ , and R ３ At least one of them is not a hydrogen atom. In certain embodiments, R １ and R ２ These are, independently, a hydrogen atom, a halogen atom, and C １～２ Alkyl, C １～２ Alkoxy, C １～２ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～２ Alkyl, C １～２ Alkoxy, C １～２ Alkylthio is hydroxy and -NR ５ R ６ It may also be replaced with R １ and R ２ At least one of them is not a hydrogen atom, R ３ It is a hydrogen atom.

[0047] In some embodiments, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is -NR ５ R ６ It may be substituted with a substituent selected from the following.

[0048] In equation (I), R ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys. In some embodiments, R ４ C １～２ Alkyl and C １～２ Selected from the group consisting of alkoxys. In a particular embodiment, R ４ C １～２ It is alkyl. In some embodiments, R ４ It is methyl.

[0049] In equation (I), R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl groups. In some embodiments, R ５ and R ６ Each is independently selected from a hydrogen atom, methyl, and ethyl. In certain embodiments, R ５ and R ６ It is ethyl.

[0050] The structures and names of specific compounds preferably used in the present invention are shown below.

[0051] In some embodiments, the pyridopyrimidine compound is at least one selected from compound 1, compound 2, compound 4, and compound 5. Among these, compounds selected from the group consisting of compound 1 and compound 2 are preferred due to their excellent deactivating effect. The pyridopyrimidine compound may also be compound 1.

[0052] In the present invention, salts of the compound represented by formula (I) above can be used. The "salt" can be any pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" is not particularly limited and includes the salts mentioned above. Furthermore, pyridopyrimidine compounds also include hydrates, various solvates, and crystalline polymorphs.

[0053] The pyridopyrimidine compound of the present invention may be a commercially available product or one synthesized by a known chemical synthesis method.

[0054] 3. Deactivator of Myofibroblasts One embodiment of the present invention relates to a deactivator of myofibroblasts comprising a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof. The above pyridopyrimidine compound has a deactivating effect on myofibroblasts and functions as a deactivator of myofibroblasts. Myofibroblasts deactivated by the deactivator of myofibroblasts adopt a quiescent state-like (deactivated) cell morphology.

[0055] In some embodiments, myofibroblasts are ACTA2-positive myofibroblasts. In some embodiments, myofibroblasts are activated fibroblasts in various organs. In some embodiments, myofibroblasts are activated hepatic stellate cells, activated cardiac fibroblasts, activated renal fibroblasts, activated pulmonary fibroblasts, activated cutaneous fibroblasts, activated gastrointestinal fibroblasts, activated ocular fibroblasts, activated intrauterine fibroblasts, or activated bone marrow fibroblasts. In some embodiments, myofibroblasts are activated hepatic stellate cells, activated cardiac fibroblasts, or activated renal fibroblasts. In some embodiments, myofibroblasts are activated hepatic stellate cells. The compounds of the present invention can exert a deactivating effect on these activated fibroblasts, regardless of the organ.

[0056] According to one embodiment of the present invention, by administering the pyridopyrimidine compound of the present invention, activated myofibroblasts can be deactivated and restored to normal myofibroblasts (quiescent-like cell morphology), thereby improving fibrosis and / or normalizing or restoring organ function.

[0057] In some forms, the deactivator of this form reduces cytoskeletal fibers. In some forms, the deactivator of this form leads to improvement of fibrosis and / or normalization of organ function. In some forms, the deactivator of this form promotes the expression of the deactivation marker TCF21. In some forms, the deactivator of this form promotes the expression of liver regeneration induction markers (e.g., PTN and MDK). In some forms, the deactivator of this form promotes the expression of quiescent markers (e.g., NGFR, LRAT, NES, LHX2, etc.). In some forms, the deactivator of this form removes or reduces damage caused by fibrosis from organs.

[0058] In some embodiments, the pyridopyrimidine compounds have an inhibitory effect on myofibroblast activation and function as myofibroblast activation (fibrosis) inhibitors. Therefore, other embodiments of the present invention also relate to myofibroblast activation (fibrosis) inhibitors containing pyridopyrimidine compounds. In some embodiments, administration of pyridopyrimidine compounds can suppress myofibroblast activation, acting as myofibroblast activation inhibitors (anti-fibrotic agents), thereby suppressing collagen expression and / or inhibiting fibrosis. In some embodiments, the activation inhibitors of this embodiment reduce the expression of fibrosis markers (e.g., COL1A1, COL1A2, COL3A1, ACTA2, TGFB1, PAI1). In some embodiments, the activation inhibitors of this embodiment suppress collagen expression and / or tissue fibrosis. In some embodiments, the activation inhibitors of this embodiment reduce cytoskeletal fibers.

[0059] 4. Therapeutic or Prophylactic Agents One embodiment of the present invention also relates to therapeutic or prophylactic agents using the pyridopyrimidine compound described above. The pyridopyrimidine compound can be used to treat or prevent fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway by promoting deactivation of myofibroblasts and / or improvement of the metabolism of cardiomyocytes. One embodiment of the present invention provides a therapeutic or prophylactic agent for a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. The present invention may also be in the following embodiments: - Use of the pyridopyrimidine compound for the manufacture of a pharmacopoeia for the treatment or prevention of a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway. - A method for treating or preventing a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, comprising administering a therapeutically effective amount of the pyridopyrimidine compound to a subject in need thereof. - The pyridopyrimidine compound for use in the treatment or prevention of a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway. - Use of the pyridopyrimidine compound for use in the treatment or prevention of a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway.

[0060] (1) Treatment or prevention of fibrosis The pyridopyrimidine compound can be used to treat or prevent fibrosis by acting on myofibroblasts to promote deactivation. One embodiment of the present invention relates to a therapeutic or prophylactic agent for fibrosis comprising a compound represented by formula (I) or a pharmaceutically acceptable salt thereof. The present invention may also be in the following embodiments: - Use of the pyridopyrimidine compound for the manufacture of a medicament for the treatment or prevention of fibrosis. - A method for treating or preventing fibrosis, comprising administering a therapeutically effective amount of the pyridopyrimidine compound to a subject in need thereof. - The pyridopyrimidine compound for use in the treatment or prevention of fibrosis. - Use of the pyridopyrimidine compound for use in the treatment or prevention of fibrosis.

[0061] This disclosure demonstrates that the pyridopyrimidine compound exhibits a deactivating effect on activated hepatic stellate cells and can be used as a therapeutic or prophylactic agent for fibrosis. As shown in the examples below, the pyridopyrimidine compound of the present invention exhibits deactivating and inhibitory effects on activated hepatic stellate cells and showed fibrosis improvement and organ function normalization effects in animal models of fibrosis in the liver. Furthermore, as shown in the examples below, the pyridopyrimidine compound also showed fibrosis improvement and organ function normalization effects in the kidneys and hearts in animal models of fibrosis. It is known that ACTA2-positive myofibroblasts induce fibrosis in many organs and tissues, and since the pyridopyrimidine compound of the present invention has a deactivating effect on activated hepatic stellate cells, it can be an effective therapeutic or prophylactic agent for fibrosis in many other organs and tissues.

[0062] Deactivation of myofibroblasts, such as activated hepatic stellate cells, and suppression of fibroblast activation have been reported to be effective in treating fibrosis. In organs where fibrosis has regressed, fibroblast-like cells (myofibroblasts) are known to undergo apoptosis or enter a deactivated cellular state similar to a quiescent state. Deactivated fibroblast-like cells (myofibroblasts) stop producing and proliferating fibers, express cellular environmental factors such as pleiotrophin and midkine, and contribute not only to the improvement of fibrosis but also to the normalization of organ tissue. In a mouse model of hepatic fibrosis, it has been shown that introducing the transcription factor TCF21 into activated hepatic stellate cells, which are fibroblast-like cells, promotes deactivation of hepatic stellate cells, improving fibrosis and liver function.

[0063] Furthermore, as shown in the examples described below, the inventors have found that the pyridopyrimidine compounds of the present invention control the plasticity of activated myofibroblasts and induce deactivation. Specifically, the pyridopyrimidine compounds of the present invention inhibit the Rho-GTPase / ROCK signaling pathway (Figure 24), thereby suppressing actin polymerization (F-actin formation by G-actin binding) and inhibiting the nuclear translocation of the transcriptional co-factor MRTF-A. As a result, the transcriptional activity of SRF (Serum Response Factor) decreases. Through this mechanism, the compounds of the present invention not only slow the progression of fibrosis but also promote reverse remodeling of already formed fibrous tissue, contributing to the normalization of tissue structure (Figures 24B, 26). Furthermore, the pyridopyrimidine compounds of the present invention can improve tissue hardening and restore reduced organ function (e.g., cardiac contractility) even in the chronic phase where the disease has progressed and fibrous tissue has accumulated (see Figure 28). Notably, with the pyridopyrimidine compounds of the present invention, the restored organ function can be maintained even if drug administration is discontinued after a certain period (e.g., two weeks) (see Figure 28). This suggests that the compounds of the present invention not only temporarily inhibit fibrosis but also irreversibly or stably change (deactivate) the cellular characteristics to a normal state.

[0064] In some embodiments, the pyridopyrimidine compound inhibits the Rho-GTPase signaling pathway in activated fibroblasts (myofibroblasts). In certain embodiments, the pyridopyrimidine compound inhibits cytoskeletal reorganization (actin polymerization) by inhibiting the Rho-GTPase signaling pathway. In some embodiments, the pyridopyrimidine compound inhibits the nuclear translocation of the transcription co-factor MRTF-A. In certain embodiments, the pyridopyrimidine compound suppresses the transcription of fibrosis-related genes by SRF (Serum Response Factor) by inhibiting the nuclear translocation of MRTF-A. In some embodiments, the pyridopyrimidine compound controls the plasticity of fibroblasts, causing cells in an activated state with enhanced collagen production to transition to a quiescent-like state with reduced collagen production. In some embodiments, the pyridopyrimidine compound can induce normalization of tissue structure (reverse remodeling) even in advanced fibrotic tissue. In some embodiments, therapeutic or prophylactic agents of this form are used to treat fibrosis or associated organ failure in advanced and chronic stages. Because compounds of this form have the effect of controlling the plasticity of fibroblasts and inducing deactivation (reverse remodeling effect), they can not only inhibit progression (prevent) but also reduce already accumulated collagen and restore the function of dysfunctional organs (treat).

[0065] For information regarding the treatment or prevention of fibrosis based on the deactivation effect in this invention, see, for example, the following literature. (1) Kisseleva, T. Nature Reviews Gastroenterology & Hepatology, 2021,18,151-166. (2) Jun J.-II, J Clin Invest. 2018,128,97-107. (3) Zisser, A. Biomedicines, 2021,9,365. (4) Ueha, S. Front. Immunol., 2012,3,71. (5) Yazdani, S. Advanced Drug Delivery Reviews, 2017,121,101-116. (6) Junien, JL Hepatology Communications 2017,1,524-537. (7) Bellusci, S. Cell Stem Cell 2017,21,166-177. (8) Lafyatis, R. Nature Reviews Rheumatology, 2019,15,705-730. (9) Wynn, TA Nature, 2022, 587, 555-566. (10) Radstake, T. Nature Reviews Rheumatology, 2018,14,657-673. (11) Varga, J. Nature Reviews Rheumatology, 2019,15,208-224. (12) Reilly, O. Nature Reviews Rheumatology, 2021,17,596-697. (13) Horton, MRJ Clin. Invest. 2021:131,e143226. (14) Selman, M. Nature Reviews Drug Discovery, 2017,16,755-772. (15) Wells, AU Nature Reviews Disease Primers, 2017,3,1-19. (16) Nakano, Y. Hepatology, 2020,71,1437-1452. (17) Lv,Ke et al.,Front.Immunol.,2022;13:1042983 (18) Pakshir, P et al., Journal of Cell Science,2020,133(13),jcs227900 (19) Niu, C et al., Front. Cell Dev. Biol., 24 October 2024, 12:1490315 .

[0066] In some embodiments, fibrosis is fibrosis occurring in organs or tissues (organ (tissue) fibrosis). Examples of fibrosis include fibrosis occurring in the heart, brain, digestive tract, skin, lungs, kidneys, liver, pancreas, hematopoietic organs, retroperitoneum, mediastinum, joints, muscles, blood vessels, eyes, breasts, or reproductive organs. Examples of such organ or tissue fibrosis include, but are not limited to, cardiac fibrosis, gastrointestinal fibrosis, intestinal fibrosis, Crohn's disease, hepatic fibrosis, cirrhosis, chronic liver disease, scleroderma, scar formation, idiopathic interstitial pneumonia, pulmonary fibrosis, renal fibrosis, nephrogenic systemic fibrosis, chronic kidney disease, chronic pancreatitis, pancreatic cancer, cystic fibrosis, myelofibrosis, retroperitoneal fibrosis, mediastinal fibrosis, myofibrosis, retinal fibrosis, bleb fibrosis, uterine fibrosis, uterine fibromas, ovarian fibrosis, glaucoma, subretinal fibrosis associated with age-related macular degeneration, or articular fibrosis. In some embodiments, fibrosis can be of the gastrointestinal tract, skin, lungs, liver, pancreas, kidneys, or heart. In some embodiments, fibrosis is fibrosis occurring in the liver, kidneys, or heart. Examples of such fibrosis include hepatic fibrosis, cirrhosis, chronic liver disease, renal fibrosis, nephrogenic systemic fibrosis, chronic kidney disease, and cardiac fibrosis.

[0067] In some embodiments, fibrosis is characterized by increased expression of fibrosis markers in the subject. In some embodiments, fibrosis is characterized by extracellular matrix (ECM) formation in the subject.

[0068] In some embodiments, the therapeutic or prophylactic agent of this embodiment promotes the expression of the deactivation marker TCF21. For example, administration of the therapeutic or prophylactic agent of this embodiment may result in a higher TCF21 expression level compared to no administration (negative control 100%), for example, 120% or more, 140% or more, 160% or more, 180% or more, 200% or more, etc.

[0069] In some embodiments, the therapeutic or prophylactic agent of this embodiment has at least one of the following features: (i) reducing the expression level of at least one fibrosis marker [e.g., selected from extracellular matrix components such as collagen (e.g., COL1A1, COL1A2, COL3A1), laminin, and fibronectin; actin (e.g., ACTA2); transforming growth factor (TGFB) (TGFB1); PAI1; and WNT (e.g., Wnt1)]. In certain embodiments, the at least one fibrosis marker includes at least one selected from COL1A1, COL1A2, COL3A1, ACTA2, TGFB1, and PAI1. In certain embodiments, the at least one fibrosis marker includes at least one selected from COL1A1, COL1A2, POSTN, CCN2, MEOX1, and ACTA2. For example, the therapeutic or prophylactic agent of this embodiment may, upon administration, result in a lower expression level of fibrosis markers compared to the case without administration (100% negative control), for example, 80% or less, 70% or less, 60% or less, 50% or less, etc. (ii) Increase the expression level of at least one quiescent hepatic stellate cell marker (e.g., LHX2, LRAT, NES, and NGFR; preferably LHX2, LRAT). For example, the therapeutic or prophylactic agent of this embodiment may, upon administration, result in a higher expression level of quiescent hepatic stellate cell markers compared to the case without administration (100% negative control), for example, 120% or more, 140% or more, 160% or more, 180% or more, 200% or more, etc. (iii) Increase the expression level of at least one liver regeneration induction marker (e.g., MDK, PTN). For example, the therapeutic or prophylactic agent of this form, upon administration, may result in a higher expression level of liver regeneration induction markers compared to the case without administration (negative control 100%), for example, 120% or more, 140% or more, 160% or more, 180% or more, 200% or more, etc. (iv) Reduces the accumulation of F-actin. For example, the formation of F-actin by polymerization of G-actin is suppressed. (v) Transitions activated myofibroblasts into a quiescent state.

[0070] In some embodiments, the therapeutic or prophylactic agent of this embodiment has at least one of the following features: (1) reduction or inhibition of the formation or deposition of extracellular matrix (ECM) proteins (e.g., collagen); (2) reduction in the number of myofibroblasts (e.g., activated hepatic stellate cells); (3) reduction in the cytoplasm of myofibroblasts (e.g., activated hepatic stellate cells); (4) reduction or absence of cytoskeleton content (e.g., actin); (5) improvement of fibrosis (e.g., reduction or absence of extracellular matrix such as collagen); (6) normalization or restoration of organ (tissue) function.

[0071] In some embodiments, the therapeutic or prophylactic agent of this form is used to treat or prevent hepatic fibrosis, and administration reduces hepatomegaly. In some embodiments, the therapeutic or prophylactic agent of this form is used to treat or prevent hepatic fibrosis, and administration results in an increase in serum total protein levels, an increase in serum albumin levels, and / or a decrease in serum ALT levels. In some embodiments, the therapeutic or prophylactic agent of this form is used to treat or prevent cardiac fibrosis, and administration reduces the expression of heart failure markers (ANP and BNP). In some embodiments, the therapeutic or prophylactic agent of this form is used to treat or prevent cardiac fibrosis, and administration reduces cardiac hypertrophy. In some embodiments, the therapeutic or prophylactic agent of this form is used to treat or prevent cardiac fibrosis, and administration improves myocardial contractility.

[0072] (2) Treatment or prevention of organ failure associated with fibrosis The pyridopyrimidine compounds described above can be used to treat or prevent various organ failures that occur in association with the fibrosis described above. The target organ failures are not particularly limited, but include, for example, liver failure, kidney failure, heart failure, respiratory failure (lung failure), pancreatic failure, intestinal failure, bone marrow failure, skin dysfunction, visual impairment, or uterine dysfunction. Specifically, these include liver failure associated with hepatic fibrosis or cirrhosis, kidney failure associated with renal fibrosis or chronic kidney disease, and heart failure accompanied by fibrosis. The pyridopyrimidine compounds (PD) of the present invention can restore organ reserve by improving tissue fibrosis and contribute to recovery from or inhibition of the progression of these dysfunctional states.

[0073] (3) Treatment or prevention of organ failure associated with downregulation of the AMPK signaling pathway The pyridopyrimidine compounds can be used to treat or prevent organ failure associated with downregulation of the AMPK signaling pathway. In some embodiments, the organ failure associated with downregulation of the AMPK signaling pathway is at least one organ failure selected from the group consisting of heart failure, liver failure, kidney failure, respiratory failure (pulmonary failure), pancreatic failure, intestinal failure, and brain dysfunction. In some embodiments, the organ failure is at least one organ failure selected from the group consisting of heart failure, liver failure, and kidney failure. In some embodiments, the organ failure associated with downregulation of the AMPK signaling pathway is heart failure. In some embodiments, the heart failure is heart failure with fibrosis. In some embodiments, the heart failure is heart failure without fibrosis. In one embodiment, the organ failure is heart failure with mitochondrial dysfunction. In some embodiments, the organ failure is multiple organ failure. In organ failure conditions such as heart failure, it is known that downregulation of the AMPK signaling pathway progresses, including energy metabolism deficiency such as a decrease in the function of cardiomyocytes themselves and a decrease in AMPK activity. The pyridopyrimidine compound (PD) described above induces AMPK activation by inhibiting kinases located upstream of AMPK. By restoring the decreased AMPK activity, the pyridopyrimidine compound (PD) improves these conditions and can be used to treat or prevent organ failure such as heart failure. Furthermore, the pathological mechanism by which such downregulation of the AMPK signaling pathway, including energy metabolism deficiency and decreased AMPK activity, is related to decreased organ function is common not only to the heart but also to other major organs. For example, decreased AMPK activity in tissues has been reported in liver failure, kidney failure, respiratory failure (pulmonary failure), pancreatic failure, intestinal failure, and brain dysfunction, and this is a common factor in organ function decline. Therefore, the pyridopyrimidine compound described above, which induces AMPK activation, can be broadly applied not only to heart failure but also to these organ failures that share common pathological conditions.

[0074] The following explanation uses heart failure as an example. For instance, the pyridopyrimidine compound (PD) mentioned above is thought to exert excellent therapeutic effects through a "dual mechanism of action" that acts on both cardiomyocytes and fibroblasts through different mechanisms, simultaneously controlling them (Figure 20).

[0075] Firstly, the pyridopyrimidine compound can improve energy metabolism in cardiomyocytes. As shown in Figure 20 (left), the pyridopyrimidine compound (PD) can promote phosphorylation (activation) of AMPK (AMP-activated protein kinase) in stressed cardiomyocytes. Activated AMPK can increase the expression or activity of PGC-1α (Ppargc1a), which is downstream of AMPK, thereby improving mitochondrial biosynthesis and function. This improves mitochondrial metabolism in cardiomyocytes, improves energy metabolism (Energetic Rescue; CM), and can contribute to the recovery of cardiac function. As shown in the examples below, this metabolic improvement effect is due to oxidative stress (H ２ O ２ ) This protects the function of myocardial cells and maintains their contractility even under harsh conditions such as heavy loads.

[0076] In some embodiments, the therapeutic or prophylactic agents of this embodiment increase mitochondrial metabolism in cardiomyocytes. In some embodiments, the therapeutic or prophylactic agents of this embodiment increase the expression of PGC-1α (Ppargc1a) in cardiomyocytes. Since PGC-1α is a master regulator of mitochondrial biosynthesis, induction of its expression suggests an improvement in the energy production capacity of cardiomyocytes. In some embodiments, the therapeutic or prophylactic agents of this embodiment decrease the expression level of at least one heart failure marker selected from NPPA (ANP) and NPPB (BNP).

[0077] In some embodiments, the therapeutic or prophylactic agents of this embodiment induce the activation of AMPK in cardiomyocytes. In some embodiments, the therapeutic or prophylactic agents of this embodiment increase the phosphorylation level of AMPK in cardiomyocytes. In particular, they have the effect of restoring AMPK activity that has decreased or stagnated under stress such as oxidative stress (e.g., hydrogen peroxide loading). According to some embodiments, the present invention provides an AMPK activator (preferably an AMPK activator in cardiomyocytes) comprising the pyridopyrimidine compound described above.

[0078] In some embodiments, the therapeutic or prophylactic agent of this form improves the mitochondrial respiratory capacity (oxygen consumption rate: OCR) of cardiomyocytes. In some embodiments, it increases maximal respiratory capacity and respiratory reserve. In some embodiments, the therapeutic or prophylactic agent of this form suppresses the production of mitochondrial reactive oxygen species (mtROS) in cardiomyocytes. Since oxidative stress causes myocardial damage and arrhythmias, reducing mtROS contributes to cardioprotection. In some embodiments, the therapeutic or prophylactic agent of this form maintains or restores the mitochondrial membrane potential of cardiomyocytes. A decrease in membrane potential is a sign of mitochondrial dysfunction and apoptosis, but the therapeutic or prophylactic agent of this form has the function of maintaining normal membrane potential even under oxidative stress. In some embodiments, the therapeutic or prophylactic agent of this form suppresses or improves the decrease in myocardial contractility caused by damaging stimuli such as oxidative stress. In some embodiments, the therapeutic or prophylactic agents of this embodiment, upon administration, result in at least one of the following: an increase in calcium transient amplitude, an increase in contraction velocity, and an improvement in contraction deformation distance.

[0079] Secondly, the pyridopyrimidine compound has an antifibrotic remodeling effect on myofibroblasts (activated cardiac fibroblasts). As shown in Figure 20 (right), the pyridopyrimidine compound (PD) deactivates activated fibroblasts by inhibiting the Rho-GTPase / MRTF-A pathway. Specifically, inhibition of the Rho-GTPase / MRTF-A pathway suppresses actin polymerization (F-actin formation by G-actin binding), inhibits nuclear translocation of MRTF-A, a transcriptional co-factor, and as a result, the transcriptional activity of SRF (Serum Response Factor) decreases, deactivating fibroblasts from an activated state to a quiescent state, and improving tissue fibrosis (antifibrotic remodeling). Furthermore, the compounds of the present invention can inhibit the Smad pathway (Smad2 phosphorylation) and the activation (phosphorylation) of Src tyrosine kinase in TGF-β signaling. This blocks a representative activation pathway of fibroblasts and has the effect of improving fibrosis.

[0080] In some embodiments, the therapeutic or prophylactic agents of this embodiment induce at least one of the following in cardiac fibroblasts: inhibition of nuclear translocation of MRTF-A, reduction of SRF transcriptional activity, or inhibition of Src phosphorylation.

[0081] The treatment or prevention of organ failure related to downregulation of the AMPK signaling pathway based on the activation effect of AMPK in the present invention is not limited to the following, but for example, the following references may be consulted: (1) Clark, AJ et al. Kidney International, 2021, 99, 828-840. (2) Yibcharoenporn, C. et al. Drug Design, Development and Therapy, 2025, 19, 3029-3058. (3) Salminen, A. Biogerontology, 2024, 25, 83-106. (4) Marcondes-de-Castro, IA et al. Journal of Gastroenterology and Hepatology, 2023, 38, 1868-1876. (5) Fang, C. et al. Frontiers in Physiology, 2022, 13, 970292.

[0082] 5. Pharmaceutical Compositions One embodiment of the present invention provides a pharmaceutical composition comprising the pyridopyrimidine compound described above. The pyridopyrimidine compound can be administered alone, but can also be administered as part of a pharmaceutical composition. The pharmaceutical composition comprises at least the pyridopyrimidine compound as an active ingredient. Some embodiments provide a pharmaceutical composition comprising the pyridopyrimidine compound of the above embodiment and one or more pharmaceutically acceptable carriers. The amount of the active ingredient (pyridopyrimidine compound) combined with the pharmaceutically acceptable carrier is generally a therapeutically effective dose. The pyridopyrimidine compound may be administered simultaneously with one or more other therapeutic agents, or before or after the administration of other therapeutic agents.

[0083] 6. Dosage Form When the pyridopyrimidine compound is administered as a therapeutic or prophylactic agent, the target of administration is a mammal. The mammal is not particularly limited, but examples include humans, non-human primates, domesticated animals, laboratory animals, and livestock, and is preferably a human. In this specification, therapeutic or prophylactic agents include pharmaceutical compositions, therapeutic or prophylactic agents, deactivators, etc. In some embodiments, the target may be a subject suffering from a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure related to downregulation of the AMPK signaling pathway, and the treatment or prevention of the disease, disorder, or condition in the subject. In some embodiments, the target is a subject suffering from a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure related to downregulation of the AMPK signaling pathway, and is a subject requiring treatment of the disease, disorder, or condition. In some embodiments, subjects are those suffering from a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, and who require prevention of said disease, disorder, or condition. In some embodiments, when subjects are suffering from a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, subjects who require treatment for said disease, disorder, or condition may be excluded. Furthermore, when subjects are suffering from a disease, disorder, or condition selected from the group consisting of fibrosis, organ failure with fibrosis, and organ failure associated with downregulation of the AMPK signaling pathway, subjects who require prevention of said disease, disorder, or condition may also be excluded.

[0084] When the above pyridopyrimidine compounds are administered as therapeutic or prophylactic drugs, they can be administered either orally or parenterally. Oral administration can be in the form of tablets, capsules, granules, powders, sustained-release preparations, or syrups. Parenteral administration can be in the form of injections, suppositories, eye drops, pulmonary formulations (e.g., those using a nebulizer), nasal formulations, or transdermal formulations (e.g., ointments, creams). In the case of injections, they can be administered systemically or locally by intravenous injection (e.g., drip infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0085] These formulations can be manufactured by conventional methods using pharmaceutically acceptable excipients as pharmaceutical compositions in the desired dosage form.

[0086] The dosage and administration interval of the active ingredient, pyridopyrimidine compound, can be appropriately selected depending on the recipient, route of administration, disease, age, weight, and symptoms. For example, the dosage of pyridopyrimidine compound may be approximately 0.01 mg to approximately 50 mg per kg of body weight per day. However, the dosage is not limited to these amounts. The daily dose may be administered in one dose or divided into multiple doses. The dosage may also be calculated based on the free form.

[0087] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. The following experiments were conducted to confirm that pyridopyrimidine compounds are effective as therapeutic or prophylactic agents for fibrosis. DMSO (Sigma) was used as a negative control solvent. The structures and abbreviations of the pyridopyrimidine compounds used in the experiments are as follows: [Pyridopyrimidine Compounds]

[0088] [Example 1. Ability to induce deactivation of activated hepatic stellate cells] (1) Preparation of activated hepatic stellate cells derived from human iPS cells (days 5-7) Human iPS cells were differentiated into quiescent hepatic stellate cells according to the method described in Koui Y, et al. Stem Cell Reports 2021. Collagen-coated 12 well plates (VTC-P12: VIOLAMO) were coated with collagen (Cellmatrix type I-C: Nitta Gelatin) to create collagen-coated 12 well plates, and the obtained quiescent hepatic stellate cells were placed on these plates in a 2 × 10⁻¹⁶ arrangement. 4 Cells were seeded in cells / well and cultured for 5-7 days to induce activation. StemPro34 SFM (Gibco) was used as the basic culture medium for activation. For the first 3 days of culture, the cells were cultured in a medium to which Y27632 (Wako) was added to a concentration of 10 μM, and for 4-7 days of culture, the cells were cultured in the basic medium alone. (2) Evaluation of the ability to induce deactivation of activated hepatic stellate cells (gene expression) After the culture in (1) above, pyridopyrimidine compound PD1 was added to the basic medium to a concentration of 5-500 nM and cultured for 3 days. RNA was purified from the cultured cells using NucleoSpin RNA Plus (Takara bio). This RNA was converted to cDNA using Prime Script RT Ki (Takara Bio), and real-time quantitative PCR (LightCycle 96 Roche) was performed using TB Green Premix EX TaqII (Takara Bio) and primers for various genes. Deactivation was evaluated using the expression levels of fibrosis markers (ACTA2, COL1A1), deactivation markers (TCF21), quiescent hepatic stellate cell markers (LHX2, LRAT), and liver regeneration induction markers (MDK, PTN) as indicators (Figure 1).

[0089] (3) Evaluation of the ability to induce deactivation of activated hepatic stellate cells (cell morphology) TrypLE Express / EDTA (Gibco) was applied to the cultured cells described in (1) above, and the cells were detached from the bottom of the culture and collected as single cells. Each of the collected cells was suspended in the same culture medium as before detachment, seeded on a collagen-coated plate, and cultured for 24 hours. Subsequently, the cytoskeletal molecule F-Actin was stained with Phalloidin (Alexa Fluor® 680 Phalloidin: A22286), and cell morphology (Figure 2 top) and fluorescence intensity were photographed using a fluorescence microscope (Keyence, BZ-X810) (Figure 2 bottom).

[0090] (Results) As shown in Figure 1, the addition of pyridopyrimidine compound PD1 resulted in a concentration-dependent decrease in the expression of fibrosis markers (ACTA2 and COL1A1) genes, and a concentration-dependent increase in the expression of deactivation marker (TCF21) gene, quiescent hepatic stellate cell markers (LHX2 and LRAT) genes, and liver regeneration induction markers (MDK and PTN) genes. Figure 2 (top) shows that the addition of pyridopyrimidine compound PD1 resulted in a smaller cytoplasm and a change to a quiescent-like morphology. Figure 2 (bottom) shows that the addition of pyridopyrimidine compound PD1 significantly reduced the accumulation of F-Actin. These results confirm that pyridopyrimidine compound PD1 has the ability to induce deactivation of activated hepatic stellate cells.

[0091] [Example 2. Inhibitory ability to inhibit activation of activated hepatic stellate cells (activation process)] (1) Preparation of human iPS cell-derived activated hepatic stellate cells (day 5) Except that the culture period of quiescent hepatic stellate cells was 5 days (basic medium only for culture days 4-5), the same procedure as in 1(1) above was used to induce activation of hepatic stellate cells and obtain activated hepatic stellate cells (day 5). (2) Evaluation of inhibitory ability to inhibit activation of hepatic stellate cells (gene expression) After culturing as in (1) above, pyridopyrimidine compound PD1 was added to the basic medium to a concentration of 100 nM and cultured for 2 days. RNA was purified from the cultured cells using NucleoSpin RNA Plus (Takara bio). This RNA was converted to cDNA using Prime Script RT Ki (Takara Bio), and real-time quantitative PCR (LightCycle 96 Roche) was performed using TB Green Premix EX TaqII (Takara Bio) and primers for various genes. The ability to suppress activation of hepatic stellate cells was evaluated using the expression levels of genes for fibrosis markers (ACTA2, COL1A1, COL1A2, COL3A1, COL4A1), pro-fibrosis factors (TGFB1, TGFB2, TGFB3), chemokine factors (CXCL12), matrix metalloproteinases (MMP7, MMP13), and the deactivation marker (TCF21) as indicators (Figure 3).

[0092] (Results) Figure 3(A) shows that the cell characteristics change upon addition of PD1. Figure 3(B) shows that day 7 (DMSO) (negative control; DMSO added 5 days after culture) showed increased expression of fibrosis markers compared to day 5 (activated hepatic stellate cells after 5 days of culture), suggesting that day 5 was in the process of hepatic stellate cell activation. Day 7 (PD1) (PD1 added 5 days after culture) showed lower expression of fibrosis marker genes and higher expression of deactivation marker genes compared to day 7 (DMSO). This confirms that adding PD1 to hepatic stellate cells in the activation process reduces the expression of fibrosis markers and fibrosis-promoting factors, and increases the expression of deactivation markers. These results confirm that the pyridopyrimidine compound PD1 has the ability to suppress activation of hepatic stellate cells and induce deactivation of activated hepatic stellate cells when added during the activation process.

[0093] [Example 3. Ability to induce deactivation of senescent hepatic stellate cells] (1) Senescent hepatic stellate cells (Xray day 8) derived from human iPS cells were prepared in the same manner as in 1(1) above, except that activated hepatic stellate cells cultured for 5 days were irradiated with radiation (X-rays) (10 Gray) and cultured for a further 8 days (basic medium only for culture days 4 to 13). (2) Evaluation of the ability to suppress activation of senescent hepatic stellate cells (gene expression) After the culture in (1) above, pyridopyrimidine compound PD1 was added to the basic medium to a concentration of 100 nM and cultured for 2 days. Similar to 1(2) above, the ability to induce deactivation in senescent hepatic stellate cells was evaluated using the expression levels of fibrosis markers (ACTA2, COL1A1, COL1A2, COL3A1, COL4A1, TGFB1, TGFB2, TGFB3, CXCL12), quiescent hepatic stellate cell markers (MMP7, MMP13), and the deactivation marker (TCF21) as indicators (Figure 4(A) and Figure 4(B)). (3) Cellular senescence of activated hepatic stellate cells Similar to (1) above, activated hepatic stellate cells cultured for 5 days from quiescent hepatic stellate cells were irradiated with radiation (X-ray) and cultured for a further 1, 3, 5, 7, 10, and 12 days to obtain cells (X-ray d1, d3, d5, d7, d10, d12). Wester blot analysis was used to analyze the expression of cellular senescence markers (p21 and p16) and β-actin proteins in each cell (Figure 4(C)).

[0094] (Results) Figure 4(A) shows that the cell characteristics change upon addition of PD1. In Figure 4(C), an increase in p16 protein expression was observed in cells from Xray d5 (5 days after X-ray irradiation) onward, confirming that cellular senescence was induced, suggesting that Xray day 8 cells were senescent hepatic stellate cells in an activated state. Figure 4(B) shows that Xray day 10 cells (PD1) with added PD1 showed decreased expression of fibrosis markers and pro-fibrosis factors, and increased expression of deactivation markers compared to the negative controls Xray day 10 (DMSO) and Xray day 8 (before PD1 addition). The number of samples for each cell type was 3. These results confirm that pyridopyrimidine compounds (PD1) have the ability to induce deactivation when added to activated hepatic stellate cells that have undergone cellular senescence. Therefore, it is suggested that the pyridopyrimidine compound (PD1) also has a deactivation-inducing effect on senescent hepatic stellate cells.

[0095] [Example 4. Activity Comparison with Existing Pulmonary Fibrosis Treatment Drugs] The activity was compared using existing pulmonary fibrosis treatment drugs, Pirespa (obtained from Celec Biotech) and Ofev (obtained from Celec Biotech). (1) Preparation of human iPS cell-derived activated hepatic stellate cells (day 5) Activation of hepatic stellate cells was induced in the same manner as in 1(1) above to obtain activated hepatic stellate cells (day 5). (2) Evaluation of the ability to induce deactivation of activated hepatic stellate cells (gene expression) After culturing in (1) above, pyridopyrimidine compound PD1, Pirespa, or Ofev was added to the basic medium to a concentration of 100 nM and cultured for 2 days. Similar to 1(2) above, the ability to induce deactivation of activated hepatic stellate cells was evaluated using the expression levels of genes for fibrosis markers (COL1A1, COL1A2, COL3A1), fibrosis-promoting factors (TGFB1), deactivation markers (TCF21), and liver regeneration-inducing markers (PTN) as indicators (Figure 5).

[0096] As shown in Figure 5, the pyridopyrimidine compound PD1 more strongly suppressed the expression of fibrosis markers and pro-fibrosis factors, and more strongly promoted the expression of deactivation markers and liver regeneration induction markers, compared to the existing pulmonary fibrosis treatments Pirespa and Ofev. These results suggest that the pyridopyrimidine compound PD1 has a stronger deactivation-inducing ability than the existing pulmonary fibrosis treatments Pirespa and Ofev.

[0097] [Example 5. Activity of various pyridopyrimidine compounds] The ability to induce deactivation of activated hepatic stellate cells was evaluated using pyridopyrimidine compounds PD1 to PD5. (1) Preparation of human iPS cell-derived activated hepatic stellate cells (day 5) Activation of hepatic stellate cells was induced in the same manner as in 1(1) above to obtain activated hepatic stellate cells (day 5). (2) Evaluation of the ability to induce deactivation of activated hepatic stellate cells (gene expression) After culturing in (1) above, pyridopyrimidine compounds (PD1, PD2, PD3, PD4, or PD5) were added to the basic medium to a concentration of 20 to 500 nM and cultured for 2 days. In the same manner as in 1(2) above, the ability to induce deactivation of activated hepatic stellate cells was evaluated using the gene expression levels of fibrosis markers (ACTA2, COL1A1) and deactivation markers (TCF21) as indicators (Figures 6 to 8). (3) Evaluation of the ability to induce deactivation of activated hepatic stellate cells (cell morphology) The cells cultured as described in (1) above were photographed using an inverted microscope to observe their morphology (Figure 9).

[0098] (Results) As shown in Figures 6-8, the pyridopyrimidine compounds (PD1, PD2, PD3, PD4, PD5) all decreased the expression of the fibrillation marker (ACTA2, COL1A1) genes in a concentration-dependent manner upon addition, and increased the expression of the deactivation marker (TCF21) gene in a concentration-dependent manner. There was no significant difference in the inhibitory effect on ACTA2 expression among the pyridopyrimidine compounds. On the other hand, there were some differences in the inhibitory effect on COL1A1 expression among the pyridopyrimidine compounds (effect strength: PD2, PD1 > PD3, PD4, PD5). There were also some differences in the promoting effect on the deactivation marker (TCF21) gene among the pyridopyrimidine compounds (PD2, PD1 > PD3, PD4, PD5). From Figure 9, inhibition of cell proliferation or induction of cell death was confirmed with PD2 and PD1. The number of viable cells was highest in the order of PD2 < PD1 < PD3, PD4, PD5. These results confirm that pyridopyrimidine compounds (PD1-PD5) have the ability to induce deactivation of activated hepatic stellate cells.

[0099] [Example 6. Therapeutic effect on a hepatic fibrosis model mouse] Nineteen 8-week-old female C57Bl6 / J mice (CREA Japan) were used. Hepatic fibrosis due to chronic liver damage was induced by adding thioacetamide (TAA) to drinking water to a concentration of 600 mg / L and administering it for 8 weeks. While continuing the administration of TAA in drinking water, pyridopyrimidine compound PD1 or the negative control DMSO was administered intraperitoneally once daily for 1 week. Pyridopyrimidine compound PD1 was prepared to a concentration of 5 mg / kg (body weight). For example, a mouse weighing 25 g was administered 0.125 mg of PD1. In this case, since a DMSO solution of 10 mg / mL of PD1 was used, the amount of DMSO administered was 12.5 μL. The DMSO solution of PD1 was diluted with corn oil and administered in a total volume of 200 μL using an injection syringe. Negative controls were administered 200 μL of DMSO diluted with corn oil using an injection syringe. After the final dose, the mice were switched to normal drinking water (TAA-free) and sacrificially killed 48 hours later (Figure 10). Serum was collected from these mice, and their livers were removed and used in experiments as appropriate.

[0100] (1) Evaluation of the effect on improving liver fibrosis (Sirius red staining) It is common practice to stain liver tissue with Sirius red, a reagent that stains collagen fibers, as an indicator of liver fibrosis. The effect of administering a pyridopyrimidine compound (PD1) on improving liver fibrosis was investigated. (Preparation of liver tissue sections) The collected liver tissue was cut into small pieces and fixed overnight at 4°C in Mildform R 10N (Fujifilm Wako Pure Chemical Industries). The fixed, finely cut liver tissue was transported to Advantech Co., Ltd., where paraffin specimens were prepared by outsourcing.

[0101] (Sirius Red Staining) A Sirius Red staining solution was prepared by dissolving 0.5% Direct Red 80 (Sigma) and 0.1% Fastgreen (Sigma) in saturated picric acid (Fujifilm Wako Pure Chemical Industries). The Sirius Red staining solution was placed on the tissue sections and left to stand at room temperature for 5 minutes, staining the collagen fibers reddish-brown and the entire tissue green. After washing the stained tissue sections with water for 30 seconds, they were immersed in 100% ethanol for 1 minute three times, then immersed in xylene for 1 minute four times, and finally mounted on coverslips using Marinol. The stained and mounted tissue sections were photographed using a microscope BZ-X810 (Keyence). For each sample, images were captured using a 10x objective lens, capturing four or more fields of view. The area of ​​the Sirius Red stain-positive, reddish-brown region was calculated using ImageJ (National Institutes of Health, USA). Statistical significance testing was performed using One-Way ANOVA (Figure 11). This was outsourced.

[0102] (2) Marker gene expression analysis After extracting RNA from the collected liver tissue using Trizole, cDNA was prepared (PrimeScript II 1st strand cDNA Synthesis Kit (Takara)), and the expression of marker genes (COL1A1, COL1A2, COL3A1) was analyzed by real-time PCR (Figure 12). The ddCt method was used for the analysis.

[0103] (3) Evaluation of hepatomegaly It is known that the liver enlarges in chronic liver disease (J Biol Chem. 2011 11;286(6):4485-92, etc.). Changes in body weight and liver weight after administration of pyridopyrimidine compound (PD1) were investigated. The obtained measurements were statistically significant using One-Way ANOVA (Figure 13).

[0104] (4) Evaluation of liver function (serum test) Total protein, albumin, and alanine transsaminase (ALT) levels in serum, which are indicators of liver function, were measured using Spotchem EZ (Arkray) and its dedicated test strip (Spotchem II Liver Function 1). The obtained measurements were validated using One-Way ANOVA for statistical significance (Figure 13).

[0105] (Results) As shown in Figure 11, collagen fibers observed in Control (DMSO administration group) disappeared upon administration of PD1. From Figure 12, it was confirmed that the expression of fibrosis marker genes (Col1a1, Col1a2, Col3a1) was significantly reduced upon administration of the pyridopyrimidine compound (PD1). The liver / body weight ratio increased with the accumulation of fibers. As shown in Figure 13, the liver / body weight ratio was significantly reduced with PD1 administration, confirming a reduction in hepatomegaly. Furthermore, as shown in Figure 13, serum total protein levels, albumin levels, and ALT levels were all significantly improved with PD1 administration. These results indicate that administration of the pyridopyrimidine compound (PD1) improves liver fibrosis and simultaneously restores liver function.

[0106] [Example 7. Therapeutic effect on a mouse model of renal fibrosis induced by unilateral ureteral ligation] Twelve 8-week-old male C57BL / 6J mice (CREA Japan) were used. Renal fibrosis induced by unilateral ureteral ligation (UUO) was induced by ligating the left ureter. From the day of ligation of the left ureter, pyridopyrimidine compound (PD1) or negative control DMSO was administered intraperitoneally once daily for five consecutive days. The pyridopyrimidine compound (PD1) was prepared to a concentration of 7.7 mg / kg (body weight). For example, a mouse weighing 25 g was administered 0.1925 mg of PD1. In this case, since a 77 mg / 9 mL PD1 DMSO solution was used, the amount of DMSO administered was 22.5 μL. The PD1 DMSO solution was diluted with Corn oil and administered in a total volume of 200 μL using an injection syringe. Negative controls were administered 200 μL of DMSO diluted with corn oil using an injection syringe. These mice were sacrificially killed 24 hours after the final dose. Kidneys were removed from these mice and used in experiments as appropriate.

[0107] (1) Marker gene expression analysis RNA was extracted from the collected kidney tissue, cDNA was prepared, and the expression of fibrosis marker genes (Acta2, Col1a1, Col1a2, Col3a1, Tghb1, Pai1) was analyzed by real-time PCR (Figure 15).

[0108] (Results) Figure 15 shows that administration of the pyridopyrimidine compound (PD1) significantly reduced the expression of fibrosis marker genes (Col1a1, Col1a2, Col3a1, Tgfb1). This result indicates that administration of the pyridopyrimidine compound (PD1) improves renal fibrosis.

[0109] [Example 8. Therapeutic effect on a mouse model of heart failure induced by transverse aortic coarctation (TAC) (early administration of PD)] The efficacy of pyridopyrimidine compound (PD1) was evaluated using a mouse model of heart failure induced by transverse aortic coarctation (TAC) (Figure 16).

[0110] Fifteen 9-week-old male C57BL6 / J mice (CREA Japan) were used. Transverse aortic constriction (TAC) was performed, and echocardiographic evaluations were conducted weekly starting from the first week postoperatively. Three mice with large variations in cardiac function were excluded from the echocardiographic evaluations at the first and second weeks postoperatively, and the remaining 12 mice were divided into two groups: a negative control group of 7 mice and a pyridopyrimidine compound PD1 administration group of 5 mice. The negative control group received intraperitoneal administration of either DMSO or pyridopyrimidine compound PD1 once daily for 3 weeks. The pyridopyrimidine compound PD1 was prepared to a concentration of 5 mg / kg (body weight). For example, a mouse weighing 25 g received 0.125 mg of PD1. In this case, since a DMSO solution of 10 mg / mL PD1 was used, the amount of DMSO administered was 12.5 μL. PD1 mice were given a DMSO solution diluted with corn oil and administered in a total volume of 100 μL using a syringe. Negative controls were administered in a DMSO solution diluted with corn oil and administered in a total volume of 100 μL using a syringe. After a 3-week administration period, the hearts of these sacrificial mice were removed and used for histological evaluation and snRNA-seq experiments.

[0111] Cardiac function was assessed (IVS, LVPW, LVDd, LVDs, FS) by transthoracic echocardiography (Vevo2100, Primetech) in awake mice. Specifically, the left ventricle was imaged from the left parasternal border, and after M-mode measurement, the interventricular septal thickness (IVS), left ventricular posterior wall thickness (LVPW), left ventricular end-diastolic diameter (LVDd), and end-systolic diameter (LVDs) were measured. FS was calculated using the following formula: FS = (LVDd - LVDs) × 100 / LVDd

[0112] (2) Evaluation of the effect on improving cardiac fibrosis. Cardiac tissue specimens (paraffin-embedded sections) were stained with FAST green picrosilius red, and after imaging with a Keyence BZ-X800 microscope, the collagen fiber staining rate (Fibrosis area, %) was calculated by dividing the area stained with picrosilius red by the total area of ​​the section.

[0113] (3) Analysis of heart failure marker gene expression (ANP, BNP) ANP and BNP are known as heart failure markers. ANP and BNP are natriuretic peptides that have diuretic and antihypertensive effects and are hormones secreted by the heart. Natriuretic peptides have the effect of reducing the load on the heart, and their levels become high when the heart is under load. We examined the changes in the expression levels of heart failure marker genes after administration of pyridopyrimidine compound (PD1).

[0114] (Measurement method) The expression of natriuretic peptide genes in cardiomyocytes was compared and evaluated by single-nuclei RNA sequencing analysis of the hearts of Control and PD-1-administered mice.

[0115] (Results) Transverse aortic constriction (TAC) is a surgical procedure that ligates the aortic arch exiting the left ventricle, resulting in a decrease in aortic output and increased pressure in the left ventricle. Cardiac hypertrophy is observed from two weeks postoperatively, and severe cardiac hypertrophy, fibrosis, and pulmonary edema are seen at four weeks (see Control group in Figure 17). LVDd (left ventricular end-diastolic diameter) and LVDs (left ventricular end-systolic diameter) are indicators of cardiac hypertrophy, while FS (left ventricular diameter shortening ratio) is known as an indicator of systolic function. As shown in Figure 17, PD1 administration significantly suppressed the increase in LVDd (left ventricular end-diastolic diameter) and LVDs (left ventricular end-systolic diameter) observed in the negative control group (Control group) from two weeks postoperatively onward. This confirms that PD1 administration suppresses cardiac hypertrophy. Furthermore, Figure 17 shows that PD1 administration suppressed the decrease in FS (left ventricular diameter shortening) observed from two weeks post-surgery onward, and improved systolic function. As shown in Figure 18, the fibrotic area decreased in the PD1 administration group. This confirms that PD1 administration improves cardiac fibrosis. As shown in Figure 19, the expression levels of heart failure markers (ANP and BNP) decreased significantly in the PD1 administration group. This confirms that PD1 administration improves cardiac function. These results demonstrate that administration of pyridopyrimidine compounds (PD1) improves cardiac fibrosis and simultaneously restores cardiac function.

[0116] [Example 9. Therapeutic effect on pressure-overload heart failure model mice] Similar to Example 8, a pressure-overload heart failure model was created by performing aortic coarctation (TAC) on C57BL / 6J mice. In this example, pyridopyrimidine compound PD1 or a solvent (Control, DMSO) was administered intraperitoneally once daily from 2 to 5 weeks post-TAC, and analysis was performed at 5 weeks post-surgery (PD1 administration group (PD) n=5, PD1 non-administration group (Control) n=7) (Figure 21A). Pyridopyrimidine compound PD1 was prepared and administered at a dose of 5 mg / kg (body weight). At 5 weeks post-surgery, (1) cardiac function was evaluated (LVDd, LVDs, FS), (2) the effect of improving cardiac fibrosis was evaluated, and (3) the effect of suppressing myocardial hypertrophy was evaluated. (1) Evaluation of cardiac function (LVDd, LVDs, FS) Left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), and left ventricular diameter shortening rate (FS) were measured by transthoracic echocardiography over time using the same method as in Example 8. (2) Evaluation of the effect on improving cardiac fibrosis Cardiac tissue was stained with picrosilius red, microscopic images were taken, and the collagen fiber staining rate (Fibrosis area, %) was calculated using the same method as in Example 8. (3) Evaluation of the effect on suppressing myocardial hypertrophy Cardiac tissue was stained with WGA, and the cross-sectional area of ​​myocardial cells was measured by microscopic images using the same method as in Example 8.

[0117] (Results) Transthoracic echocardiography over time revealed that in the Control group, the left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) increased with the postoperative course, and the left ventricular diameter shortening ratio (FS) decreased. On the other hand, these changes were significantly suppressed in the PD administration group, and left ventricular remodeling and contractility decline were significantly suppressed, with contractility being maintained (Figure 21B). Evaluation of cardiac tissue by picrosilius red staining showed that the PD administration group had a significantly lower fraction of fibrotic area compared to the Control group (Figure 21C). Furthermore, analysis of myocardial tissue sections by WGA fluorescence staining showed that the increase in cardiomyocyte cross-sectional area was suppressed in the PD administration group, confirming the suppression of left ventricular myocardial hypertrophy (Figure 21D). These results suggest that the pyridopyrimidine compound (PD) of the present invention has the effect of suppressing left ventricular remodeling, improving fibrosis, and suppressing left ventricular myocardial hypertrophy during the cardiac hypertrophy formation phase.

[0118] [Example 10. Single-nucleus transcriptome analysis] Similar to Example 8, C57BL / 6J mice underwent aortic coarctation (TAC) to create a pressure-load heart failure model. From 2 to 5 weeks post-TAC, the pyridopyrimidine compound PD1 or a solvent (DMSO, Control group) was administered intraperitoneally once daily. The Sham group was administered a solvent (DMSO + corn oil). Single nuclei were isolated from mouse cardiac tissue at 2 and 5 weeks post-TAC (PD1 administration group: pTAC2W + PD, pTAC5W + PD, and Control group: pTAC2W, pTAC5W), as well as the Sham group, for a total of 5 groups (n=2 in each group), and snRNA-seq analysis was performed (Figure 22A). All cells were clustered using UMAP, cell types were identified, and their composition ratios were analyzed (Figure 22B). Furthermore, fibroblast (FB) and cardiomyocyte (CM) populations were extracted from the identified total cell population, and subcluster analysis was performed to analyze the detailed state changes of each cell type due to PD1 administration. The analysis was performed using the programming language R. Gene expression data obtained by snRNA-seq was preprocessed and normalized using Seurat, and the expression levels of each gene were quantified. Co-expression gene network analysis was performed using hdWGCNA, and gene ontology analysis was performed using clusterProfiler.

[0119] (Results) (1) Analysis of cardiac fibroblasts (FB) (Subcluster analysis) In the subcluster analysis of fibroblasts, it was confirmed that cluster 0 accounted for a large proportion of fibroblasts in the PD administration group (pTAC 5W + PD), while the proportion of fibroblasts classified as cluster 2 was relatively high in the Control group (pTAC 5W) (Figures 23A and 23B).

[0120] (Gene Analysis) Gene expression analysis revealed that representative genes highly expressed in activated cardiac fibroblasts (Col1a1, Col1a2, Ccn2, Postn, Meox1) were highly expressed in cluster 2 (Figure 23C). This suggests that PD suppresses the expression of fibroblast (FB) activation-related genes. Considering this together with the cell state transition diagram in Figure 26, cluster 0 corresponds to the initial stage of activation (quiescent-like state), and cluster 2 corresponds to the fully activated state with enhanced collagen production (Activated State). The increase in the proportion of cluster 0 and decrease in cluster 2 in the PD1-treated group suggests that PD1 inhibits the complete activation of fibroblasts (transition to the collagen-producing state), keeping fibroblasts in a quiescent-like state (cluster 0) or a state close to the quiescent state (cluster 1). In other words, these analysis results demonstrate that the pyridopyrimidine compound (PD) of the present invention deactivates myofibroblasts and transforms them into a state with reduced collagen production capacity.

[0121] (Co-expression gene network analysis / GO analysis) Co-expression gene network analysis and Gene Ontology analysis were performed on the gene group that was upexpressed in cluster 0, and the results suggested the involvement of the Rho-GTPase signaling pathway (Figure 24A). This supports the idea that PD suppresses fibroblast activation via the Rho-GTPase pathway (Figure 24B).

[0122] (2) Plasticity of cardiac fibroblasts (RNA velocity analysis) Using the same snRNA-seq data, the direction of cell state transitions was estimated by RNA velocity analysis. As a result, in the PD-administered group, a cell population showing a vector from activated to quiescent, i.e., "reverse remodeling," was identified (framed area in Figure 25). This result suggests that cardiac fibroblasts do not become irreversibly activated, but rather possess "plasticity" that allows them to be deactivated by administration of the pyridopyrimidine compound (PD) of the present invention (Figure 26).

[0123] (3) Analysis of cardiomyocytes (CM) In the subcluster analysis of cardiomyocytes, a characteristic cluster (Cluster 2) appeared in the PD-treated group (pTAC5W + PD) (Figures 27A and 27B). Gene expression analysis revealed that in Cluster 2, the expression of genes related to mitochondrial metabolism, such as Ppargc1a (PGC-1α), was elevated (Figure 27C). This suggests that PD administration promotes the expression of mitochondrial metabolism-related genes in cardiomyocytes, improving the energy metabolism of cardiomyocytes.

[0124] [Example 11. Therapeutic effect on a mouse model of advanced heart failure (administration started 6 weeks post-TAC surgery)] To verify whether the "plasticity" suggested in Example 10 is exhibited as a therapeutic effect, an experiment was conducted in which PD1 administration was started 6 weeks post-TAC surgery, a time when heart failure and fibrosis had already been established (non-administration group n=7, administration group n=6) (Figure 28A). The experiment and analysis were performed in the same manner as in Example 9, except that the administration period was from 6 weeks to 8 weeks post-TAC surgery (observation up to 12 weeks).

[0125] (Results) Transthoracic echocardiography over time showed that even when administration was started at a stage where heart failure had progressed (6 weeks post-TAC surgery), PD1 significantly suppressed the deterioration of left ventricular function (FS, etc.) (Figure 28B). Furthermore, histological evaluation also showed that it significantly suppressed the progression of fibrosis (Figure 28C). These results demonstrate that the pyridopyrimidine compound (PD) of the present invention exhibits fibroblast plasticity and is effective in treating the disease state after more advanced remodeling has occurred (advanced stage).

[0126] [Example 12. In vitro analysis using mouse cardiac fibroblasts] The effect of PD1 under TGF-β stimulation was verified in in vitro analysis using mouse cardiac fibroblasts (Figures 29A and 31A). Specifically, mouse cardiac fibroblasts were prepared by mincing neonatal mouse cardiac tissue, enzymatically digesting it with collagenase and dispase, and then seeding the resulting cell suspension into a culture dish and culturing it.

[0127] (Results) In mouse cardiac fibroblasts, PD1 administration suppressed the increase in Col1a1 gene expression induced by TGF-β (Figure 30A). Furthermore, analysis by Western blotting and immunohistochemistry confirmed that PD1 suppresses the phosphorylation of Src and Smad2 (pSrc, pSmad2), as seen in the band images (Figure 30B) and quantitative results (Figure 30C). Specifically, the percentage of phosphorylated Src and Smad2 in the activated state (%pSrc, %pSmad2) decreased in the PD1 administration group, confirming that PD1 inhibits the phosphorylation of Smad and Src, which are representative activation pathways in fibroblasts, and thus exhibits a fibrosis-improving effect. In addition, immunohistochemistry analysis confirmed that PD1 inhibits actin polymerization and MRTF-A nuclear translocation, as seen in the immunohistochemistry images (Figure 29B) and quantitative fluorescence intensity results (Figure 29C).

[0128] (Discussion) The snRNA-seq analysis in Example 10 suggested that the Rho-GTPase signaling pathway is involved in fibroblast activation. To verify this, we performed an analysis using this example (in vitro primary fibroblast culture evaluation system) and confirmed that administration of the PD compound (PD1) actually altered various molecules related to the Rho-GTPase signaling pathway (Src phosphorylation, MRTF-A, etc.). Furthermore, in the primary fibroblast culture evaluation system, PD administration was able to suppress the gene expression of collagen, a representative collagen fiber. These results strongly support the mechanism by which PD1 deactivates fibroblasts by inhibiting the Rho-GTPase pathway.

[0129] [Example 13. In vitro analysis using human cardiac fibroblasts] The same study as in Example 12 was performed using human cardiac fibroblasts (Human CFB) isolated from the cardiac tissue of heart failure patients (Figure 31A). Specifically, human fibroblasts were isolated from left ventricular apical samples obtained at the time of left ventricular assist device implantation.

[0130] (Results) Immunostaining analysis revealed that in human cardiac fibroblasts, the PD1-administered group showed significant suppression of MRTF-A nuclear translocation and actin polymerization (phaloidin staining intensity) (Figure 31B). Furthermore, gene expression analysis showed that PD1 significantly suppressed the upregulation of major fibrosis-related genes (COL1A1, POSTN, ACTA2) induced by TGF-β stimulation (Figure 31C). These results strongly suggest that the anti-fibrotic effect and mechanism (inhibition of the Rho / MRTF-A pathway) of the pyridopyrimidine compound (PD) of the present invention are conserved not only in mouse cells but also in human cells, and that clinical efficacy can be expected.

[0131] [Example 14. Verification of the effect of improving mitochondrial metabolism in iPS cell-derived cardiomyocytes] The effect of administering PD1 alone to human iPS cell-derived cardiomyocytes (iPSC-CM) and hydrogen peroxide (H) was verified. ２ O ２ The protective effect (efficacy study) of ) under oxidative stress load was investigated. Human iPS cell-derived cardiomyocytes (iPSC-CMs) were obtained by differentiating iPS cells generated from peripheral blood cells of healthy individuals into cardiomyocytes.

[0132] (1) Activation of mitochondrial metabolic pathways (under non-oxidative stress) PD1 was administered to iPSC-CMs under normal culture conditions, and RNA-seq analysis was performed.

[0133] (Results) RNA-seq analysis of human iPSC-CMs revealed that mitochondrial metabolism-related pathways were among the top genes expressed in the PD1-treated group (Figures 32A and 32B). This suggests that PD1 fundamentally improves the mitochondrial metabolic function of cardiomyocytes, regardless of the presence or absence of stress.

[0134] (2) Maintaining function under oxidative stress (AMPK pathway and respiratory function) Next, oxidative stress (H ２ O ２The effects of PD1 were investigated under conditions of oxidative stress. Specifically, proteins were extracted from iPS-derived cardiomyocytes, and Western blotting was performed using antibodies against each target protein to evaluate the amount of protein under each condition. In addition, mitochondrial respiratory capacity was measured using a Seahorse XF flux analyzer in iPS-derived cardiomyocytes 24 hours after the start of oxidative stress.

[0135] (Results) Analysis by Western blotting showed that H ２ O ２ Phosphorylated AMPK (pAMPK) levels, which did not decrease or change with load alone, significantly increased in the PD1-treated group (Figure 33A). Phosphorylated AMPK is an important regulator for the control of mitochondrial function and metabolism. The significant increase in these phosphorylated AMPK levels in the PD1-treated group suggests that PD1 activates the energy metabolism (mitochondrial function) of cardiomyocytes. In addition, mitochondrial respiratory capacity (OCR) was measured using a Seahorse XF flux analyzer, and H ２ O ２ Respiratory capacity, which had decreased due to the load, was restored and maintained by PD1 administration (Figure 33B). In particular, significant improvements were observed in spare respiratory capacity and maximal respiration, confirming that metabolic function was activated (Figure 33C).

[0136] [Example 15: Effect of improving contractile function and mitochondrial metabolic function in iPS cell-derived cardiomyocytes] Similar to Example 14, H ２ O ２ We evaluated the contractile and mitochondrial functions of human iPSC-CMs under load. Specifically, we added a calcium fluorescent dye to iPS-derived cardiomyocytes seeded in a 96-well plate and observed calcium dynamics using FDSS / μCELL. Contractile motion was also evaluated using the Cell Motion Imaging System SI8000. Furthermore, we evaluated mitochondrial function using a mitochondrial membrane potential probe and a fluorescent dye for detecting mitochondrial superoxide.

[0137] (Results) (1) Improvement of contractile function Based on the results of calcium imaging and contractile analysis, H ２ O ２ Under loading conditions, the PD1 administration group (H ２ O ２ +PD) is the non-administered group (H ２ O ２ Compared to the other group, the calcium amplitude (an indicator of contractile force) was significantly increased (Figure 34A). In addition, significant improvements were observed in contraction velocity and deformation distance in the PD1-treated group (Figure 34B).

[0138] (2) Improvement of mitochondrial metabolic function When mitochondrial ROS (mtROS) was fluorescently stained, H ２ O ２ ROS levels, which increased due to stress, were significantly suppressed in the PD1-treated group (Figure 35). Furthermore, analysis using a mitochondrial membrane potential indicator (MT-1) confirmed the maintenance of membrane potential (increased fluorescence intensity) in the PD1-treated group (Figure 36). These results indicate that the pyridopyrimidine compound of the present invention improves mitochondrial metabolism through activation of the AMPK pathway and improves the function of cardiomyocytes under oxidative stress.

[0139] The above results demonstrate that the pyridopyrimidine compounds of the present invention have deactivating and inhibitory effects on activated hepatic stellate cells, thereby improving fibrosis and / or normalizing or restoring tissue function, and that the pyridopyrimidine compounds of the present invention are useful in the treatment or prevention of fibrosis.

[0140] The scope of the present invention is not limited to the foregoing description, and the invention may be modified and implemented in any way that does not impair the spirit of the invention, in addition to the examples given above. All documents and publications mentioned herein, regardless of their purpose, are incorporated herein by reference in their entirety.

Claims

1. A therapeutic or preventive agent for a disease, disorder or condition selected from the group consisting of fibrosis, organ failure associated with fibrosis, and organ failure associated with down-regulation of the AMPK signaling pathway, the therapeutic or preventive agent comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. [Wherein, R １ , R ２ , and R ３ are each independently a hydrogen atom, a halogen atom, C １～４ alkyl, C １～４ alkoxy, C １～４ alkylthio, and morpholin-4-yl, and the C １～４ alkyl, C １～４ alkoxy, and C １～４ alkylthio may be substituted with a substituent selected from hydroxy and -NR ５ R ６ ; R ４ is selected from the group consisting of C １～４ alkyl and C １～４ alkoxy; R ５ and R ６ are each independently selected from the group consisting of a hydrogen atom and C １～４ alkyl. ] 2. R １ , R ２ , and R ３ At least one of them is not a hydrogen atom, but R ４ C １～２ The therapeutic or prophylactic agent according to claim 1, wherein the agent is alkyl.

3. The aforementioned compound, A therapeutic or prophylactic agent according to claim 1 or 2, selected from the group consisting of and pharmaceutically acceptable salts thereof.

4. R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is -NR ５ R ６ A therapeutic or prophylactic agent according to any one of claims 1 to 3, which may be substituted with substituents selected from the following.

5. The therapeutic or prophylactic agent according to any one of claims 1 to 4, wherein the disease, disorder, or condition is fibrosis.

6. The therapeutic or prophylactic agent according to claim 5, wherein the disease, disorder, or condition is fibrosis occurring in the heart, brain, digestive tract, skin, lungs, kidneys, liver, pancreas, hematopoietic organs, retroperitoneum, mediastinum, joints, muscles, blood vessels, eyes, breasts, or reproductive organs.

7. The therapeutic or prophylactic agent according to claim 5, wherein the disease, disorder, or condition is fibrosis occurring in the liver, kidney, or heart.

8. The therapeutic or prophylactic agent according to any one of claims 1 to 7, wherein the organ failure is at least one selected from liver failure, kidney failure, and heart failure.

9. A therapeutic or prophylactic agent according to any one of claims 1 to 8, which promotes the expression of TCF21.

10. A therapeutic or prophylactic agent according to any one of claims 1 to 9, comprising: (i) reducing the expression level of at least one fibrosis marker; (ii) increasing the expression level of at least one quiescent hepatic stellate cell marker; (iii) increasing the expression level of at least one hepatic regeneration induction marker; (iv) reducing the accumulation of F-actin; and / or (v) transitioning activated myofibroblasts to a quiescent state.

11. The therapeutic or prophylactic agent according to any one of claims 1 to 4, wherein the disease, disorder, or condition is heart failure.

12. The therapeutic or prophylactic agent according to claim 11, which (i) increases mitochondrial metabolism in cardiomyocytes, (ii) induces activation of AMPK in cardiomyocytes, and / or (iii) induces at least one of inhibition of nuclear translocation of MRTF-A, reduction of SRF transcriptional activity, or inhibition of Src phosphorylation in cardiac fibroblasts.

13. The therapeutic or prophylactic agent according to claim 11 or 12, which (i) reduces the expression level of at least one fibrosis marker gene selected from COL1A1, COL1A2, POSTN, CCN2, MEOX1, and ACTA2, (ii) increases the expression level of the metabolism-related marker gene PPARGC1A, and / or (iii) reduces the expression level of at least one heart failure marker selected from NPPA (ANP) and NPPB (BNP).

14. A therapeutic or prophylactic agent according to any one of claims 1 to 13, which inhibits the Rho-GTPase signaling pathway in myofibroblasts.

15. A myofibroblast deactivator comprising a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof. [In the formula, R １ , R ２ , and R ３ These are, independently, a hydrogen atom, a halogen atom, and C １～４ Alkyl, C １～４ Alkoxy, C １～４ Selected from the group consisting of alkylthio and morpholine-4-yl, the C １～４ Alkyl, C １～４ Alkoxy, and C １～４ Alkylthio is hydroxy, and -NR ５ R ６ R may be substituted with a substituent selected from the following: ４ C １～４ Alkyl and C １～４ Selected from the group consisting of alkoxys, R ５ and R ６ These are, independently, hydrogen atoms and C １～４ Selected from the group consisting of alkyl groups.

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