JP3700854B2 - Fibrosis inhibitor - Google Patents

Description

  The present invention relates to a novel fibrosis inhibitor and a medicament for suppressing the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension containing them.

  In Japan, there are many patients with viral hepatitis such as hepatitis B and hepatitis C. According to a trial calculation by the Ministry of Health, Labor and Welfare, the number of patients with chronic hepatitis is estimated to be about 1.5 million and cirrhosis about 300,000 or more. Patients who have progressed to cirrhosis have diminished liver function and have died, resulting in liver failure. In this case, there is no treatment other than liver transplantation at the current medical level. In other words, liver fibrosis is caused by chronic liver damage due to various causes such as viral hepatitis, alcoholic hepatitis, etc., but removal of the cause causing liver damage is currently the first treatment to prevent liver fibrosis Is the law. Therefore, the development of therapeutic methods such as drugs that suppress effective fibrosis is an extremely important research theme for current liver disease.

  Recent advances in molecular biological research have dramatically deepened the understanding of hepatic fibrosis at the cellular and molecular level, and activated hepatic stellate cells and the cytokines involved in them play a major role in liver fibrosis. Is becoming clear. In addition, in the current research, with the goal of suppressing activated hepatic stellate cell proliferation as a treatment for liver fibrosis and suppressing extracellular matrix production, gene therapy and suppression of collagen production by drugs in several experimental models, etc. Although it effectively suppresses fibrosis, an effective and safe treatment for humans has not yet been established.

  Prevention and countermeasures against these viral hepatitis are gradually progressing, and some patients show dramatic effects with interferon therapy for chronic hepatitis C, or with interferon therapy for chronic hepatitis C. There are reports that improvement of fibrosis was observed (Non-patent Documents 1 and 2). However, the present condition is that the effective method other than suppressing fibrosis secondary by suppressing hepatocyte necrosis has not been found yet.

  Therefore, in the current clinical setting, the number of cases in which liver fibrosis progresses and progresses to cirrhosis / liver failure and has an unfortunate outcome has not decreased. Moreover, many patients who have progressed to cirrhosis developed liver cancer, and liver fibrosis is a major factor in the development of liver cancer. In Japan, the death rate from liver cancer is increasing year by year. In this way, suppression of liver fibrosis suppresses the progression to cirrhosis / liver failure and prevents the development of liver cancer, and if this is put to practical use, it will bring great benefits to the public, so liver fibrosis research is now the central research theme of liver disease It has become.

  Recent advances in research on liver fibrosis have dramatically deepened the understanding of liver fibrosis at the cellular and molecular level, and activated hepatic stellate cells and cytokines involved in them play a major role in liver fibrosis. In particular, it has become clear that TGFβ1 plays a central role in the pathogenesis of liver fibrosis (Non-patent Document 3). That is, when the release of TGFβ1 increases due to persistent hepatocellular injury / inflammation, the activation and proliferation of hepatic stellate cells into myofibroblast-like cells is promoted, and the production of extracellular matrix is promoted. Suppresses the synthesis of degradation enzymes and causes accumulation of fibrous tissue.

Therefore, therapies for liver fibrosis aim to suppress activated hepatic stellate cell proliferation and suppress extracellular matrix production. To date, gene therapy and therapeutic agents for fibrosis are largely classified as hepatic fibrosis suppression methods at the experimental level.
In gene therapy, for the purpose of suppressing the production of TGFβ1, it has been reported that hepatic fibrosis could be suppressed by expressing a hepatocyte growth factor (HGF) having an inhibitory effect on TGFβ1 expression by gene transfer (Non-Patent Document 4 and 5, Patent Document 1). It has also been reported that liver fibrosis could be suppressed by introducing a mutant TGFβ receptor gene and specifically suppressing the action of TGFβ1 in vivo (Non-patent Document 6).

  In addition, as a fibrosis therapeutic agent, it has been reported in clinical cases that interferon α described above suppresses the expression of TGFβ1 in liver tissue and improves liver fibrosis (Non-patent Document 1). Experimentally, there is a report that a prolyl hydroxylase inhibitor (HOE077), which is a collagen fiber production inhibitor, has suppressed liver fibrosis in a rat liver fibrosis model (Non-patent Document 7). There is also a report of suppression of liver fibrosis by anti-TGFβ1 antibody (Patent Document 2), ε-aminocaproic acid (Patent Document 3) and anti-heat shock protein antibody (Patent Document 4).

Furthermore, recent reports include free radical scavenger Flavonoid quercetin (Non-patent document 8), vitamin E (Non-patent document 9), polyphenols (Non-patent document 10), and antialdosterone agent canrenone (Non-patent document 11). ), Cordyceps sinensis (Non-Patent Document 12) and L-cysteine (Non-Patent Document 13), which are herbs, have been reported to suppress liver fibrosis.
There are several reports on experimentally controlling liver fibrosis as described above, but none of them have been put into practical use. In particular, with the discovery of effective fibrosis therapeutic agents, the development of highly compliant treatments such as oral administration is particularly desired for this type of disease with many onset cases.

  On the other hand, nitric oxide (NO) has been found as the body of vascular endothelial cell-derived vasorelaxing factor, and it has been clarified that it has various functions in various organs in addition to the physiological regulating action of the circulatory organ. In the liver, it is suggested that NO is involved in pathological conditions such as cirrhosis and hepatic ischemia / reperfusion injury (Patent Documents 5 and 6 and Non-Patent Documents 14 to 17). In the case of hepatic ischemia / reperfusion injury, the present inventors have experimentally studied and reported that administration of an inducible NO synthase inhibitor significantly suppresses hepatic ischemia / reperfusion injury. (Non-patent documents 18 to 20).

  On the other hand, the expression of the inducible NO synthase (iNOS) is enhanced in cirrhosis, and the hyperdynamic circulation state in patients with cirrhosis is caused by endotoxemia associated with cirrhosis, which induces a large amount of inducible NO synthase. It has been reported that type NO synthase-derived NO is produced and hyperdynamic circulation occurs due to this vasodilatory effect (Non-patent Document 14). However, all of these reports relate to pathological conditions such as hemodynamics of cirrhosis, and there are no reports of research results on the relationship between TGFβ1 production and liver fibrosis and inducible NO synthase-derived NO. That is, the causal relationship between inducible NO synthase-derived NO and liver fibrosis and TGFβ1 production is still unclear.

Patent Documents 5 and 6 describe substances that inhibit the activity of iNOS. However, there is no example that has led to suppression of TGFβ1 production and / or liver fibrosis by suppressing generation of inducible NO synthase-derived NO.
In addition, the compound used in this invention and its pharmaceutically acceptable salt are described in patent document 6 as a substance which inhibits the activity of iNOS. However, this document does not specifically disclose the pharmacological action of such compounds on TGFβ1 production, liver fibrosis inhibition and the like as described above.

In any case, cirrhosis is caused by liver fibrosis, and although it has been clarified that TGFβ1 expression is involved in such onset, an effective drug for suppressing liver fibrosis and progression of cirrhosis No effective drug has yet been found to suppress this.
In addition, there are stellate cells in the pancreas as in the liver, TGFβ is involved in fibrosis, and it is reported that pancreatic fibrosis may develop due to pancreatic fibrosis by the same mechanism as liver fibrosis (Non-Patent Documents 21 to 29). However, the present condition is that the effective drug for suppressing pancreatic fibrosis and the effective drug for suppressing the progression of chronic pancreatitis have not yet been found.
Furthermore, although it is known that fibrosis occurs with pulmonary hypertension, an effective drug for suppressing such fibrosis has not yet been found.

Japanese National Patent Publication No. 5-503076 JP-A-9-194360 JP-A-4-18028 JP 7-61936 A JP-A-9-235276 International Publication No. 02/0255541 Pamphlet

Bataller, M.D. et al., "Hepatic Stellate Cells as a Target for the Treatment of Liver Fibrosis", Seminars in Liver Disease, U.S.A. November 3, 2001, vol. 21, 437- 451. Ulloa, L et al., "Inhibition of transforming growth factor-β / SMAD signaling by the interferon-γ / STAT pathway", U.K., 25 February 1999, Vol. 397, 710- 713. Ueno et al., “Mechanism of hepatic fibrosis formation”, Hepatobiliary pancreas, Japan, May 2002, Vol. 44, No. 5, pp. 567-574. Ueki, T. et al., "Hepatocyte growth factor gene therapy of liver cirrhosis in rats", Nature Medicine, Nature America Inc., U.S.A. February 1999, Vol. 5, No. 2, 226-230. Iimuro et al., “Mechanism of action of HGF on liver fibrosis”, Hepatobiliary pancreas, Japan, May 2002, Vol. 44, No. 5, pp. 625-631. Qi Z. et al., "Blockade of type β transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat", Proc. Natl. Acad. Sci., USA, USA, March 1999, βVol. 96, 2345- 2349, Medical Sciences.

Saida et al., “Inhibition of fibrosis”, Hepatobiliary pancreas, Japan, May 2002, Vol. 44, No. 5, pp. 613-618. Pavanto A. et al., "Effects of quercetin on liver damage in rats with carbon tetrachloride-induced cirrhosis", [online], April 2003, National Library of Medicine, [searched June 13, 2003], Internet <URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12741479&dopt=Abstract> Zhan Y. et al., "Effects of vitamin E on the proliferation and collagen synthesis of rat hepatic stellate cells treated with IL-2 or TNF-alpha", [online], March 2003, National Library of Medicine, [Heisei Jun 13, 2015], Internet <URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12781063&dopt=Abstract>

Zhong Z. et al., "Polyphenols from Camellia sinensis Attenuate Experimental Cholestasis-Induced Liver Fibrosis in Rats", [online], June 2003, National Library of Medicine, [searched June 13, 2003], Internet < URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12791596&dopt=Abstract> Caligiuri A. et al., "Antifibrinogenic Effects of Canrenone, an Antialdosteronic Drug, on human Hepatic Stellate Cells", American Gastroenterology, Gasteroenterological Association, U.S.A. February 2003, Vol.124, 504-520. Liu Y. et al., "Inhibitive effects of cordyceps sinensis on experomental hepatic fibrosis and its possible mechanism", World Journal of Gastroenterology, The WJG Press, China, March 15, 2003, Vol. 9, No. 3, 529 -533. Horie T. et al., "L-Cysteine administration prevents liver fibrosis by suppressing hepatic stellate cell proliferation and activation", Biochemical and Biophysical Research Communications, Elsevier Science, USA, March 15, 2003, Vol. 305, 94-100 . Arkenau H. et al., "Elevated nitric oxide levels in patients with chronic liver disease and correlate with disease stage and parameters of hyperdynamic circulation", [online], November 2002, National Library of Medicine, [June 2003] Search 13 days], Internet <URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=127436367&dopt=Abstract>

Sanz-Cameno P. et al., "Enhanced intrahepatic inducible nitric oxide synthase expression and nitrotyrosine accumulation in primary biliary cirrhosis and autoimmune hepatitis", [online], December 2002, National Library of Medicine, [June 13, 2003 Day search], Internet <URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12445411&dopt=Abstract> McNaughton L. et al., "Distribution of nitric oxide synthase in normal and cirrhotic human liver", [online], December 13, 2002, National Library of Medicine, [searched June 13, 2003], Internet < URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12482944&dopt=Abstract> Hassan MI. Et al., "Evaluation of nitric oxide (NO) levels in hepatitis C virus (HCV) infection: relationship to schistosomiasis and liver cirrhosis among Egyptian patients", [online], 2002, National Library of Medicine, [Heisei Search June 13, 2015], Internet <URL: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12515909&dopt=Abstract> Meguro M, Katsuramaki T, Kimura H, Isobe M, Nagayama M, Kukita K, Nui A, Hirata L., "Apotosis and necrosis after warm ischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714", Transplantation 75 : 703-710, 2003 Meguro M, Katsuramaki T, Nagayama, M KimuraH, Isobe M, KimuraY, Matsuno T, Nui A, Hirata H., "A novel inhibitor of inducible nitric oxid synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig liver ", Transplantation 73: 1439-1446, 2002 Isobe M, Katsuramaki T, Kimura H, Nagayama M, Matsuno T., "Beneficial effects of inducible nitric oxide synthase inhibitor on reperfusion injury in the pig liver", Transplantation 68: 803-813, 1999 Kuno A et al., "Angiotensin-converting enzyme inhibitor attenuates pancreatic inflammation and fibrosis in male Wistar Bonn / Kobori rats", Gastroenterology. 2003 Apr; 124 (4): 1010-9. Tashiro M et al., "Oleic acid-induced pancreatitis alters expression of transforming growth factor-beta1 and extracellular matrix components in rats", Pancreas. 2003 Mar; 26 (2): 197-204. Phillips PA et al., "Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover", Gut. 2003 Feb; 52 (2): 275-82. Shinjia T et al., "Establishment of a novel collagenase perfusion method to isolate rat pancreatic stellate cells and investigation of their gene expression of TGF-beta1, type I collagen, and CTGF in primary culture or freshly isolated cells", Acta Med Okayama. 2002 Aug; 56 (4): 211-8. Shek FW et al., "Expression of transforming growth factor-beta 1 by pancreatic stellate cells and its implications for matrix secretion and turnover in chronic pancreatitis", Am J Pathol. 2002 May; 160 (5): 1787-98., Yoshikawa H et al., "Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats", Am J Physiol Gastrointest Liver Physiol. 2002 Mar; 282 (3): G549-58. Schuppan D et al., "Fibrosis of liver, pancreas and intestine: common mechanisms and clear targets?", Acta Gastroenterol Belg., Germany, 2000 Oct-Dec; 63 (4): 366-70. Su SB et al., "Expression of transforming growth factor-beta in spontaneous chronic pancreatitis in the WBN / Kob rat" Dig Dis Sci. 2000 Jan; 45 (1): 151-9. Apte MV et al., "Pancreatic stellate cells are activated by proinflammatory cytokines: implications for pancreatic fibrogenesis", J Gut. 1999 Apr; 44 (4): 534-41. Criado M et al., "Role of prostanoids and nitric oxide inhibition in rats with experimental hepatic fibrosis" J. Physiol. Biochem., 2000 Sep; 56 (3): 181-8.

  Therefore, an object of the present invention is to provide a compound having a fibrosis-inhibiting activity or a pharmaceutically acceptable salt thereof and a fibrosis-inhibiting agent containing them as an active ingredient, cirrhosis and / or chronic pancreatitis containing such an inhibitor and / or Another object of the present invention is to provide a medicament for suppressing the progression of pulmonary hypertension, and a method for inhibiting the progression of liver cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension using these.

  In order to solve the above-mentioned problems, and as a result of intensive studies focusing on the relationship between an iNOS inhibitor and liver fibrosis, the present inventors surprisingly made fibrosis with an iNOS inhibitor having a specific structure. Has been found to be drastically suppressed, and further the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension has been found, and further research has been carried out to complete the present invention.

式中、Ｘは

であり、
Ｒ^１はハロゲンによって置換されているベンゾフラニル、またはハロゲンによって置換されているスチリルであり、
Ｒ^２は炭素数１〜６のアルキルまたはハロアルキル、１個または２個以上の、炭素数１〜６のアルキル、アルコキシ、ハロアルキルもしくはハロアルコキシまたはハロゲンによって任意に置換されていてもよいヘテロ環基、１個または２個以上の、炭素数１〜６のアルキル、アルコキシもしくはハロアルキルまたはハロゲンによって任意に置換されていてもよいアリール基であり、
Ｙは−Ｏ−、−Ｓ−または−ＳＯ_２−である、
で表される化合物または薬学的に許容されるその塩を有効成分として含有し、ＴＧＦβ１の増加または星細胞の活性化によって誘導される線維化を抑制する、線維化抑制剤に関する。 That is, the present invention provides compounds of formula I

Where X is

And
R ¹ is benzofuranyl substituted with halogen, or styryl substituted with halogen;
R ² is an alkyl or haloalkyl having 1 to 6 carbon atoms, 1 or 2 or more alkyl groups having 1 to 6 carbon atoms, alkoxy, haloalkyl or haloalkoxy, or a heterocyclic group optionally substituted with halogen, An aryl group optionally substituted by one or more C 1-6 alkyl, alkoxy or haloalkyl or halogen;
Y is —O—, —S— or —SO ₂ —.
Or a pharmaceutically acceptable salt thereof as an active ingredient , and a fibrosis inhibitor that suppresses fibrosis induced by an increase in TGFβ1 or activation of stellate cells .

である、前記線維化抑制剤に関する。
さらに、本発明は、式Ｉにおいて、Ｙが−Ｏ−である、前記線維化抑制剤に関する。
また、本発明は、式Ｉにおいて、Ｒ^２が１個または２個以上の置換基を有するヘテロ環基である、前記線維化抑制剤に関する。
さらに、本発明は、式Ｉにおいて、Ｒ^２が１個または２個以上の置換基を有する含窒素ヘテロ環基である、前記線維化抑制剤に関する。
また、本発明は、式Ｉにおいて、Ｒ^２が１個または２個以上の置換基を有するピリミジニル基である、前記線維化抑制剤に関する。 The present invention also provides that in formula I, R ¹ is 2- (4-chlorophenyl) ethenyl, and X is

It is related with the said fibrosis inhibitor.
Furthermore, the present invention relates to the fibrosis inhibitor, wherein Y is —O— in formula I.
The present invention also relates to the fibrosis inhibitor, wherein in Formula I, R ² is a heterocyclic group having one or more substituents.
Furthermore, the present invention relates to the fibrosis inhibitor, wherein in Formula I, R ² is a nitrogen-containing heterocyclic group having one or more substituents.
In addition, the present invention relates to the fibrosis inhibitor, wherein in Formula I, R ² is a pyrimidinyl group having one or more substituents.

で表される化合物（２Ｅ）−３−（４−クロロフェニル）−Ｎ−［（１Ｓ）−２−オキソ−２−｛［２−オキソ−２−（４−｛［６−（トリフルオロメチル）−４−ピリミジニル］オキシ｝−１−ピペリジニル）エチル］アミノ｝−１−（２−ピリジルメチル）エチル］−２−プロペンアミドである、前記線維化抑制剤に関する。
また、さらに本発明は、前記線維化抑制剤の、肝硬変および／または慢性膵炎および／または肺高血圧症の進行を抑制するための医薬の製造における使用に関する。
さらにまた、本発明は、前記線維化抑制剤を含む、肝硬変および／または慢性膵炎および／または肺高血圧症の進行を抑制するための医薬に関する。
そして、本発明は、錠剤、ピル、被覆錠剤、カプセル、粉末、顆粒、シロップ、ジュース、溶液、懸濁液およびドロップから選択される経口投与形態を有する、前記医薬に関する。 Furthermore, this invention relates to the said fibrosis inhibitor which has hepatic fibrosis inhibitory activity and / or pancreatic fibrosis inhibitory activity, and / or lung fibrosis inhibitory activity.
The present invention also provides that the compound represented by Formula I is represented by Formula II:

(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[6- (trifluoromethyl)] -4-pyrimidinyl] oxy} -1-piperidinyl) ethyl] amino} -1- (2- pyridylmethyl ) ethyl] -2-propenamide.
Furthermore, the present invention relates to the use of the fibrosis inhibitor in the manufacture of a medicament for suppressing the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension.
Furthermore, the present invention relates to a medicament for suppressing the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension, comprising the fibrosis inhibitor.
And this invention relates to the said pharmaceutical which has an oral dosage form selected from a tablet, a pill, a coated tablet, a capsule, a powder, a granule, a syrup, juice, a solution, a suspension, and a drop.

  The compound which is an active ingredient of the fibrosis-suppressing agent of the present invention and a pharmaceutically acceptable salt thereof can prevent fibrosis directly or by reducing the production of NO derived from inducible NO synthase. Suppress. In addition, the compound and the salt thereof also suppress liver fibrosis and / or pancreatic fibrosis and / or lung fibrosis by suppressing fibrosis, chronic pancreatitis that develops by the same mechanism as cirrhosis, and / or Or it has a special effect of suppressing the progression of pulmonary hypertension.

  Nitric oxide (NO) was found as the main body of vascular endothelial cell-derived vasorelaxing factor, and it has been revealed that it has various functions in various organs other than the physiological regulation action of the circulatory organ. Enzymes that produce NO can be broadly classified into constitutive NO synthases that contribute to the maintenance of homeostasis and inducible NO synthases induced by various invasions. Acts as a cytoprotective, and inducible NO synthase-derived NO is thought to act cytotoxically. NO produced from the inducible NO synthase is said to be about 1000 times the NO produced by the constitutive NO synthase, and a large amount of NO derived from the inducible NO synthase is produced. Binds to active oxygen and becomes a highly toxic substance, and exerts a damaging action in various tissues.

  The present inventors have also studied NO in a hepatic ischemia / reperfusion injury model, and inducible NO synthase was expressed in inflammatory cells such as Kuppfer cells and leukocytes after ischemia / reperfusion and produced therefrom. Inducible NO synthase-derived NO combines with active oxygen to produce highly toxic peroxinitrite, causing hepatocellular injury after reperfusion, and administration of an inducible NO synthase inhibitor makes this reperfusion injury prominent It has been proved to be suppressed in pig liver ischemia reperfusion experiments and pig liver transplantation experiments. In addition to organ ischemia and reperfusion, inducible NO synthase is induced in inflammatory cells such as macrophages and leukocytes by various invasions such as sepsis and surgery, and a large amount of inducible NO synthase-derived NO produced therefrom is tissue It has been reported to work disability.

On the other hand, in the liver, it has been reported that NO is involved in a hyperdynamic circulation state occurring in cirrhosis patients other than hepatic ischemia / reperfusion injury. That is, in hyperdynamic circulation in cirrhosis patients, endotoxemia associated with cirrhosis causes inducible NO synthase to be expressed in Kupffer cells, etc., and a large amount of inducible NO synthase-derived NO produced thereby causes vasodilation. Hyperdynamic circulation is said to occur. However, the involvement of inducible NO synthase-derived NO in liver fibrosis has not been studied at all.
On the other hand, there has been a report experimentally examining the involvement of L-NAME, which is a constitutive NO synthase inhibitor, in liver fibrosis (Non-patent Document 30). However, the results obtained in this experiment indicated that L-NAME promoted liver fibrosis. That is, the action of L-NAME is completely different from the action of the composition of the present invention. Moreover, constitutive NO synthase-derived NO acts as a cytoprotective substance as described above, and its production amount is only 1/1000 of inducible NO synthase-derived NO. Therefore, even if hepatic fibrosis can be experimentally suppressed by inhibiting such a constitutive NO synthase, the risk of causing side effects is far greater than its effect, so constitutive NO synthesis Enzyme inhibitors are extremely difficult to use clinically.
That is, there have been no reports on the inhibition of liver fibrosis by inhibiting inducible NO synthase.

In the present invention, a substance having “fibrosis-inhibiting activity” means that when the substance is administered to organ cells such as hepatocytes, pancreatic cells or lung cells, it is compared with the case where the substance is not administered. Thus, it means that the fibrosis in the administered hepatocytes, pancreatic cells or lung cells is suppressed.
In the present invention, a substance having "TGFβ1 production inhibitory activity" means that when the substance is administered to a tissue, organ or living body, the administered tissue, compared to the case where the substance is not administered, It means that TGFβ1 production in an organ or a living body is suppressed.
In addition, in the present invention, a substance "has an activity of suppressing the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension" means that the substance shows signs of hepatitis or cirrhosis and / or Transition to liver cirrhosis in the administered subject when administered to a subject showing signs of chronic pancreatitis and / or a subject showing signs of pulmonary hypertension compared to non-administration of the substance Or it means that the progression of cirrhosis and / or the progression of chronic pancreatitis and / or pulmonary hypertension are respectively suppressed.

In addition, the “pharmaceutically acceptable salt” in the present invention means the salt in the case where the salt exerts a desired medicinal effect from the substance when the salt of the substance is administered to the subject. .
In the present invention, “a subject showing signs of hepatitis or cirrhosis, a subject showing signs of chronic pancreatitis or a subject showing signs of pulmonary hypertension” includes hepatitis or cirrhosis, chronic pancreatitis or pulmonary hypertension. Affected humans and animals are each included.

  According to the present invention, fibrosis in the liver and / or pancreas and / or lung can be suppressed. Further, according to the present invention, it is possible to suppress the progression of liver cirrhosis and / or the progression of chronic pancreatitis and / or the suppression of the progression of pulmonary hypertension from hepatitis, which has been impossible in the past, by administration such as oral administration of drugs. , Possible without serious side effects.

または

である。これらのうち、

は、高い線維化抑制効果を与えるため好ましい。
Ｒ^１はハロゲンによって置換されているベンゾフラニル、またはハロゲンによって置換されているスチリルである。これらのうち、２−（４−クロロフェニル）エテニルは、高い線維化抑制効果を与えるため好ましい。したがって、Ｘが

であり、Ｒ^１２が下記式で表される２−（４−クロロフェニル）エテニルである本願発明の抑制剤は、とくに好ましい。

The compound of formula I which is an active ingredient of the fibrosis inhibitor of the present invention includes each enantiomer and racemate. In the compound of formula I, X is

Or

It is. Of these,

Is preferable because it provides a high fibrosis-suppressing effect.
R ¹ is benzofuranyl substituted by halogen or styryl substituted by halogen. Among these, 2- (4-chlorophenyl) ethenyl is preferable because it provides a high fibrosis inhibitory effect. Therefore, X is

The inhibitor of the present invention in which R ¹² is 2- (4-chlorophenyl) ethenyl represented by the following formula is particularly preferable.

Y is —O—, —S— or —SO ₂ —. Among these, those in which Y is —O— are preferable because they give a high fibrosis inhibiting effect.
Moreover, in the compound I which is an active ingredient of the fibrosis inhibitor of the present invention and a pharmaceutically acceptable salt thereof, R ² is (1) an alkyl or haloalkyl having 1 to 6 carbon atoms,
(2) 1 or 2 or more, a C1-C6 alkyl, alkoxy, haloalkyl or haloalkoxy or a heterocyclic group optionally substituted by halogen, or (3) 1 or 2 or more An aryl group optionally substituted by alkyl, alkoxy or haloalkyl or halogen having 1 to 6 carbon atoms,
It is.

Among the heterocyclic groups included in R ² , one or more heterocyclic groups optionally having 1 to 6 carbon atoms, alkoxy, haloalkyl, haloalkoxy, or halogen are optionally substituted. preferable.
Among the heterocyclic groups, the substituent having a substituent selected from the group consisting of pyridyl, pyrazinyl, thiazolyl, pyridazinyl and pyrimidinyl and one or more alkyls having 1 to 6 carbon atoms, haloalkyl or haloalkoxy or halogen Heterocyclic groups are preferred, and nitrogen-containing heterocyclic groups of pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl are more preferred.
Further, among the substituted or unsubstituted heterocyclic groups, a heterocyclic group having one or more substituents is preferable, and a nitrogen-containing heterocyclic group having one or more substituents is more preferable. . Particularly preferred are those wherein R ² is pyrimidinyl having one or more substituents.

As said substituent of the said heterocyclic group, a C1-C6 haloalkyl or haloalkoxy or halogen is preferable, and the said haloalkyl is especially preferable. Further preferred substituent is a trifluoromethyl group.
R ² is most preferably 6- (trifluoromethyl) -4-pyrimidinyl.
Therefore, the most preferable —Y—R ² is —O-6- (trifluoromethyl) -4-pyrimidinyl represented by the following formula.

Among the fibrosis inhibitors of the present invention, those having an activity of inhibiting liver fibrosis can more effectively inhibit the progression of hepatitis to cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension. preferable.
That is, the fibrosis inhibitor can be used for suppressing the progression of cirrhosis and / or the progression of chronic pancreatitis and / or the progression of pulmonary hypertension.

In particular, the compound of formula I is (2E) -3- (4-chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[6- (trifluoro According to the fibrosis inhibitor that is methyl) -4-pyrimidinyl] oxy} -1-piperidinyl) ethyl] amino} -1- (2- pyridylmethyl ) ethyl] -2-propenamide or a salt thereof, progression of cirrhosis And / or the progression of chronic pancreatitis and / or pulmonary hypertension can be very effectively suppressed.
Therefore, the most preferred active ingredient of the fibrosis inhibitor of the present invention is a compound of the following formula II.

  Among the compounds of formula I, those classified as bases are converted into the corresponding acid addition salts using acids, for example, by reaction with an equal amount of base and acid in an inert solvent such as ethanol and subsequent evaporation. Can be converted. Suitable acids for this reaction are particularly those that provide pharmaceutically acceptable salts. Accordingly, as an acid addition salt, an inorganic acid, for example, a hydrohalic acid such as sulfuric acid, nitric acid, hydrochloric acid or hydrobromic acid, phosphoric acid such as orthophosphoric acid, or sulfamic acid, and also an organic acid, particularly an aliphatic, fatty acid. Cyclic, aromatic alicyclic, aromatic or polycyclic monobasic or polybasic carboxylic acids, sulfonic acids or sulfuric acids such as formic acid, acetic acid, propionic acid, pivalic acid, diethyl acetic acid, malonic acid, Succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethane Sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalene mono- and disulfonic acid, and lauryl sulfone , Salt and the like.

On the other hand, among the compounds of formula I, those classified as acids are related by the reaction of an equivalent amount of acid and base in an inert solvent such as ethanol, followed by distillation, using a base. Can be converted to acid addition salts. Suitable bases for this reaction are in particular those which give physiologically acceptable salts.
Thus, the acid of the compound of formula I can be used with a base (eg sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate) and the corresponding metal salt, in particular an alkali metal salt or alkaline earth metal salt, or the corresponding Can be converted to an ammonium salt.
An organic base is particularly suitable for this reaction. For example, an organic base that gives a physiologically acceptable salt such as ethanolamine is preferred.

  According to the medicament containing the fibrosis-suppressing agent of the present invention, an active ingredient can be effectively delivered, so that the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension from hepatitis is extremely effective. Can be suppressed.

  The medicament for suppressing the progression of cirrhosis and / or the progression of chronic pancreatitis and / or pulmonary hypertension according to the present invention comprises a fibrosis inhibitor comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient. It is. These medicaments can be effectively used as human medicine or veterinary medicine. Suitable excipients for formulating the medicament of the present invention are suitable for enteral (eg oral), parenteral or topical administration and are organic or inorganic substances which do not react with the compounds such as water, vegetable oil, benzyl alcohol , Alkylene glycol, polyethylene glycol, glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc or petrolatum.

  Suitable for oral administration are in particular tablets, pills, coated tablets, capsules, powders, granules, syrups, juices, solutions, suspensions or drops, suitable for rectal administration are suppositories, suitable for parenteral administration Solutions, preferably oil-based or aqueous solutions, also suspensions, emulsions or implants, and suitable for topical application are ointments, creams or powders, or made transdermally in patches Is.

  As a dosage form of the pharmaceutical agent of the present invention, oral administration is preferable from the viewpoint of the activity of the fibrosis inhibitor of the present invention in oral administration and the ease of administration. Since such oral administration can be carried out very easily by the subject itself, it is also preferable from the viewpoint of improving compliance, such as saving the trouble of going to hospital.

The compound of formula I and the salt thereof, which are the active ingredients of the fibrosis inhibitor according to the present invention, may be lyophilized, and the purified lyophilizate may be used, for example, for the production of injection preparations. The indicated formulations may be sterilized and / or auxiliaries such as lubricants, preservatives, stabilizers, and / or wetting agents, emulsifiers, salts to alter osmotic pressure, buffer substances , Coloring and flavoring agents, and / or a number of additional active ingredients, such as one or more vitamins.
Suitable formulations for administration in the form of an aerosol or spray are, for example, solutions, suspensions or emulsions of the active ingredient of the formula I in a pharmaceutically acceptable solvent.

  In general, the fibrosis-suppressing agent of the present invention is preferably administered at a dose of 1 to 500 mg, particularly preferably 5 to 100 mg as an amount of the active ingredient per dose unit. The daily dose is about 0.01 to 100 mg per kg body weight, preferably about 0.1 to 80 mg per kg body weight, and particularly preferably about 1 to 70 mg per kg body weight. However, the specific dose to each patient will depend on a wide range of factors such as the effectiveness of the specific compound used, age, weight, general health status, gender, diet, time and method of administration, excretion rate, Depending on the severity of the particular disease to which the pharmaceutical combination and treatment is applied. Oral administration is preferred.

  Therefore, the present invention further relates to the manufacture of a medicament for suppressing the progression of cirrhosis and / or the progression of chronic pancreatitis and / or pulmonary hypertension, particularly by a non-chemical method. About the use of. In this case, the compounds and their salts which are the active ingredients of these fibrosis-inhibiting agents are combined with at least one solid, liquid and / or semi-liquid excipient or auxiliary, and if desired, one or two Combination with the above further active ingredients can convert to a suitable dosage form.

  As shown below, the compound which is an active ingredient of the fibrosis inhibitor of the present invention and a pharmaceutically acceptable salt thereof are typically obtained by reaction with the corresponding carboxylic acid and carboxylate as follows. It is done.

Intermediate 1 Benzyl ({(2S) -2-[(tert-butoxycarbonyl) amino] -3- (2-pyridyl) propanoyl} amino) acetate
(2S) -2-[(tert-butoxycarbonyl) amino] -3- (2-pyridyl) propionic acid (55.0 g), glycine benzyl ester tosylate (69.7 g) and diphenylphosphoryl azide (46.7 ml) N, To the N-dimethylformamide solution (550 ml), N, N-diisopropylethylamine (75.6 ml) was added dropwise at 4 ° C. The mixture was warmed to room temperature and stirred for 3 hours. The resulting mixture was poured into ice-cold saturated aqueous sodium bicarbonate (700 ml). The mixture was extracted twice with ethyl acetate (total volume 1.3 L) and washed with water, saturated aqueous ammonium chloride, aqueous sodium bicarbonate (300 ml x 2) and brine (40 ml). The organic layer was dried over anhydrous magnesium sulfate and concentrated to obtain the target compound as light brown crystals (77.4 g).
ESI-MS: 414.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.48 (dd, J = 5, 2 Hz, 1H), 7.82 (br, 1H), 7.60 (td, J = 8, 2 Hz, 1H), 7.40-7.29 (m , 5H), 7.21 (d, J = 8Hz, 1H), 7.14 (dd, J = 8,5Hz, 1H), 6.33 (br, 1H), 5.15 (s, 2H), 4.62 (br, 1H), 4.04 (d, J = 6Hz, 2H), 3.36-3.18 (m, 2H), 1.43 (s, 9H).

Intermediate 2 benzyl {[(2S) -2 amino-3- (2-pyridyl) propanoyl] amino} acetate dihydrochloride benzyl ({(2S) -2-[(tert-butoxycarbonyl) -amino] -3- (2-Pyridyl) propanoyl} Amino) acetate (73.8 g) in ethyl acetate (150 ml) was added dropwise 4N hydrogen chloride in ethyl acetate (669 ml) at 10 ° C. over 30 minutes. The mixture was warmed to room temperature and stirred for 3 hours. The mixture was diluted with ethyl acetate (300 ml). The produced precipitate was collected by filtration, washed with ethyl acetate (700 ml), and dried under vacuum to obtain the target compound.
¹ H-NMR (300MHz, DMSO-d ₆ ) δ9.31 (t, J = 6Hz, 1H), 8.83 (d, J = 5Hz, 1H), 8.70 (br, 3H), 8.40 (t, J = 8Hz , 1H), 7.96 (d, J = 8Hz, 1H), 7.87 (t, J = 5Hz, 1H), 7.41-7.30 (m, 5H), 5.14 (s, 2H), 4.53 (br, 1H), 4.05 (dd, J = 18,6Hz, 1H), 3.99 (dd, J = 18,6Hz, 1H), 3.64 (dd, J = 15,5Hz, 1H), 3.54 (dd, J = 15,8Hz, 1H) .

Intermediate 3 Benzyl {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] -amino} -3- (2-pyridyl) propanoyl] amino} acetate
To a dichloromethane solution (400 ml) of 4-chlorocinnamic acid (49.2 g), oxalyl chloride (30.5 ml) and 1 drop of N, N-dimethylformamide were added, and the mixture was stirred at room temperature for 6 hours. The mixture was distilled to dryness and the acid chloride residue was dissolved in dichloromethane (900 ml). To this solution was added benzyl {[(2S) -2-amino-3- (2-pyridyl) propanoyl] amino} acetate dihydrochloride (104 g) at 10 ° C. and triethylamine (116 ml) over 40 minutes. The mixture was warmed to room temperature and stirred for 2 hours. The reaction mixture was distilled to dryness, and the residue was washed with water (500 ml x 5) and tetrahydrofuran-n-hexane (1: 2,500 ml x 10) and dried under vacuum to give the target compound as a white solid As 115 g.
ESI-MS: 478.3 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ8.51 (dd, J = 5,2Hz, 1H), 8.27 (t, J = 5Hz, 1H), 7.83 (d, J = 6Hz, 1H), 7.64 (td , J = 8,2Hz, 1H), 7.57 (d, J = 16Hz, 1H), 7.44 (d, J = 9Hz, 2H), 7.39-7.28 (m, 8H), 7.19 (dd, J = 8,5Hz , 1H), 6.47 (d, J = 16Hz, 1H), 5.14 (s, 2H), 4.98 (app q, J = 6Hz, 1H), .4.07 (d, J = 5Hz, 2H), 3.36 (dd, J = 15,5Hz, 1H), 3.26 (dd, J = 15,6Hz, 1H).

Intermediate 4 {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid benzyl {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} -acetate (10.8, g) in methanol suspension (200 ml) To the mixture was added 1N aqueous sodium hydroxide solution (22.6 ml), and the mixture was stirred at room temperature for 5 hours. The mixture was concentrated in vacuo and the residue was treated with aqueous citric acid (10%, 100 ml). The formed white precipitate was collected by filtration, washed with tetrahydrofuran-hexane (1: 1,150 ml), and dried under vacuum to obtain the target compound as a white solid (8.25 g).
ESI-MS: 388.2 (M + H)
¹ H-NMR (300 MHz, DMSO-d ₆ 12.6) δ (br, 1H), 8.50-8.36 (m, 3H), 7.68 (td, J = 8, 2Hz, 1H), 7.56 (d, J = 9Hz, 2H), 7.47 (d, J = 9Hz, 2H), 7.34 (d, J = 16Hz, 1H), 7.30 (d, J = 8Hz, 1H), 7.20 (dd, J = 8, 5Hz, 1%), 6.68 (d, J = 16Hz, 1H), 4.96-4.86 (m, 1H), 3.78 (dd, J = 18,6Hz, 1H), 3.74 (dd, J = 18,6Hz, 1H), 3.24 (dd, J = 14, 5Hz, 1H), 2.99 (dd, J = 14, 10Hz, 1H).

Intermediate 5 benzyl {[(2S) -2-{[(5-chloro-1-benzofuran-2-yl) carbonyl] amino} -3- (2-pyridyl) propanoyl] amino} acetate benzyl {[(2S) The above compound was obtained from 2-amino-3- (2-pyridyl) propanoyl] amino} acetate dihydrochloride and 5-chloro-1-benzofuran-2-carboxylic acid in the same manner as in Intermediate 3.
ESI-MS: 492.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.70-8.49 (m, 3H), 7.71-7.62 (m, 2H), 7.52-7.18 (m, 10H), 5.16 (s, 2H), 5.11-5.02 ( m, 1H), 4.19-4.03 (m, 2H), 3.44 (dd, J = 15,4Hz, 1H), 3.35 (dd, J = 15,7Hz, 1H).

Intermediate 6 {[(2S) -2-{[(5-Chloro-1-benzofuran-2-yl) carbonyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid Benzyl 1-[(2S) -2-{[(5-Chloro-1-benzofuran-2-yl) carbonyl] amino} -3- (2-pyridyl) propanoyl] -amino} acetate yields the above compound by a method similar to Intermediate 4. It was.
ESI-MS: 402.2 (M + H)
¹ H-NMR (300 MHz, DMSO-d ₆ ) δ 12.6 (br, 1H), 9.00 (d, J = 8 Hz, 1 H), 8.49 (dd, J = 5, 2 Hz, 1 H), 8.46 (t, J = 6Hz, 1H), 7.88 (d, J = 2Hz, 1H), 7.72 (d, J = 9Hz, 1H), 7.67 (td, J = 8,2Hz, 1H), 7.53 (d, J = lHz, 1H ), 7.48 (dd, J = 9, 2Hz, 1H), 7.33 (d, J = 8Hz, 1H), 7.20 (dd, J = 8, 5Hz, 1H), 5.04-4.94 (m, 1H), 3.80 ( dd, J = 18, 6Hz, 1H), 3.74 (dd, J = 18, 6Hz, 1H), 3.32 (dd, J = 14, 4Hz, 1H), 3.22 (dd, J = 14, 10Hz, 1H).

Intermediate 7 tert-Butyl 4- (1,3-thiazol-2-yloxy) -1-piperidinecarboxylate Sodium hydroxide (approximately 60% oil suspension, 5.67 g) in dimethoxyethane slurry (40 ml) -Butyl 4-hydroxy-1-piperidinecarboxylate (23.9 g) in dimethoxyethane (60 ml) was added over 30 minutes and the mixture was stirred for an additional hour. To the mixture was added 2-bromothiazole (15.0 g) and the mixture was refluxed for 4 hours.
After cooling, the resulting light brown suspension was diluted with ether (300 ml) and the mixture was filtered through a celite pad. The filtrate was washed with brine, dried over anhydrous magnesium sulfate and concentrated under vacuum. Silica gel column chromatography of oil residue
Purification by (eluent: 15% ethyl acetate in n-hexane) gave the target compound as a yellow oil (22.1 g).
ESI-MS: 285.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.11 (d, J = 4 Hz, 1 H), 6.67 (d, J = 4 Hz, 1 H), 5.13 (septet, J = 4 Hz, 1 H), 3.73 (ddd, J = 14,7,4Hz, 2H), 3.32 (ddd, J = 14,8,4Hz, 2H), 2.08-1.96 (m, 2H), 1.88-1.74 (m, 2H), 1.47 (s, 9H).

Reference example 1
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (1,3thiazol-2-yloxy) -1-piperidinyl] ] Ethyl} amino) -1- (2-pyridylmethyl) -ethyl] acrylamide
tert-Butyl 4- (1,3-thiazol-2-yloxy) -1-piperidinecarboxylate (2.93 g) in ethyl acetate (12 ml) and 4N hydrogen chloride in ethyl acetate (15 ml) at 10 ° C. Added dropwise. The mixture was stirred at room temperature for 1 hour and the mixture was further concentrated in vacuo and dried. The yellow solid residue was collected from {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) in N, N-dimethylformamide (40 ml). ) Propanoyl] amino} acetic acid (4.0 g), 1-hydroxybenzonitrile (1.53 g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.17 g) were added to an ice-cold mixture. N, N-diisopropylethylamine (3.9 ml) was added dropwise to the mixture. The mixture was stirred at room temperature for 2 hours and the resulting mixture was poured into ice-cold saturated aqueous solution of sodium bicarbonate (200 ml). The mixture was extracted three times with ethyl acetate (total amount 400 ml) and washed with saturated aqueous ammonium chloride (100 ml), water (70 ml x 3) and brine (20 ml). The organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: 1% methanol in chloroform) and recrystallized in ethyl acetate to obtain the target compound as a pale yellow solid (4.57 g).
ESI-MS: 554.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1H), 8.04-7.97 (m, 1H), 7.96-7.88 (m, 1H), 7.63 (td, J = 8 , 2Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 9Hz, 2H), 7.34 (d, J = 9Hz, 2H), 7.25 (d, J = 8Hz, 1H), 7.18 (dd, J = 8,5Hz, 1H), 7.10 (d, J = 4Hz, 1H), 6.68 (d, J = 4Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.25-5.15 ( br m, 1H), 5.06-4.99 (m, 1H), 4.03 (d, J = 4Hz, 2H), 3.83-3.52 (m, 3H), 3.43 (dd, J = 15,5Hz, 1H), 3.37- 3.31 (m, 1H), 3.26 (dd, J = 15, 6Hz, 1H), 2.08-1.81 (m, 4H).

Intermediate 8 tert-butyl 4- (2-pyrazinyloxy) -1-piperidinecarboxylate Sodium hydroxide (ca. 60% oil suspension, 114 mg) in dimethyl sulfoxide slurry (5 ml) with tert-butyl-4-hydroxy-1 -Piperidinecarboxylate (576 mg) was added dropwise. A considerable amount of foam was formed and the solution turned light orange. To this solution was added chloropyrazine (377 mg) and the mixture was stirred at room temperature for 12 hours. The resulting mixture was partitioned with saturated aqueous sodium bicarbonate and ethyl acetate. The organic layer was washed with water and brine, dried over anhydrous magnesium sulfate and concentrated under vacuum. The residual solid was triturated with a mixture of ethyl acetate and n-hexane to obtain the target compound as white crystals (472 mg).
ESI-MS: 280.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ8.20 (d, J = lHz, 1H), 8.10 (d, J = 3Hz, 1H), 8.05 (dd, J = 3, lHz, 1H), 5.20 (septet , J = 4Hz, 1H), 3.84-3.72 (m, 2H), 3.30 (ddd, J = 14,8,4Hz, 2H), 2.05-1.93 (m, 2H), 1.81-1.68 (m, 2H), 1.48 (s, 9H).

Reference Example 3
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (2 pyrazinyloxy) -1-piperidinyl] ethyl} amino)- 1- (2-Pyridylmethyl) -ethyl] acrylamide
tert-butyl 4- (2-pyrazinyloxy) -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2 The above compound was obtained from -pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 549.4 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1H), 8.22-8.20 (m, 1H), 8.13 (d, J = 3 Hz, 1H), 8.05 (dd, J = 3, lHz, 1H), 8.03-7.88 (m, 2H), 7.64 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 8Hz, 2H ), 7.35 (d, J = 8Hz, 2H), 7.27 (d, J = 8Hz, 1H), 7.19 (dd, J = 8, 5Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.33- 5.22 (m, 1H), 5.07-4.99 (m, 1H), 4.05 (appd, J = 4Hz, 2H), 3.92-3.78 (m, 1H), 3.68-3.52 (m, 2H), 3.48-3.22 (m , 3H), 2.07-1.92 (m, 2H), 1.88-1.78 (m, 2H).

Intermediate 9 tert-butyl 4- (4-pyridyloxy) -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 4-chloropyridine hydrochloride in the same manner as in Intermediate 8.
ESI-MS: 279.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.42 (d, J = 6 Hz, 2 H), 6.80 (d, J = 6 Hz, 2 H), 4.58 (septet, J = 4 Hz, 1 H), 3.69 (ddd, J = 14,8,4Hz, 2H), 3.37 (ddd, J = l4 / 8s4Hz, 2H), 2.01-1.88 (m, 2H), 1.84-1.69 (m, 2H), 1.47 (s, 9H).

Reference Example 5
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (4-pyridyloxy) -1-piperidinyl] ethyl] amino ) -1- (2-Pyridylmethyl) ethyl] acrylamide
tert-butyl 4- (4-pyridyloxy) -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- ( The above compound was obtained from 2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 548.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1H), 8.43 (dd, J = 5, 2 Hz, 2H), 8.04-7.86 (m, 2H), 7.64 (td , J = 8,2Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.26 (d, J = 8Hz , 1H), 7.18 (dd, J = 8,5Hz, 1H), 6.79 (dd, J = 5,2Hz, 2H), 6.50 (d, J = 16Hz, 1H), 5.06-4.98 (m, 1H), 4.70-4.62 (m, 1H), 4.12-3.96 (m, 2H), 3.84-3.22 (m, 6H), 1.98-1.79 (m, 4H).

Intermediate 10 tert-butyl 4- (3-pyridyloxy) -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 3-fluoropyridine in the same manner as in Intermediate 8.
ESI-MS: 279.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.33-8.30 (m, 1H), 8.22 (dd, J = 4, 3Hz, 1H), 7.23-7.20 (m, 2H), 4.51 (septet, J = 4Hz, 1H), 3.71 (ddd, J = 14,7,4Hz, 2H), 3.35 (ddd, J = 14,8,4Hz, 2H), 2.00-1.88 (m, 2H), 1.83-1.70 (m, 2H) , 1.47 (s, 9H).

Reference Example 7
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (3-pyridyloxy) -1-piperidinyl] ethyl} amino ) -1- (2-Pyridylmethyl) ethyl] acrylamide
tert-butyl 4- (3-pyridyloxy) -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- ( The above compound was obtained from 2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 548.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1 H), 8.33-8.30 (m, 1 H), 8.24 (dd, J = 4, lHz, 1 H), 8.06-7.87 ( m, 2H), 7.64 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H) , 7.29-7.16 (m, 5H), 6.50 (d, J = 16Hz, 1H), 5.06-4.99 (m, 1H), 4.634.57 (m, 1H), 4.13-3.97 (m, 2H), 3.81- 3.23 (m, 6H), 1.99-1.81 (m, .4H).

Intermediate 11 tert-Butyl 4-[(6-methoxy-2-pyridyl) oxy] -1-piperidine-carboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 2-chloro-6-methoxypyridine in the same manner as in Intermediate 8.
ESI-MS: 309.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ7.49 (t, J = 8Hz, 1H), 6.29 (d, J = 8Hz, 2H), 5.17 (septet, J = 4Hz, 1H), 3.88 (s, 3H ), 3.81-3.70 (m, 2H), 3.31 (ddd, J = 14, 8, 4Hz, 2H), 2.04-1.92 (m, 2H), 1.82-1.67 (m, 2H), 1.47 (s, 9H) .

Reference Example 9
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-methoxy-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl 4-[(6-methoxy-2-pyridyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 578.3 (M + H)
¹ H-NMR (300 MHz, CDCI ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1H), 8.04-7.86 (m, 2H), 7.63 (td, J = 8, 2 Hz, K1H), 7.61 (d, J = 16Hz, 1H), 7.53-7.42 (m, 3H), 7.35 (d, J = 8Hz, 2H), 7.26 (d, J = 8Hz, 1H), 7.18 (dd, J = 8,5Hz, 1H) , 6.50 (d, J = 16Hz, 1H), 6.29 (d, J = 8Hz, 2H), 5.32-5.20 (m, 1H), 5.06-4.98 (m, 1H), 4.03 (app d, J = 4Hz, 2H), 3.86 (s, 3H), 3.82-3.21 (m, 6H), 2.02-1.74 (m, 4H).

Intermediate 12 tert-butyl 3-[(5-chloro-2-pyridyl) oxy] -1-piperidine-carboxylate
The above compound was obtained from tert-butyl 3-hydroxy-1-piperidinecarboxylate and 2,5-dichloropyridine in the same manner as in Intermediate 8.
ESI-MS: 313.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.07 (d, J = 3 Hz, 1 H), 7.52 (dd, J = 9, 3 Hz, 1 H), 6.66 (d, J = 9 Hz, 1 H), 4.99 (br s , 1H), 3.90-3.10 (br m, 4H), 2.10-1.70 (br m, 3H), 1.60-1.50 (m, 1H), 1.34 (br s, 9H).

Reference Example 11
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {3-[(5-chloro-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl 3-[(5-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate and 1 [(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid by the same method as in Reference Example 1.
ESI-MS: 582.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.58-8.51 (m, 1H), 8.10-7.81 (m, 3H), 7.66-7.41 (m, 5H), 7.34 (d, J = 8Hz, 2H), 7.25 (d, J = 8Hz, 1H), 7.20-7.14 (m, 1H), 6.69-6.60 (m, 1H), 6.53-6.44 (m, 1H), 5.10-4.96 (m, 2H), 4.16-3.80 (m, 2H), 3.77-3.20 (m, 6H), 2.10-1.50 (m, 4H).

Intermediate 13 tert-butyl 4- (2-pyridyloxy) -1-piperidinecarboxylate
To a solution of 2-hydroxypyridine (2.0 g), tert-butyl 4-hydroxy-1-piperidinecarboxylate (5.3 g) and triphenylphosphine (8.3 g) in tetrahydrofuran (63 ml), diethyl azodicarboxylate (5.5 g) Was slowly added at 8 ° C., and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo to give a crude composition of the above target compound, which was purified by silica gel chromatography.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.16-8.10 (m, 1H), 7.60-7.52 (m, 1H), 6.88-6.81 (m, 1H), 6.71 (d, J = 8Hz, 1H), 5.27-5.17 (m, 1H), 3.85-3.70 (m, 2H), 3.35-3.23 (m, 2H), 2.05-1.90 (m, 2H), 1.80-1.65 (m, 2H), 1.47 (s, 9H ).

Intermediate 14 tert-butyl 4-[(6-methyl-2-pyridyl) oxy] -1-piperidine-carboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 2-hydroxy-6-methylpyridine in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.44 (dd, J = 8, 8 Hz, 1 H), 6.68 (d, J = 8 Hz, 1 H), 6.49 (d, J = 8 Hz, 1 H), 5.26-5.16 ( m, 1H), 3.82-3.68 (m, 2H), 3.353.24 (m, 2H), 2.42 (s, 3H), 2.02-1.90 (m, 2H), 1.79-1.64 (m, 2H), 1.47 ( s, 9H).

Reference Example 15
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-methyl-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl 4-[(6-methyl-2-pyridyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid by the same method as in Reference Example 1.
ESI-MS: m / z 562 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (d, J = 5 Hz, 1H), 8.04-7.85 (m, 2H), 7.63 (dd, J = 2, 16 Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.50-7.40 (m, 3H), 7.35 (d, J = 8Hz, 2H), 7.30-7.15 (m, 2H), 6.70 (d, J = 8Hz, 1H), 6.55-6.45 ( m, 2H), 5.35-5.25 (m, 1H), 5.07-4.98 (m, 1H), 4.07-4.00 (m, 2H), 3.86-3.72 (m, 1H), 3.70-3.52 (m, 2H), 3.48-3.20 (m, 3H), 2.41 (s, 3H), 2.01-1.71 (m, 4H).

Intermediate 15 tert-butyl 4-[(6-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from ter-butyl 4-hydroxy-1-piperidinecarboxylate and 6-chloro-2-hydroxypyridine in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.50 (dd, J = 8, 8 Hz, 1H), 6.87 (d, J = 8 Hz, 1H), 6.61 (d, J = 8 Hz, 1H), 5.26-5.15 (m, 1H), 3.80-3.68 (m, 2H), 3.353.25 (m, 2H), 2.03-1.90 (m, 2H), 1.78-1.65 (m, 2H), 1.47 (s, 9H).

Reference Example 17
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-chloro-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] acrylamide
ter-Butyl 4-[(6-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid by the same method as in Reference Example 1.
ESI-MS: m / z 582 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (d, J = 5 Hz, 1 H), 8.04-7.87 (m, 2 H), 7.69-7.49 (m, 3 H), 7.46 (d, J = 8 Hz, 2 H ), 7.36 (d, J = 8Hz, 2H), 7.30-7.15 (m, 2H), 6.90 (d, J = 8Hz, 1H), 6.64 (dd, J = 2,8Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.35-5.25 (m, 1H), 5.07-4.99 (m, 1H), 4.08-4.00 (m, 2H), 3.90-3.75 (m, 1H), 3.67-3.50 (m, 2H) ), 3.49-3.20 (m, 3H), 2.06-1.90 (m, 2H), 1.87-1.73 (m, 2H).

Intermediate 16 tert-butyl 4-{[5- (trifluoromethyl) -2-pyridyl] oxy} -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 5-trifluoromethyl-2-hydroxypyridine in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.40 (br s, 1 H), 7.76 (dd, J = 2, 8 Hz, 1 H), 6.69 (d, J = 8 Hz, 1 H), 5.33-5.23 (m, 1H), 3.84-3.70 (m, 2H), 3.35-3.23 (m, 2H), 2.05-1.92 (m, 2H), 1.80-1.66 (m, 2H), 1.48 (s, 9H).

Reference Example 19
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[5- (trifluoromethyl) -2-pyridyl] Oxy} -1-piperidinyl) ethyl] amino} -1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl 4-{[5- (trifluoromethyl) -2-pyridyl] oxy} -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl)- The above compound was obtained from 2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 616 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) b 8.55 (d, J = 5 Hz, 1H), 8.40 (br s, 1H), 8.04-7.95 (m, 1H), 7.91 (d, J = 8 Hz, 1H), 7.77 (dd, J = 2,8Hz, 1H), 7.63 (dd, J = 2,16Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.35 ( d, J = 8Hz, 2H), 7.29-7.15 (m, 2H), 6.80 (dd, J = 2,8Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.41-5.30 (m, 1H) 5.06-4.97 (m, 1H), 4.08-4.00 (m, 2H), 3.92-3.20 (m, 6H), 2.06-1.92 (m, 2H), 1.86-1.71 (m, 2H).

Intermediate 17 tert-butyl 4-[(5-chloro-2-pyridyl) oxy] -1-piperidine-carboxylate
The above compound was obtained from ter-butyl 4-hydroxy-1-piperidinecarboxylate and 5-chloro-2-hydroxypyridine in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.06 (d, J = 2 Hz, 1 H), 7.51 (dd, J = 2, 8 Hz, 1 H), 6.67 (d, J = 8 Hz, 1 H), 5.20-5.10 ( m, 1H), 3.83-3.70 (m, 2H), 3.333.20 (m, 2H), 2.02-1.90 (m, 2H), 1.77-1.64 (m, 2H), 1.47 (s, 9H).

Reference Example 21
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(5-chloro-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl 4-[(5-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid by the same method as in Reference Example 1.
ESI-MS: m / z 582 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (d, J = 5 Hz, 1 H), 8.05 (d, J = 2 Hz, 1 H), 7.98 (br s, 1 H), 7.90 (d, J = 8 Hz, 1H), 7.63 (dd, J = 2, 16Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.52 (dd, J = 2, 8Hz, 1H), 7.45 (d, J = 8Hz, 2H) , 7.35 (d, J = 8Hz, 2H), 7.28-7.14 (m, 2H), 6.66 (dd, J = 2,8Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.27-5.17 (m , 1H), 5.06-4.97 (m, 1H), 4.06-3.98 (m, 2H), 3.90-3.75 (m, 1H), 3.70-3.20 (m, 5H), 2.03-1.70 (m, 4H).

Intermediate 18 tert-butyl 4-phenoxy-1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and phenol in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.32-7.20 (m, 2H), 6.98-6.80 (m, 3H), 4.50-4.41 (m, 1H), 3.75-3.64 (m, 2H), 3.39- 3.27 (m, 2H), 1.99-1.66 (m, 4H), 1.47 (s, 9H).

Reference Example 23
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-phenoxy-1-piperidinyl) ethyl-3amino} -1- ( 2-Pyridylmethyl) ethyl] acrylamide
tert-butyl-4-phenoxy 1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl The above compound was obtained from amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 547 (M1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (d, J = 5 Hz, 1H), 8.05-7.82 (m, 2H), 7.62 (dd, J = 2, 16 Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.12-7.38 (m, 6H), 6.96 (dd, J = 8,8Hz, 1H), 6.90 (d, J = 8Hz, 2H), 6.49 (dd, J = 2,16Hz, 1H), 5.06-4.97 (m, 1H), 4.60-4.50 (m, 1H), 4.06-3.99 (m, 2H), 3.81-3.52 (m, 3H), 3.48 -3.20 (m, 3H), 1.90-1.79 (m, 4H).

Intermediate 19 tert-butyl 4- (2,2,2-trifluoroethoxy) -1-piperidinecarboxylate
To a tetrahydrofuran solution (450 ml) of tert-butyl 4-hydroxy-1-piperidine-carboxylate (30.0 g), 2,2,2-trifluoroethanol (149 g) and triphenylphosphine (58.6 g) was added diethylazodicarboxylate. Lat (38.9 g) was added slowly at 8 ° C. The mixture was stirred at room temperature for 2 hours and at 60 ° C. for 8 hours. The reaction mixture was concentrated in vacuo to give a composition of the above target compound, which was purified by silica gel chromatography.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 3.90-3.58 (m, 5H), 3.20-3.10 (m, 2H), 1.90-1.78 (m, 2H), 1.65-1.50 (m, 2H), 1.46 ( s, 9H).

Reference Example 25
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (2,2,2-trifluoroethoxy) -1- Piperidinyl] ethyl} amino) -1- (2-pyridylmethyl) ethyl] acrylamide
tert-butyl-4- (2,2,2trifluoroethoxy) -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino The above compound was obtained from} -3- (2-pyridyl) propanoyl] -amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 553 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (d, J = 5 Hz, 1H), 8.05-7.82 (m, 2H), 7.62 (dd, J = 2, 16 Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.34 (d, J = 8Hz, 2H), 7.28-7.13 (m, 2H), 6.49 (dd, J = 2,16Hz, 1H), 5.05 -4.95 (m, 1H), 4.10-3.92 (m, 2H), 3.90-3.63 (m, 4H), 3.60-3.35 (m, 3H), 3.30-3.16 (m, 2H), 1.88-1.56 (m, 4H).

Intermediate 20 tert-butyl 4-isopropoxy-1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 2-iodopropane in the same manner as in Intermediate 7.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 3.87-3.73 (m, 2H), 3.75-3.65 (m, 1H), 3.55-3.45 (m, 1H), 3.10-2.47 (m, 2H), 1.85-1.40 (m, 4H), 1.26 (s, 9H), 1.15 (d, J = 7Hz, 6H).

Reference Example 27
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-{[2- (4-isopropoxy-1-piperidinyl) -2-oxoethyl] amino} -2-oxo-1- ( 2-Pyridylmethyl) ethyl] acrylamide
tert-butyl 4-isopropoxy-1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) The above compound was obtained from propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 513 (M + 1)
¹ H-NMR (300MHz, CDCl ₃ ) δ8.55 (d, J = 5Hz, 1H), 8.01-7.81 (m, 2H), 7.66-7.55 (m, 2H), 7.45 (d, J = 8Hz, 2H ), 7.35 (d, J = 8Hz, 2H), 7.14-7.27 (m, 2H), 6.49 (dd, J = 2,16Hz, 1H), 5.05-4.97 (m, 1H), 4.04-3.96 (m, 2H), 3.90-3.31 (m, 6H), 3.29-3.10 (m, 2H), 1.82-1.45 (m, 4H), 1.14 (d, J = 7Hz, 6H).

Intermediate 21 tert-butyl 4-butoxy-1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy 1-piperidinecarboxylate and 1-iodobutane in the same manner as in Intermediate 7.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 3.82-3.70 (m, 2H), 3.48-3.36 (m, 3H), 3.12-3.00 (m, 2H), 1.87-1.76 (m, 2H), 1.46 ( s, 9H), 1.60-1.30 (m, 4H), 0.98-0.80 (m, 5H).

Reference Example 29
(2E) -N-[(1S) -2-{[2- (4-Butoxy-1-piperidinyl) -2-oxoethyl] -amino} -2-oxo-1- (2-pyridylmethyl) ethyl]- 3- (4-Chlorophenyl) acrylamide
tert-butyl 4-butoxy-1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl The above compound was obtained from amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 527 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (d, J = 5 Hz, 1H), 8.01-7.82 (m, 2H), 7.62 (dd, J = 2, 16 Hz, 1H), 7.60 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.32-7.14 (m, 2H), 6.49 (dd, J = 2,16Hz, 1H), 5.05 -4.95 (m, 1H), 4.05-3.95 (m, 2H), 3.84-3.69 (m, 1H), 3.59-3.11 (m, 8H), 1.84-1.70 (m, 2H), 1.44-1.29 (m, 4H), 0.91 (t, J = 7Hz, 3H).

Intermediate 22 tert-butyl 4-[(6-chloro-2-pyrazinyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 2,6-dichloropyrazine in the same manner as in Intermediate 8.
SI-MS: 314.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.13 (s, 1H), 8.10 (s, 1H), 5.21 (septet, J = 4Hz, 1H), 3.82-3.70 (m, 2H), 3.32 (ddd, J = 14, 9, 4Hz, 2H), 2.05-1.93 (m, 2H), 1.82-1.68 (m, 2H), 1.48 (s, 9H).

Reference Example 31
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-chloro-2-pyrazinyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(6-chloro-2-pyrazinyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 583.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ8.56 (dd, J = 5,2Hz, 1H), 8.16 (s, 1H), 8.11 (d, J = 1.5Hz, 1H), 8.02 (s, 1H) , 7.96-7.89 (m, 1H), 7.64 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 9Hz, 2H), 7.35 (d, J = 9Hz, 2H), 7.26 (d, J = 8Hz, 1H), 7.19 (dd, J = 8,5Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.33-5.22 (m, 1H), 5.07-4.99 (m, 1H), 4.05 (appd, J = 4Hz, 2H), 3.92-3.76 (m, 1H), 3.67-3.51 (m, 2H), 3.48-3.21 (m, 3H), 2.06-1.92 (m, 2H), 1.88-1.73 (m, 2H).

Intermediate 23 tert-butyl 4-[(6-chloro-3-pyridazinyl) oxy] -1piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 3,6-dichloropyridazine in the same manner as in Intermediate 8.
ESI-MS: 314.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.38 (d, J = 9 Hz, 1 H), 6.94 (d, J = 9 Hz, 1 H), 5.44 (septet, J = 4 Hz, 1 H), 3.87-3.73 (br m , 2H), 3.26 (ddd, J = 14, 9, 4Hz, 2H), 2.13-2.02 (m, 2H), 1.83-1.67 (m, 2H), 1.48 (s, 9H).

Reference Example 33
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-chloro-3pyridazinyl) oxy] -1-piperidinyl} -2-oxoethyl) amino ] -2-Oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(6-chloro-3-pyridazinyl) oxy] -1-piperidinecarboxylate and {[(2S) -2 {[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino The above compound was obtained from} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 583.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ 8.51-8.46 (m, 2H), 8.15-8.08 (m, 1H), 7.82-7.78 (m, 1H), 7.69 (td, J = 8,2Hz, 1H) 7.56 (d, J = 9Hz, 2H), 7.47 (d, J = 9Hz, 2H), 7.39-7.29 (m, 3H), 7.20 (dd, J = 8,5Hz, 1H), 6.68 (d, J = 16Hz, 1H), 5.43-5.32 (m, 1H), 5.00-4.90 (m, 1H), 4.04-3.97 (m, 2H), 3.95-3.84 (m, 1H), 3.73-3.62 (m, 1H) 3.41-3.22 (m, 3H), 3.07-2.96 (m, 1H), 2.14-1.95 (m, 2H), 1.80-1.53 (m, 2H).

Intermediate 24 tert-butyl 4-[(6-methoxy-3-pyridazinyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy 1-piperidinecarboxylate and 3-chloro-6-methoxypyridazine in the same manner as in Intermediate 7.
ESI-MS: 310.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ6.96-6.88 (m, 2H), 5.31 (tt, J = 8,4Hz, 1H), 4.04 (s, 3H), 3.89-3.75 (br m, 2H) , 3.22 (ddd, J = 14, 8, 4 Hz, 2H), 2.13-2.01 (m, 2H), 1.81-1.66 (m, 2H), 1.47 (s, 9H).

Reference Example 35
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-methoxy-3-pyridazinyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(6-methoxy-3-pyridazinyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 579.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1 H), 8.06-7.84 (m, 2 H), 7.64 (td, J = 8, 2 Hz, 1 H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.26 (d, J = 8Hz, 1H), 7.18 (dd, J = 8, 5Hz, 1H), 6.97-6.86 (m, 2H), 6.50 (d, J = 16Hz, 1H), 5.42-5.31 (m, 1H), 5.06-4.98 (m, 1H), 4.06-3.89 (m, 3H), 4.04 (s, 3H), 3.66-3.56 (m, 1H), 3.51-3.36 (m, 2H), 3.35-3.20 (m, 2H), 2.16-2.00 (m, 2H), 1.87-1.72 (m, 2H ).

Intermediate 25 tert-butyl 4-[(3-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from ter-butyl 4-hydroxy 1-piperidinecarboxylate and 2,3-dichloropyridine in the same manner as in Intermediate 7.
ESI-MS: 313.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.02 (dd, J = 5, 2 Hz, 1 H), 7.63 (dd, J = 8, 2 Hz, 1 H), 6.83 (dd, J = 8, 5 Hz, 1 H), 5.32 (tt, J = 8,4Hz, 1H); 3.71 (ddd, J = 14,8,4Hz, 2H), 3.41 (ddd, J = 14,8,4Hz, 2H), 2.02-1.89 (m, 2H ), 1.87-1.74 (m, 2H), 1.48 (s, 9H).

Reference Example 37
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(3-chloro-2-pyridyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(3-chloro-2-pyridyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 582.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1H), 8.06-7.86 (m, 3H), 7.68-7.57 (m, 3H), 7.46 (d, J = 8 Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.29-7.23 (m, 1H), 7.18 (dd, J = 8, 5Hz, 1H), 6.85 (dd, J = 8, 5Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.44-5.34 (m, 1H), 5.08-4.99 (m, 1H), 4.14-3.95 (m, 2H), 3.85-3.54 (m, 3H), 3.49-3.20 ( m, 3H), 2.01-1.80 (m, 4H).

Intermediate 26 tert-butyl 4-[(6-chloro-4-pyrimidinyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from ter-butyl 4-hydroxy-1-piperidinecarboxylate and 4,6-dichloropyrimidine in the same manner as in Intermediate 7.
ESI-MS: 314.2 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ 8.55 (s, 1H), 6.76 (s, 1H), 5.31 (tt, J = 8,4Hz, 1H), 3.84-3.70 (m, 2H), 3.29 (ddd , J = 14, 8, 4 Hz, 2H), 2.05-1.93 (m, 2H), 1.81-1.66 (m, 2H), 1.47 (s, 9H).

Reference Example 39
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(6-chloro-4-pyrimidinyl) oxy] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(6-chloro-4-pyrimidinyl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] The above compound was obtained from amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 583.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.57-8.56 (m, 1H), 8.55 (s, 1H), 8.05-7.99 (m, 1H), 7.96-7.89 (m, 1H), 7.64 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 9Hz, 2H), 7.96-7.89 (m, 1H), 7.64 (td, J = 8,2Hz, 1H ), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 9Hz, 2H), 7.35 (d, J = 9Hz, 2H), 7.26 (d, J = 8Hz, 1H), 7.19 (dd, J = 8,5Hz, 1H), 6.76 (appd, J = 2Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.42-5.32 (m, 1H), 5.07-4.98 (m, 1H), 4.05 (d, J = 4Hz, 2H), 3.93-3.77 (m, 1H), 3.65-3.47 (m, 2H), 3.47-3.21 (m, 3H), 2.07-1.91 (m, 2H), 1.87-1.71 ( m, 2H).

Intermediate 27 tert-butyl 4-{[6- (trifluoromethyl) -4-pyrimidinyl] oxy} -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 6- (trifluoromethyl) -4-pyrimidinol in the same manner as in Intermediate 13.
ESI-MS: 348.3 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ 8.86 (s, 1H), 7.06 (s, 1H), 5.39 (tt, J = 8,4Hz, 1H), 3.85-3.72 (m, 2H), 3.30 (ddd , J = 14,8,4Hz, 2H), 2.08-1.95 (m, 2H), 1.84-1.68 (m, 2H), 1.48 (s, 9H).

Reference Example 41
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[6- (trifluoromethyl) -4-pyrimidinyl] Oxy} -1-piperidinyl) ethyl] amino} -1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-{[6- (trifluoromethyl) -4-pyrimidinyl] oxy} -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl)- The above compound was obtained from 2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 617.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.86 (s, 1H), 8.57 (dd, J = 5, 2 Hz, 1H), 8.08-8.01 (m, 1H), 7.98-7.89 (m, 1H), 7.64 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 9Hz, 2H), 7.35 (d, J = 9Hz, 2H), 7.27 (d, J = 8Hz, 1H), 7.19 (dd, J = 8,5Hz, 1H), 7.07 (s, 1H), 6.50 (d, J = 16Hz, 1H), 5.51-5.41 (m, 1H), 5.06-4.98 (m, 1H), 4.05 (d, J = 4Hz, 2H), 3.96-3.80 (m, 1H), 3.68-3.50 (m, 2H), 3.50-3.21 (m, 3H), 2.10-1.95 (m, 2H), 1.90-1.74 (m, 2H).

Intermediate 28 tert-butyl 4-{[2- (trifluoromethyl) -4-pyrimidinyl] oxy} -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 4-hydroxy-2- (trifluoromethyl) pyrimidine in the same manner as in Intermediate 13.
ESI-MS: 348.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (d, J = 6 Hz, 1 H), 6.86 (d, J = 6 Hz, 1 H), 5.39 (tt, J = 8, 4 Hz, 1 H), 3.83-3.71 ( m, 2H), 3.33 (ddd, J = 14, 8, 4Hz, 2H), 2.08-1.96 (m, 2H), 1.84-1.68 (m, 2H), 1.48 (s, 9H).

Intermediate 29 tert-butyl 4-[(5-chloro-2-pyrazinyl) oxy] -1-piperidinecarboxylate
The above compound was obtained from tert-butyl-4-hydroxy 1-piperidinecarboxylate and 5-chloro-2-pyrazinol in the same manner as in Intermediate 13. 5-Chloro-2-pyrazinol was prepared by the method described in J. Org. Chem. 29, 2491-2492 (1964).
ESI-MS: 314.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.07 (d, J = lHz, 1H), 7.99 (d, J = lHz, 1H), 5.14 (tt, J = 8, 4 Hz, 1H), 3.83-3.70 ( m, 2H), 3.29 (ddd, J = 14, 8, 4Hz, 2H), 2.03-1.91 (m, 2H), 1.66-1.80 (m, 2H), 1.48 (s, 9H).

Intermediate 30 2-Bromo-5-methyl-1,3-thiazole
To a solution of 2-amino-5-methyl-1,3-thiazole (11.7 g) in acetonitrile (200 ml) was added dropwise tert-butyl nitrite (8.33 ml) at 0 ° C., and copper (II) bromide (5 g) Was added over 5 minutes. After stirring at 0 ° C. for 3 hours, the mixture was concentrated and partitioned with 1N hydrochloric acid and ethyl acetate. The organic layer was washed with water, saturated aqueous sodium hydrogen carbonate solution and brine, dried over anhydrous magnesium sulfate, filtered through a celite pad and concentrated in vacuo to give the desired compound as an oil (3.24 g )
ESI-MS: 177.8 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.25 (s, 1H), 2.44 (s, 3H).

Intermediate 31 tert-butyl 4-[(5-methyl-1,3-thiazol-2-yl) oxy] -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 2-bromo-5-methyl-1,3-thiazole in the same manner as in Intermediate 7.
ESI-MS: 299.3 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 5.06 (tt, J = 8, 4Hz, 1H), 3.78-3.66 (m, 2H), 3.30 (ddd, J = 14, 8, 4Hz, 2H), 2.31 (s, 3H), 2.06-1.95 (m, 2H), 1.85-1.72 (m, 2H), 1.47 (s, 9H).

Reference Example 45
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(5-methyl-1,3thiazol-2-yl) oxy] -1-piperidinyl} -2-oxoethyl) amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-Butyl 4-[(5-methyl-1,3-thiazol-2-yl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl The above compound was obtained from) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 568.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1H), 8.04-7.97 (m, 1H), 7.96-7.87 (m ,, 1H), 7.63 (td, J = 8 , 2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.34 (d, J = 8Hz, 2H), 7.25 (d, J = 8Hz, 1H), 7.18 (dd, J = 8,5Hz, 1H), 6.73 (app s, 1H), 6.50 (d, J = 16Hz, 1H), 5.13 (br s, 1H), 5.06-4.98 (m, 1H), 4.03 (app d, J = 4Hz, 2H), 3.83-3.49 (m, 3H), 3.43 (dd, J = 15,5Hz, 1H), 3.37-3.20 (m, 2H), 2.31 (d, J = lHz, 3H), 2.05-1.79 (m, 4H).

Intermediate 32 tert-butyl 4-[(5-chloro-1,3-thiazol-2-yl) oxy] -1-piperidinecarboxylate
A solution of tert-butyl 4-[(1,3-thiazol-2-yl) oxy] -1-piperidinecarboxylate (8.0 g) in acetic acid (80 ml) was added dropwise to N-chlorosuccinimide (4.5 g). The mixture was warmed to 60-70 ° C. and stirred for 5 hours. The resulting mixture was poured into a mixture of (80 g) and water (80 ml) and extracted with ethyl acetate. The organic layer was concentrated under vacuum and the residue was dissolved in ethyl acetate. The solution was basified with 1N sodium hydroxide solution. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography (eluent: 9% ethyl acetate in n-hexane) to obtain the target compound as white crystals (5.56 g).
ESI-MS: 319.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.93 (s, 1H), 5.11 (tt, J = 8, 4Hz, 1H), 3.79-3.63 (m, 2H), 3.31 (ddd, J = 14, 8, 4Hz, 2H), 2.07-1.94 (m, 2H), 1.87-1.72 (m, 2H), 1.47 (s, 9H).

Reference Example 47
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(5-chloro-1,3thiazol-2-yl) oxy] -1-piperidinyl} -2-oxoethyl) amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-Butyl 4-[(5-chloro-1,3-thiazol-2-yl) oxy] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl The above compound was obtained from) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
(ESI-MS: 588.KM + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1H), 8.06-7.98 (m, 1H), 7.95-7.88 (m, 1H), 7.63 (td, J = 8 , 2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.46 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.25 (d, J = 8Hz, 1H), 7.18 (dd, J = 8,5Hz, 1H), 6.92 (s, 1H), 6.50 (d, J = 16Hz, 1H), 5.235.13 (m, 1H), 5.05-4.97 (m, 1H), 4.03 (app d, J = 4Hz, 2H), 3.83-3.49 (m, 3H), 3.48-3.20 (m, 3H), 2.06-1.78 (m, 4H).

Intermediate 33 tert-butyl (3R) -3-[(5-chloro-2-pyridyl) oxy] -1-pyrrolidinecarboxylate
The above compound was obtained from tert-butyl (3R) -3 hydroxy-1-pyrrolidinecarboxylate and 2,5-dichloropyridine in the same manner as in Intermediate 7.
ESI-MS: 299.1 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ8.08 (s, 1H), 7.53 (d, J = 9Hz, 1H), 6.69 (d, J = 9Hz, 1H), 5.49 (br s, 1H), 3.71 -3.40 (m, 4H), 2.14 (br s, 2H), 1.47 (s, 9H).

Intermediate 34 tert-butyl (3S) -3-[(5-chloro-2-pyridyl) oxy] -1-pyrrolidinecarboxylate
The above compound was obtained from tert-butyl (3S) -3hydroxy-1-pyrrolidinecarboxylate and 5-chloro-2-pyridinol by the same method as in Intermediate 13.
ESI-MS: 299.1 (M + H)
¹ H-NMR (300MHz, CDCl ₃ ) δ 8.08 (s, 1H), 7.53 (d, J = 9Hz, 1H), 6.68 (d, J = 9Hz, 1H), 5.48 (br s, 1H), 3.69- 3.42 (m, 4H), 2.14 (br s, 2H), 1.46 (s, 9H).

Intermediate 35 tert-butyl (3R) -3-[(6-chloro-2-pyrazinyl) oxy] -1-pyrrolidinecarboxylate
The above compound was obtained from tert-butyl (3R) -3hydroxy-1-pyrrolidinecarboxylate and 2,6-dichloropyrazine in the same manner as in Intermediate 7.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.17 (s, 1H), 7.12 (s, 1H), 5.54 (br s, 1H), 3.73-3.43 (m, 4H), 2.26-2.12 (m, 2H) , 1.48 (s, 9H).

Intermediate 36 tert-butyl (3R) -3-[(5-methyl-1,3-thiazol-2-yl) oxy] -1-pyrrolidinecarboxylate
The above compound was obtained from tert-butyl (3R) -3 hydroxy-1-pyrrolidinecarboxylate and 2-bromo-5-methyl-1,3 thiazole in the same manner as in Intermediate 7.
ESI-MS: 285.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 6.75 (s, 1H), 5.46-5.40 (m, 1H), 3.77-3.38 (m, 4H), 2.31 (s, 3H), 2.40-2.00 (m, 2H), 1.47 (s, 9H).

Intermediate 37 tert-butyl 3-{[6- (trifluoromethyl) -4-pyrimidinyl] oxy} -1-azetidinecarboxylate
The above compound was obtained from tert-butyl 3-hydroxy 1-azetidinecarboxylate and 6- (trifluoromethyl) -4-pyrimidinol in the same manner as in Intermediate 13.
ESI-MS: 320.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.87 (s, 1H), 7.14 (.s, 1H), 5.44 (tt, J = 7, 4Hz, 1H), 4.37 (dd, J = 10, 7Hz, 2H), 4.01 (dd, J = 10, 4Hz, 2H), 1.45 (s, 9H).

Intermediate 38 3,5-dichloro-2- (4-piperidinyloxy) pyridine dihydrochloride
To a tetrahydrofuran solution of tert-butyl-4-hydroxy-1-piperidinecarboxylate (0.5 g), 3,5-dichloro-2-pyridone (0.41 g) and triphenylphosphine (0.98 g) was added diethyl azodicarboxylate ( 0.65 g) was added slowly at 8 ° C. and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under vacuum to give a crude composition.
To a methanol solution (5 ml) of the crude composition was added 4N hydrogen chloride in ethyl acetate (6.2 ml) at 8 ° C. The mixture was stirred at room temperature for 1 hour and concentrated under vacuum. The solid residue was washed with ethyl acetate to obtain the target compound (585 mg).
¹ H-NMR (300MHz, CDCl ₃ ) δ7.99 (d, J = 2Hz, 1H), 7.68 (d, J = 2Hz, 1H), 5.45-5.37 (m, 1H), 3.45-3.29 (m, 4H ), 2.44-2.13 (m, 4H).

Reference Example 59
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(3,5-dichloro-2-pyridyl) oxy] -1-piperidinyl} -2- Oxoethyl) amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
{[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid (0.19 g), 3,5- Dichloro-2- (4-piperidinyloxy) pyridine dihydrochloride (0.16 g), 1-hydroxybenzonitrile (0.084 g) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.11 g) N, N-Dipropylethylamine (0.27 ml) was added dropwise at 8 ° C. to an N, N-dimethylformamide solution (2.5 ml) of the above mixture. The mixture was stirred at room temperature for 6 hours, poured into ice water and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium bicarbonate and brine. Dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography to obtain the target compound (231 mg).
¹ H-NMR (300 MHz, CDCl ₃ ) δ8.61-8.53 (m, 1H), 8.07-7.85 (m, 3H), 7.707.57 (m, 3H), 7.45 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.30-7.15 (m, 2H), 6.51 (dd, J = 2,15Hz, 1H), 5.38-5.28 (m, 1H), 5.07-4.98 (m, 1H) 4.13-3.95 (m, 2H), 3.85-3.20 (m, 6H), 2.00-1.79 (m, 4H).
ESI-MS: m / z 616 (M + 1)

Intermediate 39 tert-Butyl 4-([3- (Trifluoromethyl) -2-pyridyloxy-1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 3- (trifluoromethyl) -2-hydroxypyridine in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.33-8.24 (m, 1H), 7.90-7.81 (m, 1H), 6.69.90 (m, 1H), 5.48-5.33 (m, 1H), 3.63-3.44 (m, 4H), 1.99-1.77 (m, 4H), 1.47 (s, 9H).

Reference Example 61
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[3 (trifluoromethyl) -2-pyridyl] oxy } -1-Piperidinyl) ethyl] amino} -1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-{[3- (trifluoromethyl) -2-pyridyl] oxy} -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl)- The above compound was obtained from 2-propenoyl] amino} -3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 616 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.59-8.53 (m, 1H), 8.30-8.25 (m, 1H), 8.067.83 (m, 3H), 7.66-7.55 (m, 2H), 7.45 (d , J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.29-7.14 (m, 2H), 7.00-6.93 (m, 1H), 6.50 (dd, J = 15,3Hz, 1H), 5.55-5.45 (m, 1H), 5.06-4.98 (m, 1H), 4.14-3.90 (m, 3H), 3.60-3.35 (m, 4H), 3.30-3.20 (m, 1H), 2.00-1.79 (m , 4H).

Intermediate 40 4- (4-Fluorophenoxy) piperidine
To a tetrahydrofuran solution of tert-butyl 4-hydroxy-1-piperidinecarboxylate (2.0 g), 4-fluorophenol (1.1 g) and triphenylphosphine (3.9 g) was added diethyl azodicarboxylate (2.6 g) at 8 ° C. Was added slowly and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under vacuum to give a crude composition.
To a solution of the crude composition in ethyl acetate (5 ml) and methanol (5 ml) was added 4N hydrogen chloride in ethyl acetate (25 ml) at 8 ° C. The mixture was stirred at room temperature for 1 hour. The mixture was poured into saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated under vacuum to obtain the target compound (1.08 g).

Reference Example 63
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-({2- [4- (4-fluorophenoxy) -1-piperidinyl] -2-oxoethyl} amino) -2-oxo -1- (2-Pyridylmethyl) ethyl] -2-propenamide
4- (4-Fluorophenoxy) piperidine and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -2- (2-pyridyl) propanoyl] amino} The above compound was obtained from acetic acid by the same method as in Reference Example 59.
ESI-MS: m / z 565 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.60-8.53 (m, 1H), 8.07-7.85 (2H, m), 7.68-7.56 (m, 2H), 7.45 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.31-7.15 (m, 2H), 7.04-6.93 (m, 2H), 6.90-6.80 (m, 2H), 6.50 (dd, J = 15,2Hz, 1H) , 5.06-4.97 (m, 1H), 4.50-4.40 (m, 1H), 4.11-3.95 (m, 2H), 3.79-3.20 (m, 6H), 1.94-1.75 (m, 4H).

Intermediate 41 4- [4- (Trifluoromethyl) phenoxy] piperidine hydrochloride
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 4- (trifluoromethyl) phenol in the same manner as in Intermediate 38.
¹ H-NMR (300 MHz, DMSO-d ₆ ) δ 7.67 (d, J = 8 Hz, 2H), 7.19 (d, J = 8 Hz, 2H), 4.85-4.737 (m, 1H), 3.29-3.00 (m, 4H), 2.20-2.05 (m, 2H), 1.981.78 (m, 2H).

Reference Example 65
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-[(2-oxo-2- {4- [4- (trifluoromethyl) phenoxy] -1-piperidinyl } Ethyl) amino] -1- (2-pyridylmethyl) ethyl] -2-propenamide
4- [4- (Trifluoromethyl) phenoxy] piperidine hydrochloride and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2- The above compound was obtained from pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 59.
ESI-MS: m / z 615 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.60-8.53 (m, 1H), 8.10-7.85 (m, 2H), 7.68-7.51 (m, 4H), 7.45 (d, J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.30-7.15 (m, 2H), 6.95 (d, J = 8Hz, 2H), 6.50 (dd, J = 15,2Hz, 1H), 5.07-4.97 (m, 1H ), 4.68-4.58 (m, 1H), 4.09-3.97 (m, 2H), 3.86-3.20 (m, 6H), 1.99-1.78 (m, 4H).

Intermediate 42 tert-butyl 4- (4-chlorophenoxy) -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-hydroxy-1-piperidinecarboxylate and 4-chlorophenol in the same manner as in Intermediate 13.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 7.31-7.14 (m, 2H), 6.74-6.60 (m, 2H), 4.474.36 (m, 1H), 3.75-3.61 (m, 2H), 3.40-3.26 (m, 2H), 1.97-1.84 (m, 2H), 1.80-1.66 (m, 2H), 1.47 (s, 9H).

Reference Example 67
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-({2- [4- (4-chlorophenoxy) -1-piperidinyl] -2-oxoethyl} amino) -2-oxo -1- (2-Pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4- (4-chlorophenoxy) -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- ( The above compound was obtained from 2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: m / z 581 (M + 1)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.60-8.51 (m, 1H), 8.09-7.84 (m, 2H), 7.68-7.55 (m, 2H), 7.4.5 (d, J = 8Hz, 2H ), 7.35 (d, J = 8Hz, 2H), 7.30-7.14 (m, 4H), 6.83 (d, J = 8Hz, 2H), 6.50 (d, J = 15Hz, 1H), 5.05-4.97 (m, 1H), 4.55-4.45 (m, 1H), 4.11-3.94 (m, 2H), 3.80-3.20 (m, 6H), 1.91-1.73 (m, 4H).

Intermediate 43 tert-butyl 4- (2-pyridylthio) -1-piperidinecarboxylate
To a dimethoxyethane slurry (4 ml) of sodium hydroxide (approximately 60% oil suspension, 397 mg) was added 2-pyridinethiol (1.05 g) in dimethoxyethane (6 ml) at 0 ° C. and the mixture was allowed to cool to room temperature. And stirred for 1 hour. To the mixture was added tert-butyl-4-bromo-1-piperidinecarboxylate (2.74 g) and the mixture was refluxed for 2 hours. After cooling, the resulting suspension was diluted with ether and filtered through a celite pad. The filtrate was washed with brine, dried over anhydrous magnesium sulfate and concentrated under vacuum.
The residue was purified by silica gel chromatography (eluent: 14% ethyl acetate in n-hexane) to obtain the target compound as a white solid (2.2 g).
ESI-MS: 295.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.42 (dd, J = 5, 2 Hz, 1 H), 7.47 (td, J = 8, 2 Hz, 1 H), 7.16 (d, J = 8 Hz, 1 H), 6.99 ( dd, J = 8,5Hz, 1H), 4.06-3.83 (m, 3H), 3.16-3.02 (m, 2H), 2.12-2.00 (m, 2H), 1.71-1.56 (m, 2H), 1.46 (s , 9H).

Reference Example 69
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (2-pyridylthio) -1-piperidinyl] ethyl} amino) -1- (2-Pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4- (2-pyridylthio) -1-piperidinecarboxylate and {[(2S) -2-1 [(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2 The above compound was obtained from -pyridyl) propanoyl] amino} -acetic acid in the same manner as in Reference Example 1.
ESI-MS: 564.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1H), 8.41 (dd, J = 5, 2 Hz, 1H), 8.02-7.96 (m, 1H), 7.95-7.88 ( m, 1H), 7.63 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.52-7.42 (m, 3H), 7.35 (d, J = 8Hz, 2H), 7.28 -7.23 (m, 1H), 7.18 (dd, J = 8,5Hz, 1H), 7.15 (d, J = 8Hz, 1H), 7.00 (dd, J = 8,5Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.06-4.97 (m, 1H), 4.26-4.04 (m, 2H), 4.02 (appd, J = 4Hz, 2H), 3.70-3.59 (m, 1H), 3.43 (dd, J = 15,5Hz, 1H), 3.30-3.10 (m, 3H), 2.19-2.02 (m, 2H), 1.73-1.54 (m, 2H).

Reference Example 71
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (2-pyridylsulfonyl) -1-piperidinyl] ethyl} amino ) -1- (2-Pyridylmethyl) ethyl] -2-propenamide
2- (4-Piperidinylsulfonyl) pyridine hydrochloride and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) The above compound was obtained from propanoyl] -amino} acetic acid in the same manner as in Reference Example 59.
ESI-MS: 596.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.75-8.80 (m, 1H), 8.58-8.52 (m, 1H), 8.127.82 (m, 4H), 7.68-7.55 (m, 3H), 7.45 (d , J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.28-7.15 (m, 2H), 6.54-6.44 (m, 1H), 5.04-4.96 (m, 1H), 4.684.56 ( m, 1H), 3.99 (appd, J = 4Hz, 2H), 3.88-3.67 (m, 2H), 3.473.35 (m, 1H), 3.29-3.19 (m, 1H), 3.11-2.97 (m, 1H ), 2.74-2.61 (m, 1H), 2.12-1.90 (m, 2H), 1.86-1.68 (m, 2H).

Intermediate 44 tert-butyl 4-[(5-chloro-2-pyridyl) thio] -1-piperidinecarboxylate
The above compound was obtained from tert-butyl 4-mercapto-1-piperidinecarboxylate and 2,5-dichloropyridine in the same manner as in Intermediate 7.
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.38 (dd, J = 2, lHz, 1H), 7.45 (dd, J = 8, 2Hz, 1H), 7.10 (dd, J = 8, lHz, 1H), 4.01-3.85 (m, 3H), 3.14-3.00 (m, 2H), 2.10-1.98 (m, 2H), 1.70-1.58 (m, 2H), 1.46 (s, 9H).

Reference Example 73
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(5-chloro-2-pyridyl) thio] -1-piperidinyl} -2-oxoethyl) Amino] -2-oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl 4-[(5-chloro-2-pyridyl) thio] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] Amino} -3- (2-pyridyl) -propanoyl} amino] acetic acid was used to obtain the above compound in the same manner as in Reference Example 1.
ESI-MS: 598.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1H), 8.37 (d, J = 2 Hz, 1H), 8.03-7.97 (m, 1H), 7.95-7.89 (m , 1H), 7.63 (td, J = 8,2Hz, 1H), 7.61 (d, J = 16Hz, 1H), 7.49-7.42 (m, 3H), 7.35 (d, J = 8Hz, 2H), 7.28- 7.23 (m, 1H), 7.18 (dd, J = 8,5Hz, 1H), 7.09 (d, J = 8Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.06-4.97 (m, 1H) , 4.27-4.14 (m, 1H), 4.10-3.97 (m, 3H), 3.703.58 (m, 1H), 3.43 (dd, J = 15, 5Hz, 1H), 3.30-3.07 (m, 3H), 2.17-2.00 (m, 2H), 1.72-1.54 (m, 2H).

Reference Example 75
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-({2-oxo-2- [4- (4-pyridylthio) -1-piperidinyl] ethyl} amino) -1- (2-Pyridylmethyl) ethyl] -2-propenamide
4- (4-Piperidinylthio) pyridine dihydrochloride and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) propanoyl]- The above compound was obtained from amino} acetic acid in the same manner as in Reference Example 59.
ESI-MS: 564.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.56 (dd, J = 5, 2 Hz, 1H), 8.45 (d, J = 6 Hz, 2H), 8.08-7.88 (m, 1H), 7.65 (td,. J = 8,2Hz, 1H), 7.62 (d, J = 16Hz, 1H), 7.47 (d, J = 8Hz, .2H), 7.36 (d, J = 8Hz, 2H), 7.29-7.24 (m, 1H ), 7.20 (dd, J = 8, 5Hz, 1H), 7.15 (d, J = 6Hz, 1H), 6.50 (d, J = 16Hz, 1H), 5.064.97 (m, 1H), 4.27-4.13 ( m, 1H), 4.03 (appd, J = 4Hz, 2H), 3.75-3.52 (m, 2H), 3.49-3.39 (m, 1H), 3.31-3.11 (m, 3H), 2.14-2.01 (m, 2H ), 1.72-1.55 (m, 2H).

Reference Example 77
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-t (4chlorophenyl) thio] -1-piperidinyl} -2-oxoethyl) amino] -2- Oxo-1- (2-pyridylmethyl) ethyl] -2-propenamide
tert-butyl-4-[(4-chlorophenyl) thio] -1-piperidinecarboxylate and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino}- The above compound was obtained from 3- (2-pyridyl) propanoyl] amino} acetic acid in the same manner as in Reference Example 1.
ESI-MS: 597.2 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.55 (dd, J = 5, 2 Hz, 1 H), 8.03-7.85 (m, 2 H), 7.63 (td, J = 8, 2 Hz, 1 H), 7.60 (d, 16Hz, 1H), 7.45 (d, J = 8Hz, 2H), 7.37-7.22 (m, 7H), 7.18 (dd, J = 8,5Hz, 1H), 6.49 (d, J = 16Hz, 1H), 5.04 -4.96 (m, 1H), 4.30-4.16 (m, 1H), 3.99 (appd, d = 4Hz, 2H), 3.713.59 (m, 1H), 3.47-3.37 (m, 1H), 3.29-3.16 ( m, 2H), 3.15-2.91 (m, 2H), 2.00-1.88 (m, 2H), 1.59-1.42 (m, 2H).

Reference Example 79
(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-[(2- {4-[(4-chlorophenyl) sulfonyl] -1-piperidinyl} -2-oxoethyl) amino] -2 -Oxo-{(2-pyridylmethyl) ethyl] -2-propenamide
4-[(4-Chlorophenyl) sulfonyl] piperidine hydrochloride and {[(2S) -2-{[(2E) -3- (4-chlorophenyl) -2-propenoyl] amino} -3- (2-pyridyl) The above compound was obtained from -propanoyl] amino} acetic acid in the same manner as in Reference Example 59.
ESI-MS: 629.1 (M + H)
¹ H-NMR (300 MHz, CDCl ₃ ) δ 8.57-8.51 (m, 1H), 8.05-7.83 (m, 2H), 7.827.74 (m, 2H), 7.67-7.52 (m, 4H), 7.45 (d , J = 8Hz, 2H), 7.35 (d, J = 8Hz, 2H), 7.28-7.13 (m, 2H), 6.53-6.43 (m, 1H), 5.03-4.93 (m, 1H), 4.71-4.57 ( m, 1H), 3.98 (appd, J = 4Hz, 2H), 3.86-3.75 (m, 1H), 3.46-3.34 (m, 1H), 3.29-3.19 (m, 1H), 3.16-2.90 (m, 2H ), 2.65-2.50 (m, 1H), 2.15-1.95 (m, 2H), 1.69-1.49 (m, 2H).

  In addition, the method of manufacturing another reference example is described in Patent Document 6 incorporated in the present specification as a reference material.

Method 1 Cirrhosis model Cirrhosis was created by intraperitoneally administering thioacetamide (TAA) at a dose of 200 mg / kg twice weekly to a 200 g body weight rat for 12 weeks.
2 NO Synthase Inhibitor and Method of Administration An inducible NO synthase inhibitor of the formula II (provided by Fujisawa Pharmaceutical Co., Ltd., hereinafter abbreviated as FR ) was orally administered twice daily at a dose of 32 mg / kg. . The compound of formula II was administered in the form of a solution dissolved in polyethylene glycol.
3 Experimental groups Three experimental groups were set.
TAA single group: TAA only administration (n = 7), TAA + FR administration group: TAA and FR administration (n = 8), FR single administration group: FR only administration (n = 5).
4 Samples were sacrificed at 12 weeks, and liver weight and spleen weight were measured. A part of the liver tissue was preserved with frozen sections and formalin fixation. At the same time, blood was collected and serum was separated and stored at -80 ° C.

5 Measurement Items / Histological Examination HE staining, Azan-Mallory staining / Immunohistological examination iNOS staining, TGFβ1 staining / blood / biochemical examination As the blood count, the number of red blood cells, white blood cells, and platelets were measured. AST and AST T. as biochemical tests. Bil was measured. Fibrinogen and PT-INR were measured as coagulation factors.
-NOx measurement Serum NOx was measured according to the conventional method (Greiss method) for NO production amount estimation.
-Hyaluronic acid Hyaluronic acid level (HA) was measured as an index indicating hepatic fibrosis and sinusoidal disorder.
-TGFβ measurement TGFβ was measured as a cytokine involved in liver fibrosis.
The measured values were expressed as mean ± standard deviation, and statistical treatment was considered significant with P <0.05 using one-way analysis of variance and t-test.

Results 1 Liver morphological findings In the intraperitoneal findings at the time of sacrifice, all patients in the FR-administered group were morphologically normal liver, but all patients in the TAA group showed cirrhosis and liver atrophy, ascites retention and splenomegaly. Admitted. On the other hand, although hepatic fibrosis was observed in the TAA + FR group, fibrosis was milder and the degree of liver atrophy was slight compared with the TAA group, and ascites and splenomegaly were not observed (FIG. 1).
In the histological examination, fibrosis and inflammatory cell infiltration were ++ in all rats in the TAA group, but in the TAA + FR group, all were mild and about + compared to the TAA group (Table 1).

2 Liver weight, spleen weight Liver weight to body weight ratio (liver weight / body weight) is 2.4 ± 0.6% in the TAA group, 3.5 ± 0.5% in the TAA + FR group, and 3. ± in the FR group. At 3 ± 0.3%, the TAA group showed significant liver atrophy (FIG. 2). Similarly, the ratio of spleen weight to body weight (spleen weight / body weight) is 0.42 ± 0.08% in the TAA group, 0.28 ± 0.03% in the TAA + FR group, and 0.18 ± 0.01 in the FR group. %, The TAA group had significantly enlarged spleen (FIG. 3).

2 Azan-Mallory staining The degree of liver fibrosis was examined by Azan-Mallory staining (Fig. 4). Since the TAA group progressed to cirrhosis, a marked increase in the fiber component was observed, but fibrosis was suppressed in the TAA + FR group. When the degree of fibrosis was calculated by area ratio, the TAA group was 25.45%, but the TAA + FR group was 9.60%, and the degree of fibrosis was significantly low.

3 Blood and biochemical tests The red blood cell count was 787.6 ± 39.4 × 106 / μl in the FR group, the TAA + FR group 779.5 ± 68.8 × 106 / μl, and the TAA group 656.4 ± 51.8 × 106 / μl. At μl, the TAA + FR group was significantly higher than the TAA group. The number of leukocytes was 5340 ± 726 / μl in the FR group, 12500 ± 1464 μl in the TAA + FR group, 12457 ± 4125 μl in the TAA group, and there was no difference between the TAA + FR group and the TAA group (FIG. 5). Platelet count is 88.1 ± 5.7 × 104 / μl in the FR group, TAA + FR group is 81.7 ± 11.8 × 104 / μl, TAA group is 50.8 ± 14.1 × 104 / μl, TAA + FR group is TAA It was significantly higher than the group (Fig. 5).

  In biochemical tests, AST and ALT were 152.0 ± 26.5 IU / L in the FR group, 34 ± 3.5 IU / L, and 315.7 ± 33.4 IU / L in the TAA + FR group, 98.2 ± 13. 4 IU / L, TAA group 350.4 ± 29.4 IU / L, 104.6 ± 20.5 IU / L, and there was no difference between the TAA + FR group and the TAA group (FIG. 6). T.A. Bil is 0.10 ± 0, 13 mg / ml in the FR group, 0.35 ± 0.14 mg / ml in the TAA + FR group, 0.67 ± 0.13 mg / ml in the TAA group, and the TAA + FR group is compared to the TAA group. Significantly lower (Figure 6).

  Fibrinogen as a clotting factor is 280.8 ± 15.1 mg / ml in the FR group, 201.7 ± 29.4 mg / ml in the TAA + FR group, 175.4 ± 37.8 mg / ml in the TAA group, and tends to be high in the TAA + FR group However, there was no significant difference (FIG. 7). PT-INR was 1.83 ± 0.11 in the FR group, 2.81 ± 0.69 in the TAA + FR group, 2.98 ± 0.46 in the TAA group, and the TAA + FR group was significantly lower than the TAA group (FIG. 7). ).

4 Serum NOx level Serum NOx was 69.4 ± 19.4 in the FR group, 72.2 ± 13.1 in the TAA + FR group, and 92.3 ± 67.0 in the TAA group, and there was no significant difference between the three groups ( FIG. 8).

5 Hyaluronic acid level The hyaluronic acid level is 36.4 ± 4.3 in the FR group, 110.0 ± 31.6 in the TAA + FR group, 294.8 ± 150.0 in the TAA group, and the TAA group in the TAA + FR group between the 3 groups. (FIG. 9).

6 TGFβ1
TGFβ1 was 56.4 ± 52.6 in the FR group, 87.7 ± 20.9 in the TAA + FR group, 110.0 ± 7.4 in the TAA group, and the TAA group was significantly higher than the FR group. In the TAA group and the TAA + FR group, the TAA + FR group tended to be low (FIG. 10). That is, it was speculated that the compound of the formula II had TGFβ1 production inhibitory activity.

7 iNOS immunohistochemical study Although iNOS expression was confirmed by immunostaining in the TAA group, iNOS expression was attenuated in the TAA + FR group (FIG. 11).

8 TGFβ1 immunohistological examination In the TAA group, TGFβ1 was strongly stained at the site where fibrosis occurred, but was attenuated in the TAA + FR group (FIG. 12).

(Summary)
In the process of studying suppression of reperfusion injury by an inducible NO synthase inhibitor in hepatic ischemia reperfusion injury, the inducible NO synthase inhibitor is an extremely useful treatment in the state of invasion. We have recognized that it is a means. Therefore, in order to pursue new possibilities and developability of this inducible NO synthase inhibitor, NO has been investigated in the liver fibrosis where no relation to NO has been studied so far, especially NO derived from inducible NO synthase. Was involved in liver fibrosis.

  In the present invention, using the orally administrable inducible NO synthase inhibitor (compound of formula II) found by Fujisawa Pharmaceutical, the present inventors can mainly suppress TGFβ1 production and liver fibrosis by the compound. This was investigated using a rat cirrhosis model. Inducible NO synthase is inactive when it is a monomer and must be dimer to activate, but the compound of formula II is an enzyme that suppresses dimerization of inducible NO synthase. It is a drug that inhibits the activity.

  In preparing rat cirrhosis, TAA was prepared by intraperitoneal administration twice a week. This model becomes chronic hepatitis at 8 weeks after administration and complete cirrhosis at 12 weeks. Hepatic atrophy, splenomegaly, and ascites were observed. Inflammatory cell infiltration and strong fibrosis were observed histologically, and it is considered to be a model histologically close to human cirrhosis. In this model, when the compound of formula II was orally administered twice daily, it was possible to obtain an epochal result that liver fibrosis was suppressed by administration of the compound of formula II. That is, administration of the compound of formula II suppressed the progression to cirrhosis, ascites and splenomegaly were not observed, and the liver fibrosis rate was reduced from 25% to 9.6%. In addition, a decrease in platelet count, T.P. Improvements in abnormal blood and biochemical tests in cirrhosis such as increased Bi were also observed. In addition, an increase in the level of hyaluronic acid, which is an index of hepatic sinusoidal disorder, was markedly suppressed. From the above results, a new fact was found that an inducible NO synthase inhibitor suppresses liver fibrosis.

As described above, it has been revealed that TGFβ1 plays a central role in the pathogenesis of liver fibrosis. That is, when the release of TGFβ1 increases due to the persistence of inflammation such as hepatitis, the production of extracellular matrix is promoted, and the synthesis of degradation enzymes in the extracellular matrix is suppressed, leading to the accumulation of fibrous tissue. . In this study, the present inventors examined the expression of TGFβ1 by immunostaining. In the TAA group, TGFβ1 was markedly stained in line with the fibrosis, but the TAA + FR administration group decreased the staining. It was. Serum TGFβ1 tended to be low in the TAA + FR administration group although there was no significant difference between the two groups. Thus, it is considered that FR may suppress TGFβ1 production by some mechanism, thereby suppressing collagen production from stellate cells .

  Chronic inflammation due to TAA may induce inducible NO synthase, and the NO produced therefrom may promote the production of TGFβ1. Certainly, in the immunohistochemical examination, inducible NO synthase was markedly expressed in the TAA administration group, but was attenuated in the TAA + FR administration group. However, the inventors measured serum NOx, but found no difference between the FR group, the TAA group, and the TAA + FR administration group. In general, inducible NO synthase is expressed in cirrhosis, but there are reports that this is a secondary result induced by accompanying endotoxemia. On the other hand, inducible NO synthase-derived NO that is expressed by invasion newly induces the production of cytokines such as TNFα and interferon γ, so it is also considered that inducible NO synthase-derived NO causes chronic production of TGFβ1 in minute amounts. It is done.

The compounds of formula II and other related compounds used in this experiment decrease TGFβ1 production by decreasing inducible NO synthase-derived NO production chronically and continuously, and consequently suppress liver fibrosis It is also possible.
On the other hand, it is also possible that the compound of formula II and other related compounds may directly act to suppress TGFβ1 production or to inhibit collagen production regardless of inducible NO synthase inhibition.

  If the compound of formula II and other related compounds are clinically applied in patients with chronic hepatitis B, chronic hepatitis C, etc., and the transition to cirrhosis is suppressed, the number of liver failure cases due to cirrhosis may be drastically reduced, The number of patients with liver transplantation is expected to decrease, and there are immeasurable benefits for patients and medical finance. In addition, patients with chronic hepatitis C develop liver cancer at a high rate in the process of developing cirrhosis, but if liver cirrhosis is suppressed by suppressing the progression of liver cirrhosis, a very important effect of preventing liver cancer is expected. Is done. If such an effect is clinically confirmed, it can be said to be a gospel for patients who are endangered by liver cancer.

  Furthermore, it is only interferon alpha that is considered clinically possible to suppress liver fibrosis at the present time, but since this drug is not an oral agent, patients must go to the hospital, fever, thrombocytopenia, suicide It also has side effects such as mental symptoms such as intentions. On the other hand, FR, which is an inducible NO synthase inhibitor used this time, can be administered as an oral agent, so the number of visits is reduced and the burden on the patient is reduced. From this point of view, if this drug is clinically applied, it will be an extremely useful drug.

  The composition and treatment method having the effect of inhibiting fibrosis of liver and / or the effect of inhibiting fibrosis of the pancreas and / or the effect of inhibiting fibrosis of the lung according to the present invention include liver fibrosis inhibition research, pancreatic fibrosis inhibition research, and lung. This is an unprecedented original in fibrosis suppression research. Therefore, the present invention greatly contributes to the development of related industries such as the pharmaceutical industry.

It is a photograph figure which shows the liver and spleen of each treatment group mouse. It is a graph which shows the liver weight of each process group mouse | mouth. It is a graph which shows the spleen weight of each process group mouse | mouth. It is a photograph figure which shows the degree of liver fibrosis of each treatment group mouse by staining with Azan-Mallory. It is a figure which shows the erythrocyte count, leukocyte count, and platelet count of each treatment group mouse. It is a figure which shows the biochemical test result of each process group mouse | mouth. It is a figure which shows the amount of coagulation factors of each treatment group mouse. It is a figure which shows the serum NOx value of each process group mouse | mouth. It is a figure which shows the hyaluronic acid value of each process group mouse | mouth. It is a figure which shows the grade of TGF (beta) 1 production of each process group mouse | mouth. It is a photograph figure which shows iNOS immunohistological examination result. It is a photograph figure which shows a TGF (beta) 1 immunohistological examination result.

Claims (11)

Formula I

Where X is

And
R ¹ is benzofuranyl substituted with halogen, or styryl substituted with halogen;
R ² is an alkyl or haloalkyl having 1 to 6 carbon atoms, 1 or 2 or more alkyl groups having 1 to 6 carbon atoms, alkoxy, haloalkyl or haloalkoxy, or a heterocyclic group optionally substituted with halogen, An aryl group optionally substituted by one or more C 1-6 alkyl, alkoxy or haloalkyl or halogen;
Y is —O—, —S— or —SO ₂ —.
Or a pharmaceutically acceptable salt thereof as an active ingredient , which suppresses fibrosis induced by an increase in TGFβ1 or activation of stellate cells . In Formula I, R ¹ is 2- (4-chlorophenyl) ethenyl and X is

The fibrosis-suppressing agent according to claim 1, wherein   The fibrosis inhibitor according to claim 1 or 2, wherein in formula I, Y is -O-. The fibrosis inhibitor according to any one of claims 1 to 3, wherein in the formula I, R ² is a heterocyclic group having one or more substituents. The fibrosis inhibitor according to any one of Claims 1 to 4, wherein in Formula I, R ² is a nitrogen-containing heterocyclic group having one or more substituents. The fibrosis inhibitor according to any one of claims 1 to 5, wherein in formula I, R ² is a pyrimidinyl group having one or more substituents.   The fibrosis inhibitor according to any one of claims 1 to 6, which has liver fibrosis inhibitory activity and / or pancreatic fibrosis inhibitory activity and / or lung fibrosis inhibitory activity. The compound of formula I is of formula II

(2E) -3- (4-Chlorophenyl) -N-[(1S) -2-oxo-2-{[2-oxo-2- (4-{[6- (trifluoromethyl)] The fibrosis inhibitor according to any one of claims 1 to 7 , which is -4-pyrimidinyl] oxy} -1-piperidinyl) ethyl] amino} -1- (2- pyridylmethyl ) ethyl] -2-propenamide. .   Use of the fibrosis-suppressing agent according to any one of claims 1 to 8 in the manufacture of a medicament for suppressing progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension.   A medicament for suppressing the progression of cirrhosis and / or chronic pancreatitis and / or pulmonary hypertension, comprising the fibrosis inhibitor according to any one of claims 1 to 8.   11. A medicament according to claim 10, having an oral dosage form selected from tablets, pills, coated tablets, capsules, powders, granules, syrups, juices, solutions, suspensions and drops.

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