KR20140017332A - Pharmaceutical composition or pharmaceutically acceptable salt thereof for the prevention or treatment of circadian clock related diseases containing 2-ethoxypropionic acid derivative as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of circadian clock related diseases containing a 2-ethoxypropionic acid derivative or pharmaceutically acceptable salt thereof as an active ingredient. The 2-ethoxypropionic acid derivative controls a function of CRY1/2 and promotes the activity of CLOCK:BMAL1 dimer for effectively controlling circadian clock molecule network damaged by genetic or environmental causes, and the composition containing the 2-ethoxypropionic acid derivative or pharmaceutically acceptable salt thereof as an active ingredient can be usefully applied to the prevention or treatment of circadian clock related diseases including sleep disorder, metabolic disorder, mood disorder, and cancer. [Reference numerals] (AA) Example 4; (BB,DD) Input; (CC,EE) Developing solvent; (FF) Bait + Example 4

Description

Pharmaceutical composition for preventing or treating circadian related diseases containing 2-ethoxypropionic acid derivative or pharmaceutically acceptable salt thereof as an active ingredient. containing 2-ethoxypropionic acid derivative as an active ingredient}

The present invention relates to a pharmaceutical composition for the prevention or treatment of a one-cycle-related disease comprising a 2-ethoxypropionic acid derivative or a pharmaceutically acceptable salt thereof.

Like all living things on Earth, humans exhibit a circadian rhythm that is aligned with the 24-hour cycle of day and night changes in physiological or behavioral terms. The most well-known human circadian behavior is sleep, but in addition, body temperature, diet, hormones in the body, blood glucose levels and other metabolic activities and cell division cycles are also regulated by these circadian rhythms. When the circadian rhythm is broken due to genetic or environmental factors, it may be caused by various physiological or mental disorders. This is called circadian rhythm-related disorder. Especially in modern industrialized society, the majority of people are exposed to various environmental biological clock disturbance causes such as excessive night light, excessive night activities such as shift work, and abnormal dietary forms. Research is gaining in importance.

Organisms have genes for the control of circadian rhythms, and circadian rhythm control genes regulate circadian rhythms. Recent studies have identified bioclock molecular networks that govern circadian rhythms. Mammalian circadian rhythm control genes include PER gene (Per1, Per2, Per3), CLOCK gene, BMAL gene (Bmal1, Bmal2, Bmal3), CRY gene (CRY1, CRY2), Rev-erb gene (Rev-erbα, Rev -erbβ) and the like, and they form a molecular network, in which the dimers of the CLOCK and BMAL1 proteins are the transcription factors at the lower levels of the Period (PER) and Cryptochrome (CRY) family of genes. It triggers expression and its gene products form a series of molecular circulatory structures that inactivate higher-level transcription factors. These molecular networks are known to affect various physiological processes by triggering circadianity of genes regulated downstream (Non-Patent Document 1). Molecular networks of these biological clock genes are found in various peripheral organs as well as in the suprachiasmatic nucleus of the hypothalamus, where the mammalian central biological clock is located. It is expressed periodically with its own daily rhythm. The biological clock molecular network expressed in such various peripheral organs is called a peripheral biological clock (peripheral clock) in contrast to the central biological clock (Non-Patent Document 1).

Because the biological clock is a universal regulator of overall behavior and physiology, circadian-associated diseases are also very extensive, including sleep disorders, cancer, metabolic disorders, and mood disorders.

In fact, emergency workers, who work alternately by altering the normal 24-hour rhythm of weather and sleep, are exposed to various diseases including metabolic disorders such as diabetes (Non-Patent Document 2), and are exposed to long-term shift work. Pathological studies of nurses have reported that the number and intensity of their shifts are higher in the incidence of breast cancer than in normal individuals (Non-Patent Document 3).

There have been many studies on the cause and treatment of circadian related diseases caused by various environmental and genetic bioclock disturbances, and one of them is the regulation of circadian rhythm through phosphorylation of vital clock genes. Since mammalian kinases casein kinase Iε (CKIε) and casein kinase Iδ (CKIδ) are known to phosphorylate vital clock genes to regulate circadian rhythms, studies have been conducted to control circadian rhythms by inhibiting CKI genes. (Non-Patent Documents 4 and 5). However, the effect is minimal because inhibition of the CKI gene does not have a direct effect on the biological clock molecular network.

Another study is Rev-erbα, a key gene for the biological clock molecule, which modulates circadian rhythms through agonists and antagonists of Rev-erb α, which inhibits the expression of BMAL1. In Patent Documents 6 and 7), again, Rev-erb α's main role is to slow down the activity of CLOCK: BMAL1 dimers in the biological clock molecular network and to fine-tune the circadian rhythms effectively for the clock to work properly. There is difficulty in doing.

Therefore, at the present time when the physiological and clinical importance of the circadian rhythm is highlighted, there is an urgent need for the development of drugs capable of directly and effectively controlling the biological clock molecular network for the prevention and treatment of various circadian rhythm-related diseases.

The inventors of the present invention developed a drug that directly acts on the core clock of the biological clock through a cell-based assay that measures the activity of the dimer of CLOCK: BMAL1, while 2-ethoxypropionic acid derivatives inhibit the action of CRYs. It was confirmed that there is an effect of regulating the clock molecule network, and completed the present invention.

T. Stratmann., Et al., J. Biol. Rhythms., 21, (2006), 494. F.A. Scher., Et al., Proc. Natl. Acad. Sci. USA, 106, (2009), 4453. E.S. Schernhammer., Et al., J. natl. Caner. Inst., 93, (2001), 1563. T. Hirota., Et al., PLoS Biol. 8, (2010), e1000559. Z. Chen., Et al., Proc. Natl Acad. Sci., 109, (2012), 101. Q.J. Meng., Et al., J. Cell. Sci., 121, (2008), 3629. D. Kojetin., Et al., ACS Chem. Biol., 6, (2011), 131. A, Schirmacher, et al., Biological Rhythm Research, 42, (2011), 407. J.P. Wisor., Et al., BMC Neurosci., 3, (2002), 20. R.E. Mistlberger., Et al., Sleep, 6, (1983), 217. I. Tobler., Et al., Neurosci. Lett., 42, (1983), 49. L. Trachsel., Et al., Brain Res., 589, (1992), 253. K. Spiegel., Et al., Nat. Rev. Endocrinol., 5, (2009), 253. F.W. Turek, et al., Science., 308, (2005), 1043. K.A. Lamia., Et al., Proc. Natl. Acad. Sci USA., 105, (2008), 15172. R.D. Rudic., Et al., PLoS Biol., 2, (2004), e377. S. Shimba., Et al., Proc. Natl. Acad. Sci. USA, 102, (2005), 12071. I.M. Bur., Et al., J. Biol. Chem., 284, (2009), 9066. S. Okano., Et al., Neurosci. Lett., 451, (2009), 246. J.P. Gouin., Et al., J. affec. disorders. 126, 161 K.A. Lamia., Et al., Nat. Med., 16, (2011), 1152. E.E Zhang., Et al., Nat. Med., 16, (2010), 1152. V.Y. Gorbacheva., Et al., Proc. Natl. Acad. Sci. USA, 102, (2005), 3407. N. Ozturk., Et al., Proc. Natl. Acad. Sci. USA., 106, (2009), 2841 G.T. van der Horst., et al., Nature, 398, (1999), 627. P. Chomczynski., Et al., Nat. Protoc., 1, (2008), 581.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of circadian related diseases, including 2-ethoxypropionic acid derivatives or acceptable salts thereof.

In order to achieve the above object,

It provides a pharmaceutical composition for the prevention or treatment of circadian-associated diseases comprising a 2-ethoxypropionic acid derivative represented by the general formula (1) or an acceptable salt thereof.

[Formula 1]

(X, Z, n in the general formula 1 is as defined in the present specification)

The 2-ethoxypropionic acid derivative according to the present invention can effectively regulate the biological clock molecular network by inhibiting the action of CRYs and promoting the activity of CLOCK: BMAL1 dimer, so that the 2-ethoxypropionic acid derivative or its acceptable salts as an active ingredient The composition comprising can be usefully used for the prevention or treatment of circadian-associated diseases sleep disorders, metabolic disorders, mood disorders, cancer.

1 is a graph showing the transcriptional activity effect of E-box-Luc for each compound according to Experimental Example 1 of the present invention.
The code | symbol shown in said FIG. 1 is as follows.
(a): Summary of E-box-Luc activity assessment results
(b): E-box-Luc Modulation Compound Status
2 is a graph showing the transcriptional activity effect of StAR-Luc for each compound according to Experimental Example 1 of the present invention.
The code | symbol shown in said FIG. 2 is as follows.
(a): Summary of StAR-Luc activity assessment results
(b): Status of StAR-Luc Modulating Compounds
3 is a graph showing the E-box transcription activity of each Bmal1 deficient embryonic fibroblast cell line and wild type embryonic fibroblast cell line according to Experimental Example 1 of the present invention.
4 is a graph showing the effect of E-box-Luc transcriptional activity of Compound (1b) and Compound (1i) according to Experimental Example 1 of the present invention.
The code | symbol shown in said FIG. 4 is as follows.
(a): Screening result in step 1
(b): Screening result in step 2
(c) Derivation of the core common structure of compound (1b) of Example 2 and compound (1i) of Example 9)
5 is a graph showing E-box-Luc activity for the concentration of Examples 1 to 4 according to Experimental Example 2 of the present invention.
6 is a graph showing E-box-Luc activity for the concentration of Examples 5 to 9 according to Experimental Example 2 of the present invention.
7 is a photograph analyzing the pull-down activity according to Experimental Example 3 of the present invention.
The code | symbol shown in said FIG. 7 is as follows.
(a): Chemical structure of Bait, a compound of Examples 4 and 8 used for pull-down activity assay
(b): Bait binding of key genes of biological clock molecular machinery
(c): binding of Bait to CRY1 / 2 and selective competition inhibition by compound (1d) of Example 4)
8 is a graph showing the results of the CRYs dependency verification test according to the compound treatment according to Experimental Example 4 of the present invention.
The code | symbol shown in said FIG. 8 is as follows.
(a): E-box-Luc Reporter Activity Assessment
(b): Evaluation of Per1 mRNA expression level
(c): Per2 mRNA expression level evaluation
(d): Evaluation of Rev-erbα mRNA expression level

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating circadian-associated diseases, including 2-ethoxypropionic acid derivative represented by the following formula (1) or an acceptable salt thereof.

In Formula 1,

X is hydrogen; halogen; C1 to C4 straight or branched chain alkyl unsubstituted or substituted with one or more halogens; Hydroxy; Or unsubstituted C1 to C4 straight or branched alkoxy, wherein X is substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;

또는

이고, 상기 Z는 방향족 고리의 메타 또는 파라 위치 중 어느 하나의 위치에 치환되고; Z가

인 경우 0 내지 5의 정수이며; R₁은 수소 또는 비치환된 C1 내지 C4의 직쇄 또는 측쇄 알킬이고; 및Z is

or

Z is substituted at either the meta or para position of the aromatic ring; Z is

When is an integer from 0 to 5; R ₁ is hydrogen or unsubstituted C1 to C4 straight or branched alkyl; And

인 경우 1 내지 5의 정수이다.
n is the Z

Is an integer of 1 to 5.

Preferably, in Formula 1

X is hydrogen; Fluoro; Chloro; Bromo; Iodo; methyl; ethyl; profile; Isopropyl; Butyl; Isobutyl; t-butyl; Trifluoromethyl; Trichloromethyl; Fluoromethyl; Chloromethyl; Hydroxy; Methoxy; Ethoxy; Propoxy; Butoxy; Or trifluoromethoxy, wherein X may be substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;

또는

이고, 상기 Z는 방향족 고리의 메타 또는 파라 위치 중 어느 하나의 위치에 치환되고; ; R₁은 수소; 메틸; 에틸; 프로필; 부틸; 이소프로필, 이소부틸 또는 t-부틸이고; 및Z is

or

Z is substituted at either the meta or para position of the aromatic ring; ; R ₁ is hydrogen; methyl; ethyl; profile; Butyl; Isopropyl, isobutyl or t-butyl; And

인 경우 1 내지 3의 정수이고, Z가

인 경우 0 내지 2의 정수인 것을 특징으로 하는 2-에톡시프로피온산 유도체 또는 이의 허용가능한 염을 포함하는 일주기 연관성 질환의 예방 또는 치료용 약학적 조성물이다.
n is the Z

When is an integer of 1 to 3, Z is

If is a pharmaceutical composition for the prevention or treatment of circadian-associated diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof, characterized in that an integer of 0 to 2.

More preferably, in Formula 1,

X is hydrogen; Fluoro; Chloro; Bromo; Iodo; Trifluoromethyl; Hydroxy; Methoxy or trifluoromethoxy, wherein X is substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;

또는

이고, 상기 Z는 방향족 고리의 메타 또는 파라 위치 중 어느 하나의 위치에 치환되고; R₁은 메틸; 에틸 또는 프로필이고; 및 Z is

or

Z is substituted at either the meta or para position of the aromatic ring; R ₁ is methyl; Ethyl or propyl; And

인 경우 1 내지 3의 정수이고, Z가

인 경우 0 내지 2의 정수인 것을 특징으로 하는 2-에톡시프로피온산 유도체 또는 이의 허용가능한 염을 포함하는 일주기 연관성 질환의 예방 또는 치료용 약학적 조성물이다.
n is the Z

When is an integer of 1 to 3, Z is

If is a pharmaceutical composition for the prevention or treatment of circadian-associated diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof, characterized in that an integer of 0 to 2.

Most preferably,

2-ethoxypropionic acid derivative of Formula 1 is:

1) (R) -3- (3-((E) -1-benzyloxyiminoethyl) phenyl) -2-ethoxypropionic acid;

2) (R) -3- (3-((E) -1- (4-fluorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

3) (R) -3- (3-((E) -1- (4-methoxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

4) (R) -3- (3-((E) -1- (4-bromobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

5) (R) -3- (3-((E) -1- (4-chlorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

6) (R) -3- (3-((E) -1- (4-hydroxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

7) (R) -3- (3-((E) -1- (4-trifluoromethylbenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

8) (R) -3- (3-((E) -1- (4-trifluoromethoxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

9) (R) -3- (3-((E) -1- (3-chlorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

10) (R) -3- (3-((E) -1- (4-iodobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;

11) (R) -2-ethoxy-3- (3- (5- (4-fluorophenyl) -4,5-dihydroisoxazol-3-yl) phenyl) propionic acid;

12) (R) -2-ethoxy-3- (3- (5- (4-trifluoromethylphenyl) -4,5-dihydroisoxazol-3-yl) phenyl) propionic acid;

13) (R) -2-ethoxy-3- (3- (5- (2-chlorophenyl) -4,5-dihydroisooxa-3-yl) phenyl) propionic acid; And

14) any one selected from the group consisting of (R) -2-ethoxy-3- (3- (5- (3-chlorophenyl) 4,5-dihydroisoxazol-3-yl) phenyl) propionic acid It is a pharmaceutical composition for the prophylaxis or treatment of circadian related diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof.

The 2-ethoxypropionic acid derivative of Formula 1 according to the present invention includes pharmaceutically acceptable salts thereof, hydrates and solvates that can be prepared therefrom.

As used in the present invention, when referring to the compound represented by the formula (1) it can be understood to include their pharmaceutically acceptable salts. Such pharmaceutically acceptable salts can be prepared by methods of preparing commonly used salts.

The 'pharmaceutically acceptable salts' refer to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric iron, ferrous iron, lithium, magnesium, manganese salts, manganese, potassium, sodium, zinc and the like. Especially ammonium, calcium, magnesium, potassium or sodium salts are preferred. Solid salts may exist in one or more crystal structures, and may also exist in hydrate form. Salts derived from pharmaceutically acceptable non-toxic organic bases include primary, secondary or tertiary amines, substituted amines including naturally substituted amines, amine rings, or arginine, betaine, caffeine, choline, N, N '-Dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, Such as hydravamin, isopropyl amine, lysine, methylglucamine, popoline, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Basic ion exchange resins.

When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. The acid is acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isetionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muk Acids, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like. Citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, fumaric acid or tartaric acid are particularly preferred.

Hydrates of compounds represented by Formula 1 of the present invention or pharmaceutically acceptable salts thereof may be understood to include stoichiometric or nonstoichiometric amounts of water bound by non-covalent intermolecular forces. The hydrate may contain at least 1 equivalent, typically 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

The solvate of the compound represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof may be understood to include a stoichiometric or non-stoichiometric amount of solvent bonded by non-covalent intermolecular force. Preferred as the solvent include solvents which are volatile, non-toxic or suitable for administration to humans.

Diurnal related diseases according to the present invention include, but are not limited to, sleep disorders, metabolic disorders, mood disorders and cancer.

Inhibition of the biological clock molecular network due to genetic or environmental factors can cause various physiological and mental disorders. The circulatory related diseases may be represented by parallax, sleep disorders, metabolic disorders, mood disorders, cancers, etc. In addition, there are various kinds of diseases, and these diseases may be prevented or treated by controlling biological clock molecular networks.

Circadian-related sleep disorders are disorders in which sleep / wake rhythms appear in abnormal patterns due to genetic or environmental disturbances on the biological clock. Patients with sleep disorders tend to complain of lack of sleep because they have difficulty falling asleep or waking at socially necessary times. The cause of sleep disorders has been reported by CRYs, a subgene of the biological clock network. . Recent genetic studies of sleep disorder patients have shown that in genetic studies of sleep disorder patients with excessively excessive sleep during the day, the genetic polymorphism rs17289712 in the intron of the CRY1 gene was significantly associated with the symptoms of the disease. The relationship was shown (Non Patent Literature 8). This means that CRY plays a unique role in regulating sleep / wake rhythm by the biological clock.

In another study to examine the effect of CRY, a sub-clock sub-gene, on sleep, mice lacking both CRY1 and CRY2 genes had increased NREMS (non-REM sleepness) compared to normal mice, and deltas during non-REM sleep. The intensity | strength of the wave also showed the tendency to increase (nonpatent literature 9). This means increased deep sleep, and this pattern of sleep in mice with CRY1 and CRY2 gene depletion differs from those in animal models that artificially destroy the hypothalamus of the optic nerves and destroy their circadian rhythms. When the upper nucleus is destroyed, there is no change in the total sleeping time, but the sleeping pattern is fragmented and the intensity of the delta wave also tends to decrease (Non-Patent Document 10-12). This suggests that there may be an independent role for CRY in regulating sleep homeostasis apart from the role that CRY plays in the circadian clock.

Thus, Experiments 3 and 4 were conducted to verify the regulation of the CRYs protein activity of the 2-ethoxypropionic acid derivative represented by Chemical Formula 1 according to the present invention. As a result, the 2-ethoxypropionic acid derivative was directly bound to the CRYs protein And thus has an excellent effect of increasing the transcriptional activity of the CLOCK: BMAL1 dimer of CRYs. Therefore, the pharmaceutical composition containing the 2-ethoxypropionic acid derivative represented by the formula (1) Cycle-related sleep-related disorders associated with clock genes may be readily used in the prevention or treatment of sleep disorders.

In this case, the circadian-related sleep disorder diseases include parallax syndrome, shift work sleep disorder, progressive sleep phase syndrome, and sleep phase delay syndrome colon, but are not limited thereto.

Metabolic disorders, one of the cycle-related disorders, have been reported to be caused by functional abnormalities of the biological clock due to genetic or environmental causes. Abnormal life patterns, such as long-term shifts or lack of sleep, are known to slow down the circadianity of growth hormone and melatonin secretion in the body and increase cortisol levels (Non-Patent Document 13). It leads to an increase in body weight similar to symptoms.

In addition, the relationship between the biological clock system and metabolic disorders is more evident from the analysis of genetically engineered animal models.

In CLOCK mutant mice, it was found that leptin hyperactivity and insulin secretion were lowered, and symptoms of hyperlipidemia and hyperglycemia were shown (Non Patent Literature 14). In addition, in the BMAL1 gene deficient mice, growth and differentiation of fat cells appeared, and abnormal symptoms were also found in carbohydrate metabolism in the liver (Non Patent Literature 15-17). In addition, recent studies have shown that CRYs function as important regulators in the gluconeogenesis process in the liver, the cause of hyperglycemia. In mice lacking CRYs, abnormalities in glycemic control were observed compared to normal mice, because CRYs bind to the glucocorticoid receptor (GR) to regulate the expression of hormone-induced glucose neosynthesis genes. (Nonpatent Document 18). In addition, CRYs bind to G subunits, one of the components of G-protein coupled receptors, as well as glucocorticoid hormone receptors (GRs), thereby adhering to adenyl cyclase. It regulates the activity of, thereby showing the effect of controlling the expression of genes that act on glucose neosynthesis induced by cyclic adenosine monophosphate (cAMP) (Non-Patent Document 19).

Not only does this indicate that metabolic disorders are related to the phase shift of the biological clock, but the results of the studies show more clearly that CLOCK, deficiency of BMAL1 and CRY1 / 2 expression can lead to metabolic disorders. It also suggests that the expression of CLOCK and BMAL1 and the enhanced activity of CLOCK: BMAL1 dimers and the inhibition of CRY1 / 2 activity are effective in treatment.

Thus, in order to confirm the activity-enhancing effect of CLOCK: BMAL1 dimer and the inhibitory effect of CRY1 / 2 by 2-ethoxypropionic acid derivative represented by Formula 1 according to the present invention, the same experiment as Experimental Example 1 to Experimental Example 4 was performed. Was performed. As a result, it was confirmed that the active strengthening effect of the dimer of CLOCK: BMAL1 was excellent through Experimental Example 1 to Experimental Example 2, the activity inhibitory effect of CRY1 / 2 was excellent through Experimental Example 3 to Experimental Example 4, CRY1 / 2 Inhibition of the activity of CLOCK: BMAL1 can be seen to enhance the dimer activity. Therefore, the pharmaceutical composition containing the 2-ethoxypropionic acid derivative represented by the formula (1) or an acceptable salt thereof according to the present invention can be usefully used for the prevention or treatment of metabolic disorders caused by the functional disturbance of the biological clock. .

At this time, the circadian-associated metabolic disorders include obesity, hyperlipidemia, hyperglycemia and polyuria, but are not limited thereto.

Mood disorders can also be invented due to variations in the circadian rhythm, and research on this is being actively conducted. As with physical functions such as metabolic activity and cell division, changes in emotion, one of the higher functions of the brain, are circadian, and in the development and progression of various types of mood disorders that show pathological symptoms in the control of these emotions. The role of the biological clock is becoming important.

To determine the association between mood disorders and the biological clock, blood samples were analyzed to evaluate messenger RNA levels associated with four circadian rhythm genes in individuals with depression and without disorder. The messenger RNA represents gene activity, in which participants with a history of depression had higher gene activity, known as the CLOCK gene, than those who did not have depression, from which it can be predicted that depressive disorders are associated with the activity of the key biological clock, CLOCK. It can be (Non-Patent Document 20).

In addition, the relationship between the biological clock and mood disorders can be known through light therapy. As one of the widely used methods of treating mood disorders through artificial stimulation that can control the biological clock in addition to drug treatment, light therapy is known to be particularly effective for seasonal mood disorders. One of the most common mental illnesses in which 5 to 10 percent of the population living in temperate climates is a symptom is depression-like symptoms during the winter, when the sun rises and the days get shorter. At this time, when the patient with seasonal mood disorder is artificially exposed to strong light, the symptoms of depression are alleviated. Phototherapy performed early in the morning is more effective than that performed in the evening (Non-Patent Document 21-22). Not only does this indicate that symptoms of seasonal mood disorders are associated with phase shifts in the biological clock, but the expression of CLOCK and BMAL1, the key biological clock genes activated during daylight hours, and the activity of the dimers of CLOCK: BMAL1 in treatment It means that it works.

In order to confirm the activity-enhancing effect of the dimer of CLOCK: BMAL1 of the 2-ethoxypropionic acid derivative represented by Chemical Formula 1 according to the present invention with the facts obtained from this, the same experiments as Experimental Examples 1 and 2 were carried out. It was confirmed that the activity enhancing effect of the dimer of CLOCK: BMAL1 was excellent. Therefore, the pharmaceutical composition containing the 2-ethoxypropionic acid derivative represented by Formula 1 or an acceptable salt thereof according to the present invention may be usefully used for preventing or treating a disorder related to mood disorders through a pharmacological approach to a biological clock. Can be.

At this time, the one-cycle-related mood disorder includes depressive disorder, bipolar disorder, and seasonal disorder, but is not limited thereto.

In cancer, one of the cycle-related diseases, the cyclical change in sensitivity to anti-cancer agents shows the association of tumor clock core genes with tumors. Anti-cancer drug resistance was higher in CRYs-deficient mice compared to normal mice at all time points. This difference in resistance was attributed to the difference in functional status of CLOCK: BMAL1 dimers in hepatocytes in normal and circadian genetically engineered mice, resulting in a difference in the ability of the liver to treat the toxicity of anticancer drugs. Non-Patent Document 23). This study shows that the effects of the key bio-cycle gene CLOCK: BMAL1 dimer and sub-gene CRYs on anticancer drugs are significant.

In addition to affecting the cyclic changes in sensitivity to anticancer drugs, the biological clock gene has the effect of directly inhibiting tumor formation and growth. Further deletion of CRYs in mice lacking p53 gene, one of animal models for tumor induction, inhibits tumor formation and growth (Non-Patent Document 24). Genetic deficiencies in CRYs have been reported to completely destroy the intrinsic circadianity of individuals (Non-Patent Document 25), so that the pathways leading to disturbance of the biological clock and tumor development do not necessarily coincide, and more complex correlations may exist. In particular, it suggests that the core clock genes, in particular, have the potential to have independent functions in the cell cycle and tumor development process as well as the maintenance of circadian rhythms, and the new possibilities of temporary inhibition of CRYs in anticancer activity. Provides room for consideration.

Compound represented by the formula (1) according to the present invention selectively binds to the CRYs of the biological clock sub-gene through Experimental Example 3 has an excellent effect of inhibiting the action of CRYs, so that the drug through the drug limited to a specific time period by controlling this In addition to enhancing the efficacy and reducing the toxicity, and inhibits the production and growth of tumor cells, pharmaceutical compositions containing it as an active ingredient can be useful for the prevention or treatment of cancer caused by alteration of the circadian rhythm. Can be.

Herein, the periodic-related cancer includes, but is not limited to, colorectal cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, lung cancer, uterine cancer, colon cancer, pancreatic cancer and ovarian cancer.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The pharmaceutical compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be used. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

 In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be formulated into the compositions of the present invention with at least one excipient such as starch, calcium carbonate, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally, any parenteral administration method can be used, systemic or topical administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Taking all of the above factors into consideration it is important to administer an amount that will achieve the maximum effect with a minimum amount without side effects, which can be readily determined by the practitioner concerned.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 to 100 mg, preferably 0.5 to 10 mg daily or every other day or daily It can be administered in 1 to 3 divided doses. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

Hereinafter, the present invention will be described in detail with reference to Production Examples and Examples.

However, the following Preparation Examples and Examples are merely to illustrate the present invention, and the content of the present invention is not limited to the following Preparation Examples and Examples.

< Manufacturing example  1> benzyl Hydroxylamine Hydrochloride  Preparation of Derivatives (4)

Wherein R ₁ and n are as defined in Formula 1

Step 1: Preparation of Benzyl Alcohol Derivative (14)

As shown in Scheme 3, commercially available benzaldehyde (13) was placed in a reactor and methanol was added. After cooling the reactor to 0 ° C., sodium borohydride (1.1 equiv) was slowly added, and the temperature of the reaction solution was raised to room temperature and stirred for 1 to 2 hours. 2N hydrochloric acid was added to the reaction solution to terminate the reaction, and concentrated under reduced pressure. The residue was diluted with diethyl ether and washed with water, then the filtrate was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1: 5) to obtain the target compound (14) (yield 85 to 99%).

Step 2: 2- ( Benzyloxy ) Isoindoline -1,3- Dion  Preparation of derivative (15)

Benzyl alcohol (14), triphenylphosphine (1.1 equivalents) and N-hydroxyphthalimide (1.1 equivalents) obtained in step 1 were added to a reactor, argon was substituted therein, anhydrous THF was injected, and the reactor was zero. After cooling to 占 폚, diethylazodicarboxylate (DEAD) (1.2 equiv) was slowly added dropwise. The temperature of the reaction solution was raised to room temperature and stirred for 2 to 12 hours. Water was added to the reaction solution to terminate the reaction. After concentration under reduced pressure, the residue was diluted with ethyl acetate and washed with water. The filtrate was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1: 5) to obtain the target compound (15) (yield 90 to 99%).

Step 3: benzyl Hydroxylamine Hydrochloride  Preparation of Derivatives 4

The 2- (benzyloxy) isoindoline-1,3-dione derivative (15) obtained in the above step 2 was added to the reactor, and acetonitrile was added thereto. Hydrazine (1.1 equivalent) was slowly added dropwise. The reaction solution was stirred at room temperature for 10 minutes to 1 hour. The reaction solution was filtered to remove the solid precipitate, and diethyl ether and hydrochloric acid were added to the filtrate to obtain the target compound (4) (yield 10 to 99%) in the salt state.

< Manufacturing example  2> Preparation of Styrene Derivatives (7)

(Wherein R ₂ and n are as defined in Chemical Formulas 1 to 3)

Methyltriphenylphosphonium bromide (1.5 equivalents) was added to the reactor, argon was substituted for the inside, anhydrous THF was injected, and normal butyllithium (1.6 M solution, 1.3 equivalents) was slowly added dropwise. After the temperature of the reaction solution was raised to room temperature and stirred for 2 hours, benzaldehyde (13), a starting material of Preparation Example 1, was dissolved in anhydrous THF and added to the reactor through a cannula. The reaction solution was stirred at room temperature for 1-2 hours, and the reaction was terminated by addition of saturated ammonium chloride solution and concentrated under reduced pressure. The residue was diluted with diethyl ether and washed with water, and the filtrate was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1:10) to obtain the target compound (7) (yield 40 to 90%).

Compounds used as starting materials in the following examples were obtained using a known method (KR 10-0847780).

< Example  1> (R) -3- (3-((E) -1- Benzyloxyiminoethyl ) Phenyl )-2- Ethoxypropionic acid  Preparation of (1a)

Step 1: (R)- methyl  3- (3-(-1- Benzyloxyiminoethyl ) Phenyl )-2- Ethoxy Propione Preparation of meth 5a

(R) -methyl 3- (3-acetylphenyl) -2-ethoxypropionate (3) (46 mg, 0.184 mmol) and benzylhydroxylamine hydrochloride (4a) (35 mg, 0.221 mmol) in a reactor Pyridine (3 ml) was added. The reaction solution was stirred for 1 hour while heating to reflux at 75 ℃. The reaction solution was cooled to room temperature, and then concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1: 5) to obtain the target compound (5a) (50 mg, 74%).

Step 2: (R) -3- (3-((E) -1- Benzyloxyiminoethyl ) Phenyl )-2- Ethoxypropionic acid  Preparation of (1a)

(R) -methyl 3- (3-((E) -1-benzyloxyiminoethyl) phenyl) -2-ethoxypropionate (5a) (50 mg, 0.135 mmol) obtained in step 1 and LiOH. H ₂ O (17 mg, 0.405 mmol) was added to the reactor, THF / water / methanol (3: 1: 1) (10 ml) was added, followed by stirring at room temperature for 2 hours. After completion of the reaction by adding 2N hydrochloric acid to the reaction solution, the reaction solution was concentrated under reduced pressure, the residue was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The target compound (1a) (46 mg, 99%) was obtained.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.53-7.50 (m, 2H), 7.42-7.25 (m, 7H), 5.22 (s, 2H), 4.07 (dd, 1H, J = 8.06, 4.04 Hz), 3.64-3.54 (m, 1H), 3.45-3.35 (m, 1H), 3.14 (dd, 1H, J = 14.1, 4.02 Hz), 3.00 (dd, 1H, J = 14.0, 8.13 Hz), 2.24 (s, 3H), 1.14 (t, 3H, J = 6.96 Hz).

< Example  2> (R) -3- (3-((E) -1- (4- Fluorobenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1b)

Same as Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4b) (34 mg, 0.192 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1b) (14 mg, 53%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.52-7.48 (m, 2H), 7.39-7.22 (m, 4H), 7.06-7.00 (m, 2H), 5.17 (s, 2H), 4.07 (dd, 1H) , J = 8.07, 4.02 Hz), 3.64-3.54 (m, 1H), 3.45-3.35 (m, 1H), 3.14 (dd, 1H, J = 14.0, 4.11 Hz), 3.00 (dd, 1H, J = 14.1 , 8.07 Hz), 2.23 (s, 3H), 1.14 (t, 3H, J = 6.95 Hz).

< Example  3> (R) -3- (3-((E) -1- (4- Methoxybenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1c)

The same procedure as in Example 1, except that (4-fluorobenzyl) hydroxylamine hydrochloride (4c) (51 mg, 0.267 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1c) (34 mg, 42%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.53-7.50 (m, 2H), 7.36-7.25 (m, 4H), 6.90-6.87 (m, 2H), 5.15 (s, 2H), 4.09-4.05 (m , 1H), 3.79 (s, 3H), 3.64-3.54 (m, 1H), 3.46-3.36 (m, 1H), 3.14 (dd, 1H, J = 13.9, 3.84 Hz), 3.00 (dd, 1H, J = 14.1, 8.07 Hz), 2.21 (s, 3H), 1.15 (t, 3H, J = 6.96 Hz).

< Example  4> (R) -3- (3-((E) -1- (4- Bromobenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1d)

Same as Example 1 above except that (4-fluorobenzyl) hydroxylamine hydrochloride (4d) (60 mg, 250 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1d) (52 mg, 60%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.50-7.45 (m, 4H), 7.30-7.25 (m, 4H), 5.16 (s, 2H), 4.08 (dd, 1H, J = 7.80, 4.11 Hz), 3.60-3.52 (m, 1H), 3.47-3.39 (m, 1H), 3.15 (dd, 1H, J = 14.1, 3.84 Hz), 3.00 (dd, 1H, J = 14.1, 7.86 Hz), 2.23 (s, 3H), 1.14 (t, 3H, J = 7.05 Hz).

< Example  5> (R) -3- (3-((E) -1- (4- Chlorobenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1e)

The same procedure as in Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4e) (35 mg, 0.259 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1e) (14 mg, 20%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ): 7.51-7.48 (m, 2H), 7.35-7.25 (m, 6H), 5.18 (s, 2H), 4.07 (dd, 1H, J = 7.77, 3.93 Hz) , 3.61-3.53 (m, 1H), 3.46-3.38 (m, 1H), 3.14 (dd, 1H, J = 14.1, 4.20 Hz), 3.00 (dd, 1H, J = 14.1, 7.86 Hz), 2.23 (s , 3H), 1.14 (t, 3H, J = 6.96 Hz).

< Example  6> (R) -3- (3-((E) -1- (4- Hydroxybenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  (1f) Preparation

Same as Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4f) (32 mg, 0.110 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1f) (18 mg, 58%) was obtained by the method).

¹ H-NMR (300 MHz, CDCl ₃ ) 7.52-7.49 (m, 2H), 7.31-7.20 (m, 4H), 6.82-6.79 (m, 2H), 5.13 (s, 2H), 4.08 (dd, 1H) , J = 7.98, 3.93 Hz), 3.60-3.52 (m, 1H), 3.48-3.38 (m, 1H), 3.15 (dd, 1H, J = 14.1, 3.86 Hz), 3.00 (dd, 1H, J = 14.0 , 7.79 Hz), 2.21 (s, 3H), 1.15 (t, 3H, J = 6.96 Hz).

< Example  7> (R) -3- (3-((E) -1- (4- Trifluoromethylbenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  (1g) Preparation

Same as Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4 g) (130 mg, 0.570 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1 g) (89 mg, 57%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.61-7.48 (m, 6H), 7.30-7.22 (m, 2H), 5.26 (s, 2H), 4.07 (dd, 1H, J = 7.97, 4.13 Hz), 3.63-3.53 (m, 1H), 3.45-3.35 (m, 1H), 3.13 (dd, 1H, J = 14.1, 4.02 Hz), 2.99 (dd, 1H, J = 14.2, 7.97 Hz), 2.26 (s, 3H), 1.13 (t, 3H, J = 6.96 Hz).

< Example  8> (R) -3- (3-((E) -1- (4- Trifluoromethoxybenzyloxyimino ) Ethyl) phenyl) -2- Ethoxypropionic acid  (1h) Preparation

Same as Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4h) (33 mg, 0.134 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1h) (41 mg, 87%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.52-7.49 (m, 2H), 7.43-7.40 (m, 2H), 7.31-7.17 (m, 4H), 5.21 (s, 2H), 4.07 (dd, 1H) , J = 7.79, 4.13 Hz), 3.63-3.53 (m, 1H), 3.47-3.37 (m, 1H), 3.14 (dd, 1H, J = 14.0, 3.93 Hz), 3.00 (dd, 1H, J = 14.0 , 7.98 Hz), 2.24 (s, 3H), 1.14 (t, 3H, J = 6.96 Hz).

< Example  9> (R) -3- (3-((E) -1- (3- Chlorobenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1i)

Same as Example 1 except that (4-fluorobenzyl) hydroxylamine hydrochloride (4i) (76 mg, 0392 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1i) (37 mg, 50.5%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.51-7.48 (m, 2H), 7.38 (m, 1H), 7.30-7.24 (m, 5H), 5.18 (s, 2H), 4.07 (dd, 1H, J = 7.68, 4.02 Hz), 3.59-3.54 (m, 1H), 3.45-3.43 (m, 1H), 3.18-3.12 (m, 1H), 3.03-2.96 (m, 1H), 2.25 (s, 3H), 1.14 (t, 3H, J = 6.96 Hz).

< Example  10> (R) -3- (3-((E) -1- (4- Iodobenzyloxyimino )ethyl) Phenyl )-2- Ethoxypropionic acid  Preparation of (1j)

Same as Example 1 above except that (4-fluorobenzyl) hydroxylamine hydrochloride (4j) (80 mg, 0.280 mmol) obtained in Preparation Example 1 was used instead of benzylhydroxylamine hydrochloride (4a). The target compound (1j) (15 mg, 34%) was obtained by the method.

¹ H-NMR (300 MHz, CDCl ₃ ) 7.68-7.13 (m, 8H), 5.15 (s, 2H), 4.09 (br m, , 3.16-3.13 (br m, IH), 3.00 (br m, IH), 2.23 (s, 3H), 1.14 (t, 3H, J = 6.50 Hz).

< Example  11> (R) -2- Ethoxy -3- (3- (5- (4- Fluorophenyl ) -4,5- Dihydroisoxazole -3 days) Phenyl Preparation of propionic acid (2a)

Step 1: ethyl 2- Ethoxy -3- (3- (4,5- Dihydro -5- Phenylisoxazole -3 days) Phenyl ) Propionate  (8a)

Ethyl 2-ethoxy-3- (3-benzaldehyde- (E) -oxime) propionate (6) (225 mg, 0.848 mmol) and 4-fluorostyrene (7a) obtained in Preparation Example 2 (0.11 ml) , 0.933 mmol) was added to the reactor, CH ₂ Cl ₂ (15 ml) was added, and sodium hypochlorite (10-13% solution, 2 ml) was added thereto, followed by stirring at room temperature for 2 hours. The reaction solution was diluted with CH ₂ Cl ₂ , washed with water, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1: 5) to obtain the title compound (8a) (304 mg, 93%).

¹ H-NMR (300 MHz, CDCl _{3 ,} mixture of rotamers) δ 7.58-7.51 (m, 2H), 7.37-7.30 (m, 4H), 7.04 (t, 2H, J = 8.6 Hz), 5.70 (dd, 1H, J = 10.8, 8.0 Hz), 4.17 (q, 2H, J = 7.1 Hz), 3.99 (dd, 1H, J = 7.9, 5.5 Hz), 3.79-3.70 (m, 1H), 3.65-3.54 (m , 1H), 3.37-3.23 (m, 2H), 3.07-2.94 (m, 2H), 1.22 (t, 3H, J = 7.14 Hz), 1.13 (td, 3H, J = 7.0, 2.8 Hz).

Step 2: 2- Ethoxy -3- (3- (4,5- Dihydro -5- (4- Fluorophenyl ) Isoxazole -3 days) Phenyl Preparation of propionic acid (9a)

Ethyl 2-ethoxy-3- (3- (4,5-dihydro-5- (4- fluorophenyl ) isoxazol-3-yl) phenyl) propionate (8a) ( 300 mg, 0.782 mmol) and LiOH.H ₂ O (98 mg, 2.334 mmol) were added to the reactor, THF / distilled water / methanol (3: 1: 1) (24 ml) was added thereto, followed by stirring at room temperature for 1 hour. . After completion of the reaction by adding 2N hydrochloric acid, the reaction solution was concentrated under reduced pressure, the residue was diluted with ethyl acetate and washed with water. Next, after drying with anhydrous magnesium sulfate and filtering, the filtrate was concentrated under reduced pressure to give the target compound (9a) (276 mg, 100%).

¹ H-NMR (300 MHz, CDCl _{3 ,} mixture of rotamers) δ 7.58-7.52 (m, 2H), 7.37-7.28 (m, 4H), 7.04 (t, 2H, J = 8.6 Hz), 5.70 (dd, 1H, J = 10.8, 8.3 Hz), 4.09 (q, 1H, J = 3.3 Hz), 3.75 (dd, 1H, J = 16.7, 11.0 Hz), 3.66-3.55 (m, 1H), 3.50-3.42 (m , 1H), 3.28 (dd, 1H, J = 16.7, 11.0 Hz), 3.18-2.98. (M, 2H), 1.16 (td, 3H, J = 7.0, 2.9 Hz).

Step 3: (2R) -2- Ethoxy -3- (3- (4,5- Dihydro -5- (4- Fluorophenyl ) Isoxazole -3 days) Phenyl ) -N-((R) -2- Hydroxy -One- Phenylethyl ) Propanamide  Preparation of 10a

2-ethoxy-3- (3- (4,5-dihydro-5- (4- fluorophenyl ) isoxazol-3-yl) phenyl) propionic acid (9a) (311 mg, obtained in step 2) 0.870 mmol), N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDCI) (184 mg, 0.957 mmol), (R)-(-)-2-phenylglycinol ( 409 mg, 2.982 mmol) and 1-hydroxybenzotriazole hydrate (HOBT) (130 mg, 0.957 mmol) were placed in a reactor, and after argon substitution, anhydrous CH ₂ Cl ₂ (15 ml) was added. After cooling the reactor to 0 ° C., diisopropylethylamine (0.17 ml, 0.957 mmol) was slowly added dropwise, the temperature was raised to room temperature and stirred for 12 hours. The reaction was terminated by addition of saturated ammonium chloride solution, diluted with CH ₂ Cl ₂ and washed with water. Next, the mixture was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (2: 1) to obtain the target compound (10a) (139 mg, 34%).

¹ H-NMR (300 MHz, CDCl _{3 ,} mixture of rotamers) δ 7.63-7.58 (m, 1H), 7.44-7.15 (m, 8H), 7.08-6.97 (m, 4H), 5.70-5.56 (m, 1H ), 5.02-4.95 (m, 1H), 3.85-3.80 (m, 2H), 3.72-3.38 (m, 3H), 3.22-3.08 (m, 2H), 3.02-2.91 (m, 1H), 1.27-1.15 (m, 3 H).

Step 4: (R) -2- Ethoxy -3- (3- (4,5- Dihydro -5- (4- Fluorophenyl ) Isoxazole -3 days) Phenyl Preparation of Propionic Acid (11a)

(2R) -2-ethoxy-3- (3- (4,5-dihydro-5- (4- fluorophenyl ) isoxazol-3-yl) phenyl) -N- (obtained in step 3 above. (R) -2-hydroxy-1-phenylethyl) propaneamide (10a) (139 mg, 0.292 mmol) was added to the reactor and 1,4-Dioxane / distilled water (10: 1) (8 ml) was added. , Concentrated sulfuric acid (2 ml) was slowly added dropwise. The reaction solution was heated to reflux at 100 ° C. for 6 hours, then cooled to room temperature and concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a concentrated mixture (137 mg) of the target compound (11a).

Step 5: (R)- methyl  2- Ethoxy -3- (3- (4,5- Dihydro -5- (4- Fluorophenyl ) Isoxazole -3 days) Phenyl ) Propionate  Preparation of (12a)

(R) -2-ethoxy-3- (3- (4,5-dihydro-5- (4- fluorophenyl ) isoxazol-3-yl) phenyl) propionic acid (11a) obtained in step 4 above. Mixture of (137) mg) was added to the reactor, methanol (10 ml) was added, and trimethylsilyl chloride (0.15 ml, 1.149 mmol) was slowly added dropwise. The reaction solution was heated to reflux at 70 ° C. for 3 hours, and then saturated sodium bicarbonate solution was added to terminate the reaction. Then, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, filtered and the filtrate was concentrated under reduced pressure. The concentrated compound was subjected to column chromatography with ethyl acetate: hexane (1: 5) to give the target compound (12a) (91 mg, 84%).

¹ H-NMR (300 MHz, CDCl _{3 ,} mixture of rotamers) δ 7.59-7.52 (m, 2H), 7.37-7.29 (m, 4H), 7.074 (t, 2H, J = 8.6 Hz), 5.70 (dd, 1H, J = 8.2, 10.8 Hz, 4.02 (dd, 1H, J = 5.3, 7.7 Hz), 3.80-3.59 (m, 2H), 3.71 (s, 3H), 3.36-3.23 (m, 2H), 3.06 -2.99 (m, 2H), 1.12 (td, 3H, J = 7.0, 2.7 Hz).

Step 6: (R) -2- Ethoxy -3- (3- (4,5- Dihydro -5- (4- Fluorophenyl ) Isoxazole -3 days) Phenyl Preparation of propionic acid (2a)

(R) -methyl 2-ethoxy-3- (3- (4,5-dihydro-5- (4- fluorophenyl ) isoxazol-3-yl) phenyl) propionate obtained in step 5 above (12a) (92 mg, 0.248 mmol) and LiOH.H ₂ O (31 mg, 0.744 mmol) were added to the reactor, THF / distilled water / methanol (3: 1: 1) (9 ml) was added, followed by 5 at room temperature. Stir for hours. After completion of the reaction by adding 2N hydrochloric acid, the reaction solution was concentrated under reduced pressure, the residue was diluted with ethyl acetate and washed with water. Next, the mixture was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound (2a) (91 mg, 90%).

¹ H-NMR (300 MHz, CDCl _{3 ,} mixture of rotamers) δ 7.60-7.51 (m, 2H), 7.37-7.28 (m, 4H), 7.04 (t, 2H, J = 8.6 Hz), 5.70 (dd, 1H, J = 8.2, 10.8 Hz, 4.09 (q, 1H, J = 3.5 Hz), 3.75 (dd, 1H, J = 16.7-11.0 Hz), 3.66-3.56 (m, 1H), 3.48-3.38 (m 1H), 3.28 (1, 1H, J = 8.4 Hz), 3.17-2.98 (m, 2H), 1.15 (td, 3H, J = 7.0, 2.2 Hz).

< Example  12> (R) -2- Ethoxy -3- (3- (5- (4- Trifluoromethylphenyl ) -4,5- Dihydroisoxazole -3 days) Phenyl Preparation of propionic acid (2b)

 Target compound (2b) (72 mg) was carried out in the same manner as in Example 10, except that 4-trifluoromethylstyrene (7b) (238 mg, 1.388 mmol) was used instead of 4-fluorostyrene (7a). , 14%).

¹ H-NMR (300 MHz, CDCl ₃ ) 7.64-7.48 (m, 6H), 7.36-7.31 (m, 2H), 5.78 (dd, 1H, J = 10.8, 7.89 Hz), 4.09 (dd, 1H, J = 7.50, 4.20 Hz), 3.82 (dd, 1H, J = 16.7, 11.2 Hz), 3.63-3.58 (m, 1H), 3.47-3.42 (m, 1H), 3.29 (dd, 1H, J = 16.7, 7.70 Hz), 3.15 (dd, 1H, J = 14.2, 4.11 Hz), 3.02 (dd, 1H, J = 14.4, 7.41 Hz), 1.16 (dt, 3H, J = 3.18, 7.01 Hz)

< Example  13> (R) -2- Ethoxy -3- (3- (5- (2- Chlorophenyl ) -4,5- Dihydroisooxa -3 days) Phenyl Preparation of propionic acid (2c)

Target compound (2c) (63 mg, 15% by the same method as Example 10 except for using 2-chlorostyrene (7c) (172 mg, 1.241 mmol) instead of 4-fluorostyrene (7a) )

¹ H-NMR (300 MHz, CDCl ₃ ) 7.57-7.52 (m, 3H), 7.39-7.20 (m, 5H), 6.01 (dd, 1H, J = 11.1, 7.05 Hz), 4.07 (dd, 1H, J = 7.88, 4.22 Hz), 3.92 (dd, 1H, J = 16.8, 11.2 Hz), 3.65-3.55 (m, 1H), 3.47-3.37 (m, 1H), 3.23-3.10 (m, 2H), 3.01 ( dd, 1H, J = 14.2, 7.77 Hz), 1.16 (dt, 3H, J = 3.36, 7.05 Hz).

< Example  14> (R) -2- Ethoxy -3- (3- (5- (3- Chlorophenyl 4,5- Dihydroisoxazole -3 days) Phenyl Preparation of propionic acid (2d)

Except for using 4-chlorostyrene (7d) (171 mg, 1.234 mmol) instead of 4-fluorostyrene (7a) was carried out in the same manner as in Example 10 to give the target compound (2d) (63 mg, 15% )

¹ H-NMR (300 MHz, CDCl ₃ ) 7.58-7.52 (m, 2H), 7.37-7.24 (m, 6H), 5.70 (dd, 1H, J = 11.0, 7.86 Hz), 4.10 (dd, 1H, J) = 7.43, 4.31 Hz), 3.78 (dd, 1H, J = 16.7, 11.2 Hz), 3.63-3.55 (m, 1H), 3.51-3.41 (m, 1H), 3.29 (dd, 1H, J = 16.7, 7.86 Hz), 3.16 (dd, 1H, J = 14.1, 4.20 Hz), 3.02 (dd, 1H, J = 14.2, 7.41 Hz), 1.16 (dt, 3H, J = 2.02, 6.95 Hz).

< Experimental Example  1> E- box  And StAR  Screening for selection of active compounds ( screening )

The following experiment was performed to select compounds that modulate E-box mediated transcriptional activity from the compound represented by Formula 1 according to the present invention.

The promoter of the StAR gene, a gene that plays an important role in the synthesis of adrenal steroids, contains a functional E-box sequence, whereby StAR gene expression is a direct transcriptional control of CLOCK and BMAL1, the key genes and transcription factors of biological clock molecules. (Non-patent document 25). Therefore, in the present invention, two kinds of E-box-Luc and StARp-Luc reporters including the E-box sequence were introduced and used as an index for quantitative measurement of transcriptional activity by the core clock gene.

The E-box-Luc reporter is a reporter that combines the SV40 promoter with the E-box sequence present in the mouse StAR promoter sequence to regulate the expression of luciferase, and the StARp-Luc reporter is based on the transcription start position of the mouse StAR gene. It is a reporter that clones a promoter sequence of about 2.5 kb and thus regulates expression of luciferase. The StARp-Luc reporter was used in the screening process as a model of circadian genes expressed in vivo and as another indicator of transcriptional activity by E-box.

Step 1: Establish Reporter Sustained Expression Cell Line

As a preliminary experiment for selecting a transcriptional activity regulating compound by E-box according to the present invention, a cell line stably sustaining expression of two kinds of reporters of E-box-Luc and StARp-Luc was prepared.

The mouse fibroblast-derived cell line NIH3T3 and the adrenal cortex-derived cell line Y1 were used. All cell lines were cultured in Dulbecco's modified eagles medium (invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (invitrogen). An incubator with 5% and 37 degrees Celsius humidification was used. Twenty four hours prior to transfection, 1 × 10 ⁶ NIH3T3 or Y1 cells were aliquoted into 35 mm culture dishes and cultured. Transduction was performed according to Invitrogen's producer manual of lipofectamine, and the following two kinds of plasmids were simultaneously introduced into each cell line. One of the luciferase reporter plasmids was E-box-Luc for the NIH3T3 cell line and StARp-Luc for the Y1 cell line, which takes into account tissue-specific expression. The other one used a plasmid (pcDNA3.1) that confers neomycin resistance to the transduced cell line. The amount of plasmid used for transduction was 600 mg of reporter per culture dish and 200 mg of neomycin resistant plasmid, and all plasmids used for transduction were purified according to the producer's manual of the Plasmid Maxi kit (Qiagen). . Two days after transduction, the neomycin analogue G-418 (invitrogen) was added to the medium at a final concentration of 1 mg / ml and dispensed at 2.5-day intervals during each selection for about three weeks. The concentration of G-418 was sequentially lowered to finally 250 μg / ml. Evaluation of the reporter seating of the resistant cell population was performed using a bioluminescence microscope imaging device (Cellgraph, Atto). For bioluminescence measurement, the culture medium of the resistant cell population was replaced with a medium containing 300 nM luciferin, and then the degree of luminescence of each cell population was imaged at the individual cell level by a high-sensitivity CCD camera to display sufficient light intensity. Were selected. After measurement, the selected populations were separated from the culture dish by treatment with 0.05% trypsin-EDTA solution, and then transferred to a new 35 mm culture dish and incubated. The isolated cultured cells were dispensed in stages of large sized culture incubation in a culture medium containing 250 μg / ml G-418 and finally grown to 10 cm culture dishes. The bioluminescence intensity of the cell line was measured every aliquot to confirm the stability of reporter expression.

Step 2: Primary Active Compound Screening screening )

In order to select the transcriptional activity control compound by the E-box according to the present invention, after treating the compound in the cell line in which the two types of reporters are individually seated in step 1, the change in luciferase activity is measured on the E-box as a measure. Transcriptional activity by quantitatively was evaluated.

Two reporter expressing cell lines were cultured by dispensing into 48 well culture plates 24 hours prior to compound treatment. Compounds in cultured cells were treated in media with a final concentration of 20 μΜ. As a control, cells treated with the same amount of dimethyl sulfoxide (DMSO) were used. After 24 hours of compound treatment, the cell lines were lysed with 100 μL of passive lysis buffer (Promega) per well for 15 minutes, and 20 μL of the sample in the lysate was subjected to the same amount of luciferase activity. After reacting with an assay reagent (Lucifase assay reagent, Promefa), the activity of luciferase was measured by a luminometer (TD-20 / 20 Luminometer, Turner systems). The activity of the measured luciferase was corrected by the protein content of the lysate and the protein content was measured using a Bradford dyeing reagent from Bio-rad. The activity of each compound was converted as a ratio to the average of luciferase activity measured by the control in the same culture plate measured, and the results are shown in FIGS. 1 and 2.

As shown in Figures 1 and 2, the compound showing the activity was evaluated by dividing according to the reinforcing effect or inhibitory effect, and the compound having a 50% diacid change in the reporter activity was selected. 76 compounds exhibited E-box-Luc activity enhancing effect and 182 compounds exhibited StARp-Luc activity enhancing effect. 24 compounds exhibiting E-box-Luc activity inhibiting effect, StARp-Luc activity inhibiting effect The compound which shows was 61 types. Among them, compounds showing significant activity change were used to perform secondary active compound screening.

Step 3: Secondary Active Compound Screening ( screening )

In order to select a compound that modulates E-box-mediated transcriptional activity according to the present invention, a compound showing a significant reporter activity modulation effect was selected in step 2, and the same for E-box-Luc and StARp-Luc. Among the compounds having an effect, a compound having a statistically significant effect with a change range of 50% or more for both reporters was selected, and secondary active compound screening was performed on the selected compound.

In the second screening process, compounds that selectively regulate transcriptional activity in biological clock molecular apparatuses were selected based on the selectivity of the efficacy of the compounds. The efficacy of compounds against mouse Embryonic Fibroblast (MEF) obtained from BMAL1 gene deficient mice with broken biological clock molecular mechanisms can be compared with wild-type cell lines to isolate the efficacy of compounds that depend on biological clock molecular mechanisms. there was.

Embryonic fibroblasts from wild-type mice and BMAL1 gene deficient mice were treated with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin (invitrogen) and 1% L-glutamine. Cultured in (Dulbecco's modified eagles medium, invitrogen). All of them used an incubator in which 5% of carbon dioxide and 37 degrees Celsius humidification state were maintained. Twenty four hours prior to transduction, wild-type or BMAL1 deficient embryonic fibroblasts were cultured in aliquots of 1 × 10 ⁵ / well into 24 well culture plates. Transduction was performed using Metafectene easy (Biotex), and the transduction process was performed according to the manual distributed by the producer. According to the type of plasmid used in the cultured cells divided into two groups was performed transient expression. Group A is E-box-Luc (250 ng / well) and pRL-basic (750 ng / well). Group B is GL3-basic (500 ng / well) and pRL-basic (750 ng / well). well) is a group introduced. All plasmids were purified according to the producer's manual of the Plasmid maxi kit (Qiagen). Three wells of A and B groups were allocated to one compound, respectively, and prepared in the same manner for wild type and BMAL1 deficient embryonic fibroblast lines. After 24 hours of transduction, incubation was performed in a medium to which 100 nM of dexamethasone (DEX) was added for 2 hours to synchronize the intracellular biological clock. Twelve hours after the end of synchronization, the compounds selected in the primary screening group were treated at a concentration of 20 μM, and after 24 hours, the luciferase activity of the cells was determined using a dual-luciferase reporter activity assay system ( luciferase reporter assay system, Promega). Each well was dissolved in 100 µl passive Lysis buffer (Promefa) for 15 minutes and 20 µl of the lysate was reacted with the same amount of Luciferase assay reagent (promefa). The activity of luciferase was then measured by a luminometer (TD-20 / 20 Luminometer, Turner systems). The same amount of Stop & GloR reaction reagent (Stop & GloR reagent, Promega) was added to the dissolved solution, and the activity of Renila luciferase was measured by the same measuring instrument. The change of E-box-Luc activity by the compound was corrected by the well-corrected error of the renilla luciferase by pRL-basic, which was measured independently for each well, and the error between the compound treatment groups was calculated by the same method. Was calibrated using the average of luciferase activity of the GL3-basic group. Finally, the activity of the compound was calculated as the ratio of the average of the E-box-Luc activity values analyzed in the control group treated with the same concentration of dimethyl sulfoxide (DMSO) for the group transduced through the same procedure. The results are shown in FIG. 3, and a comparison of the primary and secondary screening results is shown in FIG. 4.

As shown in Figures 3 and 4, the compounds (1b and 1i) prepared in Examples 2 and 9 have a similar effect and selectivity in the normal embryonic cell line while having an enhanced effect on E-box transcription activity The embryonic cell line from which BMAL1 was removed did not show this effect. This is because the compounds according to the present invention enhance the transcriptional activity of E-box by enhancing CLOCK and BMAL1 protein dimer activities that activate E-box mediated transcription. In addition, when comparing the results of the primary and secondary screening of the compound (1b) and the compound (1i) prepared in Example 2 and Example 9, it can be seen that they are an active substance having an activity to enhance the E-box transcription activity And the structures of the compounds they share play a key role in enhancing transcriptional activity.

Therefore, since the compound according to the present invention has an excellent effect of enhancing the dimer activity of CLOCK: BMAL1, the composition having the active ingredient is useful for the prevention or treatment of circadian-associated diseases such as sleep disorders, metabolic disorders, mood disorders, and cancer. Can be used.

< Experimental Example  2> E- according to concentration box  Active measurement

The following experiment was carried out to investigate the effect of enhancing the transcriptional activity of CLOCK: BMAL1 dimer according to the concentration of 2-ethoxypropionic acid derivative according to the present invention.

E-box-Luc reporter expressing cell lines were isolated and cultured in 24 well plates 24 hours prior to compound treatment. Example 1, Examples 3 to 8, and the compound (1b to 1j) and the structural analogues of Examples 2 to 9 excellent in enhancing the E-box transcription activity in Experimental Example 1 to the cultured cell line, and Example 10 (1a, 1c-1h and 1j) was serially diluted in dimethylsulfoxide solvent, and finally the cells had a concentration of 20 μM, 6.67 μM, 2.22 μM, 0.74 μM, 0.25 μM, 0.082 μM, 0.027 μM As a control, the same amount of dimethyl sulfoxide was treated and used.

After treatment with the compound according to the present invention, after 24 hours, the cells were analyzed using a reporter assay reagent (Promega) in the same manner as in Step 3 of Experimental Example 1, and the Bradford assay was analyzed. Reporter activity was corrected by obtaining the total protein amount. The change in transcriptional activity by treatment of the compound according to the invention is expressed as a percentage of the activity of the control.

Concentration-response correlations were analyzed by converting them into standard curves using Four Parameter Logistic Equation, and the calculation process utilized statistical analysis functions provided by SigmaPlot 8.0, Systat Software Inc. The concentration-response curves are shown in FIGS. 5 and 6, and the maximum activity and EC ₅₀ values of each derivative were calculated based on the concentration-response curves and are shown in Table 1 below.

X Max. (%) EC ₅₀ ([mu] M) Example 1 (1a) -H 193.12 4.02 Example 2 (1b) -F (p) 200.55 1.25 Example 3 (1c) -OMe (p) 221.80 3.79 Example 4 (1d) -Br (p) 209.05 0.33 Example 5 (1e) -Cl (p) 208.10 4.77 Example 6 (1f) -OH (p) 138.88 13.22 Example 7 (1 g) -CF ₃ (p) 143.07 5.68 Example 8 (1h) -OCF ₃ (p) 140.93 8.70 Example 9 (1i) -Cl (m) 192.40 2.22 Example 10 (1j) -I (p) 195.94 1.91

As shown in FIGS. 5 to 6 and Table 1, the compound (1d) of Example 4 has a maximum activity of 209.05%, EC ₅₀ value 0.33 μM was confirmed to be the best when comparing the EC ₅₀ with the maximum activity value It was. Compound (1d) of Example 4 has the lowest EC ₅₀ value, it can be seen that the high activity in a small amount, the activity at this time also has a high effect of the E-box-mediated transcriptional activity of the cell line It can be said that the strengthening effect is large.

Therefore, the compound according to the present invention has an excellent effect of enhancing CLOCK: BMAL1 dimer activity at low concentrations, so the composition having the active ingredient is useful for the prevention or treatment of circadian diseases such as sleep disorders, metabolic disorders, mood disorders, and cancer. Can be used.

< Experimental Example  3> Protein-Compound Bond Measurement

In order to see whether the candidate compound selected in Experimental Example 2 according to the present invention is bound to the biological clock molecular instrument core proteins and its selectivity, the following experiment was performed.

The HEK293T cell line was incubated in Dulbecco's modified eagles medium (invitrogen) with 10% fetal bovine serum and 1% penicillin / streptomycin (invitrogen), and 5% Celsius carbon dioxide. The cells were cultured in an incubator maintained at 37 degrees of humidification. A total of 2 X 10 ⁷ cell lines were isolated and cultured for one protein of the subject 24 hours before the transfection for overexpression of the target protein, and the cultured cells were labeled with a specific peptide for recognition of vital clock genes. 32 μg of each of the plasmids (myc-BMAL1, myc-CLOCK, V5-Per1, V5-Per2, flag-Cry1, flag-Cry2) overexpressed. All plasmids were purified according to the producer's manual of the Plasmid maxi kit (Qiagen). Transfection was performed using lipofectamine (Invitrogen) and the procedure was performed according to the producer's manual. 48 hours after transduction, 2 ml of lysis buffer (50 mM Tris-Hcl) pH 7.4, 2 mM EDTA, 1 mM MgCl ₂ , 0.2% NP-40, 0.1% sodium By adding deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 X protease inhibitor cocktail (P2714, Sigma Aldrich) The cells were homogenized. The homogenized lysate was ground by an ultrasonic generator and centrifuged for 10 minutes at a rate of 7000 g at 4 degrees Celsius. After taking the supernatant from the resultant, 2 ml of binding buffer (100 mM Tris-Hcl) pH 7.4, 300 mM sodium chloride, 0.2% NP-40, 2 mM sodiumol sovanadate , 2 mM sodium fluoride, 2 X protease inhibitor cocktail were added to the bead buffer, 50 mM Tri-Hcl pH 7.4, 150 mM sodium chloride (NaCl). ), Neutravidin agarose resin prepared in 50% slurry in 0.1% NP-40, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 X protease inhibitor mixture ( 200 μl of NeurtoAvidin Agarose Resin, P29700, Thermo Scientific) was added and the reaction was carried out for 1 hour and 30 minutes with slow stirring at 4 ° C. After the pretreatment, the resin was removed by centrifugation and the supernatant 1 4 groups of ml Each compound was treated as follows: A) Dimethylsulfoxide only B) Bait 20 μM C) Bait 20 μM + Compound (1d) 40 μM, D) Bait 20 μM + Compound (1h) 40 μM. The final dimethylsulfoxide concentration of all groups was uniformly adjusted to 0.5%. After reacting for 30 minutes while the compound was treated, 50 μl of Neutravidin Agarose Resin prepared in 50% slurry was added to each group and reacted for 2 hours. After the reaction, the agarose resin was washed six times with 4 ml of bead buffer. The protein bound to the washed resin was dissolved in SDS sample buffer and isolated via 8% SDS-PAGE. The isolated protein was transferred from a PAGE gel to a PVDF membrane (membrane, Immobilin P, Millipore) and settled through an electrophoretic device (Mini Trans-Blot Electrophoretic Transfer Cell, bio-red). Protein-adhered PVDF membranes consisted of Tris-Buffered Saline and Tween-20, 50 mM Tris-HCl, 150 mM in 5% bovine serum albumin (Millpore). Sodium chloride, 0.05% Tween-20, pH 7.6 ~ 7.8) was treated with low speed stirring for 1 hour. After treatment, the cells were washed three times with triaque Tween-20 buffer, and then treated with trisge Tween-20 buffer containing 3% bovine serum albumin, anti-myc (9E10, Santa Cruz Biotechnology) and anti-V5 (V8137, Sigma Aldrich). ), anti-flag (M2, Sigma Aldrich) g antibody was dissolved in a 1: 2000 ratio and the reaction solution was treated with low speed for 1 hour. After treatment, it was washed three times with Tris-based Tween-20 buffer solution, and anti-secondary antibody in which horse radish peroxidase was bound to Tris-based Tween-20 buffer solution containing 3% bovine serum albumin. -Mouse (Jackson ImmunoResearch) or anti-rabbit (Jackson ImmunoResearch) antibodies were treated with a reaction solution in which they were dissolved at a rate of 1: 10000, respectively, and stirred at low speed for 30 minutes. After treatment, the cells were washed three times with Tris-based Tween-20 buffer solution, and the PVDF membrane was then exposed to a medical X-ray film through Enhanced Chemoluminescence (ECL) to visualize the detected target protein. I was. The results are shown in Fig.

As shown in FIG. 7, the physical binding of Bait derived in Example 4 to CLOCK, BMAL1, PER1 and PER2 (PER1 / 2), and CRY1 and CRY2 (CRY1 / 2), which are core clock biological proteins, were examined. As a result, it was confirmed that only CRY1 / 2 binds. The binding between Bait and CRY1 / 2 is greatly inhibited by competition when the compound (1d) of Example 4 according to the present invention is present together, so it can be seen that the compound of Example 4 and CRY1 / 2 are directly bonded. . On the other hand, compound (1h) of Example 8, which is weakly active, does not exhibit this competition, and thus, compound (1d) and derived Bait share a binding site for CRY1 / 2 and also have high selectivity. Is showing.

Therefore, the compound according to the present invention has an excellent effect of inhibiting its activity by binding to CRY1 / 2 among the key factors of the biological clock molecular mechanism, so that the composition having the active ingredient is a circadian disease that regulates the circadian clock rhythm. It can be useful for preventing or treating metabolic disorders, mood disorders, and cancer.

< Experimental Example  4> according to compound treatment CRYs  Dependency verification experiment

In order to verify whether the compound (1d) of Example 4 according to the present invention actually regulates transcriptional activity by E-box via CRY1 / 2, the following experiment was performed.

Comparing embryonic fibroblasts from mice lacking the CRY1 / 2 gene with those of wild-type embryonic fibroblasts, the effect of compound (1d) of Example 4 was expressed by the expression of the CLOCK: BMAL1 subgene with E-box on the promoter. The effect on the activity of and the E-box-Luc reporter was measured. Compound (1d) and compound (1h) of Example 8 and compound (2b) of Example 12 were used as a candidate compound (1d) and a structural analogue and low activity thereof. Compared with control. The activity of the reporter was measured by the same process as in the third step screening, and gene expression patterns were analyzed by real time RT-PCR.

Embryonic fibroblasts from wild-type or CRY1 / 2 deficient mice were treated with Dulbecco's modified eagle medium, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (invitrogen). invitrogen) and incubator in a humidified state of 5% carbon dioxide and 37 degrees Celsius. Twenty four hours before the sample was obtained, two cell lines were cultured by dispensing at a density of 3 × 10 ⁵ / well in 6-well plates. The cultured cells were incubated in a medium containing 100 μM of DEX for 2 hours to synchronize the biological clock mechanism. After 2 hours, the mixture was again exchanged with the original medium for 12 hours, and then the compound was added to the medium at a final concentration of 6.7 μM / ml and then treated for 24 hours. After 24 hours, cells were washed with 1 × di-phosphate buffer (D-PBS) and stored at -70 degrees Celsius or used to extract RNA.

Acid guanidinium thicyanate-phenol-chloroform extraction method was used to extract total RNA in cells (Non-Patent Paper 26). The cryopreserved samples were transferred to 600 μl of D-solution per well (Solution D, 4 M Guaniidinium thiocyanate, 25 mM sodium citrate, 0.5% N-laurosil sarcosine (N). -Laurosylsarcosine), 0.1 M 2-mercaptoethanol, pH 7.0) for 5 minutes and transferred to a 1.5 ml tube. To the solution was added 600 μl acid phenol (pH 4.0), 180 μl isoamyl-chloroform 1:49 isoamyl-chloroform, 90 μl of 2 M sodium acetate (pH 4.0), followed by stirring. (vortex) was used to mix the whole solution for 1 minute at low temperature. The mixed solution was centrifuged for 20 minutes at a speed of 12000 rpm at 4 degrees Celsius, and the supernatant was taken up and shaken up and down by adding 600 μl of isopropanol (isopropanol) and precipitated at -20 degrees Celsius for 1 hour. The precipitated mixture was centrifuged for 20 minutes at 12000 rpm at 4 degrees Celsius and the supernatant was removed. The precipitated pellet was washed by adding 700 μl of 80% ethanol and centrifuging. After washing, ethanol was removed and the pellet was dissolved using 100 μl of distilled water. The concentration of RNA was measured using a spectrophotometer and used for the next reaction. After obtaining 2 μg of RNA from each sample, cDNA was obtained by reverse transcription using MMLV-reverse transcriptase (Reverse transcriptase, Promega, Madison, Wis.). This cDNA was measured using quantitative real-time PCR using SYBR Green I (Sigma-Aldrich). Gene expression levels of Per1, Per2, and Rev-erbA, the biological clock genes regulated by E-box, were corrected by the expression level of TATA-box binding protein (TBP), one of the housekeeping genes. It is expressed as a ratio to the gene levels measured in the control group. The measurement result is shown in FIG.

As shown in FIG. 8, in the experimental results, in the wild embryonic fibroblast cell line treated with the compound of Example 4 (1d), Per1, Per2, Rev regulated by the activity of the E-box-Luc reporter and E-box on the promoter. In the embryonic fibroblast cell line with an increased expression of -erbA, but lacking the CRY1 / 2 gene, an increase in the activity of the E-box-Luc reporter and the expression of Per1, Per2, Rev-erbA due to the compound of Example 4 were observed. Not observed. In the case of the low-active compounds (1h and 2b) synthesized in Examples 8 and 12, there is no effect of enhancing E-box transcriptional activity. From this, the compound (1d) of Example 4 did not directly bind and enhance the activity of CLOCK: BMAL1, but rather inhibited its inhibitory activity by binding to CRY1 / 2, a negative feedback factor, resulting in the CLOCK: BMAL1 dimer. It can be seen that it has an effect of enhancing transcriptional activity.

Therefore, the compound according to the present invention not only has an excellent effect of binding to CRY1 / 2 and inhibiting its activity, but also has an excellent effect of enhancing the transcriptional activity of the dimer of CLOCK: BMAL1, thereby making the composition an active ingredient. It can be usefully used for the prevention or treatment of circadian diseases such as sleep disorders, metabolic disorders, mood disorders and cancer.

Examples of preparations for the therapeutic agents of the present invention are illustrated below.

< Formulation example > Preparation of Pharmaceutical Formulations for Oral Administration

1. Manufacturing of powder

2 g of 2-ethoxypropionic acid derivative

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

100 mg of 2-ethoxypropionic acid derivative

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

100 mg of 2-ethoxypropionic acid derivative

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

10 ug / ml 2-ethoxypropionic acid derivative

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 ml

The 2-ethoxypropionic acid derivative was dissolved in an appropriate volume of sodium chloride BP for injection, and the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted with sodium chloride BP for injection and thoroughly mixed. . The solution was filled into a 5 ml Type I ampoule made of clear glass, dissolved under glass, enclosed under an upper grid of air, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

Claims (10)

A pharmaceutical composition for preventing or treating a cycle-associated disease comprising a 2-ethoxypropionic acid derivative represented by Formula 1 or an acceptable salt thereof;
[Chemical Formula 1]

(In Formula 1,
X is hydrogen; halogen; C1 to C4 straight or branched chain alkyl unsubstituted or substituted with one or more halogens; Hydroxy; Or unsubstituted C1 to C4 straight or branched alkoxy, wherein X is substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;
Z is

or

Z is substituted at either the meta or para position of the aromatic ring; R ₁ is hydrogen or unsubstituted C1 to C4 straight or branched alkyl; And
n is the Z

When is an integer of 1 to 5, Z is

Is an integer of 0 to 5.)
The method of claim 1,
X is hydrogen; Fluoro; Chloro; Bromo; Iodo; methyl; ethyl; profile; Isopropyl; Butyl; Isobutyl; t-butyl; Trifluoromethyl; Trichloromethyl; Fluoromethyl; Chloromethyl; Hydroxy; Methoxy; Ethoxy; Propoxy; Butoxy; Or trifluoromethoxy, wherein X may be substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;
Z is

or

Z is substituted at either the meta or para position of the aromatic ring; R ₁ is hydrogen; methyl; ethyl; profile; Isopropyl; Butyl; Isobutyl or t-butyl; And
n is the Z

When is an integer of 1 to 3, Z is

In the case of the pharmaceutical composition for the prevention or treatment of circadian-associated diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof, characterized in that an integer of 0 to 2.

The method of claim 1,
X is hydrogen; Fluoro; Chloro; Bromo; Iodo; Trifluoromethyl; Hydroxy; Methoxy or trifluoromethoxy, wherein X is substituted at any one or two or more of the ortho, meta or para positions of the aromatic ring;
Z is

or

Z is substituted at either the meta or para position of the aromatic ring; R ₁ is methyl, ethyl or propyl; And
n is the Z

When is an integer of 1 to 3, Z is

In the case of the pharmaceutical composition for the prevention or treatment of circadian-associated diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof, characterized in that an integer of 0 to 2.
The method of claim 1,
2-ethoxypropionic acid derivative of Formula 1 is:
1) (R) -3- (3-((E) -1-benzyloxyiminoethyl) phenyl) -2-ethoxypropionic acid;
2) (R) -3- (3-((E) -1- (4-fluorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
3) (R) -3- (3-((E) -1- (4-methoxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
4) (R) -3- (3-((E) -1- (4-bromobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
5) (R) -3- (3-((E) -1- (4-chlorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
6) (R) -3- (3-((E) -1- (4-hydroxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
7) (R) -3- (3-((E) -1- (4-trifluoromethylbenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
8) (R) -3- (3-((E) -1- (4-trifluoromethoxybenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
9) (R) -3- (3-((E) -1- (3-chlorobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
10) (R) -3- (3-((E) -1- (4-iodobenzyloxyimino) ethyl) phenyl) -2-ethoxypropionic acid;
11) (R) -2-ethoxy-3- (3- (5- (4-fluorophenyl) -4,5-dihydroisoxazol-3-yl) phenyl) propionic acid;
12) (R) -2-ethoxy-3- (3- (5- (4-trifluoromethylphenyl) -4,5-dihydroisoxazol-3-yl) phenyl) propionic acid;
13) (R) -2-ethoxy-3- (3- (5- (2-chlorophenyl) -4,5-dihydroisooxa-3-yl) phenyl) propionic acid; And
14) (R) -2-ethoxy-3- (3- (5- (3-chlorophenyl) 4,5-dihydroisoxazol-3-yl) phenyl) propionic acid
A pharmaceutical composition for preventing or treating circadian-associated diseases comprising a 2-ethoxypropionic acid derivative or an acceptable salt thereof, which is any one selected from the group consisting of:
The method of claim 1,
The circulatory related diseases are sleep disorders, metabolic disorders, mood disorders or cancer, the pharmaceutical composition for preventing or treating circadian related diseases, characterized in that.
6. The method of claim 5,
The sleep disorder is parallax syndrome, shift work sleep disorder, progressive sleep phase syndrome or sleep phase delay syndrome, characterized in that the pharmaceutical composition for the prevention or treatment of sleep disorder diseases.
6. The method of claim 5,
The metabolic disorders are obesity, hyperlipidemia, hyperglycemia or polyuria is a pharmaceutical composition for preventing or treating sleep disorders, characterized in that.
6. The method of claim 5,
The mood disorder is a depressive disorder, bipolar disorder or seasonal disorders characterized in that the prevention or treatment of the mood disorders pharmaceutical composition.
6. The method of claim 5,
The cancer is lung cancer, breast cancer, uterine cancer, pancreatic cancer or lymph cancer, characterized in that the pharmaceutical composition for preventing or treating cancer.
The 2-ethoxypropionic acid derivative represented by Chemical Formula 1 of claim 1 prevents the activity of CRYs, which is a transcription factor of a biological clock molecule network, and prevents circadian-associated diseases, wherein the activity of CLOCK: BMAL1 dimer is enhanced. Therapeutic pharmaceutical composition.

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