WO2008093940A1

WO2008093940A1 - Quinoline derivatives as caspase-3 inhibitor, preparation process for the same and pharmaceutical composition comprising the same

Info

Publication number: WO2008093940A1
Application number: PCT/KR2008/000202
Authority: WO
Inventors: Je Wook Han; Sung Gyu Kim; Min Ki Shin; Sang Kwon Sohn; Yoon Sung Jung; Ni Na Ha; Jong Hwan Kim
Original assignee: Yungjin Pharmaceutical Co., Ltd.
Priority date: 2007-01-31
Filing date: 2008-01-11
Publication date: 2008-08-07
Also published as: KR100844926B1

Abstract

Provided is a quinoline derivative represented by the following Formula (1) for use in treating a caspase- mediated disease by inhibition of caspase-3 activity. Further provided are a method for preparing the quinoline derivative or a pharmaceutically acceptable salt thereof and a pharmaceutical composition containing the same.

Description

[DESCRIPTION] [Invention Title]

QUINOLINE DERIVATIVES AS CASPASE-3 INHIBITOR, PREPARATION PROCESS FOR THE SAME AND PHARMACEUTICAL COMPOSITION COMPRISING THE SAME

[Technical Field]

The present invention relates to a quinoline derivative with inhibitory effects against caspase-3 activity, a pharmaceutically acceptable salt thereof, a method for preparing the quinoline derivative or the pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same.

[Background Art]

The regulation over the number of mammalian cells is known to be determined by the balance between cell proliferation and cell death. One type of cell death called "necrotic cell death" (also called "necrosis") is characterized as a pathological form of cell death caused by traumatic or cellular injury. Such necrosis causes damage to other tissues e.g., induction of inflammation. In contrast to necrosis, the other pathological form of cell death processes in an orderly regulated pattern. Such a form of cell death is often referred to as "apoptosis" (Barr, et al . , Bio/Technology, 12:487-497, 1994; Steller, et al., 267:1445-1449, 1995). Apoptosis is programmed cell death and is capable of removing exhausted or aged cells without causing any damage to other tissues. Accordingly, apoptosis is a physiologically essential and important process which maintains normal development and homeostasis of organisms by killing damaged or overgrown cells.

There are many causes of apoptosis. Of these, caspases are proteins which play the most important roles in apoptosis and fourteen types of caspases have been so far identified. These caspases are cysteine proteases and use a varierty of main cellular proteins as substrates. Apoptosis involves a process in which cells fragmentized by a class of caspase are absorbed in the form of small particles in other cells or are killed by cells such as macrophages without undesired phenomena e.g. inflammation .

Caspases are divided into two main groups, i.e. initiator caspases and effector caspases. The initiator caspases serve to initiatilly receive apoptosis signals and then transmit the signals to effector caspases and representative examples thereof include caspase-8 and -9. The effector caspases serve to cleave various cellular components and representative examples thereof include caspase-3, -6 and -7. Of these, caspase-3 has been the most actively researched, which serves as the final acceptor in signal transduction cascades . A great deal of research ascertained that apoptosis can be suppressed by inhibiting expression level or activity of caspase-3. Caspase-3 is a cysteine protease of 32kDa and is an effector caspase which plays a critical role during morphogenetic cell death in mammalian brains. Defective mechanism of caspase-related apoptosis is implicated in a variety of diseases, representatively cancers. Namely, cancer is one of disesases caused by failure of apoptosis. On the other hand, excessive apoptosis causes various neurologic disorders.

Representative examples of caspase-3 mediated diseases include Alzheimer's disease (Gervais F. G. et al., Cell, 97 (3) : 395-406, 1999; Walter J. et al., Proc

Natl Acad Sci U S A 96 ( 4 ) : 1391 -6, 1999; Barnes N. Y. et al., J Neurosci 18 (15) : 5869-80, 1998; Kim T. W. et al.,

Science 277 (5324 ): 373-6, 1997), Huntington' s disease

(Goldberg Y. P. et al., Nat Genet. 13(4):442-9, 1996; Wellington C. L. et al., J Biol Chem. 273 (15) : 9158-67, 1998; Sanchez I. et al., Neuron. 22 (3) : 623-33, 1999), Parkinson's disease (Dodel R. C. et al., MoI Brain Res 64(l):141-8, 1999; Takai N. et al., J Neurosci Res 54 (2) :214-22, 1998), ALS (amyltrophic lateral sclerosis) (Pasinelli P. et al., Proc Natl Acad Sci U S A. 95 (26) :15763-8, 1998), AIDS (Kruman I. I. et al., Exp Neurol. 154 (2) : 276-88, 1998), stroke/ischemia (Hara H. et al., Proc Natl Acad Sci U S A. 94 (5) : 2007-12, 1997; Namura S. et al., J Neurosci. 18 (10) : 3659-68, 1998; Schulz J. B. et al., Ann Neurol. 45(4):421-9, 1999), traumatic brain injury (Yakovlev A. G. et al., J Neurosci. 17 (19) : 7415-24 , 1997; Kermer P. et al., J Neurosci. 18 (12 ): 4656-62, 1998; Chaudhary P. et al, MoI Brain Res. 67(l):36-45, 1999), spinal cord injury (Crowe M. J. et al., Nat Med. 3(l):73-6, 1997; Shuman S. L. et al., J Neurosci Res. 50 (5) : 798-808 , 1997), osteoarthritis and the like.

Accordingly, there have been continuous attempts to develop potent drugs employing caspase-3 inhibitors, for use in treating caspase-mediated diseases such as Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke/ischemia, traumatic brain injury, spinal cord injury and osteoarthritis. Up to date, aspartic acid-based, peptide-based and gamma-keto acid-based compounds were reported as caspase-

3 inhibitors (WO 93/05071, WO 96/03982, U.S. Pat. No.

5,585,357, WO 00/32620, WO 00/55157 and U.S. Pat. No. 6,153,591) .

However, these compounds cannot exhibit sufficient inhibitory activity against caspase-3. Accordingly, there is a continuous need to develop a material that exhibits more superior inhibitory activity against caspase-3.

[Disclosure] [Technical Problem]

It is one aspect of the present invention to provide a quinoline derivative with superior inhibitory activity against caspase-3 or a pharmaceutically acceptable salt thereof.

It is another aspect of the present invention to provide a method for preparing the quinoline derivative or pharmaceutically acceptable salt thereof.

It is another aspect of the present invention to provide a pharmaceutical composition comprising the quinoline derivative or the pharmaceutically acceptable salt thereof. It is yet another aspect of the present invention to provide the use of the quinoline derivative or pharmaceutically acceptable salt thereof for preparing a drug for treating caspase-mediated diseases based on inhibition of caspase-3 activity.

[Technical Solution]

The present invention has been made to solve the foregoing problems and in accordance with one aspect of the present invention, there is provided a quinoline derivative of the following Formula 1 for use in treating caspase-mediated diseases by inhibition of caspase-3 activity:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, C_L-₆ alkoxy, Ci-6 alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to 15-membered aryl;

R is in which R₂ is C or

N; R₃ is - (CH₂) n~ in which n is 0, 1, 2, 3 or 4; R₄ is C, N, O or S; and R₅ is hydrogen, halogen, Ci_₆ alkyl, Ci_₆ alkoxy, Cχ_₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl;

A is C, N, O or S; and

Y is hydrogen, halogen, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Cχ-6 halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof.

Preferably, the quinoline derivative or a pharmaceutically acceptable salt thereof has a structure of Formula 1 wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, nitro or Ci-6

halogenoalkyl; R is or in which R₂

is N; R₃ is -(CH₂J₂-; R₄ is C, N, 0 or S; and R₅ is hydrogen, Cχ-₆ alkyl or Cχ-₆ alkoxyalkyl; A is N; and Y is hydrogen.

More preferably, the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof may be 5-fluoro-3- [ (7-nitro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-4-oxo-3- [ (4-piperazin-l-yl-7- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid, 5-fluoro-4-oxo-3- [ (4- piperazin-l-yl-8-trifluoromethyl-quinoline-3-carbonyl) - amino] -pentanoic acid trifluoroacetic acid, 5-fluoro-4- oxo-3- [ (4-piperidin-l-yl-7-trifiuoromethyl-quinoline-3- carbonyl) -amino] -pentanoic acid trifluoroacetic acid, 5- fluoro-3- [ (8-fluoro-4-piperidin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (4-morpholin-4-yl-7-trifluoromethyl- quinoline-3-carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro--4-oxo-3- [ (4-thiomorpholin- 4-yl-7-trifluoromethyl-quinoline-3-carbonyl) -amino] - pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (8- nitro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] -4- oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- ({ 4- [4- (2-methoxy-ethyl) -piperazin-1-yl] -8-nitro-quinoline-3- carbonyl } -amino) -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ ( 8-fluoro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (7-fluoro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3-{ [4- (4-methyl-piperazin-l-yl) -8-nitro- quinoline-3-carbonyl] -amino}-4-oxo-pentanoic acid trifluoroacetic acid or 3-{ [4- (4-ethyl-piperazin-l-yl) -8- nitro-quinoline-3-carbonyl] -amino } -5-fluoro-4-oxo- pentanoic acid trifluoroacetic acid.

The caspase-mediated disease that may employ the quinoline derivative of the Formula 1 or a pharmaceutically acceptable salt thereof may be Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.

In addition, the quinoline derivative of the Formula 1 or a pharmaceutically acceptable salt may be hydrochloride, bromate, sulfate, nitrate, perchlorate, fumarate, maleate, phosphate, glycolate, lactate, salicylate, succinate, p-toluenesulfonate, tartrate, trifluoroacetate, acetate, citrate, methanesulfonate, formate, benzoate, malonate, naphthalene-2-sulfonate, benzensulfonate, an alkali-metal salt or an alkaline earth metal salt.

In accordance with another aspect of the present invention, there is provided a method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-6 alkyl, Ct-e alkoxy, d-β alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is -(CH₂)_n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, O or S; and R₅ is hydrogen, halogen, C₁-₆ alkyl, Ci-₆ alkoxy, Ci-ε alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and

Y is hydrogen, halogen, Ci_6 alkyl, Ci-6 alkoxy, Ci-6 alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci_6 halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein Xi, X₂, X₃ and X₄ are defined as above;

wherein Xi, X₂, X₃ and X₄ are defined as above; cyclizing the compound of Formula 3 in the presence of diphenylether to obtain a compound represented by Formula 4 below:

wherein Xi, X₂, X₃ and X₄ are defined as above; halogenating the compound of Formula 4 to obtain a compound represented by Formula 5 below:

wherein Xi, X₂, X₃ and X₄ are defined as above and Z is halogen; reacting the compound of Formula 5 with RH in which R is defined as above, to obtain a compound represented by Formula 6 below:

wherein Xi, X₂, X₃, X₄ and R are defined as above; hydrolyzing the compound of Formula 6 to obtain a compound represented by Formula 7 below:

wherein Xi, X₂, X₃, X₄ and R are defined as above; reacting the compound of Formula 7 with a compound represented by the following Formula 8 to obtain a compound represented by Formula 9 below:

wherein A and Y are defined as above;

wherein Xi, X₂, X₃, X4, R, A and Y are defined as above; and carbonylating the compound of Formula 9 to obtain the compound of Formula 1 above.

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-₆ alkyl, Ci_₆ alkoxy, Ci-₆ alkoxyalkyl , C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to

15-membered aryl; R is

in which

R₂ is C or N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is N; and R₅ is hydrogen; A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein Xi, X2, X₃ and X4 are defined as above;

wherein Xi, X₂, X₃ and X₄ are defined as above and Z is halogen; reacting the compound of Formula 5 with RiH wherein

Ri is

or in which R₂ and R₃ are defined as above, to obtain a compound represented by Formula 10 below:

wherein Xi, X₂, X₃, X₄ and Ri are defined as above; hydrolyzing the compound of Formula 10 to obtain a compound represented by Formula 11 below:

wherein Xi, X₂, X₃, X₄ and Ri are defined as above; reacting the compound of Formula 11 with a compound represented by the following Formula 12 to obtain a compound represented by Formula 13 below:

wherein A is defined as above;

wherein X₁, X₂, X₃, X₄, Ri and A are defined as above; carbonylating the compound of Formula 13 to obtain a compound represented by Formula 14 below:

wherein X₁, X₂, X₃, X₄, Ri and A are defined as above; and deprotecting protective groups I and II of the compound of Formula 14 to obtain the compound of Formula 1 above .

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, C₁-₆ alkyl, Cχ-₆ alkoxy, Ci_₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to

15-membered aryl; R is , or in which

R₂ is C or N; R₃ is - (CH₂) _n~ in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Cχ_₆ alkyl, Cχ-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

(2) wherein Xi, X₂, X₃ and X₄ are defined as above;

wherein X₁, X₂, X₃ and X4 are defined as above and Z is halogen; reacting the compound of Formula 5 with RH in which R is defined as above, to obtain a compound represented by Formula 6 below:

wherein X₁, X₂, X₃, X₄ and R are defined as above; hydrolyzing the compound of Formula 6 to obtain a compound represented by Formula 7 below:

wherein X₁, X₂, X₃, X₄ and R are defined as above; reacting the compound of Formula 7 with a compound represented by the following Formula 12 to obtain a compound represented by Formula 15 below:

^• Protective Group II :i2: wherein A is defined as above;

wherein Xi, X2, X₃, X₄, R and A are defined as above; carbonylating the compound of Formula 15 to obtain a compound represented by Formula 16 below:

I'

wherein Xi, X₂, X₃, X₄, R and A are defined as above; and deprotecting a protective group II of the compound of Formula 16 to obtain the compound of Formula 1 above. In accordance with another aspect of the present invention, there is provided a pharmaceutical composition containing a pharmaceutically effective amount of the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof for use in treating a caspase- mediated disease based on inhibition of caspase-3 activity.

The caspase-mediated disease using the pharmaceutical composition may be Alzheimer' s disease, Huntington's disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.

Hereinafter, the construction and operation of preferred embodiments of the present invention will be explained. However, these embodiments are given for the purpose of illustration and are not intended to limit scope of the present invention.

In one aspect, the present invention is directed to a quinoline derivative represented by the following Formula 1 or a pharmaceutically acceptable salt thereof, for use in treating caspase-mediated diseases based on inhibition of caspase-3 activity:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Cχ-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to

15-membered aryl; R is , or in which

R.₂ is C or N; R₃ is - (CH₂) _n~ in which n is O, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci-6 alkyl, Ci-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen, halogen, Ci-₆ alkyl, Cχ-6 alkoxy, Ci-₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci-6 halogenoalkyl or 5- to 15-membered aryl.

As will be mentioned in the following Examples or Experimental Examples, as a result of repeated experiments, the present inventors ascertained that the quinoline derivative of Formula 1 or the pharmaceutically acceptable salt thereof exhibit considerably excellent inhibitory activity against caspase-3. Accordingly, the quinoline derivative of Formula 1 or the pharmaceutically acceptable salt thereof is suitable for use in treating caspase-3 mediated diseases. Meanwhile, a definition of the terms used herein will be given below.

First, the term "halogen" refers to a radical such as fluorine, chlorine or bromine.

The term "alkyl" refers to a straight-chained or branched hydrocarbon radical and examples thereof include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl and the like. The term "halogenoalkyl" refers to a group in which at least one hydrogen atom contained in the alkyl is substituted by halogen.

The term "alkoxy" refers to a straight-chained or branched alkyl radical linked to oxygen and examples thereof include methoxy, ethoxy, propoxy, iso-propoxy, butoxy, iso-butoxy and the like.

The term "alkoxyalkyl" refers to a radical in which at least one hydrogen atom contained in the alkyl is substituted by alkoxy. The alkoxy may be straight-chained or branched.

The term "cycloalkyl" refers to a non-aromatic hydrocarbon ring and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term "aryl" refers to a mono- or poly-cyclic aromatic ring, and in particular is intended to include not only homoaromatic rings having carbon atoms exclusively, but also heteroaromatic rings having nitrogen, sulfur or oxygen atoms in addition to carbon atoms. Further, the term "aryl" used herein includes (hetero) aromatic rings in which one or more hydrogen are substituted by the following group selected from acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thio, alkyl, cycloalkyl, alkoxy, aryloxy, sulfoxy and guanido.

Examples of the aryl include phenyl, napthyl, pyridinyl pyrimidinyl, quinolinyl, benzothienyl, indolyl, pyrazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl, imidazopyridinyl, isothiazolyl, cinnolinyl, carbazolyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazianyl, pteridinyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl , benzoxazinyl, dihydrobenzisothiopyranyl, benzopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, chromonyl, chromanonyl, pyridyl-N-oxide, tetrahydroquinolinyl-N- oxide, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl-N-oxide, pyrimidinyl-N-oxide, pyrazinyl-N-oxide, quinolinyl-N-oxide, indolyl-N-oxide, indolinyl-N-oxide, isoquinolyl-N-oxide, quinazolinyl-N- oxide, quinoxalinyl-N-oxide, phthalazinyl-N-oxide, irαidazolinyl-N-oxide, isoxazolyl-N-oxide, oxazolyl-N- oxide, thiazolyl-N-oxide, indolizinyl-N-oxide, indazolyl- N-oxide, benzothiazolyl-N-oxide, benzoimidazolyl-N-oxide, oxadiazolyl-N-oxide, thiadiazolyl-N-oxide, triazolyl-N- oxide and tetrazolyl-N-oxide .

Preferably, in the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof, Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, nitro

or Ci-₆ halogenoalkyl; R is

xn

which R₂ is C or N; R₃ is -(CH₂) 2-; R4 is C, N, O or S; and R₅ is hydrogen, Ci-₆ alkyl or Ci-₆ alkoxyalkyl; A is N; and Y is hydrogen.

More preferably, the quinoline derivative of Formula 1 or the pharmaceutically acceptable salt thereof may be 5-fluoro-3- [ (7-nitro-4~piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-4-oxo-3- [ (4-piperazin-l-yl-7- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid, 5-fluoro-4-oxo-3- [ (4- piperazin-l-yl-8-trifluoromethyl -quinoline-3-carbonyl) - amino] -pentanoic acid trifluoroacetic acid, 5-fluoro-4- oxo-3- [ (4-piperidin-l-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -pentanoic acid trifluoroacetic acid, 5- fluoro-3- [ (8-fluoro-4-piperidin~l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (4-morpholin-4-yl-7-trifluoromethyl- quinoline-3-carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-4-oxo-3- [ (4-thiomorpholin- 4-yl-7-trifluoromethyl-quinoline-3-carbonyl) -amino] - pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (8- nitro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] -4- oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- ({ 4- [4- (2-methoxy-ethyl) -piperazin-1-yl] -δ-nitro-quinoline-3- carbonyl} -amino) -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3-[ (8-fluoro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3- [ (7-fluoro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid, 5-fluoro-3-{ [4- (4-methyl-piperazin-l-yl) -8-nitro- quinoline-3-carbonyl] -amino} -4-oxo-pentanoic acid trifluoroacetic acid or 3-{ [4- (4-ethyl-piperazin-l-yl) -8- nitro-quinoline-3-carbonyl] -amino } -5-fluoro-4-oxo- pentanoic acid trifluoroacetic acid.

The quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof exhibit superior inhibitory activity against caspase-3 and is thus more suitable for use in treating caspase-mediated diseases.

Representative examples of caspase-mediated diseases that may employ the quinoline derivative of Formula 1 or pharmaceutically acceptable salt thereof include Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury, osteoarthritis and the like. Owing to potent inhibitory activity against caspase-3, the quinoline derivative of Formula 1 or pharmaceutically acceptable salt thereof may be used to treat any caspase-related disease known to be mediated by caspase-3, in addition to the exemplified disesases . In addition, the quinoline derivative of Formula 1 may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salt may be a salt of pharmaceutically acceptable organic acid, inorganic acid or base. Examples of suitable organic and inorganic acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycollic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, trifluoroacetic acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Examples of suitable bases include alkali metal e.g. sodium, alkaline earth metal e.g. magnesium and ammonium.

In another aspect, the present invention is directed to a method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, C₁-₆ alkyl, C]_₆ alkoxy, Cχ-₆ alkoxyalkyl, C3-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to

15-membered aryl; R is , or in which

R.2 is C or N; R3 is -(CH2)_n~ in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci_₆ alkyl, Ci-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃--₆ cycloalkyl, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen, halogen, Ci_₆ alkyl, Ci_₆ alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein X₁, X₂, X₃ and X₄ are defined as above;

wherein X₁, X₂, X₃ and X₄ are defined as above; cyclizing the compound of Formula 3 in the presence of diphenylether to obtain a compound represented by Formula 4 below:

wherein X₁, X₂, X₃ and X₄ are defined as above; halogenating the compound of Formula 4 to obtain a compound represented by Formula 5 below :

wherein A and Y are defined as above;

wherein X₁, X₂, X₃, X₄, R, A and Y are defined as above; and carbonylating the compound of Formula 9 to obtain the compound of Formula 1 above.

The respective processes of the preparation method will be illustrated below.

First, the compound of Formula 2 is reacted with diethylethoxymethylenemalonate to obtain the compound represented by Formula 3. In this process, the compound of Formula 3 may be prepared by reacting the compound of Formula 2 with diethylethoxymethylenemalonate without using any additional solvent while heating at 100-150°C. Alternatively, the compound of Formula 3 may be prepared by reacting the compound of Formula 2 with diethylethoxymethylenemalonate, while heating in an organic solvent such as toluene, chlorobenzene or xylene at a boiling point of the organic solvent. Then, the compound of Formula 3 is cyclized in the presence of diphenylether to obtain the compound of Formula 4. In this process, the compound of Formula 4 may be prepared by cyclizing the compound of Formula 3 while heating in the presence of diphenylether as an organic solvent at 26O⁰C to a melting point of the organic solvent .

Subsequently, the compound of Formula 4 is halogenated to obtain the compound of Formula 5. In this process, the compound of Formula 5 is prepared by reacting the compound of Formula 4 with a halogenating agent such as phosphorus oxychloride, phosphorus trichloride or phosphorus pentachloride .

Then, the compound of Formula 5 is reacted with RH to obtain the compound of Formula 6. In this process, for example, the compound of Formula 6 is prepared by reacting the compound of Formula 5 with RH in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide at ambient temperature to the boiling point of the organic solvent. Alternatively, the reaction of the compound of Formula 5 with RH may be carried out in the presence of an amine compound and examples of suitable amine compounds include triethylamine, diisopropylethylamine, dimethylaniline, pyridine and quinoline.

Then, the compound of Formula 6 is hydrolyzed to obtain the compound of Formula 7. In this process, for example, the compound of Formula 7 is prepared by hydrolyzing the compound of Formula 6 with a base such as lithium hydroxide, sodium hydroxide, potassium hydroxide or sodium bicarbonate in the presence of a mixed solvent of water and an organic solvent such as methanol, ethanol, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide at ambient temperature to the boiling point of the organic solvent.

Then, the compound of Formula 7 is reacted with the compound of Formula 8 to obtain the compound of Formula 9. In this process, for example, the compound of Formula 9 is prepared by reacting the compound of Formula 7 with the compound of Formula 8 in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide at ambient temperature to the boiling point of the organic solvent. Alternatively, the reaction of the compound of Formula 7 with the compound of Formula 8 may be carried out in the presence of 1- hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM) or l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) .

Then, the compound of Formula 9 is carbonylated to obtain the quinoline derivative of Formula 1. In this process, for example, the quinoline derivative of Formula 1 is prepared by carbonylating the compound of Formula 9 via oxidation with a Dess Martin reagent in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide . In another aspect, the present invention is directed to a method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Ci_₆ alkoxy, Cχ-6 alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to

15-membered aryl; R is , or in which

R2 is C or N; R₃ is -(CHa)_n" in which n is 0, 1, 2, 3 or 4; R₄ is N; and R₅ is hydrogen; A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethyl enemalonate to obtain a compound represented by Formula 3 below:

wherein X₁, X₂, X₃ and X₄ are defined as above;

R₁ is

OtouB U (12)

wherein A is defined as above;

wherein Xi, X₂, X₃, X₄, Ri and A are defined as above; carbonylating the compound of Formula 13 to obtain a compound represented by Formula 14 below:

wherein Xi, X₂, X₃, X₄, Ri and A are defined as above; and deprotecting protective groups I and II of the compound of Formula 14 to obtain the compound of Formula 1 above .

In this preparation method, the compound of Formula 5 is prepared from the compound of Formula 2 in the same manner as in the aforementioned another aspect. Then, for example, the compound of Formula 5 is reacted with RiH in which a protective group e.g. tert- butoxy is introduced at a R₅ position, to obtain the compound of Formula 10. In this process, for example, the compound of Formula 10 is prepared by reacting the compound of Formula 5 with RiH in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide in the presence of an amine compound such as triethylamine, diisopropylethylamine, dimethylaniline, pyridine or quinoline at ambient temperature to the boiling point of the organic solvent.

Then, the compound of Formula 11 is prepared by hydrolyzing the compound of Formula 10 under the same conditions as in the preparation of the compound of Formula 7 of the aforementioned another aspect.

Then, for example, the compound of Formula 12 in which a protective group II such as tert-butyl is introduced at a Y position, is reacted with the compound of Formula 11 to obtain the compound of Formula 13. In this process, for example, the compound of Formula 13 is prepared by reacting the compound of Formula 11 with the compound of Formula 12 in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide in the presence of 1- hydroxybenzotriazole (HOBt) , N-methylmorpholine (NMM) or l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) at ambient temperature to the boiling point of the organic solvent.

Then, the compound of Formula 13 is carbonylated to the compound of Formula 14. In this process, for example, the quinoline derivative of Formula 14 is prepared by carbonylating the compound of Formula 13 via oxidation with a Dess Martin reagent in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide . Then, protective groups I and II of the compound of Formula 14 are deprotected to obtain the compound of Formula 1. In this process, the deprotection of the protective groups I and II is carried out by reacting the compound of Formula 14 with acid e.g. trifluoroacetic acid. Such deprotection may be performed in an organic solvent e.g. dichloromethane.

In the preparation method according to the aforementioned another aspect, there is prepared a quinoline derivative of Formula 1 wherein R₄ is N, and R₅ and Y are hydrogen, or a pharmaceutically acceptable salt thereof. By introducing any substituent at a R₅ or Y position of the quinoline derivative or a pharmaceutically acceptable salt thereof, it is possible to prepare other quinoline derivatives or pharmaceutically acceptable salts thereof that fall within the scope of the compound of Formula 1.

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Ci_6 alkoxy, Cχ-₆ alkoxyalkyl , C₃-₆ cycloalkyl, nitro, amino, C1-6 halogenoalkyl or 5- to

15-membered aryl; R is or in which

R.2 is C or N; R₃ is -(C^)_n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, C_±-β alkyl, Ci-₆ alkoxy, Cχ-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein Xi, X₂, X₃ and X₄ are defined as above;

wherein Xi, X₂, X₃, X4 and R are defined as above; hydrolyzing the compound of Formula β to obtain a compound represented by Formula 7 below:

wherein Xi, X2, X₃, X4 and R are defined as above; reacting the compound of Formula 7 with a compound represented by the following Formula 12 to obtain a compound represented by Formula 15 below:

' Protective Group II :i2: wherein A is defined as above;

wherein Xi, X₂, X₃, X₄, R and A are defined as above; carbonylating the compound of Formula 15 to obtain a compound represented by Formula 16 below:

wherein X₁, X₂, X₃, X₄, R and A are defined as above; and deprotecting a protective group II of the compound of Formula 16 to obtain the compound of Formula 1 above.

In this preparation method, the compound of Formula 7 is prepared from the compound of Formula 2 in the same manner as in the aforementioned other aspects.

Then, the compound of Formula 12 in which a protective group II e.g. tert-butyl is introduced at a Y position, is reacted with the compound of Formula 7 to obtain the compound of Formula 15. In this process, for example, the compound of Formula 15 is prepared by reacting the compound of Formula 7 with the compound fo Formula 12 in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide or dimethylsulfoxide in the presence of 1- hydroxybenzotriazole (HOBt) , N-methylmorpholine (NMM) or l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) at ambient temperature to the boiling point of the organic solvent.

Then, the compound of Formula 15 is carbonylated to obtain the compound of Formula 16. In this process, for example, the quinolone derivative of Formula 16 is prepared by carbonylating the compound of Formula 15 via oxidation with a Dess Martin reagent in an organic solvent such as dichloromethane, chloroform, tetrahydrofuran, dioxane, anisole, acetonitrile, propionitrile, dimethylformamide, formamide or dimethylsulfoxide .

Then, a protective group II of the compound of Formula 16 is deprotected to obtain the quinoline derivative of Formula 1. In this process, the deprotection of the protective group II is carried out by reacting the compound of Formula 16 with acid e.g. trifluoroacetic acid. Such deprotection may be performed in an organic solvent e.g. dichloromethane. In this preparation method according to the aforementioned another aspect, there is prepared a quinoline derivative of Formula 1 wherein Y is hydrogen, or a pharmaceutically acceptable salt thereof. By introducing any substituent at a Y position of the quinoline derivative or a pharmaceutically acceptable salt thereof, it is possible to prepare other quinoline derivatives or pharmaceutically acceptable salts thereof that fall within the scope of the compound of Formula 1. In another aspect, the present invention is directed to a pharmaceutical composition containing a pharmaceutically effective amount of the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof, for use in treating a caspase-mediated disease based on inhibition of caspase-3 activity.

The pharmaceutical composition comprises the quinoline derivative of Formula 1 with potent inhibitory activity against caspase-3 or a pharmaceutically acceptable salt thereof as an active ingredient and is thus more suitable for use in treating caspase-mediated diseases e.g. Alzheimer's disease, Huntington's disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury, osteoarthritis and the like. In addition to the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. Examples of excipients that can be used for the pharmaceutical composition include, but are particularly not limited to: ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g. human serum albumin), buffer substances (e.g. phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, wool fat and the like.

In addition, the pharmaceutical composition may be administered to subjects such as mammals, e.g. humans by means of oral, parenteral, suction, topical, rectal, intranasal, buccal or intravaginal route. Alternatively, the the pharmaceutical composition may be contained in an implant depot. The parenteral administration may be carried out by subcutaneous, intradermal, intravenous, intramuscular, intraarterial, intrasynovial, intracisternal, intrathecal, intratumoral or intracranial injection, or suction. The pharmaceutical composition may be in the form of a sterile injectable preparation e.g. sterile injectable aqueous or oleaginous suspension. The suspension may be formulated using suitable dispersing agents, wetting agents (e.g. tween 80) or suspending agents in accordance with a method well-known in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. The sterile injectable preparation may comprise vehicles or solvents such as mannitol, water, Ringer's solution or aqueous isotonic salt solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any fixed oil product may be used, including synthetic mono- or diglycerides . In addition, fatty acids such as oleic acid or glyceride derivatives thereof may be also useful for injectable preparations, in addition to pharmaceutically acceptable natural oils (in particular, polyoxyethylated natural oils) such as olive oil or castor oil. These oil solutions or suspensions may contain a long chain-alcohol diluent or dispersant. The pharmaceutical composition may be orally administered in any orally acceptable dosage form including capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral administration, the commonly used excipient is lactose and corn starch. In addition, the tablets may contain lubricants e.g. magnesium stearate. In the case of capsules for oral administration, usable diluents are lactose and anhydrous corn starch. In the case of aqueous suspensions for oral administration, active ingredients may be mixed with emulsifying agent and suspending agents. In addition, if necessary, aqueous suspensions may contain specific sweetening, flavoring or coloring agents . The pharmaceutical composition may also be formulated in the form of suppositories for rectal administration. The pharmaceutical composition can be prepared by mixing the active ingredients with a suitable non-irritating excipient which is solid at ambient temperature but liquid at the rectal temperature. Examples of non-irritating excipients include cocoa butter, beeswax, polyethylene glycol and the like.

The topical administration of the pharmaceutical composition is particularly useful for the treatment of readily topically applicable sites or organs. For example, upon topical application to the skin, the pharmaceutical composition may be formulated in the form of suitable ointments containing the pharmaceutical composition suspended or dissolved in carriers, or be in the form of sprays. Examples of carriers for topical administration that can be used for the pharmaceutical composition include mineral oils, liquid vaseline, white vaseline, compounds such as propylene glycol, polyoxyethylene or polyoxypropylene, emulsion wax, water and the like.

The pharmaceutical composition may be formulated in the form of suitable lotions or creams containing the pharmaceutical composition suspended or dissolved in carriers. Suitable carriers include, but are not limited to mineral oils, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, water and the like.

The pharmaceutical composition may be also formulated in the form of rectal suppositories or suitable enemata clysters so that it can be topically administered through lower intestinal tracts. In addition, the pharmaceutical composition may be formulated as topically-applicable transdermal patches or ocular drops .

The pharmaceutical composition may be also administered by nasal aerosols or suction. The pharmaceutical composition is prepared in accordance with a technique well-known in pharmaceutics. For example, the pharmaceutical composition may be prepared as a solution in saline employing suitable preservatives such as benzyl alcohol, absorption promoters to improve bioavailability, fluorocarbon or other solubilizing agents or dispersing agents .

The amount of the active ingredient administered through the pharmaceutical composition will vary depending upon a varierty of factors such as the subject in need of, the disease severity, the particular mode of administration, sex and doctor's prescription. An effective amount of the active ingredient will be readily determined by those skilled in the art. The effective amount may be generally in the range of 0.001 to 100 mg/kg/day, preferably 0.001 to LO mg/kg/day.

In another aspect, the present invention is directed to the use of a quinoline derivative of the following Formula 1 or a pharmaceutically acceptable salt thereof, for preparing drugs for treating caspase- mediated diseases based on inhibition of caspase-3 activity.

wherein Xi, X₂, X₃ and X4 are each independently hydrogen, halogen, Ci-₆ alkyl, Ct-6 alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-e halogenoalkyl or 5- to 15-membered aryl;

R is in which R₂ is C or

N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, O or S; and R₅ is hydrogen, halogen, Ci_₆ alkyl, Ci-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃-S cycloalkyl, nitro, amino, Ci-6 halogenoalkyl or 5- to 15-membered aryl;

A is C, N, 0 or S; and

Y is hydrogen, halogen, Ci-₆ alkyl, Ci_₆ alkoxy, Ci_₆ alkoxyalkyl, C₃-₆ cycloalkyL, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl.

The caspase-mediated diseases include Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury and osteoarthritis.

[Mode for Invention] Example 1: Preparation of 5-fluoro-3- [ (7-nitro-4- piperazin-l-yl-quinoline-3-carbonyl) -amino] -4-oxo- pentanoic acid trifluoroacetic acid (8)

[Reaction Scheme 1]

ether

Preparation process 1: Preparation of 2- [ (3-nitro- phenylamino) -methylene] -malonic acid diethyl ester (1)

Diethylethoxymethylenemalonate (150 g, 694 mmol, 1.1 eq) was added to a solution of 3-nitroaniline (87 g, 631 mmol, 1 eq) in xylene (600 ml) and the mixture was stirred at 13O⁰C for 4 hours. The completion of the reaction was confirmed by thin-layer chromatography. The reaction product was cooled and filtered to obtain the target compound 1 (155 g, yield = 80 %) . 1H NMR (CDCl₃, 300 MHz) δ: 11.06 (d, J = 9.8 Hz,

IH), 8.49 (d, J = 13.2 Hz, IH), 8.00 - 7.96 (m, 2H), 7.56 - 7.51 (m, IH), 7.43 - 7.24 (m, IH), 4.35 - 4.24 (m, 4H), 1.41 - 1.28 (m, 6H) .

Preparation process 2: Preparation of 7-nitro~4- oxo-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester

(2)

The compound 1 (85 g, 280 mmol, 1 eq) was slowly added to refluxing diphenyl ether (250 ml) and the mixture was stirred for one hour. After cooling, the mixture was decanted and filtered to obtain the target compound 2 (6O g, yield = 82 %) . ¹H NMR (DMSO, 300 MHz) δ: 8.75 - 8.50 (m, IH) , 838 - 8.13 (m, IH) , 7.88 - 7.84 (m, 2H) , 7.66 - 7.62 (m, IH) , 4.25 - 4.19 (m, 2H), 1.38 - 1.18 (m, 3H) .

Preparation process 3: Preparation of 4-chloro-7- nitro-quinoline-3-carboxylic acid ethyl ester (3)

Phosphorus oxychloride (123 g, 800 mmol, 10 eq) was added to the compound 2 (20 g, 80 mmol, 1 eq) and the mixture was stirred under reflux for 12 hours . The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was cooled, neutralized with 20% NaOH and extracted with chloroform.

The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 3 (8.4 g, yield =

37 %) .

¹H NMR (CDCl₃, 400 MHz) δ: 9.30 (s, IH), 9.00 (s, IH), 8.59 - 8.56 (m, IH), 8.45 - 8.42 (m, IH), 4.53 (q, J = 7.2 Hz, 2H), 1.49 (t, J= 7.2 Hz, 3H). aaa

Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl ) ~7-nitro-quinoline-3- carboxylic acid ethyl ester (4) Tert-butoxy piperazine (5 g, 27.7 mmol, 1.1 eq) and trimethylamine (5.1 g, 50.4 mmol, 2 eq) were added to a solution of the compound 4 (7 g, 25.2 mmol, 1 eq) in dichloromethane (100 ml) and the mixture was stirred at room temperature for 12 hours. After the completion of the reaction was confirmed by thin-layer chromatography, the reaction mixture was extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate: hexane = 1 : 2) to obtain the target compound 4 (10.2 g, yield = 92 %) .

¹H NMR (CDCl₃, 300 MHz) δ : 9.01 (s, 0.5H), 8.95 (s, 0.5H), 8.31 (s, IH), 8.31 - 8.30 (m, IH), 7.33 - 7.31 (m, IH), 7.26 (s, IH), 4.49 (q, J = 7.1 Hz, 2H), 3.73 - 3.70 (m, 4H), 3.31 - 3.28 (m, 4H), 1.52 - 1.43 (m, 12H).

Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -7-nitro-quinoline-3- carboxylic acid (5) Lithium hydroxide (1.18 g, 27.9 mmol, 1.5 eq) was added to a solution of the compound 4 (8 g, 18.6 mmol, 1 eq) in ethanol (40 ml) and water (40 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 5 (6.8 g, yield = 90 %) .

¹H NMR (CD₃OD, 300 MHz) δ: 8.92 - 8.77 (m, 2H), 8.50 - 8.41 (m, IH), 8.33 - 8.29 (m, IH), 3.71 - 3.70 (m, 4H), 3.43 - 3.30 (m, 4H), 1.50 (s, 9H).

Preparation process 6: Preparation of 4- [3- (1-tert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propyl carbamoyl) -7-nitro-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (6) N-methylmorpholine (5.7 g, 56 mmol, 5 eq) , 1- hydroxybenzotriazole (2.3 g, 16.8 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.6 g, 13.4 mmol, 1.2 eq) were added to a solution of the compound 5 (4.5 g, 11.2 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was stirred for one hour. An amine reactant (2.3 g, 11.2 mmol, 1 eq) indicated in Reaction Scheme 1 above was added to the reaction mixture and the resulting mixture was stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 6 (3 g, yield = 46 %) .

¹H NMR (CDCl₃, 300 MHz) δ: 8.97 - 8.41 (m, 2H), 8.34 - 8.21 (m, 2H), 7.40 - 7.33 (m, IH), 4.67 - 4.50 (m, 3H), 4.51 - 4.48 (m, IH), 3.70 (brs, 4H), 3.33 - 3.29 (m, 4H), 2.82 - 2.73 (m, 2H), 1.50 - 1.45 (m, 18H).

Preparation process 7: Preparation of 4- [3- (1- tert- butoxycarbonylmethyl-3-fluoro-2-oxo-propylcarbamoyl) -7- nitro-quinolin-4-yl] -piperazine-1-carboxylic acid tert- butyl ester (7)

A Dess Martin reagent (2.6 g, 6.1 mmol, 1.2 eq) was added to a solution of the compound 6 (3 g, 5.1 mmol, 1 eq) in dichloromethane (30 ml) and the mixture was stirred for two hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 7 (1.8 g, yield = 60 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.93 (d, J = 1.9 Hz, IH),

8.91 (s, IH), 8.31 - 8.26 (m, 2H), 7.46 (d, J = 7.4 Hz, IH), 5.25 - 5.19 (m, 2H), 5.16 - 5.06 (m, IH), 3.71 (s,

4H), 3.30 (t, J = 4.9 Hz, IH), 3.15 - 3.07 (m, 2H), 1.51

(s, 9H) , 1.44 (s, 9H) .

Preparation process 8 : Preparation of 5-fluoro-3- [ (7-nitro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] - 4-oxo-pentanoic acid trifluoroacetic acid (8)

Trifluoroacetic acid (10 ml, 38 eq) was added to a solution of the compound 7 (1.8 g, 3.05 mmol, 1 eq) in dichloromethane (10 ml) and the mixture was stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 8 (1.3 g, yield = 77 %) . 1H NMR (CD₃OD, 500 MHz) δ: 8.89 - 8.83 (m, 2H), 8.45

- 8.40 (m, 2H), 5.20 - 5.05 (m, IH), 4.65 - 4.35 (m, 2H), 3.65 -3.49 (m, 8H), 3.00 - 2.55 (m, 2H). Example 2: Preparation of 5-fluoro-4-oxo-3- [ (4- piperazin-l-yl-7-trifluoromethyl-quinoline-3-carbonyl) - amino] -pentanoic acid trifluoroacetic acid (16)

[Reaction Scheme 2]

Preparation process 1: Preparation of 2-[(3- trifluoromethyl-phenylamino) -methylene] -malonic acid diethyl ester (9)

Diethylethoxymethylenemalonate (19.3 g, 89.4 mmol, 1.2 eq) was added to 3- (trifluoromethyl) aniline (10 g, 62.1 mmol, 1 eq) and the mixture was stirred at 130⁰C for 4 hours . The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was filtered to obtain the compound 9 (20 g, yield = 97 %) . ¹H NMR (CDCl₃, 500 MHz) δ: 11.1 (d, J = 13.3 Hz, IH), 8.51 (d, J = 13.4 Hz, IH), 7.60 - 7.49 (m, IH), 7.41 - 7.28 ( (m, 2H), 4.33 - 4.26 (m, 6H), 1.40 - 1.35 (m, 9H) .

Preparation process 2: Preparation of 4-oxo-7- trifluoromethyl-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester (10)

The compound 9 (10 g, 30.2 mmol, 1 eq) was slowly added to refluxing diphenyl ether (30 ml) and the mixture was stirred for one hour. After cooling, the reaction mixture was slowly decanted and filtered to obtain the compound 10 (6 g, yield = 70 %) .

¹H NMR (DMSO, 500 MHz) δ: 8.70 - 8.53 (m, IH), 8.34 (d, J = 8.6 Hz, 0.5H), 7.97 - 7.67 (m, 2H), 7.41 - 7.38 (m, IH), 7.15 - 7.00 (m, IH), 4.24 - 4.20 (m, 2H), 1.30 - 1.23 (m, 3H) .

Preparation process 3: Preparation of 4-chloro-7- trichloromethyl-quinoline-3-carboxylic acid ethyl ester (11)

Phosphorus oxychloride (26.8 g, 175 mmol, 10 eq) was added to the compound 10 (5 g, 17.5 mmol, 1 eq) and the mixture was stirred under reflux for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was cooled, neutralized with 20% NaOH and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 11 (3 g, yield = 57 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 1.30 - 1.23 (m, 3H), 8.57 - 8.54 (m, IH), 8.46 (s, IH), 7.77 (t, J = 7.9 Hz, IH), 4.55 - 4.50 (m, 2H), 1.50 - 1.46 (m, 3H).

Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl ) -7-trifluoromethyl- quinoline-3-carboxylic acid ethyl ester (12) Tert-butoxy piperazine (1.4 g, 7.3 mmol, 1.1 eq) and triethylamine (1.3 g, 13.2 mmol, 2 eq) were added to a solution of the compound 11 (2 g, 6.6 mmol, 1 eq) in dichloromethane (20 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography and the reaction mixture was then extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate: hexane = 1 : 2) to obtain the target compound 12 (2.2 g, yield = 73 %) .

¹H NMR (CDCl₃, 500 MHz) δ : 8.98 (s, IH), 8.38 (s, IH), 8.29 (d, J = 8.8 Hz, IH), 7.74 - 7.72 (m, IH), 4.48 (q, J = 7.1 Hz, 2H), 3.71 (s, 4H), 3.29 (s, 4H), 1.51 (s, 9H) , 1.45 (t, J = 7.1 Hz, 3H) .

Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -7-trifluoromethyl- quinoline-3-carboxylic acid (13)

Lithium hydroxide (0.56 g, 13.2 mmol, 3 eq) was added to a solution of the compound 12 (2 g, 4.4 mmol, 1 eq) in ethanol (40 ml) and water (40 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was then neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 13 (1.7 g, yield = 90 %) .

¹H NMR (CD₃OD, 500 MHz) δ: 8.84 - 8.74 (m, IH), 8.45 - 8.37 (m, IH), 8.18 (d, J = 3.6 Hz, IH), 7.76 - 7.72 (m, IH) , 3 . 67 - 3 . 56 (m, 4H ) , 3 . 38 - 3 . 12 (m, 4H ) , 1 . 48 - 1 . 43 (m, 9H) .

Preparation process 6: Preparation of 4- [3- ( 1- tert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propylcarbamoyl ) - 7-trifluoromethyl-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (14)

N-methylmorpholine (0.6 g, 5.9 iranol, 5 eq) , 1- hydroxybenzotriazole (0.24 g, 1.77 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride

(0.27 g, 1.41 mmol, 1.2 eq) were added to a solution of the compound 13 (0.5 g, 1.18 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was stirred for one hour. An amine reactant (0.25 g, 1.18 mmol, 1 eq) indicated in Reaction Scheme 2 above was added to the reaction mixture and the resulting mixture was stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 14 (0.4 g, yield = 55 %) . ¹H NMR (CDCl₃, 500 MHz) δ : 8.89 - 8.83 (m, IH) ,

8.36 - 8.01 (m, 2H) , 7.72 - 7.70 (m, IH) , 7.37 - 7.35 (m,

IH) , 4.62 - 4.50 (m, 3H) , 4.16 - 4.15 (m, IH) , 3.70 -

3.62 (m, 4H) , 3.31 (s, 4H) , 2.96 - 2.79 (m, 2H) , 1.51 - 1.42 (m, 18H) .

Preparation process 7: Preparation of 4- [3- (1-fcert- butoxycarbonylmethyl-3-fluoro-2-oxo-propylcarbamoyl ) -7- trifluoromethyl-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (15)

A Dess Martin reagent (0.3 g, 0.74 πunol, 1.5 eq) was added to a solution of the compound 14 (0.3 g, 0.49 itimol, 1 eq) in dichloromethane (30 ml) and the mixture was stirred for two hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 15 (0.2 g, yield = 67 %) .

¹H NMR (CDCl₃, 500 MHz) δ : 8.89 (s, IH), 8.38 (s, IH), 8.26 (d, J = 8.7 Hz, IH), 7.73 - 7.70 (m, IH), 7.50 (d, J = 7.4 Hz, IH), 5.25 - 5.06 (m, 3H), 3.70 (s, 4H), 3.30 (s, 4H), 3.15 - 3.04 (m, 2H), 1.51 - 1.26 (m, 18H) .

Preparation process 8: Preparation of 5-fluoro-4- oxo-3- [ (4-piperazin-l-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid (16)

Trifluoroacetic acid (0.73 ml, 38 eq) was added to a solution of the compound 15 (0.15 g, 0.25 mmol, 1 eq) in dichloromethane (0.73 ml) and the mixture was stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 16 (0.1 g, yield = 71 %) .

¹H NMR (CD₃OD, 500 MHz) δ: 8.86 - 8.80 (m, IH), 8.42 (t, J = 8.6 Hz, IH), 8.33 (d, J = 8.8 Hz, IH), 7.90 - 7.87 (m, 3H), 4.87 - 4.51 (m, 3H), 3.59 - 3.45 (m, 8H), 2.97 - 2.62 (m, 2H) .

Example 3: Preparation of 5-fluoro-4-oxo-3- [ (4- piperazin-l-yl-8-trifluoromethyl-quinoline-3-carbonyl) - amino] -pentanoic acid trifluoroacetic acid (24) [Reaction Scheme 3]

ether

Preparation process 1: Preparation of 2-[(2- trifluoromethyl-phenylamino) -methylene] -malonic acid diethyl ester (17)

Diethylethoxymethylenemalonate (19.3 g, 89.4 mmol, 1.2 eq) was added to 2- (trif Luoromethyl) aniline (10 g, 62.1 mmol, 1 eq) , the mixture was stirred at 130°C for 4 hours . The completion of the reaction was confirmed by thin-layer chromatography. The reaction product was cooled and filtered to obtain the target compound 17 (19 g, yield = 93 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 11.36 (d, J = 12.6 Hz, IH), 8.47 (d, J = 12.8 Hz, IH), 7.66 - 7.59 (m, 2H), 7.38 (d, J = 8.3 Hz, IH), 7.28 - 7.24 (m, IH), 4.37 - 4.19 (m, 4H) , 1.39 - 1.27 (m, 6H) .

Preparation process 2: Preparation of 4-oxo-8- trifluoromethyl-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester (18)

The compound 17 (10 g, 30.2 mmol, 1 eq) was slowly added to refluxing diphenyl ether (30 ml) and the mixture was stirred for one hour. After cooling, the mixture was slowly decanted and filtered to obtain the target compound 18 (7.5 g, yield = 87 %).

¹H NMR (DMSO, 500 MHz) δ: 11.67 (brs, IH), 8.49 - 8.47 (m, 2H), 8.21 (d, J = 7.4 Hz, IH), 7.58 (t, J = 7.8 Hz, IH), 4.24 (d, J = 7.1 Hz, 3H), 1.28 (t, J = 7.1 Hz, 3H) .

Preparation process 3: Preparation of 4-chloro-8- trichloroirtethyl-quinoline-3-carboxylic acid ethyl ester (19) Phosphorus oxychloride (26.8 g, 175 mmol, 10 eq) was added to the compound 18 (5 g, 17.5 mmol, 1 eq) and the mixture was stirred under reflux for 12 hours . The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was cooled, neutralized with 20% NaOH and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 19 (4 g, yield = 76 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 9.36 (s, IH), 8.65 (d, J = 8.5 Hz, IH), 8.28 (d, J = 7.3 Hz, IH), 7.77 (t, J = 7.9 Hz, IH), 4.54 (q, J = 7.1 Hz, IH), 1.47 (d, J = 7.1 Hz, 3H) .

Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -8-trifluoromethyl- quinoline-3-carboxylic acid ethyl ester (20)

Tert-butoxy piperazine (1.4 g, 7.3 mmol, 1.1 eq) and triethylamine (1.3 g, 13.2 mmol, 2 eq) were added to a solution of the compound 19 (2 g, 6.6 mmol, 1 eq) in dichloromethane (20 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography and the reaction mixture was extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 20 (2.2 g, yield = 73 %) .

¹H NMR (CDCl₃, 500 MHz) δ : 9.06 (s, IH), 8.40 (d, J = 8.5 Hz, IH), 8.10 (d, J = 7.1 Hz, IH), 7.61 (t, J = 7.9 Hz, IH), 4.47 (d, J = 7.1 Hz, 2H), 3.70 (s, 4H), 3.28 (s, 4H), 1.51 (s, 9H), 1.43 (t, J= 7.1 Hz, 3H).

Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-l-yl) -8-trifluoromethyl- quinoline-3-carboxylic acid (21)

Lithium hydroxide (0.56 g, 13.2 mmol, 3 eq) was added to a solution of the compound 20 (2 g, 4.4 mmol, 1 eq) in ethanol (40 ml) and water (40 ml), and the mixture was then stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 21 (1.7 g, yield = 90 %) . ¹H NMR (CD₃OD, 500 MHz) δ: 8.89 (s, IH), 8.77 (d, J = 1.8 Hz, IH), 8.48 (d, J = 4.2 Hz, IH), 8.04 (d, J = 3.6 Hz, IH), 7.65 - 7.61 (m, IH), 3.67 (s, 4H), 3.36 - 3.16 (m, 4H) , 1.48 - 1.26 (m, 9H) .

Preparation process 6: Preparation of 4- [3- (1- tert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propylcarbamoyl ) - 8-trifluoromethyl-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (22) N-methylmorpholine (0.6 g, 5.9 mmol, 5 eq) , 1- hydroxybenzotriazole (0.24 g, 1.77 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.27 g, 1.41 mmol, 1.2 eq) were added to a solution of the compound 21 (0.5 g, 1.18 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was stirred for one hour. An amine reactant (0.25 g, 1.18 mmol, 1 eq) indicated in Reaction Scheme 3 above was added to the reaction mixture and the resulting mixture was stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 22 (0.5 g, yield = 69 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.97 - 8.67 (m, IH), 8.38

- 8.36 (m, IH), 8.09 - 8.00 (m, IH), 7.61 - 7.58 (m, IH), 7.30 - 7.26 (m, IH), 4.54 - 4.49 (in, 3H), 4.15 (s, IH),

3.69 (s, 4H), 3.32 - 3.29 (m, 4H), 3.03 - 2.71 (m, 2H),

1.55 - 1.44 (m, 18H) .

Preparation process 7: Preparation of 4- [3- (1- tert- butoxycarbonylmethyl-3-fluoro-2-oxo-propylcarbamoyl) -8- trifluoromethyl-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (23)

A Dess Martin reagent (0.3 g, 0.74 mmol, 1.5 eq) was added to a solution of the compound 22 (0.3 g, 0.49 mmol, 1 eq) in dichloromethane (30 ml) and the mixture was then stirred for two hours. The completion of the reaction was confirmed by thin-layer chromatography.

After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound

23 (0.17 g, yield = 57 %) . ¹H NMR (CDCl₃, 500 MHz) δ: 8.97 (s, IH), 8.38 - 8.37 (m, IH), 8.11 - 8.04 (m, IH), 7.60 (t, J = 7.9 Hz, IH), 7.43 - 7.37 (m, IH), 5.21 - 5.06 (m, 3H), 3.70 (s, 4H), 3.29 (s, 4H), 3.15 - 3.01 (m, 2H), 1.52 - 1.41 (m, 18H).

Preparation process 8: Preparation of 5-fluoro-4- oxo-3- [ (4-piperazin-l-yl-8-trif Luoromethyl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid (24) Trifluoroacetic acid (0.73 ml, 38 eq) was added to a solution of the compound 23 (0.15 g, 0.25 mmol, 1 eq) in dichloromethane (0.73 ml) and the mixture was then stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 24 (0.1 g, yield = 71 %) .

¹H NMR (CD₃OD, 300 MHz) δ: 8.82 (d, J = 4.3 Hz, IH), 8.50 (t, J = 7.9 Hz, IH), 8.18 (t, J = 8.3 Hz, IH), 7.78 - 7.73 (m, IH), 4.87 - 4.58 (m, 3H), 3.56 - 3.46 (m, 8H), 2.93 - 2.67 (m, 2H) . Example 4: Preparation of 5-fluoro-4-oxo-3- [ (4- piperidin-l-yl-7-trifluoromethyl-quinoline-3-carbonyl) - amino] -pentanoic acid trifluoroacetic acid (29)

[Reaction Scheme 4]

Preparation process 1: Preparation of 4-piperidin- l-yl-7-trifluoromethyl-quinoline-S-carboxylic acid ethyl ester (25)

Piperidine (0.62 g, 7.3 mmol, 1.1 eq) and triethylamine (1.3 g, 13.2 mmol, 2 eq) were added to a solution of the compound 11 (prepared in Preparation process 3 of Example 2; 2 g, 6.6 mmol, 1 eq) in dichloromethane (20 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography and the reaction mixture was then extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 25 (2 g, yield = 87 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.91 (s, IH), 8.33 (s, IH), 8.27 (d, J = 8.8 Hz, IH), 7.70 - 7.68 (m, IH), 4.46 (q, J = 7.2 Hz, 2H), 3.31 - 3.29 (m, 2H), 1.86 - 1.73 (m, 6H) , 1.45 (t, J = 7.1 Hz, 3H) .

Preparation process 2: Preparation of 4-piperidin- l-yl-7-trifluoromethyl-quinoline-3-carboxylic acid (26)

Lithium hydroxide (0.68 g, 16.2 mmol, 3 eq) was added to a solution of the compound 25 (1.9 g, 5.4 mmol, 1 eq) in ethanol (20 ml) and water (10 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 26 (1.6 g, yield = 90 %) . ¹H NMR (CD₃OD, 500 MHz) δ: 8.66 (s, IH) , 8.31 (d, J = 8.8 Hz, IH) , 8.13 (s, IH) , 7.71 - 7.69 (m, IH) , 3.39 - 3.37 (m, 4H) , 1.81 - 1.70 (m, 6H) .

Preparation process 3: Preparation of 5-fluoro-4- hydroxy-3- [ (4-piperidin-l-yl-7-trifluoromethyl-quinoline- 3-carbonyl) -amino] -pentanoic acid tert-butyl ester (27)

N-methylmorpholine (0.78 g, 7.7 mmol, 5 eq) , 1- hydroxybenzotriazole (0.31 g, 2.31 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.44 g, 2.31 mmol, 1.5 eq) were added to a solution of the compound 26 (0.5 g, 1.54 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was then stirred for one hour. An amine reactant (0.32 g, 1.18 mmol, 1 eq) indicated in Reaction Scheme 4 above was added to the reaction mixture and the resulting mixture was stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 27 (0.38 g, yield = 48 %) . ¹H NMR (CDCl₃, 500 MHz) δ : 8.84 - 8.79 (m, IH),

8.31 (s, IH) , 8.21 (d, J = 8.8 Hz, IH) , 7.67 - 7.65 (m,

IH) , 7.43 (d, J = 8.1 Hz, IH) , 4.59 - 4.49 (m, 3H) , 4.18

- 4.15 (m, IH) , 2.87 - 2.81 (m, 2H) , 1.83 - 1.76 (m, 6H) , 1.46 (s, 9H) .

Preparation process 4 : Preparation of 5-fluoro-4- oxo-3- [ (4-piperidin-l-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -pentanoic acid tert-butyl ester (28) A Dess Martin reagent (0.35 g, 0.83 mmol, 1.5 eq) was added to a solution of the compound 27 (0.28 g, 0.55 mmol, 1 eq) in dichloromethane (10 ml) and the mixture was stirred for two hours . The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 28 (0.2 g, yield = 70 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.86 (s, IH), 8.35 (s, IH), 8.25 (d, J = 5 Hz, IH), 7.71 - 7.69 (m, IH), 7.44 - 7.43 (m, IH), 5.27 - 5.08 (m, 3H), 3.32 - 3.30 (m, 6H), 3.11 - 307 (m, 2H), 1.85 - 1.77 (m, 6H), 1.45 (s, 9H). Preparation process 5: Preparation of 5-fluoro-4- oxo-3- [ (4-piperidin-l-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -pentanoic acid trifluoroacetic acid (29) Trifluoroacetic acid (0.5 ml, 38 eq) was added to a solution of the compound 28 (0.09 g, 0.18 mitiol, 1 eq) in dichloromethane (0.5 ml) and the mixture was stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 29 (0.04 g, yield = 40 %) .

¹H NMR (CD₃OD, 500 MHz) δ : 8.59 (m, 0.5H), 8.55 - 8.43 (m, 0.5H), 8.21 (d, J = 5.0 Hz, IH), 5.20 - 5.00 (m, IH), 4.60 - 4.40 (m, 0.5H), 4.58 - 4.45 (m, 1.5H), 3.64 - 3.45 (m, 4H), 2.94 - 2.90 (m, 1.5H), 2.62 - 2.55 (m, 0.5H) , 1.90 - 1.82 (m. 6H) .

Example 5: Preparation of 5-fluoro-3- [ (4-morpholin- 4-yl-7-trifluoromethγl-quinoline-3-carbonyl) -amino] -4- oxo-pentanoic acid trifluoroacetic acid (34)

[Reaction Scheme 5]

Preparation process 1: Preparation of 4-morpholin- 4-yl-7-trifluoromethyl-quinoline-3-carboxylic acid ethyl ester (30)

Morpholine (0.64 g, 7.3 iranol, 1.1 eq) and triethylamine (1.3 g, 13.2 mmol, 2 eq) were added to a solution of the compound 11 (prepared in Preparation process 3 of Example 2; 2 g, 6.6 mmol, 1 eq) in dichloromethane (20 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography and the reaction mixture was then extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 4) to obtain the target compound 30 (2 g, yield = 86 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.97 (s, IH), 8.38 (s, IH), 8.30 (d, J = 5 Hz, IH), 7.74 - 7.72 (m, IH), 4.49 (q, J = 5 Hz, 2H), 3.98 - 3.97 (m, 4H), 3.37 - 3.35 (m, 4H) , 1.47 (t, J = 5 Hz, 3H) .

Preparation process 2: Preparation of 4-morpholin- 4-yl-7-trifluoromethyl-quinoline-3-carboxylic acid (31) Lithium hydroxide (0.68 g, 16.2 mmol, 3 eq) was added to a solution of the compound 30 (1.9 g, 5.4 mmol, 1 eq) in ethanol (20 ml) and water (10 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 31 (1.6 g, yield = 90 %) .

¹H NMR (CD₃OD, 500 MHz) δ: 8.66 (s, IH), 8.31 (d, J = 8.8 Hz, IH), 8.13 (s, IH), 7.71 - 7.69 (m, IH), 3.39 - 3.37 (m, 4H), 1.81 - 1.70 (m, 6H). Preparation process 3: Preparation of 5-fluoro-4- hydroxy-3- [ (4-morpholin-4-yl-7-trifluoromethyl-quinoline- 3-carbonyl) -amino] -pentanoic acid tert-butyl ester (32)

N-methylmorpholine (0.78 g, 7.7 mmol, 5 eq) , 1- hydroxybenzotriazole (0.31 g, 2.30 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl ) carbodiimide hydrochloride (0.44 g, 2.3 mmol, 1.5 eq) were added to a solution of the compound 31 (0.5 g, 1.53 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was stirred for one hour. An amine reactant (0.32 g, 1.53 mmol, 1 eq) indicated in Reaction Scheme 5 above was added to the reaction mixture and the resulting mixture was stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 32 (0.5 g, yield = 69 %) . 1H NMR (CDCl₃, 500 MHz) δ: 8.92 - 8.82 (m, IH), 8.36

- 8.22 (m, 2H), 8.00 - 7.80 (m, IH), 7.72 - 7.68 (m, IH), 7.60 - 7.40 (m, 0.5H), 4.64 - 4.53 (s, 4H), 3.96 (s, 4H), 3.39 (s, 4H), 2.88 - 2.57 (m, 3H), 1.46 - 1.37 (m, 9H). Preparation process 4 : Preparation of 5-fluoro-3- [ (4-morpholin-4-yl-7-trifluoromethyl-quinoline-3- carbonyl ) -amino] -4-oxo-pentanoic acid tert-butyl ester (33) A Dess Martin reagent (0.51 g, 1.2 mmol, 1.5 eq) was added to a solution of the compound 32 (0.42 g, 0.81 mmol, 1 eq) in dichloromethane (10 ml) and the mixture was stirred for two hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 33 (0.29g, yield = 70 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.91 (s, IH), 8.39 (s, IH), 8.28 (d, J = 10 Hz, IH), 7.74 - 7.72 (m, IH), 7.54 (d, J = 5 Hz, IH), 5.25 - 5.07 (m, 3H), 3.99 - 3.97 (m, 2H), 3.38 - 3.36 (m, 2H), 3.16 - 3.09 (m, 2H), 1.46 (s, 9H) .

Preparation process 5: Preparation of 5-fluoro-3- [ (4-morpholin-4-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid (34)

Trifluoroacetic acid (0.85 ml, 38 eq) was added to a solution of the compound 33 (0.15 g, 0.29 mmol, 1 eq) in dichloromethane (0.85 ml) and the mixture was stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 34 (0.1 g, yield = 59 %) .

¹H NMR (CD₃OD, 500 MHz) δ: 8.80 - 8.60 (m, IH), 8.48 (d, J = 8.9 Hz, IH), 8.26 (d, J = 7.1 Hz, 3H), 7.94 - 7.92 (m, IH), 5.30 - 5.10 (m, IH), 4.79 - 4.39 (m, 2H), 4.03 - 3.93 (m, 4H), 3.79 - 3.65 (m, 3H), 3.20 - 2.70 (m, 1.5H) , 2.63 - 2.60 (m, 0.5H) .

Example 6: Preparation of 5-fluoro-4-oxo-3- [ (4- thiomorpholin-4-yl-7-trifluoromethyl-quinoline-3- carbonyl) -amino] -pentanoic acid trifluoroacetic acid (39)

[Reaction Scheme 6]

Preparation process 1: Preparation of 4- thiomorpholin-4-yl-7-trifluoromethyl-quinoline-3- carboxylic acid ethyl ester (35)

Thiomorpholine (0.75 g, 7.3 mmol, 1.1 eq) and triethylamine (1.3 g, 13.2 mmol, 2 eq) were added to a solution of the compound 11 (prepared in Preparation process 3 of Example 2; 2 g, 6.6 mmol, 1 eq) in dichloromethane (20 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography and the reaction mixture was then extracted with chloroform.

The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 2) to obtain the target compound 35 (2.3 g, yield = 96 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 8.99 (s, IH), 8.29 (d, J = 8.8 Hz, IH), 7.76 - 7.73 (m, IH), 4.50 (q, J = 7.13 Hz, 2H), 3.58 - 3.56 (m, 4H), 2.91 (s, 4H), 1.48 (t, J = 7.2 Hz, 3H) .

Preparation process 2: Preparation of 4- thiomorpholin-4-yl-7-trifluoromethyl-quinoline-3- carboxylic acid (36)

Lithium hydroxide (0.68 g, 16.2 mmol, 3 eq) was added to a solution of the compound 35 (2 g, 5.4 mmol, 1 eq) in ethanol (40 ml) and water (40 ml) and the mixture was stirred at room temperature for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The solvent was evaporated under reduced pressure and the residue was neutralized with IN HCl. The resulting solution was extracted with chloroform, evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 36 (1.7 g, yield = 90 %) .

¹H NMR (CD₃OD₃, 300 MHz) δ: 8.73 (s, IH), 8.35 (d, J = 8.8 Hz, IH), 8.17 (s, IH), 7.77 - 7.75 (m, IH), 3.65 - 3.63 (m, 4H) , 2.86 (s, 4H) . Preparation process 3: Preparation of 5~fluoro-4- hydroxy-3- [ (4-thiomorpholin-4-yl~7-trifluoromethyl- quinoline-3-carbonyl) -amino] -pentanoic acid tert-butyl ester (32)

N-methylmorpholine (0.74 g, 7.3 mmol, 5 eq) , 1- hydroxybenzotriazole (0.30 g, 2.19 mmol, 1.5 eq) and 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.42 g, 2.19 mmol, 1.5 eq) were added to a solution of the compound 36 (0.5 g, 1.46 mmol, 1 eq) in dimethylformamide (40 ml) and the mixture was stirred for one hour. An amine reactant (0.3 g, 1.46 mmol, 1 eq) indicated in Reaction Scheme 6 was added to the reaction mixture and the resulting mixture was stirred for 1 hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with chloroform. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 37 (0.25 g, yield = 32 %).

¹H NMR (CDCl₃, 500 MHz) δ : 8.94 - 8.86 (m, IH), 8.37 (s, IH), 8.37 - 8.26 (m, IH), 8.26 - 8.25 (m, IH), 7.74 - 7.72 (m, IH), 7.50 - 7.39 (m, IH), 4.63 - 4.50 (m, 3H) , 4.19 - 4.18 (m, IH) , 3.60 - 3.58 (m, 4H) , 2.91 - 2.84 (m, 4H) , 2.76 - 2.73 (m, 2H) , 1.44 (s, 9H) .

Preparation process 4 : Preparation of 5-fluoro-4- oxo-3- [ (4-thiomorpholin-4-yl-7-trifluoromethyl-quinoline- 3-carbonyl) -amino] -pentanoic acid tert-butyl ester (38)

A Dess Martin reagent (0.28 g, 0.65 mmol, 1.5 eq) was added to a solution of the compound 37 (0.23 g, 0.43 mmol, 1 eq) in dichloromethane (30 ml) and the mixture was stirred for two hours. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate, the resulting mixture was stirred and extracted with dichloromethane. The extract was evaporated under reduced pressure and purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 38 (O.lδg, yield = 75 %) .

Preparation process 5: Preparation of 5-fluoro-4- oxo-3- [ (4-thiomorpholin--4-yl-7-trifluoromethyl-quinoline- 3-carbonyl ) -amino] -pentanoic acid trifluoroacetic acid (39)

Trifluoroacetic acid (0.12 ml, 38 eq) was added to a solution of the compound 38 (0.02 g, 0.04 mmol, 1 eq) in dichloromethane (0.12 ml) and the mixture was stirred for one hour. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was evaporated under reduced pressure and purified by column chromatography (methanol : methylene chloride = 1 : 4) to obtain the target compound 39 (0.1 g, yield = 71 %) .

¹H NMR (CD₃OD, 500 MHz) δ: 8.52 - 8.48 (m, IH), 8.37 - 8.31 (m, IH), 7.97 (s, IH), 7.88 - 7.86 (m, IH), 4.55 - 4.24 (m, 4H), 3.51 - 3.26 (m, 4H), 3.00 - 2.57 (m, 6H),

Example 7: Preparation of 5-fluoro-3- [ (8-nitro-4- piperazin-l-yl-quinoline-3-carbonyl) -amino] -4-oxo- pentanoic acid trifluoroacetic acid (54) [Reaction Scheme 7]

Preparation process 1: Preparation of 2- [ (2-nitro- phenylamino) -methylene] -malonic acid diethyl ester (47)

Diethylethoxymethylenemalonate (34 g, 159.3 mmol) was added to a solution of 2-nitroaniline (20.0 g, 144.8 mmol) in ethanol (100 ml) and the mixture was stirred at 120⁰C for 6 hours. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was cooled, filtered and washed three times with hexane (100 ml) to obtain the target compound 47 (31 g, yield = 69 %) .

¹H NMR (CDCl₃, 300 MHz) δ: 12.6-12.53 (m, IH), 8.53 (d, J = 12.0 Hz, IH), 8.13 (d, J = 4.0 Hz, IH), 7.73-7.66 (m, 1 H), 7.52-7.48 (m, IH), 7.27-7.17 (m, IH), 4.48-4.20 (m, 4H), 1.45-1.26 (m, 6H)

Preparation process 2: Preparation of 8-nitro-4- oxo-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester (48)

The compound 47 (140.0 g, 450 mmol) was slowly added to refluxing phenyl ether (400 ml) at 260⁰C and the mixture was stirred for 3 hours. After completion of the reaction, the reaction mixture was allowed to cool to room temperature, and crystallized while stirring in petroleum ether (1.4 L) . After the resulting compound was filtered and washed with petroLeum ether, the solvent was removed under reduced pressure to obtain the target compound 48 (104.4 g, yield = 88 %). 1H NMR (CDCl₃, 300 MHz) δ: 9.01 (s, IH), 8.37 (d, J = 8.7 Hz, 1 H), 8.01 (d, J = 7.5 Hz, 1 H), 7.61 (dd, J = 8.4, 7.5 Hz, 1 H), 4.47 (q, J = 7.2 Hz, 2 H), 3.72 - 3.69 (m, 4 H), 3.31 - 3.28 (m, 4 H), 1.51 (s, 9 H), 1.43 (t, J = 7.2 Hz, 3 H) . Preparation process 3: Preparation of 4-chloro-8- nitro-quinoline-3-carboxylic acid ethyl ester (49)

Phosphorus oxychloride (175 ml, 1.91 iriol) was added to the compound 48 (50 g, 0.19 mol) and the mixture was stirred under reflux for 5 hours . The completion of the reaction was confirmed by thin-layer chromatography. After the phosphorus oxychloride was removed under reduced pressure, the concentrated compound was stirred in cooling water (0⁰C). The resulting mixture was dissolved in dichloromethane (250 ml) and neutralized with ammonia water. The organic layer was extracted and washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The resulting compound was dried over anhydrous magnesium sulfate and stirred in the presence of activated carbon for 30 minutes. After filtering and vacuum concentration, the resulting mixture was crystallized from methanol (500 mL) . The solid compound thus obtained was filtered to obtain the target compound 49 (38 g, yield = 72 %) .

¹H NMR (CDCl₃, 500 MHz) δ: 9.33 (s, 1 H), 8.66 (d, J = 8.4 Hz, 1 H), 8.65 (d, J = 7.5 Hz, 1 H), 7.80 (t, J = 8.0 Hz, 1 H), 4.53 (q, J = 7.1 Hz, 2 H), 1.47 (t, J = 7.2 Hz, 3 H) . Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -8-nitro-quinoline~3- carboxylic acid ethyl ester (50) Tert-butoxy piperazine (1.3 g, 6.77 mmol) and triethylamine (1.1 g, 11.2 mmol) were added to a solution of the compound 49 (1.6 g, 5.64 mmol) in dichloromethane (15 mL) and the mixture was stirred at room temperature for 10 hours. The completion of the reaction was confirmed by thin-layer chromatography. Water (15 ml) was added to the reaction mixture with stirring. The organic layer was extracted with chloroform and washed with a saturated aqueous sodium bicarbonate solution and saturated brine. The resulting mixture was dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum. The obtained concentrate was purified by column chromatography (ethyl acetate : hexane = 1 : 2) to obtain the target compound 50 as a yellow solid (2.13 g, yield = 88 %) . 1H NMR (CDCl₃, 300 MHz) δ : 9.01 (s, 1 H), 8.37 (d,

J = 8.7 Hz, 1 H), 8.01 (d, J = 7.5 Hz, 1 H), 7.61 (dd, J = 8.4, 7.5 Hz, 1 H), 4.47 (q, J = 7.2 Hz, 2 H), 3.72 - 3.69 (m, 4 H), 3.31 - 3.28 (m, 4 H), 1.51 (s, 9 H), 1.43 (t, J = 7.2 Hz, 3 H) . Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -8-nitro-quinoline-3- carboxylic acid (51) Lithium hydroxide (0.44 g, 10.46 mmol) was added to a solution of the compound 50 (1.5 g, 3.49 mmol) in a mixed solvent of ethanol and water (4 : 1, 20 ml) and the mixture was stirred at room temperature for 48 hours. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was neutralized with IN HCl and evaporated under reduced pressure to remove the solvent. The obtained concentrate was purified by column chromatography (methanol : ethyl acetate = 1 : 10) to obtain the target compound 51 as a solid (1.01 g, yield = 72 %) .

¹H NMR (CD₃OD, 300 MHz) δ: 8.73 (s, 1 H), 8.45 (d, J = 8.6 Hz, 1 H), 8.00 (d, J = 7.4 Hz, 1 H), 7.65 (dd, J = 8.4, 7.6 Hz, 1 H), 3.69 (brs, 4 H), 3.42-3.89 (m, 4 H), 1.49 (s, 9H).

Preparation process 6: Preparation of 4- [3- ( I- tert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propylcarbamoyl ) - 8-nitro-quinolin-4-yl] -piperazine-1-carboxylic acid tert- butyl ester (52) Methylmorpholine (0.38 g, 1.49 mmol), 1- hydroxybenzotriazole (0.2 g, 1.49 mmol) and l-ethyl-3- (3- dimethylaminopropyl) carbodiimide hydrochloride (0.29 g, 1.49 mmol) were added to a solution of the compound 51 (0.5 g, 1.24 mmol) in dimethylformamide (5 ml) and the mixture was stirred for 30 minutes. An amine reactant 51- 1 indicated in Reaction Scheme 7 above was added and the resulting mixture was then stirred for 12 hours. The completion of the reaction was confirmed by thin-layer chromatography. The reaction mixture was extracted with dichloromethane (10 ml), washed twice with water (10 ml) and once with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The obtained concentrate was purified by column chromatography (ethyl acetate : hexane = 1 : 2) to obtain the target compound 52 as a light yellow solid (0.42 g, yield = 57 %) .

¹H NMR (CD₃Cl₃, 300 MHz) δ : 8.84 (s, 0.5 H), 8.81 (s, 0.5 H), 8.31 (d, J = 8.7 Hz, 1 H), 7.94 (d, J = 6.9 Hz, 1 H), 7.56 (t, J = 8.4 Hz, 1 H), 7.45 (d, J = 8.4 Hz, 0.5 H), 7.33 (d, J = 8.7 Hz, 0.5 H), 4.64-4.53 (m, 3 H), 4.17-4.12 (m, 1 H), 3.71-3.67 (m, 4 H), 3.41-3.24 (m, 4 H), 2.79-2.71 (m, 2 H), 1.50 (s, 9H), 1.46 (s, 9 H). Preparation process 7: Preparation of 4- [3- (1- tert- butoxycarbonylmethyl-3-fluoro-2--oxo-propylcarbamoyl ) -8- nitro-quinolin-4-yl] -piperazine-1-carboxylic acid tert- butyl ester (53) A Dess Martin reagent (0.29 g, 0.68 mmol) was added to a solution of the compound 52 (0.34 g, 0.57 mmol) in dichloromethane (3.5 ml) and the mixture was stirred for 10 minutes. The completion of the reaction was confirmed by thin-layer chromatography. After addition of saturated sodium bicarbonate (3 ml), the resulting mixture was stirred for 30 minutes and extracted with dichloromethane. The extract was washed once with saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated. The concentrate thus obtained was purified by column chromatography (ethyl acetate : hexane = 1 : 1) to obtain the target compound 53 as a light yellow solid (0.29 g, yieLd = 85 %).

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.89 (s, 1 H), 8.33 (dd, J = 10, 1.2 Hz, 1 H), 8.31 (dd, J = 25, 1.2 Hz, 1 H), 7.60 (dd, J = 10, 7.6 Hz, 1 H), 7.35 (d, J = 25 Hz, 1 H), 5.19-5.18 (m, 2 H), 5.09 (q, 1 H), 3.71-3.67 (m, 4 H), 3.30-3.29 (m, 4 H), 3.15-2.84 (m, 2 H), 1.50 (s, 9H), 1.43 (s, 9 H) . Preparation process 8: Preparation of 5-fluoro-3- [ (8-nitro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] - 4-oxo-pentanoic acid trifluoroacetic acid (54)

Trifluoroacetic acid (1 ml) was added to a solution of the compound 53 (0.2 g, 0.34 mmol) in dichloromethane

(1 ml) and the mixture was stirred at 0°C for 10 minutes and at room temperature for two hours . The solvent and trifluoroacetic acid were removed by evaporation under reduced pressure. The residue was purified by column chromatography (methanol : dichloromethane = 1 : 10) to obtain the target compound 54 (0.15 g, yield = 79 %) .

¹H NMR (CD₃OD, 500 MHz) δ : 8.78 (s, 0.5 H), 8.74

(s, 0.5 H), 8.45 (d, J = 7.4 Hz, 1 H), 8.12 (t, J = 7.4

Hz, 1 H), 7.77-7.73 (m, 1 H), 5.14-5.11 (m, 1 H), 4.83- 4.81 (m, 1 H), 4.60-4.58 (m, 0.5 H), 4.51-4.47 (m, 1 H),

3.62-3.48 (m, 8 H), 2.95-2.55 (m, 2H).

Example 8: Preparation of 5-fluoro-3- [ (8-fluoro-4- piperazin-l-yl-quinoline-3-carbonyl) -amino] -4-oxo- pentanoic acid trifluoroacetic acid (63)

[Reaction Scheme 8] P henyl ether

SS S6 Sϊ

POCfj

60 £1

D ess-martin TFA penodinane

CH₂CI₂ CHjCl2

Preparation process 1: Preparation of 2- [ (2-fluoro- phenylamino) -methylene] -malonic acid diethyl ester (56)

The target compound 56 was obtained from 2- fluoroaniline 55 in the similar manner as in Preparation process 1 of Example 7.

¹H NMR (CDCl₃, 500 MHz) δ : 11.07 (d, J = 12.9 Hz, IH), 8.51 (d, J = 13.6 Hz, IH), 7.29-7.28 (m, IH), 7.17- 7.28 (m, 2 H), 7.10-7.08 (m, IH), 4.33 (q, J = 7.1 Hz, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.34 (t, J = 7.1 Hz, 3 H) .

Preparation process 2: Preparation of 8-fluoro-4- oxo-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester (57)

The target compound 57 was obtained from the compound 56 in the similar manner as in Preparation process 2 of Example 7. 1H NMR (CD₃OD, 500 MHz) δ: 12.47 (s, IH), 8.63 (s,

IH), 8.12 (d, J = 8.2 Hz, IH), 7.58-7.54 (m, IH), 7.46- 7.42 (m, 1 H), 4.35 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H) .

Preparation process 3: Preparation of 4-chloro-8- fluoro-quinoline-3-carboxylic acid ethyl ester (58)

The target compound 58 was obtained from the compound 57 in the similar manner as in Preparation process 3 of Example 7. 1H NMR (CDCl₃, 500 MHz) δ: 9.23 (s, IH), 8.21 (d, J = 8.1 Hz, IH), 7.68-7.64 (m, IH), 7.58-7.54 (m, IH), 4.52 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz, 3H). Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -8-fluoro-quinoline-3- carboxylic acid ethyl ester (59)

The target compound 59 was obtained from the compound 58 in the similar manner as in Preparation process 4 of Example 7.

¹H NMR (CDCl₃, 500 MHz) δ: 8.93 (s, IH), 7.94 (d, J = 8.3 Hz, IH), 7.51-7.42 (m, 2H), 4.47 (q, J = 7.2 Hz, 2H), 3.69 (brs, 4H), 3.29 (brs, 4H), 1.51 (s, 9H), 1.44 (t, J = 7.2 Hz, 3H) .

Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -8-fluoro-quinoline-3- carboxylic acid (60) The target compound 60 was obtained from the compound 59 in the similar manner as in Preparation process 5 of Example 7.

¹H NMR (CD₃OD, 500 MHz) δ: 8.65 (s, IH), 8.27-8.24 (m, 1 H), 7.53-7.51 (m, IH), 7.39-7.35 (m, IH), 3.65 (brs, 4H), 3.37 (brs, 4 H), 1.46 (s, 9H).

Preparation process 6: Preparation of 4- [3- (1- tert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propylcarbamoyl) - 8-fluoro-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (61)

The target compound 61 was obtained from the compound 60 in the similar manner as in preparation process 6 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.86 (s, 0.5H), 8.79 (s, 0.5 H), 7.56 (t, J = 7.4 Hz, IH), 7.54-7.37 (m, 3H), 4.62-4.49 (m, IH), 3.68 (brs, 4H), 3.31-3.29 (m, 4H), 2.85-2.70 (m, 2H), 1.50 (s, 9H), 1.46 (s, 9H).

Preparation process 7: Preparation of 4- [3- ( 1- tert- butoxycarbonylmethyl-3-fluoro-2-oxo-propylcarbamoyl ) -8- fluoro-quinolin-4-yl] -piperazine-1-carboxylic acid tert- butyl ester (62) The target compound 62 was obtained from the compound 61 in the similar manner as in Preparation process 7 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.86 (s, 1 H) , 7.90 (d,

J = 8.4 Hz, 1 H), 7.56 (d, J = 7.4 Hz, 1 H), 7.51-7.46 (m, 1 H), 7.44-7.40 (m, 1 H), 5.25-5.06 (m, 3 H), 3.69

(brs, 4 H), 3.32-3.27 (m, 4 H), 3.09 (dq, J = 17.9, 4.7

Hz, 2 H), 1.51 (s, 9H), 1.44 (s, 9 H). Preparation process 8: Preparation of 5-fluoro-3- [ (8-fluoro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] - 4-oxo-pentanoic acid trifluoroacetic acid (63)

The target compound 63 was obtained from the compound 62 in the similar manner as in Preparation process 8 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.75 (s, 0.5 H), 8.70 (d, J = 4.0 Hz, 0.5 H), 8.03-8.00 (m, 1 H), 7.68-7.62 (m, 1 H), 7.59-7.54 (m, 1 H), 4.59-4.45 (m, 1 H), 3.69-3.65 (m, 1 H), 3.59-3.43 (m, 8 H), 2.96-2.93 (m, 2 H).

Example 9: Preparation of 5-fluoro-3- [ (7-fluoro-4- piperazin-l-yl-quinoline-3-carbonyl) -amino] -4-oxo- pentanoic acid trifluoroacetic acid (72) [Reaction Scheme 9]

DfS-rtβrtin psflαeinww

CHjCla

« 1!

Preparation process 1: Preparation of 2- [ (3-fluoro- phenylamino) -methylene] -malonic acid diethyl ester (65)

The target compound 65 was obtained from 3- fluoroaniline 64 in the similar manner as in Preparation process 1 of Example 7.

¹H NMR (CDCl₃, 500 MHz) δ : 11.06 (d, J = 13.2 Hz , 1 H), 8.46 (d, J = 13.5 Hz, 1 H), 7.35-7.32 (m, 1 H), 6.91 (d, J = 8.1 Hz, 1 H), 6.87-6.84 (m, 2 H), 4.31 (q, J = 7.1 Hz, 2 H), 4.26 (q, J = 7.1 Hz, 2 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.34 (t, J= 7.1 Hz, 3 H). Preparation process 2: Preparation of 7-fluoro-4- oxo-1, 4-dihydro-quinoline-3-carboxylic acid ethyl ester (66)

The target compound 66 was obtained from the compound 65 in the similar manner as in Preparation process 2 of Example 7.

¹H NMR (DMSO-d₆, 500 MHz) δ: 12.30 (s, 1 H), 8.58 (s, 1 H), 8.12 (dd, J = 8.9, 6.3 Hz, 1 H), 7.40-7.37 (m, 1 H), 7.29-7.25 (m, 1 H), 4.21 (q, J = 7.0 Hz, 2 H), 1.28 (t, J = 7.0 Hz, 3 H) .

Preparation process 3: Preparation of 4-chloro-7- fluoro-quinoline-3-carboxylic acid ethyl ester (67)

The target compound 67 was obtained from the compound 66 in the similar manner as in Preparation process 3 of Example 7.

¹H NMR (CDCl₃, 500 MHz) δ: 9.22 (s, 1 H), 8.45 (dd, J = 9.4, 5.9 Hz, 1 H), 7.78 (dd, J = 9.5 2.5 Hz, 1 H), 7.51-7.47 (m, 1 H), 4.51 (q, J = 7.2 Hz, 2 H), 1.47 (t, J = 7.2 Hz, 3 H) .

Preparation process 4: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -7-fluoro-quinoline-3- carboxylic acid ethyl ester (68) The target compound 68 was obtained from the compound 67 in the similar manner as in Preparation process 4 of Example 7.

¹H NMR (CDCl₃, 500 MHz) δ : 8.91 (s, 1 H) , 8.19 (dd, J = 9.2, 6.0 Hz, 1 H), 7.69 (dd, J = 9.7, 2.5 Hz, 1 H),

7.35-7.31 (m, 1 H), 4.46 (q, J = 7.2 Hz, 2 H), 3.69 (brs,

4 H), 3.28 (brs, 4 H), 1.51 (s, 9 H), 1.44 (t, J = 7.2

Hz, 3 H) .

Preparation process 5: Preparation of 4-(4-tert- butoxycarbonyl-piperazin-1-yl) -7-fluoro-quinoline-3- carboxylic acid (69)

The target compound 69 was obtained from the compound 68 in the similar manner as in Preparation process 5 of Example 7.

¹H NMR (CD₃OD, 500 MHz) δ: 8.67 (s, 1 H) , 8.01 (d, J = 8.6 Hz, 1 H), 7.54-7.49 (m, 1 H), 7.42-7.39 (m, 1 H), 3.67 (brs, 4 H), 3.40-3.38 (m, 4 H), 1.48 (s, 9 H).

Preparation process 6: Preparation of 4- [3- (1-fcert- butoxycarbonylmethyl-3-fluoro-2-hydroxy-propylcarbamoyl ) -

7-fluoro-quinolin-4-yl] -piperazine-1-carboxylic acid tert-butyl ester (70) The target compound 70 was obtained from the compound 69 in the similar manner as in Preparation process 6 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.86 (s, 0.5 H), 8.78 (s, 0.5 H), 8.16-8.12 (m, 1 H), 7.70-7.67 (m, 1 H), 7.47- 7.45 (m, 0.5 H), 7.41-7.39 (m, 0.5 H), 7.34-7.30 (m, 1 H), 4.62-4.48 (m, 3 H), 4.13-4.11 (m, 1 H), 3.68 (brs, 4 H), 3.30-3.29 (m, 4 H), 2.88-2.73 (m, 2 H), 1.51 (s, 9H), 1.45 (s, 9 H) .

Preparation process 7: Preparation of 4- [3- (1-tert- butoxycarbonylmethyl-3-fluoro-2-oxo-propylcarbamoyl ) -7- fluoro-quinolin-4-yl] -piperazine-1-carboxylic acid tert- butyl ester (71) The target compound 71 was obtained from the compound 70 in the similar manner as in Preparation process 7 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.83 (s, 1 H), 8.93

(dd, J = 9.3, 5.9 Hz, 1 H), 7.70 (dd, J = 9.6, 2.6 Hz, 1 H), 7.61 (d, J = 7.4 Hz, 1 H), 7.35-7.31 (m 1 H), 5.26-

5.06 (m, 3 H), 3.69 (brs, 4 H), 3.29-3.28 (m, 4 H), 3.08

(dq, J= 17.0, 4.7 Hz, 2 H), 1.51 (s, 9H), 1.44 (s, 9 H). Preparation process 8: Preparation of 5-fluoro-3- [ (7-fluoro-4-piperazin-l-yl-quinoline-3-carbonyl) -amino] - 4-oxQ-pentanoic acid trifluoroacetic acid (72)

The target compound 72 was obtained from the compound 71 in the similar manner as in Preparation process 8 of Example 7.

¹H NMR (CD₃Cl₃, 500 MHz) δ : 8.74 (s, 0.5 H), 8.69 (d, J = 2.4 Hz, 0.5 H), 8.32-8.28 (m, 1 H), 7.68-7.63 (m, 1 H), 7.53-7.47 (m, 1 H), 5.12-5.95 (in, 1 H), 4.59-4.47 (m, 1 H), 3.59-3.46 (m, 8 H), 2.96-2.93 (m, 2 H).

Example 10: Preparation of 5-fluoro-3-{ [4- (4- methyl-piperazin-1-yl) -8-nitro-quinoline-3-carbonyl] - amino} -4-oxo-pentanoic acid trifluoroacetic acid (74) [Reaction Scheme 10]

The target compound 74 was obtained from the compound 73 in the similar manner as in Preparation process 8 of Example 7. 1H NMR (CD₃OD, 500 MHz) δ: 8.73-8.71 (m, 0.5 H),

8.68-8.67 (m, 0.5 H), 8.46-8.44 (m, 1 H), 8.11-8.09 (m, 1 H) , 7.76-7.72 (m, 1 H) , 5.22-5.13 (m, 2 H) , 3.62-3.30 (m, 8 H) , 2.90-2.87 (m, 5 H) .

Example 11: Preparation of 3-{ [4- (4-ethyl- piperazin-1-yl) -8-nitro-quinoline-3~carbonyl] -amino} -5- fluoro-4-oxo-pentanoic acid tπfluoroacetic acid (76)

[Reaction Scheme 11]

The target compound 76 was obtained from the compound 75 in the similar manner as in Preparation process 8 of Example 7.

¹H NMR (CD₃OD, 500 MHz) δ: 8.79-8.75 (m, 1 H), 8.45- 8.43 (m, 1 H), 8.13-8.11 (m, 1 H), 7.76-7.74 (m, 1 H), 5.11-5.08 (m, 1 H), 3.70-3.57 (m, 10 H), 3.34-3.31 (m, 3 H), 2.95-2.91 (m, 1 H), 1.43-1.39 (m, 3 H).

Example 12: Preparation of 5-fluoro-3- ({4- [4- (2- meth.oxy-eth.yl) —piperazin-1-yl] -8-nitrO-qpiinoline—3- carbonyl} -amino) -4-oxo-pentanoic acid trifluoroacetic acid (78)

[Reaction Scheme 12]

'17 78

The target compound 78 was obtained from the compound 77 in the similar manner as in Preparation process 8 of Example 7.

¹H NMR (CD₃OD, 500 MHz) δ: 8.79-8.78 (m, 0.5 H), 8.76-8.75 (m, 0.5 H), 8.43-8.41 (m, 1 H), 8.13-8.10 (m, 1 H), 7.77-7.73 (m, 1 H), 5.09-5.07 (m, 1 H), 3.79 (brs, 2 H), 3.69-3.66 (m, 8 H), 3.61 (brs, 2 H), 3.48 (s, 3 H), 2.95-2.92 (m, 2 H), 2.65-2.63 (m, 1 H).

Example 13: Preparation of 5-fluoro-3- [ (8-fluoro-4- piperidin-l-yl-quinoline-3-carbonyl) -amino] -4-oxo- pentanoic acid trifluoroacetic acid (80)

[Reaction Scheme 13]

The target compound 80 was obtained from the compound 79 in the similar manner as in Preparation process 8 of Example 7. ¹H NMR (CD₃OD, 500 MHz): δ 8.59-8.52 (m, 1 H), 8.01- 8.00 (m, 1 H), 7.62-7.54 (m, 1 H), 5.08 (brs, 1 H), 4.58- 4.56 (m, 1 H), 3.50-3.49 (m, 1 H), 3.41 (brs, 4 H), 3.01- 2.91 (m, 2 H), 1.86-1.78 (m, 6 H).

Experimental Example 1 : Evaluation of inhibitory activity against caspase-3 on OGD models of primary- cultured neuronal cells

Inhibitory activity against caspase-3 was evaluated for oxygen-glucose deprivation (OGD) -induced in vitro models of simulated cerebral ischemia. After 15-20 days pregnant SD rats were sacrificed, cerebrum cortex was separated from embryo and then centrifuged at 900 rpm for 9 minutes. The resulting precipitate was completely separated into single cells by addition of Dulbecco' s modified Eagle medium (DMEM) containing 10% FBS, 5% HS and 1% PS, and the single cells were seeded on PEI/laminin-coated plates at IxIO⁶ cells/ml. After 10-12 days in vitro (DIV), glucose-free DMEM was gas-bubbled with 95% NO₂ and 5% CO₂ for 20 minutes to remove oxygen present in the medium and then replaced with the medium containing culture cells. Then, the medium was incubated in a hypoxia chamber at 37⁰C for 1.5 hours so that it was exposed to oxygen-glucose deprivation and then treated with a glucose-containing DMEM. At 4 hours following the treatment, in-vivo caspase-3 assay was performed. IxIO⁶ cells were collected with a 100 ul cell lysis buffer. The lysate thus obtained was directly reacted with 20 uM DEVD-AFC as a substrate of caspase-3 at 35⁰C for 30 minutes. After the reaction, the cleavage of the substrate by caspase-3 was measured with an ELISA reader at an excitation wavelength of 400 nm and an emission wavelength of 508 nm. IC50 of respective experimental compounds was calculated using a graphPad Prism 4.0 program. The results thus obtained are shown in Table 1 below. TABLE 1

It can be confirmed from Table 1 that the compound 29 of Example 4 had a low IC50 of 10 nM and thus exhibited considerably superior inhibitory activity against caspase-3 and that the compounds^' of Examples 1, 2, 3, 7, 8, 9, 12 and 13 had an IC50 in the range of 200 nM to 15 uM and inhibitory activity against caspase-3 of the compounds are thus substantially equivalent to or superior to those of conventional caspase-3 inhibitors.

Experimental Example 2 : Evaluation of enzyme-level inhibitory activity against caspase-3

Caspase-3 known as a cystein protease was obtained by a series of processes involving expression, purification and activation in accordance with the method reported by Rotonda et al. (Nat Struct Biol., 1996, 3(7): 619). Enzyme-level inhibitory activity against caspase-3 of the experimental compounds was evaluated employing the caspase-3 thus obtained.

P12 and pl7 subunits were expressed in Escherichia coli, purified by nickel column and anion exchange chromatography and then recombined. Enzyme inactivity of the compounds was measured with Ac-DEVD-AFC as a fluorescent substrate.

In this process, the caspase-3 was reacted with 10 nM Ac-DEVD-AFC at 37⁰C in the presence of a buffer containing 50 mM NaCl, 10 mM DTT, 1 mM EDTA, 10% glycerol and 0.1% CHAPS, Synergy HT^® available from BIO TEK Instruments, Inc. was used as a fluorescence spectrometer, and the excitation and emission wavelengths herein used were 400 nm and 508 nm, respectively.

The IC50 of the experimental compounds is shown in Table 2 below. TABLE 2

[Industrial Applicability]

As apparent from the foregoing, the present invention provides a quinoline derivative with superior inhibitory activity against caspase-3 or a pharmaceutically acceptable salt thereof. Accordingly, with the quinoline derivative, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising one of them, it is possible to prepare a potent drug for treating a caspase-3 related disease e.g. Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke/ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.

Claims

[CLAIMS]

[Claim 1]

A quinoline derivative represented by the following Formula 1 for use in treating a caspase-mediated disease by inhibition of caspase-3 activity:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Ci-₆ alkoxy, Ci-6 alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Cχ-₆ halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, O or S; and R₅ is hydrogen, halogen, Ci_₆ alkyl, Ci_6 alkoxy, Ci-₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Cχ-6 halogenoalkyl or 5- to 15-membered aryl; A is C, N, O or S; and Y is hydrogen, halogen, Cj-e alkyl, Ci-₆ alkoxy, Ci-6 alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof.

[Claim 2]

The quinoline derivative according to claim 1 wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, nitro or Ci-₆ halogenoalkyl;

R is , or in which R2 is C or

N; R3 is -(CH2)2~; R4 is C, N, O or S; and R5 is hydrogen, Ci_₆ alkyl or Cχ_6 alkoxyalkyl;

A is N; and

Y is hydrogen.

[Claim 3]

The quinoline derivative according to claim 1 wherein the quinoline derivative of Formula 1 or a pharmaceutically acceptable salt thereof is selected from: 5-fluoro-3- [ (7-nitro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid;

5-fluoro-4-oxo-3- [ (4-piperazin-l-yl-7- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid;

5-fluoro-4-oxo-3- [ (4-piperazin-l-yl-8- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid; 5-fluoro-4-oxo-3- [ (4-piperidin-l-yl-7- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid;

5-fluoro-3- [ (8-fluoro-4-piperidin-l-yl-quinoline-3- carbonyl ) -amino] -4-oxo-pentanoic acid trifluoroacetic acid;

5-fluoro-3- [ (4-morpholin-4-yl-7-trifluoromethyl- quinoline-3-carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid;

5-fluoro-4-oxo-3- [ (4-thiomorpholin-4-yl-7- trifluoromethyl-quinoline-3-carbonyl) -amino] -pentanoic acid trifluoroacetic acid;

5-fluoro-3- [ (8-nitro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid; 5-fluoro-3- ( { 4- [4- (2-methoxy-ethyl) -piperazin-1- yl] -δ-nitro-quinoline-3-carbonyL } -amino) -4-oxo-pentanoic acid trifluoroacetic acid;

5-fluoro-3- [ (8-fluoro-4-piperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid;

5-fluoro-3- [ (7-fluoro-4-plperazin-l-yl-quinoline-3- carbonyl) -amino] -4-oxo-pentanoic acid trifluoroacetic acid; 5-fluoro-3-{ [4- (4-methyl-piperazin-l-yl) -8-nitro- quinoline-3-carbonyl] -amino } -4-oxo-pentanoic acid trifluoroacetic acid; and

3- { [4- (4-ethyl-piperazin-1-yl) -8-nitro-quinoline-3- carbonyl] -amino} -5-fluoro-4-oxo-pentanoic acid trifluoroacetic acid.

[Claim 4]

The quinoline derivative according to claim 1 wherein the caspase-mediated disease is Alzheimer' s disease, Huntington' s disease, Parkinson's disease, ALS,

AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.

[Claim 5] The quinoline derivative according to claim 1 wherein the quinoline derivative of the Formula 1 or a pharmaceutically acceptable salt is hydrochloride, bromate, sulfate, nitrate, perchlorate, fumarate, maleate, phosphate, glycolate, lactate, salicylate, succinate, p-toluenesulfonate, tartrate, trifluoroacetate, acetate, citrate, methanesulfonate, formate, benzoate, malonate, naphthalene-2-sulfonate, benzensulfonate, an alkali-metal salt or an alkaline earth metal salt.

[Claim 6]

A method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-₆ alkyl, Ci-e alkoxy, Ci_₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci_g halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is -(CH₂)_n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci-6 alkyl, Ci-6 alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Cj-β halogenoalkyl or 5- to 15-membered aryl; A is C, N, O or S; and

Y is hydrogen, halogen, Cχ-₆ alkyl, Ci_₆ alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-.₆ halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

(2)

wherein Xi, X2, X₃ and X4 are defined as above;

(4)

wherein Xi, X2, X₃ and X₄ are defined as above; halogenizing the compound of Formula 4 to obtain a compound represented by Formula 5 below:

wherein Xi, X₂, X₃ and X₄ are defined as above and Z is halogen; reacting the compound of Formula 5 with RH in which R is defined as above, to obtain a compound represented by Formula β below:

wherein X₁, X₂, X₃, X₄ and R are defined as above; reacting the compound of Formula 7 with a compound represented by the following Formula 8 to obtain a compound represented by Formula 9 below:

(8) wherein A and Y are defined as above;

[Claim 7]

An intermediate compound represented by the following Formula 7 for use in preparing a quinoline derivative represented by the following Formula 1:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-₆ alkyl, Cχ-₆ alkoxy, Ci_6 alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl; and

R is , or in which R₂ is C or

N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci-₆ alkyl, Ci-₆ alkoxy, Ci_₆ alkoxyalkyl, C₃-₅ cycloalkyl, nitro, amino, Cχ-6 halogenoalkyl or 5- to 15-membered aryl;

wherein Xi, X₂, X₃, X₄ and R are defined as above;

A is C, N, 0 or S; and

Y is hydrogen, halogen, Ci_₆ alkyl, Ci-₆ alkoxy, Ci_6 alkoxyalkyl, C₃-₆ cycloalkyi, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl, or a pharmaceutically acceptable salt thereof.

[Claim 8]

An intermediate compound represented by the following Formula 9 for use in preparing a quinoline derivative represented by the following Formula 1:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Ci~₆ alkoxy, Ci_₆ alkoxyalkyl, C₃-₆ cycloalkyi, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl;

R is in which R₂ is C or

N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is C,

N, 0 or S; and R₅ is hydrogen, halogen, Ci-6 alkyl, Ci-6 alkoxy, Ci_₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci_6 halogenoalkyl or 5- to 15-membered aryl;

A is C, N, 0 or S; and

Y is hydrogen, halogen, C]-₆ alkyl, Ci_₆ alkoxy, C]-₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci-6 halogenoalkyl or 5- to 15-membered aryl;

wherein Xi, X₂, X3, X₄, R, A and Y are defined as above .

[Claim 9] A method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, C₁-₆ alkyl, Ci-6 alkoxy, Ci-6 alkoxyalkyl, C₃_₅ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl;

R is or in which R₂ is C or

N; R₃ is - (CH₂) n- in which n is 0, 1, 2, 3 or 4; R₄ is N; and R₅ is hydrogen;

A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein Xi, X₂, X₃ and X₄ are defined as above;

(4)

Protective

Prote Group

Protective

Ri is or

in which R₂ and R₃ are defined as above, to obtain a compound represented by Formula 10 below:

wherein Xi, X2, X3, X4 and R] are defined as above; hydrolyzing the compound of Formula 10 to obtain a compound represented by Formula 11 below:

wherein A is defined as above;

wherein Xi, X₂, X₃, X4, Ri and A are defined as above; carbonylating the compound of Formula 13 to obtain a compound represented by Formula 14 below:

wherein Xi, X2, X₃, X4, Ri and A are defined as above; and deprotecting protective groups I and Il of the compound of Formula 14 to obtain the compound of Formula 1 above .

[Claim 10] A method for preparing a quinoline derivative represented by Formula 1 below:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-₆ alkyl, Cj-β alkoxy, Ci-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is -(CH2)_n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci-₆ alkyl, Cχ-ς alkoxy, Cχ_₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen, or a pharmaceutically acceptable salt thereof, the method comprising the steps of: reacting a compound represented by the following Formula 2 with diethylethoxymethylenemalonate to obtain a compound represented by Formula 3 below:

wherein Xi, X₂, X₃ and X₄ are defined as above;

wherein Xi, X2, X₃, X4 and R are defined as above; hydrolyzing the compound of Formula 6 to obtain a compound represented by Formula 7 below:

wherein Xi, X₂, X₃, X4 and R are defined as above; reacting the compound of Formula 7 with a compound represented by the following Formula 12 to obtain a compound represented by Formula 15 below:

Protective Group II :i2) wherein A is defined as above;

wherein Xi, X2, X₃, X4, R and A are defined as above; carbonylating the compound of Formula 15 to obtain a compound represented by Formula 16 below:

wherein Xi, X₂, X₃, X₄, R and A are defined as above; and deprotecting a protective group II of the compound of Formula 16 to obtain the compound of Formula 1 above.

[Claim 11]

A pharmaceutical composition containing a pharmaceutically effective amount of a quinoline derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof for use in treating a caspase- mediated disease based on inhibition of caspase-3 activity:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci-₆ alkyl, Ci-g alkoxy, C1-₆ alkoxyalkyl, C₃-₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is -(CH₂)_n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, O or S; and R₅ is hydrogen, halogen, Ci-₆ alkyl, Cχ-₆ alkoxy, Ci-₆ alkoxyalkyl, C₃_6 cycloalkyl, nitro, amino, Ci-6 halogenoalkyl or 5- to 15-membered aryl; A is C, N, 0 or S; and Y is hydrogen.

[Claim 12]

The pharmaceutical composition according to claim 11 wherein the caspase-mediated disease is Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.

[Claim 13]

A use of a quinoline derivative of the following Formula 1 or a pharmaceutical] y acceptable salt thereof for preparing a drug for treating a caspase-mediated disease based on inhibition of caspase-3 activity:

wherein Xi, X₂, X₃ and X₄ are each independently hydrogen, halogen, Ci_₆ alkyl, Cχ-₆ alkoxy, Ci-₆ alkoxyalkyl, C3-6 cycloalkyl, nitro, amino, Ci_₆ halogenoalkyl or 5- to 15-membered aryl;

R is , or in which R₂ is C or

N; R₃ is - (CH₂) _n- in which n is 0, 1, 2, 3 or 4; R₄ is C, N, 0 or S; and R₅ is hydrogen, halogen, Ci-₆ alkyl, Ci_₆ alkoxy, Ci_₆ alkoxyalkyl, C₃_₆ cycloalkyl, nitro, amino, Ci-₆ halogenoalkyl or 5- to 15-membered aryl; A is C, N, O or S; and Y is hydrogen.

[Claim 14]

The use of the quinoline derivative according to claim 11 wherein the caspase-mediated disease is Alzheimer's disease, Huntington' s disease, Parkinson's disease, ALS, AIDS, stroke, ischemia, traumatic brain injury, spinal cord injury or osteoarthritis.