KR20200090649A - Oxyiminomethylbenzene derivatives - Google Patents

Abstract

The present invention is an oxyiminomethylbenzene derivative. More specifically, the present invention relates to an oxyiminomethylbenzene derivative which reinforces amplitude of the circadian rhythm and stabilizes rhythm through the activity of CRYs, a transcriptional regulator of a transcriptional regulator of the circadian biological clock molecular network and the reinforcement of the activity of CLOCK:BMAL1 dimer.

Description

Oxyminomethylbenzene derivatives {Oxyiminomethylbenzene derivatives}

The present invention is an oxyiminomethylbenzene derivative, which enhances the amplitude of the circadian biorhythm and stabilizes the cycle by inhibiting the activity of CRYs, a transcriptional regulator of the circadian bioclock molecular network, and enhancing the activity of the CLOCK:BMAL1 dimer. It relates to an oxyiminomethylbenzene derivative.

Like all living things on Earth, humans have a circadian rhythm that is aligned with a 24-hour cycle in response to 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 sugar levels and other metabolic activity in the body, and cell division cycles are also regulated by this circadian rhythm. When these circadian rhythms are damaged due to genetic or environmental factors, it can be a cause of various physiological or psychological abnormalities, which are called circadian rhythm-related disorders. In modern industrialized society in particular, the majority of people are exposed to various environmental biological clock disturbances, such as excessive night lighting, excessive night activities such as shift work, and abnormal dietary forms. Research is adding to its importance.

The organism has a gene related to the control of the circadian rhythm, and the circadian rhythm control gene regulates the circadian rhythm. Recent studies have identified a molecular network of bioclocks that governs circadian rhythms. The mammalian circadian rhythm control genes include Period (PER) genes (Per1, Per2, Per3), CLOCK genes, BMAL genes (Bmal1, Bmal2), Cryptochrome (CRY) genes (Cry1, Cry2), and REV-ERB genes (Rev -erbα, Rev-erbβ), ROR genes (Rora, Rorb, Rorc), etc. are known, and they form a molecular network. In particular, the dimer of the CLOCK and BMAL1 proteins at the top level binds to the E-box, a cis-element present in the promoters of the PER and CRY genes, the feedback genes located at the lower level as transcription factors, and triggers its expression. A series of molecular cyclic structures in which products inhibit the high-level transcription factors form the core of the circadian molecular bioclock, which is called the bioclock core loop. These molecular networks are known to influence various physiological processes by triggering the circadianity of genes regulated below (Non-Patent Document 1). In addition, the molecular networks of these circadian bioclock genes are found in various peripheral organs, as well as in the suprachiasmatic nucleus (SCN), where the central biological clock of the mammal is located, and the optic nerve crossing in cells of these peripheral organs. It has a unique circadian rhythm and is periodically expressed in a similar pattern to the nucleus. The network of biological clock molecules expressed in various peripheral organs is referred to as a peripheral or local clock in contrast to a central biological clock (non-patent document 1).

Since the biological clock is a general-purpose control mechanism that controls overall behavior and physiology, the types of circadian-related diseases are also very difficult including sleep disorders, cancer, metabolic diseases, cardiovascular diseases, immune and inflammatory diseases, mood disorders, addiction disorders, and degenerative brain diseases Extensive.

In fact, emergency workers who alternately work by changing the normal 24-hour rhythm of weather and sleep are at risk of various diseases including metabolic disorders such as diabetes (non-patent document 2), and are exposed to long-term shift work. Through epidemiological studies of nurses, it has been reported that the incidence of breast cancer is higher than the normal population in the number and intensity of shift work (Non-Patent Document 3).

There have been many studies on the causes and treatments of circadian-related diseases caused by various environmental and genetic bioclock disturbances, and one of them is to regulate circadian rhythms through phosphorylation of key bioclock genes. Since it is known that the mammalian kinase casein kinase Iε (CKIε) and casein kinase Iδ (CKIδ) phosphorylate the core clock biomolecule to regulate circadian rhythms, research to control circadian rhythms targeting CKI proteins is underway (Non-Patent Document 4). However, the effect is minimal because inhibiting the CKI gene does not directly affect the bioclock molecular network.

Another study is a study of Rev-erbα/β, a gene constituting an auxiliary loop, and an agonist and an antagonist for REV-ERBα/β that suppresses the expression of BMAL1. By adjusting the circadian rhythm through (Non-patent Document 5), the main role of REV-ERBα/β is to slow the activity of the CLOCK:BMAL1 dimer in the bioclock molecular network and make the fine adjustment necessary for the clock to work properly. There are difficulties in effectively adjusting the circadian rhythm.

Recently, it has been found that KS15, a type of 2-ethoxypropionic acid derivative, can increase the transcriptional activity of the CLOCK:BMAL1 dimer through inhibition of the CRY protein, and has been confirmed to weaken the circadian molecular biorhythm. (Non-Patent Document 6).

Therefore, at the present time when the physiological and clinical importance of circadian rhythms is highlighted, there is an urgent need to develop a drug that can directly and effectively promote a biological clock molecular network for the prevention and treatment of various circadian rhythm-related diseases. to be.

Schirmacher A, Hor H, Heidbreder A, Happe S, Kelsch R, Kuhlenbaumer G, Meißa Wisor JP, O'Hara BF, Terao A, Selby CP, Kilduff TS, Sancar A, Edgar DM, Franken P. A role for cryptochromes in sleep regulation. BMC Neurosci. (2002) 3: 20. Trachsel L, Edgar DM, Seidel WF, Heller HC, Dement WC. Sleep homeostasis in suprachiasmatic nuclei-lesioned rats: effects of sleep deprivation and triazolam administration. Brain Res. (1992) 589(2): 253-261. Spiegel K, Tasali E, Leproult R, Van Cauter E. Effects of poor and short sleep on glucose metabolism and obesity risk. Nat Rev Endocrinol. (2009) 5(5): 253-261. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolic syndrome in circadian Clock mutant mice. Science. (2005) 308(5724): 1043-1045. Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheral tissue circadian clock. Proc Natl Acad Sci U S A. (2008) 105(39): 15172-15177. 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Moon JH, Cho CH, Son GH, Geum D, Chung S, Kim H, Kang SG, Park YM, Yoon HK, Kim L, Jee HJ, An H, Kripke DF, Lee HJ. Advanced Circadian Phase in Mania and Delayed Circadian Phase in Mixed Mania and Depression Returned to Normal after Treatment of Bipolar Disorder. EBioMedicine. (2016) 11:285-295. Menculini G, Verdolini N, Murru A, Pacchiarotti I, Volpe U, Cervino A, Steardo L, Moretti P, Vieta E, Tortorella A. Depressive mood and circadian rhythms disturbances as outcomes of seasonal affective disorder treatment: A systematic review. J Affect Disord. (2018) 241: 608-626. Hood S, Amir S. Neurodegeneration and the Circadian Clock. Front Aging Neurosci. (2017) 9: 170. Musiek ES, Lim MM, Yang G, Bauer AQ, Qi L, Lee Y, Roh JH, Ortiz-Gonzalez X, Dearborn JT, Culver JP, Herzog ED, Hogenesch JB, Wozniak DF, Dikranian K, Giasson BI, Weaver DR, Holtzman DM, Fitzgerald GA. Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration. J Clin Invest. (2013) 123(12): 5389-5400. 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A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells. Biochem Biophys Res Commun. (2015) 467(2): 441-446. Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A. (2004) 101(15): 5339-5346. Stratmann M, Schibler U. Properties, entrainment, and physiological functions of mammalian peripheral oscillators. J Biol Rhythms. (2006) 21(6): 494-506. Scheer FA, Hilton MF, Mantzoros CS, Shea SA. Adverse metabolic and cardiovascular consequences of circadian misalignment. Proc Natl Acad Sci US A. (2009) 106(11): 4453-4458. Schernhammer ES, Laden F, Speizer FE, Willett WC, Hunter DJ, Kawachi I, Colditz GA. Rotating night shifts and risk of breast cancer in women participating in the nurses' health study. J Natl Cancer Inst. (2001) 93(20): 1563-1568. Chen Z, Yoo SH, Takahashi JS. Development and Therapeutic Potential of Small-Molecule Modulators of Circadian Systems. Annu Rev Pharmacol Toxicol. (2018) 58: 231-252. Kojetin DJ, Burris TP. REV-ERB and ROR nuclear receptors as drug targets. Nat Rev Drug Discov. (2014) 13(3): 197-216. Chun SK, Jang J, Chung S, Yun H, Kim NJ, Jung JW, Son GH, Suh YG, Kim K. Identification and validation of cryptochrome inhibitors that modulate the molecular circadian clock. ACS Chem Biol. (2014) 9(3): 703-710. Son GH, Chung S, Choe HK, Kim HD, Baik SM, Lee H, Lee HW, Choi S, Sun W, Kim H, Cho S, Lee KH, Kim K. Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production. Proc Natl Acad Sci US A. (2008) 105(52): 20970-20975.

Schirmacher A, Hor H, Heidbreder A, Happe S, Kelsch R, Kuhlenbaumer G, Meißa Wisor JP, O'Hara BF, Terao A, Selby CP, Kilduff TS, Sancar A, Edgar DM, Franken P. A role for cryptochromes in sleep regulation. BMC Neurosci. (2002) 3: 20. Trachsel L, Edgar DM, Seidel WF, Heller HC, Dement WC. Sleep homeostasis in suprachiasmatic nuclei-lesioned rats: effects of sleep deprivation and triazolam administration. Brain Res. (1992) 589(2): 253-261. Spiegel K, Tasali E, Leproult R, Van Cauter E. Effects of poor and short sleep on glucose metabolism and obesity risk. Nat Rev Endocrinol. (2009) 5(5): 253-261. Turek FW, Joshu C, Kohsaka A, Lin E, Ivanova G, McDearmon E, Laposky A, Losee-Olson S, Easton A, Jensen DR, Eckel RH, Takahashi JS, Bass J. Obesity and metabolic syndrome in circadian Clock mutant mice . Science. (2005) 308 (5724): 1043-1045. Lamia KA, Storch KF, Weitz CJ. Physiological significance of a peripheral tissue circadian clock. Proc Natl Acad Sci US A. (2008) 105(39): 15172-15177. Rudic RD, McNamara P, Curtis AM, Boston RC, Panda S, Hogenesch JB, Fitzgerald GA. BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol. (2004) 2(11): e377. Shimba S, Ishii N, Ohta Y, Ohno T, Watabe Y, Hayashi M, Wada T, Aoyagi T, Tezuka M. Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis. Proc Natl Acad Sci US A. (2005) 102(34):12071-12076. Bur IM, Cohen-Solal AM, Carmignac D, Abecassis PY, Chauvet N, Martin AO, van der Horst GT, Robinson IC, Maurel P, Mollard P, Bonnefont X. The circadian clock components CRY1 and CRY2 are necessary to sustain sex dimorphism in mouse liver metabolism. J Biol Chem. (2009) 284(14): 9066-9073. Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, Nusinow DA, Sun X, Landais S, Kodama Y, Brenner DA, Montminy M, Kay SA. Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Nat Med. (2010) 16(10):1152-1156. Gouin JP, Connors J, Kiecolt-Glaser JK, Glaser R, Malarkey WB, Atkinson C, Beversdorf D, Quan N. Altered expression of circadian rhythm genes among individuals with a history of depression. J Affect Disord. (2010) 126(1-2): 161-166. McClung CA, Sidiropoulou K, Vitaterna M, Takahashi JS, White FJ, Cooper DC, Nestler EJ. Regulation of dopaminergic transmission and cocaine reward by the Clock gene. Proc Natl Acad Sci US A. (2005) 102(26): 9377-9381. Chung S, Lee EJ, Yun S, Choe HK, Park SB, Son HJ, Kim KS, Dluzen DE, Lee I, Hwang O, Son GH, Kim K. Impact of circadian nuclear receptor REV-ERBα on midbrain dopamine production and mood regulation. Cell. (2014) 157(4): 858-868. Schnell A, Sandrelli F, Ranc V, Ripperger JA, Brai E, Alberi L, Rainer G, Albrecht U. Mice lacking circadian clock components display different mood-related behaviors and do not respond uniformly to chronic lithium treatment. Chronobiol Int. (2015) 32(8): 1075-1089. Moon JH, Cho CH, Son GH, Geum D, Chung S, Kim H, Kang SG, Park YM, Yoon HK, Kim L, Jee HJ, An H, Kripke DF, Lee HJ. Advanced Circadian Phase in Mania and Delayed Circadian Phase in Mixed Mania and Depression Returned to Normal after Treatment of Bipolar Disorder. EBioMedicine. (2016) 11:285-295. Menculini G, Verdolini N, Murru A, Pacchiarotti I, Volpe U, Cervino A, Steardo L, Moretti P, Vieta E, Tortorella A. Depressive mood and circadian rhythms disturbances as outcomes of seasonal affective disorder treatment: A systematic review. J Affect Disord. (2018) 241: 608-626. Hood S, Amir S. Neurodegeneration and the Circadian Clock. Front Aging Neurosci. (2017) 9: 170. Musiek ES, Lim MM, Yang G, Bauer AQ, Qi L, Lee Y, Roh JH, Ortiz-Gonzalez X, Dearborn JT, Culver JP, Herzog ED, Hogenesch JB, Wozniak DF, Dikranian K, Giasson BI, Weaver DR, Holtzman DM, Fitzgerald GA. Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration. J Clin Invest. (2013) 123(12): 5389-5400. Lauretti E, Di Meco A, Merali S, Pratico D. Circadian rhythm dysfunction: a novel environmental risk factor for Parkinson's disease. Mol Psychiatry. (2017) 22(2): 280-286. Gorbacheva VY, Kondratov RV, Zhang R, Cherukuri S, Gudkov AV, Takahashi JS, Antoch MP. Circadian sensitivity to the chemotherapeutic agent cyclophosphamide depends on the functional status of the CLOCK/BMAL1 transactivation complex. Proc Natl Acad Sci US A. (2005) 102(9): 3407-3412. Ozturk N, Lee JH, Gaddameedhi S, Sancar A. Loss of cryptochrome reduces cancer risk in p53 mutant mice. Proc Natl Acad Sci US A. (2009) 106(8): 2841-2846. van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, Takao M, de Wit J, Verkerk A, Eker AP, van Leenen D, Buijs R, Bootsma D, Hoeijmakers JH, Yasui A. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature. (1999) 398 (6728): 627-630. Chun SK, Chung S, Kim HD, Lee JH, Jang J, Kim J, Kim D, Son GH, Oh YJ, Suh YG, Lee CS, Kim K. A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells. Biochem Biophys Res Commun. (2015) 467(2): 441-446. Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci US A. (2004) 101(15): 5339-5346.

Against this background, the present inventors are developing a drug that directly acts on the molecular bioclock backbone through a cell-based test method that measures the activity of the dimer of CLOCK:BMAL1, while oxyiminomethylbenzene derivative compounds act on CRY. The present invention was completed by confirming that there is an effect of enhancing the circadian activity of the biological clock molecular network by suppressing it.

Accordingly, an object of the present invention is to provide an oxyiminomethylbenzene derivative compound prepared using an organic synthesis technique.

However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those having ordinary knowledge in the art from the following description.

According to an embodiment of the present invention, an oxyiminomethylbenzene derivative represented by the following Chemical Formula 1 is provided.

[Formula 1]

(In the above formula 1, R ¹ is a substituted or unsubstituted phenyl group connected by C1-C3 alkyl, wherein the substituent is hydrogen, halogen, cyano, hydroxy, NO ₂ , C1-6 haloalkyl, C1-6 alkoxy, Represents 1 to 4 substituents selected from the group consisting of C 1-6 haloalkoxy; R ² is a methyl group; A, E is hydrogen; B, C, D are each independently COOH, COOC 1-6 alkyl, COO (pyridine- NHC1-6alkyl) or C1 alkoxy substituted with COO (histamine), or hydrogen.)

According to one side, the oxyiminomethylbenzene derivative enhances the amplitude of the circadian biorhythm by inhibiting the activity of CRYs, a transcriptional regulator of the circadian bioclock molecular network, and enhancing the activity of the CLOCK:BMAL1 dimer, and It may be to stabilize.

The oxyiminomethylbenzene derivative of the present invention can effectively promote the function of the bioclock molecular network by inhibiting the action of CRY to promote the activity of the CLOCK:BMAL1 dimer. Accordingly, the pharmaceutical composition comprising the oxyiminomethylbenzene derivative of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is a sleep-related disorder, metabolic disease, cardiovascular disease, immune and inflammatory disease, mood disorder, It can be useful for the prevention or treatment of degenerative brain disease and cancer.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a concentration of the compounds of formulas 8-9, 9-9, 8-10, and 9-10 in oxyiminomethylbenzene derivatives on luciferase reporter (E-box-LUC) measuring the activity of the CLOCK:BMAL1 dimer As a result of evaluating the dependent effect, the compounds of Formulas 8-9 and 8-10 show the results of increasing the activity of E-box-LUC in a concentration-dependent manner.
2 is an increase in E-box-LUC reporter activity by CLOCK:BMAL1 dimer (C/B) is inhibited by overexpression of CRY1, and inhibition of CLOCK:BMAL1 activity by CRY1 is among oxyiminomethylbenzene derivatives by chemical formula Results showing concentration-dependent recovery by treatment of compounds 8-9 and 8-10 are shown.
FIG. 3 shows the effect of the compounds of formulas 8-9 and 8-10 among oxyiminomethylbenzene derivatives on the cyclic expression of the PER2-LUC fusion protein observed in mouse fibroblast lines. It is the result of evaluation compared to the material KS15. Figure 3 a is to confirm the effect of the compound of 8-9, 8-10 and KS15 on the cyclic expression of PER2-LUC fusion protein, b of Figure 3 is a compound of 8-9, 8-10 and KS15 The results of statistical analysis of the effects of PER2-LUC fusion protein expression on the circadian amplitude (Amplitude, left), period (Period, middle) and stability (Robustness, right).
4 is a result showing the effect of suppressing obesity represented by the compound of Formula 8-9 in a process in which obesity is induced by supplying a high-fat diet (HFD) for 10 weeks to mice. In the process, the compound of Formula 8-9 was administered at a dose of 10 mg/kg, once every 2 days, by intraperitoneal injection to ZT06, and was administered for 8 weeks from the 3rd week of supplying a high-lipid diet, and controls (Vehicle, VEH) ) Is the same volume (200 μl) of saline containing 0.1% DMSO.
4 a is the appearance of mice receiving a diet of high lipid diet for 10 weeks and a compound of formula 8-9 for 8 weeks, and b is a diet of high lipid diet and administration of a compound of formula 8-9 for the body weight of mice. The effect is shown weekly, and c shows the weight gain from the start of the administration of the compound of Formula 8-9 to the end of the administration for each experimental group.
FIG. 5 is a quantitative RT-PCR technique for expressing the expression patterns of bioclock genes (Bmal1, Npas2, Rev-erbα, Per2) targeting SCN at the time of ZT02 and ZT12 the day after the final administration day after the same process as described in FIG. 4. It was measured by using and it is shown for each treatment group: TBP (TATA-binding protein) mRNA was used as an internal control for quantitative analysis.
FIG. 6 shows the results of sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT) as an animal behavior test between ZT04 and ZT08 the day after the final administration day after the same process as described in FIG. 4 above. It is shown by treatment group.

Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and the scope of the patent application right is not limited or limited by these embodiments. It should be understood that all modifications, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are used for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, the terms "include" or "have" are intended to indicate the presence of features, numbers, steps, actions, components, parts or combinations thereof described in the specification, one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed descriptions will be omitted.

According to an embodiment of the present invention, an oxyiminomethylbenzene derivative represented by the following Chemical Formula 1 is provided.

[Formula 1]

(In the formula 1, R ¹ is a substituted or unsubstituted phenyl group connected by C1-C3 alkyl, wherein the substituent is hydrogen, halogen, cyano, hydroxy, NO ₂ , C1-6 haloalkyl, C1-6 alkoxy, Represents 1 to 4 substituents selected from the group consisting of C 1-6 haloalkoxy; R ² is a methyl group; A, E is hydrogen; B, C, D are each independently COOH, COOC 1-6 alkyl, COO (pyridine- NHC1-6 alkyl) or C1 alkoxy substituted with COO (histamine), or hydrogen.)

More specifically, the oxyiminomethylbenzene derivative represented by Chemical Formula 1 is

1) methyl {3-[N-{[3-(trifluoromethyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetate;

2) {3-[N-{[3-(trifluoromethyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetic acid;

3) methyl (3-{N-[(4-methoxyphenyl)methoxy]ethanimidoyl}phenoxy)acetate;

4) (3-{N-[(4-methoxyphenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

5) methyl (3-{N-[(4-bromophenyl)methoxy]ethanimidoyl}phenoxy)acetate;

6) (3-{N-[(4-bromophenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

7) 2-{3-[N-(4-bromophenyl)methoxyethanimidoyl]phenoxy}-N-[(pyridin-2-yl)methyl]acetamide;

8) 2-{3-[N-(4-bromophenyl)methoxyethanimidoyl]phenoxy}-N-[2-(1H-imidazol-2-yl)ethyl]acetamide;

9) methyl (3-{N-[(4-nitrophenyl)methoxy]ethanimidoyl}phenoxy)acetate;

10) (3-{N-[(4-nitrophenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

11) methyl {3-[N-{[4-(methanesulfonyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetate;

12) {3-[N-{[4-(methanesulfonyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetic acid;

13) methyl (3-{(N-[(4-fluorophenyl)methoxy]ethanimidoyl}phenoxy)acetate;

14) (3-{N-[(4-fluorophenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

15) methyl {4-[N-{[3-(trifluoromethyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetate;

16) {4-[N-{[3-(trifluoromethyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetic acid;

17) methyl (4-{N-[(4-methoxyphenyl)methoxy]ethanimidoyl}phenoxy)acetate;

18) (4-{N-[(4-methoxyphenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

19) methyl (4-{N-[(4-bromophenyl)methoxy]ethanimidoyl}phenoxy)acetate;

20) (4-{N-[(4-bromophenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

21) 2-{4-[N-(4-bromophenyl)methoxyethanimidoyl]phenoxy}-N-[(pyridin-2-yl)methyl]acetamide;

22) 2-{4-[N-(4-bromophenyl)methoxyethanimidoyl]phenoxy}-N-[2-(1H-imidazol-2-yl)ethyl]acetamide;

23) methyl (4-{N-[(4-nitrophenyl)methoxy]ethanimidoyl}phenoxy)acetate;

24) (4-{N-[(4-nitrophenyl)methoxy]ethanimidoyl}phenoxy)acetic acid;

25) methyl {4-[N-{[4-(methanesulfonyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetate; And

26) {4-[N-{[4-(methanesulfonyl)phenyl]methoxy}ethanimidoyl]phenoxy}acetic acid;

It may be any one compound selected from the group consisting of.

The oxyiminomethylbenzene derivative represented by Formula 1 of the present invention includes pharmaceutically acceptable salts, hydrates and solvates thereof.

In the present specification, when referring to the compound represented by Formula 1, it may be understood that it is intended to include their pharmaceutically acceptable salts, and these pharmaceutically acceptable salts are commonly used as a method for preparing salts. Can be produced by.

The'pharmaceutically acceptable salt' refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid comprising an inorganic or organic base and an inorganic or organic acid. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese salts, manganese, potassium, sodium, zinc, and the like. Particularly preferred are ammonium, calcium, magnesium, potassium or sodium salts. Solid salts may exist in more than one crystal structure, 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, Hydrabamine, isopropyl amine, lysine, methylglucamine, popolin, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Basic ion exchange resin.

When the compounds of the present invention are basic, salts can 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, isethionic 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-toluene sulfonic acid and the like. Citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, fumaric acid or tartaric acid are particularly preferred.

It is to be understood that the hydrate of the oxyiminomethylbenzene derivative compound represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof contains a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Can. The hydrate may contain 1 equivalent or more, generally 1 to 5 equivalents of water. Such hydrates can be prepared by crystallizing a compound represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

It is understood that the solvate of the oxyiminomethylbenzene derivative compound represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof contains a stoichiometric or non-stoichiometric amount of a solvent that is bound by a non-covalent intermolecular force. Can be. Preferred as the solvent include volatile, non-toxic or solvents suitable for administration to humans.

The pharmaceutical composition of the present invention contains the oxyiminomethylbenzene derivative compound represented by Chemical Formula 1 as an active ingredient, and is added in a conventional non-toxic pharmaceutically acceptable carrier, adjuvant, excipient, etc. Phosphorus formulations, for example, tablets, capsules, troches, liquids, suspensions, or the like, for oral administration or parenteral administration.

Excipients that can be used in the pharmaceutical composition of the present invention may include sweeteners, binders, solubilizers, solubilizers, wetting agents, emulsifiers, isotonic agents, adsorbents, disintegrants, antioxidants, preservatives, lubricants, fillers, fragrances, etc. . Examples include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, silica, talc, stearic acid, sterin, magnesium stearate, magnesium aluminum silicate, starch, gelatin, tragacanth rubber, arginic acid, sodium And alginate, methylcellulose, sodium carboxymethylcellulose, agar, water, ethanol, polyethylene glycol, polyvinylpyrrolidone, sodium chloride, calcium chloride, orange essence, strawberry essence, and vanilla flavor.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, and activity of the drug , The sensitivity to the drug, the time of administration, the route of administration and rate of excretion, the duration of treatment, factors including the drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, condition, weight, absorption of active ingredients in the body, inactivation rate and excretion rate, disease type, and drugs used in general. As 0.1 mg/kg to 100 mg/kg per day, preferably, it may be administered in an amount of 1 to 30 mg/kg, and may be administered once or several times a day.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. Any mode of administration can be expected, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrathecal dura or cerebral vascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

The pharmaceutical composition of the present invention can inhibit the activity of the CRY molecule, thereby enhancing the circadianity of the molecular bioclock backbone. Therefore, the pharmaceutical composition of the present invention includes an oxyiminomethylbenzene derivative compound and a pharmaceutically acceptable salt thereof as an active ingredient, thereby suppressing the CRY activity possessed by the derivative or the circadian molecular bioclock backbone hypertonic effect. Through this, it can be useful for the treatment of circadian-related diseases.

The circadian-related diseases may include, but are not limited to, sleep disorders, metabolic disorders, cardiovascular disorders, immune and inflammatory disorders, mood disorders, addiction disorders, degenerative brain disorders and cancer.

In the present invention, "circulatory-related disease" refers to a disease that is caused by a variety of physiological and psychological abnormalities due to inhibition of the biological clock molecular network due to genetic or environmental factors. Examples include sleep disorders, metabolic disorders including obesity and diabetes, cardiovascular disorders, immune and inflammatory disorders, mood disorders, degenerative brain disorders, and cancer. In addition, the types of the disease are very diverse, and these diseases can be prevented or treated by regulating the circadian biological clock molecular network.

For example, the pharmaceutical composition of the present invention has an effective effect as a prophylaxis, control or treatment of circadian rhythm-related diseases such as sleep disorders, metabolic diseases, cardiovascular diseases, immune and inflammatory diseases, mood disorders, degenerative brain diseases and cancer have.

In addition, the pharmaceutical composition of the present invention alone, or surgery, hormonal treatment, drug treatment for the prevention and treatment of diseases such as sleep disorders, metabolic diseases, cardiovascular diseases, immune and inflammatory diseases, mood disorders, degenerative brain diseases and cancer And methods that use biological response modifiers.

In the present invention, the term, "periodic associative sleep disorder" refers to a disorder in which sleep/wake rhythms appear in an abnormal pattern due to genetic or environmental disturbances applied to the biological clock. It is easy to complain of sleep-related disorders because patients with sleep disorders have difficulty falling asleep or waking up at socially necessary times. As a cause of the disease, sleep disorders caused by CRY, a sub-gene of the bio-clock network, have been reported as a cause of the onset. . According to a recent genetic study of patients with sleep disorders, in a genetic study of patients with sleep disorders who get excessive excessive sleep during the daytime, a genetic polymorphism called rs17289712 in the intron of the CRY1 gene was significantly associated with the symptoms of the disease. Showed a relationship. This means that CRY plays a unique role in controlling sleep/wakeing rhythm by a biological clock (Non-Patent Document 8).

On the other hand, in another study to investigate the effect of CRY, a sub-clock of the bioclock, on mice in which both CRY1 and CRY2 genes were deleted, non-REM sleep (NREMS) increased and non-REM sleep compared to normal mice. Meanwhile, the intensity of delta waves also tended to increase (Non-Patent Document 9). This means an increase in deep sleep, and this sleep pattern of CRY1 and CRY2 gene-deficient mice shows symptoms and differences in animal models that artificially destroy the hypothalamic optic nerve cross-nucleus, destroying diurism. When the nucleus is destroyed, there is no change in the total sleep time, but the sleep pattern fragments and the intensity of delta waves also tends to decrease (Non-Patent Document 10). This suggests the possibility that an independent role in the regulation of sleep homeostasis may exist independently of the role that CRY plays in the circadian biological clock.

Accordingly, in order to verify the CRY protein activity control of the oxyiminomethylbenzene derivative represented by Formula 1 according to the present invention, as a result of performing Experimental Examples 1 to 3 shown below, the oxyiminomethylbenzene derivative compound is the activity of the CLOCK:BMAL1 dimer It was confirmed that the effect of enhancing the activity by enhancing the amplitude of the circadian molecular biorhythm at the cell level measured through the PER2-LUC fusion protein was excellent, as well as the inhibitory effect on the CRY protein that inhibits. Accordingly, the pharmaceutical composition comprising the oxyiminomethylbenzene derivative represented by Chemical Formula 1 of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is easy for the prevention or treatment of circadian-related sleep disorders related to the core gene of the bioclock. Can be used.

Studies have been reported that metabolic disorders, one of the circadian linkage disorders, can be caused by functional abnormalities of the biological clock due to genetic or environmental causes. It is known that abnormal life patterns, such as long shifts or lack of sleep, slow the circadian secretion of growth hormone and melatonin in the body and increase the level of cortisol (non-patent document 11). This leads to an increase in body weight similar to symptoms.

In addition, the relationship between the biological clock system and metabolic disorders is more clearly revealed through analysis results of genetically modified animal models. In CLOCK mutant mice, it was found that leptin hyperactivity and insulin secretion were lowered, and symptoms of hyperlipidemia and hyperglycemia appeared (Non-Patent Document 12). In addition, in the BMAL1 gene deficient mouse, disorders were observed in the growth and differentiation of adipocytes, and abnormal symptoms were also found in carbohydrate metabolism in the liver (non-patent document 13-15). In addition, recent research has revealed that CRY functions as an important regulatory factor in the process of glucose synthesis in the liver, which is the cause of hyperglycemia. In mice lacking CRY, abnormalities in blood sugar regulation were observed compared to normal mice, which was found because CRY binds to the glucocorticoid receptor (GR), thereby regulating the expression of the hormone glucose synthesis by the hormone. (Non-Patent Document 16). In addition, CRY regulates the activity of adenyl cyclase by combining with G subunit, which is one of the components of G-protein coupled receptor, as well as GR. It shows the effect of regulating the expression of genes acting on glucose biosynthesis induced by cyclic adenosine monophosphate (Non-Patent Document 17).

This is a result of a study more clearly showing that metabolic disorders are related to the phase shift of the biological clock, as well as the lack of CLOCK, BMAL1 and abnormal expression of CRY1 can cause metabolic disorders. In addition, these results suggest that CLOCK and BMAL1 expression and CLOCK:BMAL1 dimer activity enhancement and CRY activity inhibition are effective in treatment.

Thus, in order to confirm the effect of enhancing the activity of the CLOCK:BMAL1 dimer by the oxyiminomethylbenzene derivative represented by Formula 1 according to the present invention and inhibiting the CRY activity, as a result of performing Experimental Examples 1 to 3, the oxyiminomethylbenzene derivative Not only has excellent inhibitory effect on CRY protein that inhibits the activity of the CLOCK:BMAL1 dimer, but also enhances the activity by increasing the amplitude of the cell-level circadian molecular biorhythm measured through the PER2-LUC fusion protein. Was confirmed to be excellent.

In addition, through the results of Experimental Example 4, the oxyiminomethylbenzene derivative represented by Formula 1 of the present invention was repeatedly administered to mice in which obesity was induced through the supply of a high-lipid diet, and statistically significant weight loss efficacy was confirmed. Did. In addition, in Experimental Example 5, when the oxyiminomethylbenzene derivative was repeatedly administered to a mouse having an obesity due to a high-lipid diet and an abnormality in the phase of the circadian central biological clock located in the optic nerve cross-phase nucleus, the circadian molecular biorhythm was repeated. The effect of restoring was confirmed.

Accordingly, the pharmaceutical composition containing the oxyiminomethylbenzene derivative represented by Chemical Formula 1 according to the present invention or an acceptable salt thereof can be usefully used for the prevention or treatment of metabolic diseases caused by functional disturbance of the bioclock.

At this time, the circadian-related metabolic disease may be obesity, hypertension, hyperlipidemia, hyperglycemia or polyuria, but is not limited thereto.

Mood disorders and addiction disorders are also possible due to changes in circadian rhythms, and research is actively underway. Like physical functions such as metabolic activity and cell division, changes in emotions, which are one of the higher functions of the brain, exhibit circadianity and are also biological 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 clock is becoming important.

Blood samples were analyzed to evaluate the patterns of mRNA expression related to the four circadian rhythm genes in 30 people who did not have a major depression and those who did not have a disorder, in order to investigate the relationship between mood disorders and the biological clock. mRNA indicates gene activity, and participants with a history of depression had higher activity of the CLOCK gene than people without depression, from which it can be predicted that the depressive disorder is related to the activity of CLOCK, a key gene in the bioclock ( Non-Patent Document 18).

Among the mood disorders, the most striking association with the biological clock is bipolar mood disorder, or bipolar disorder. Gene-hit mice for various bioclock genes, such as Clock, Rev-erbα, Per, and Cry, show behavioral phenotypes similar to bipolar disorder (Non-Patent Documents 19-21), and especially circulate prior to the occurrence of manic episodes in bipolar patients Modulation of the biorhythm phase has been reported (non-patent document 22). The above results strongly suggest that the weakening of the activity and stability of the circadian bioclock is a key mechanism for the development of bipolar disorder.

In addition, the relationship between the biological clock and mood disorders can be seen through light therapy. In addition to drug treatment, as one of the widely used methods of treating mood disorders through artificial stimulation that can control the biological clock, phototherapy is known to be particularly effective for seasonal mood disorders. Seasonal mood disorders live in temperate climate zones with four seasons. One of the most common mental illnesses in which 5 to 10% of the population is symptomatic, symptoms such as depression appear during the winter when the sun rises and the day becomes shorter. At this time, when the patient with seasonal mood disorder is exposed to artificially strong light, the symptoms of depression are alleviated, but phototherapy performed in the early morning is more effective than that performed in the evening (Non-Patent Document 23). This not only indicates that the symptoms of seasonal mood disorders are related to the phase modulation of the biological clock, but also the expression of CLOCK and BMAL1, the core genes of the biological clock, which are activated during daylight hours with light, and the activity of the dimer of CLOCK:BMAL1. It means it works.

In view of these facts, the activity of the dimer of CLOCK:BMAL1 of the oxyiminomethylbenzene derivative compound represented by Chemical Formula 1 of the present invention and the effect of enhancing the circadian molecular biorhythm were confirmed. As a result of performing the experiments of Experimental Examples 1 to 3, the oxyiminomethylbenzene derivative has excellent inhibitory effect on the CRY protein that inhibits the activity of the CLOCK:BMAL1 dimer, as well as the cell level measured through the PER2-LUC fusion protein. It was confirmed that the effect of enhancing the activity, such as increasing the amplitude of the circadian molecular biorhythm of, is excellent. In addition, as a result of evaluating the efficacy of behavioral phenotypes related to mood disorders such as depression, anxiety, and pleasure response in mice through Experimental Example 6, the fact that administration of oxyiminomethylbenzene derivatives has antidepressant and anti-anxiety effects Proved. Therefore, a pharmaceutical composition containing an oxyiminomethylbenzene derivative represented by Chemical Formula 1 of the present invention or a pharmaceutically acceptable salt thereof is used to prevent or treat diseases related to mood disorders through pharmacological control of a circadian bioclock. It can be useful.

In this case, the circadian-related mood disorder may be, for example, major depression, bipolar mood disorder, seasonal disorder, or anxiety disorder, but is not limited thereto.

Various degenerative brain diseases are also possible due to the variation of the circadian rhythm, and research is actively underway. In patients with various degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, abnormalities in circadian rhythms including sleep disorders have been reported from the onset of the disease (non-patent document 24), which deteriorates the quality of life of patients with degenerative brain disease. This is the main cause. In particular, circadian rhythm abnormalities and related sleep disorders may be regarded as typical non-motor symptoms in Parkinson's disease and Huntington's disease. In a recent study using animal models, it has been demonstrated that weakening of the biological clock increases the risk of developing degenerative brain diseases and is a major etiology that exacerbates symptoms (Non-Patent Documents 25-26).

Considering this fact, in order to confirm the activity of dimer of CLOCK:BMAL1 of the oxyiminomethylbenzene derivative represented by Chemical Formula 1 according to the present invention and the effect of enhancing the circadian molecular biorhythm, Experimental Examples 1 and 3 were performed, As a result, it was confirmed that the effect of enhancing the circadian molecular biorhythm was excellent, such as enhancing the activity of the dimer of CLOCK:BMAL1 and increasing the amplitude of cyclic PER-LUC fusion protein expression. Therefore, the pharmaceutical composition containing the oxyiminomethylbenzene derivative of the present invention or a pharmaceutically acceptable salt thereof is usefully used to prevent or treat diseases related to degenerative brain diseases through pharmacological control of a circadian bioclock. Can be.

In this case, the circadian-related degenerative brain disease may be Alzheimer's disease, Parkinson's disease, or Huntington's disease, but is not limited thereto.

In cancer, which is one of the circadian-related diseases, circadian changes in sensitivity to anticancer drugs show the link between the core genes of the bio-clock and the tumor. In CRY gene-hit mice, anticancer drug resistance was higher than normal mice at all times. This difference in resistance was found to be due to the fact that the functional status of the CLOCK:BMAL1 dimer in hepatocytes is different in normal mice and in circadian genetically modified mice. Non-patent document 27). This study shows that the effect of CLOCK:BMAL1 dimer, a key gene in the biocycle, and CRY, which is a sub-gene, have a significant effect on anticancer drugs.

In addition to affecting the cyclic changes in sensitivity to anticancer drugs, the bioclock gene has the effect of directly inhibiting tumor formation and growth. Tumor production and growth are inhibited when CRY is further deleted in a p53 gene-deficient mouse, one of the animal models for tumorigenesis (Non-Patent Document 28). Because the gene deficiency of CRY completely destroys the intrinsic diurnality of the individual (non-patent document 29), it is not necessarily that the path to disturbance of the biological clock and the progression to tumor development are inconsistent, and that more complex correlations may exist. Show.

In particular, it suggests the possibility that key genes in bioclockwork have independent functions in cell cycle and tumor development, as well as in maintaining circadianity, and we can consider new possibilities that temporary inhibition of CRY may have in anticancer action. Provides room for. In fact, in the case of the persistent CRY inhibitor KS15, although the circadian biorhythm enhancement effect was insignificant, it exhibited an anti-cancer effect on the breast cancer cell line (non-patent document 30).

Since the oxyiminomethylbenzene derivative compound represented by Chemical Formula 1 of the present invention has an excellent effect of inhibiting the action of CRY as shown in the result of Experimental Example 1, the drug is limited to a specific time period through pharmacological control of CRY activity using the same. It has the effect of increasing the efficacy of the drug and reducing toxicity. In addition, since it suppresses the generation and growth of tumor cells, the pharmaceutical composition containing it as an active ingredient can be usefully used for the prevention or treatment of cancer caused by a circadian rhythm modification.

In this case, the circadian-related cancer may be colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, lung cancer, uterine cancer, colon cancer, pancreatic cancer, ovarian cancer, or blood cancer, but is not limited thereto.

The production method of the oxyiminomethylbenzene derivative represented by Chemical Formula 1 of the present invention is as follows.

[Scheme 1]

(A to E, R1, and R2 in Reaction Scheme 1 are as defined above.)

More specifically, the aldehyde or ketone compound substituted with A to E or R2 represented by Formula 3, which is usually obtainable with R1 substituted amine compound represented by Formula 2 or a salt thereof, which can be obtained normally, And reacting to synthesize an oxyiminomethyl compound represented by Chemical Formula 1.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example One: Oxyiminomethylbenzene Synthesis of derivative compounds (Chemical Formula 8-9, Chemical Formula 8-10)

[Scheme 2]

Step 1: Preparation of acetophenone derivative (Formula 6)

As described in Scheme 2 above, commercially available 3-hydroxyacetophenone (Formula 4, 1 g) was placed in a reactor and N,N-dimethylformaldehyde was added. After cooling the reactor to 0° C., potassium carbonate (3 eq., 3 g) and methylchloroacetate (Formula 5, 1.2 eq., 0.767 ml) were slowly added, and the temperature of the reaction solution was raised to room temperature and for 12 hours. It was stirred. After completion of the reaction, the reaction solution was filtered and the filtrate was concentrated under reduced pressure. The concentrated compound was subjected to silica gel chromatography using ethyl acetate and nucleic acid as mobile phases to obtain the desired compound, acetophenone derivative (Formula 6, 1453 mg, yield 95%).

1H-NMR (CDCl3, 500 MHz) δ 7.60 (m, 1H), 7.48 (m, 1H), 7.40 (dd, J = 8.1, 7.5 Hz, 1H), 7.15 (m, 1H), 4.70 (s, 2H ), 3.82 (s, 3H), 2.60 (s, 3H).

[Scheme 3]

Step 2: Oxyiminomethylbenzene Preparation of derivative compound (Formula 8-9)

As described in Scheme 3, acetophenone derivatives (Formula 6, 208 mg) and commercially available alkoxyamine compounds (Formula 7, 1.2 eq, 246 mg) were placed in a reactor and pyridine was added. The reaction solution was stirred for 5 hours while heating to reflux. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure. The concentrated compound was subjected to silica gel chromatography using ethyl acetate and a nucleic acid as mobile phases to obtain an oxyiminomethylbenzene derivative compound (Formula 8-9, 197 mg, yield 55%) as a target compound.

[Scheme 4]

Step 3: Oxyiminomethylbenzene derivative Hydrolyzate (Formula 8-10) Produce

As shown in Reaction Scheme 4, an oxyiminomethylbenzene derivative compound (Chemical Formula 8-9, 20 mg) was placed in a reactor, and tetrahydrofuran, water, and methanol were added. After the reactor was cooled to 0° C., lithium hydroxide (3 eq., 7 mg) was slowly added and the temperature of the reaction solution was raised to room temperature and stirred for 8 hours. After the reaction was completed, the reaction solution was neutralized with dilute hydrochloric acid, and then concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with water, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and ethyl acetate and nucleic acid were used as a mobile phase to perform silica gel chromatography to obtain a hydrolyzate of the desired compound, oxyiminomethylbenzene derivative (Formula 8-10, 19 mg, yield 99%).

Oxyiminomethylbenzene containing a hydrolyzate by performing in the same manner as in Example 1, except that A to E, R ¹ and R ² in the compound represented by Formula 1 are performed as described in [Table 1] below. Derivatives (8-1 to 14, 9-1 to 12) were synthesized. The structure and analysis results of the synthesized oxyiminomethylbenzene derivative compound are summarized in Table 1 below.

[Formula 8]

compound R ³ R ⁴ R ⁵ Analysis data
(1H/13C-NMR or LC-MS) Max (%) EC ₅₀ 8-1 CO ₂ CH ₃ H CF ₃ 1H-NMR (500 MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 2.5 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 1.5 Hz, 1H), 6.91-6.89 (m, 1H), 5.27 (s, 2H), 4.66 ( s, 2H), 3.80 (s, 3H) 171.49 27.43 8-2 CO ₂ H H CF ₃ 1H-NMR (500 MHz, CDCl3) δ 7.47 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 6.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 6.92 (t, J = 15.5 Hz, 2H), 6.74 (s, 1H),6.57 (s, 1H), 5.01 (s, 2H), 4.17 (s, 2H) 1.86 (s, 3H). 130.73 N/A 8-3 CO ₂ CH ₃ OCH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.36 (d, J = 6.5Hz, 2H), 7.34-7.27 (m, 2H), 7.25 (d, J = 2.0 Hz, 1H), 6.91-6.89 (m, 3H ), 5.16 (s, 2H), 4.67 (s, 2H), 3.81 (t, J = 2.0 Hz, 6H), 2.21 (s, 3H). 151.78 6.32 8-4 CO ₂ H OCH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.36 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.5 Hz, 2H), 6.908-6.891 (m, 3H), 5.16 (s, 2H), 4.70 (s, 2H), 3.81 (s, 3H), 2.21 (d, J = 2.0 Hz, 3) 182.06 9.88 8-5 CO ₂ CH ₃ Br H 1H-NMR (500 MHz, CDCl3) δ7.52-7.50 (m, 2H), 7.32-7.27 (m, 3H), 6.94-6.92 (m, 1H), 5.20 (s, 2H), 4.67 (s, 2H) ), 3.82 (s, 3H), 2.26 (s, 3H); 13C-NMR (CDCl3, 125 MHz) δ169.2, 157.7, 154.7, 138.0, 137.1, 131.5, 129.8, 129.5, 121.7, 119.7, 115.3, 112.4, 75.4, 65.3, 52.3, 12.8; LRMS (ESI+) m/z 391.84 [M]+. 149.13 N/A (Tox.) 8-6 CO ₂ H Br H 1H-NMR (500 MHz, CDCl3) δ7.51-7.50 (t, 2H, J = 2 Hz), 7.34-7.27 (m, 3H), 6.95-6.93 (m, 1H), 5.20 (s, 2H), 4.73 (s, 1H), 2.27 (s, 3H); 13C-NMR (CDCl3, 125 MHz) δ174.2, 157.4, 154.9, 138.0, 137.0, 131.5, 129.8, 129.6, 121.8, 120.0, 115.3, 112.4, 75.4, 64.8, 13.0; LRMS (ESI+) m/z 378.99 [M]+. 123.78 N/A (Tox.) 8-7 CO ₂ X,X=(2pyridyl) methylamine Br H 1H-NMR (CDCl3, 500 MHz) δ 8.60-8.58 (m, 1H), 7.95 (s, 1H), 7.80 (s, 1H), 7.52-7.50 (d, J = 8.5 Hz, 2H), 7.46-7.44 (m, 1H), 7.35-7.27 (m, 5H), 7.01-6.98 (m, 1H), 5.20 (s, 2H), 4.76-4.75 (d, J = 5.5 Hz, 2H), 4.60 (s, 2H ), 2.26 (s, 3H); 13C-NMR (CDCl3, 500 MHz) δ 168.46, 157.31, 155.52, 154.74, 148.40, 147.44, 138.77, 138.16, 137.08, 131.52, 129.85, 123.15, 121.76, 119.99, 115.66, 112.40, 75.45, 67.40, 43.18; LRMS (ESI+) m/z 468.49 [M]+. 129.69 N/A 8-8 CO ₂ Y,Y=histamine Br H 1H-NMR (CDCl3, 500 MHz) δ 8.95 (s, 1H), 7.51-7.46 (m, 2H), 7.29-7.22 (m, 4H), 7.03 (s, 1H), 6.89-6.88 (d, J = 6.5 Hz, 1H), 5.16 (s, 2H), 4.53 (s, 2H), 3.67 (s, 2H), 3.00 (s, 2H), 2.22 (s, 3H); 13C-NMR (CDCl3, 500 MHz) δ 157.1, 154.8, 137.0, 131.5, 130.9, 129.7, 121.7, 115.8, 115.3, 112.4, 75.3, 67.2, 24.9, 13.0; LRMS (ESI+) m/z 494.26 [M+Na]+. 121.10 N/A (Tox.) 8-9 CO ₂ CH ₃ NO ₂ H 1H-NMR (500 MHz, CDCl3) δ 7.36 (d, J = 6.5Hz, 2H), 7.34-7.27 (m, 2H), 7.25 (d, J = 2.0 Hz, 1H), 6.91-6.89 (m, 3H ), 5.16 (s, 2H), 4.67 (s, 2H), 3.81 (t, J = 2.0 Hz, 6H), 2.21 (s, 3H). 193.04 6.44 8-10 CO ₂ H NO ₂ H 1H-NMR (500 MHz, CDCl3) δ 7.36 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.5 Hz, 2H), 6.91-6.89 (m, 3H), 5.16 (s, 2H), 4.70 (s, 2H), 3.81 (s, 3H), 2.21 (s, 3H). 161.26 10.17 8-11 CO ₂ CH ₃ SO ₂ CH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.93 (d, J =8.5 Hz, 2H), 7.59 (d, J =1.5 Hz, 2H), 7.30-7.25 (m 1H), 7.24-7.22 (m 2H), 6.91-6.89 (m 1H), 5.31 (s, 2H), 4.65 (s, 2H), 3.80 (s, 3H), 3.06 (s, 3H), 2.28 (s, 3H). 104.72 N/A 8-12 CO ₂ H SO ₂ CH ₃ H 1H-NMR (500 MHz, MeOH) δ 7.96 (d, J =7.0 Hz, 2H), 7.67 (d, J =8.5 Hz, 2H), 7.30-7.21 (m 3H), 6.96-6.94 (m 1H), 6.91-6.89 (m 1 H), 5.32 (s, 2H), 4.67 (s, 2H), 3.11 (s, 3H), 2.27 (s, 3H). 98.71 N/A 8-13 CO ₂ CH ₃ F H 1H-NMR (500 MHz, CDCl3) δ 7.39-7.36 (m, 2H), 7.29 (s, 1H), 7.228 (t, J =2.5 1H), 7.059-7.024 (m, 2H), 6.90-6.88 (m , 1H), 5.18 (s, 2H), 4.65 (s, 2H), 3.81 (s, 3H). 119.93 N/A (Tox.) 8-14 CO ₂ H F H 1H-NMR (500 MHz, CDCl3) δ 7.38-7.36 (m, 2H), 7.35 (s, 1H), 7.23 (s, 1H), 7.03 (t, J =8.5 2H), 6.90 (t, J =3.0 1H), 5.17 (s, 2H), 4.66 (s, 2H), 2.20 (s, 3H). N/D N/D [Formula 9]

compound R ³ R ⁴ R ⁵ Analysis data
(1H/13C-NMR or LC-MS) Max (%) EC ₅₀ 9-1 CO ₂ CH ₃ H CF ₃ 1H-NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 7.59-7.55 (m, 4H), 7.48 (d, J = 7.5 Hz, 1H), 6.89-6.87 (m, 2H) 5.25 (s, 2H), 4.65 (s, 2H), 3.80 (s, 3H), 2.25 (s, 3H). 107.89 N/A 9-2 CO ₂ H H CF ₃ 1H-NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 7.60-7.55 (m, 4H), 7.48 (d, J = 8.0 Hz, 2H), 6.91-6.89 (m, 2H), 5.26 (s , 2H), 4.70 (s, 2H), 2.26 (s, 3H). 115.15 N/A 9-3 CO ₂ CH ₃ OCH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.60-7.59 (m, 2H), 7.36-7.35 (m, 2H), 6.90-6.88 (m, 4H), 5.14 (s, 2H), 4.65 (s, 2H) , 3.81 (d, J = 2.5 Hz, 6H), 2.21 (s, 3H) 103.87 N/A 9-4 CO ₂ H OCH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.61 (d, J = 9.0 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 6.91-6.89 (m, 4H), 5.14 (s, 2H), 4.69 (s, 2H), 3.81 (s, 3H), 2.21 (s, 3H). 102.65 N/A 9-5 CO ₂ CH ₃ Br H 1H-NMR (500 MHz, CDCl3) δ 7.58-7.56 (d, 2H, J = 9 Hz), 7.48-7.47 (d, 2H, J = 8 Hz), 7.28-7.25 (d, 2H, J = 8.5 Hz ), 6.89-6.87 (d, 2H, J = 9 Hz), 5.15 (s, 2H), 4.65 (s, 2H), 3.80 (s, 3H), 2.22(s, 3H); 13C-NMR (CDCl3, 125 MHz) δ 169.1, 158.5, 154.6, 137.3, 131.4, 130.1, 129.8, 127.5, 121.6, 114.4, 75.2, 65.2,52.3, 12.8; LRMS (ESI+) m/z 392.13 [M]+. 111.88 N/A 9-6 CO ₂ H Br H 1H-NMR (500 MHz, CDCl3) δ7.56-7.51 (m, 4H), 7.35-7.34 (d, 2H, J = 8.5), 5.12 (s, 2H), 4.39 (s, 2H), 2.16 (s , 3H); 13C-NMR (CDCl3, 125 MHz) δ159.4, 154.3, 137.7, 131.2, 130.1, 127.8, 127.0, 120.7, 114.3, 74.2, 66.3, 12.4; LRMS (ESI+) m/z 400.78 [M+Na]+. 106.70 N/A 9-7 CO ₂ X,
X=(2-pyridyl) methylamine Br H 1H-NMR (MeOD, 500 MHz) δ7.90 (s, 1H), 7.62-7.60 (d, 2H), 7.52-7.50 (d, 2H), 7.34 (s, 2H), 6.96-6.95 (d, J = 8.5 Hz, 3H), 5.16 (s, 2H), 4.52 (s, 2H), 3.56 (s, 2H), 2.86 (s, 2H), 2.24 (s, 3H); 13C-NMR (MeOD, 500 MHz) δ169.56, 158.56, 154.76, 137.70, 134.38, 133.68, 131.07, 129.95, 129.58, 127.26, 121.09, 116.35, 114.25, 74.70, 66.79, 25.80, 11.44; LRMS (ESI+) m/z 493.06 [M+Na]+. 135.47 N/A 9-8 CO ₂ Y,Y=histamine Br H 1H-NMR (CDCl3, 500 MHz) δ 8.74 (s, 1H), 8.62 (s, 1H), 8.31 (s, 1H), 7.96 (s, 1H), 7.75 (s, 1H), 7.53-7.48 (m , 4H), 7.26-7.25 (d, J = 1.0 Hz, 2H), 6.99-6.97 (d, J = 8.0 Hz, 2H), 5.14 (s, 1H), 4.97 (s, 1H), 4.52 (s, 1H), 2.21 (s, 1H); 13C-NMR (CDCl3, 500 MHz) δ 168.43, 158.03, 155.18, 154.57, 146.73, 139.55, 137.28, 131.48, 130.40, 129.81, 127.64, 123.74, 123.42, 121.67, 114.68, 75.26, 67.30, 42.81 LRMS (ESI+) m/z 490.00 [M+Na]+. 120.89 N/A 9-9 CO ₂ CH ₃ NO ₂ H 1H-NMR (500 MHz, CDCl3) δ 8.22 (d, J =9.0 Hz, 2H), 7.57-7.53 (m, 4H), 6.89 (d, J =9.0 2H), 5.30 (s, 2H), 4.64 ( s, 2H), 3.80 (s, 3H), 3.27 (s, 3H). 113.52 N/A 9-10 CO ₂ H NO ₂ H 1H-NMR (500 MHz, CDCl3) δ 8.22 (d, J =7.0 Hz, 2H), 7.59-7.54 (m, 4H), 6.90 (d, J =7.5 2H), 5.30 (s, 2H), 4.69 ( s, 2H), 2.28 (s, 3H). 108.43 N/A 9-11 CO ₂ CH ₃ SO ₂ CH ₃ H 1H-NMR (500 MHz, CDCl3) δ 7.94 (d, J =8.5 Hz, 2H), 7.59-7.56 (m, 4H), 6.89 (m 2H), 5.29 (s 2H), 4.65 (s, 2H), 3.80 (s, 3H), 3.05 (s, 3H), 2.27 (s, 3H).\ 100.39 N/A 9-12 CO ₂ H SO ₂ CH ₃ H 1H-NMR (500 MHz, MeOH) δ 7.95 (d, J =8.5 Hz, 2H), 7.65 (d, J =8.5 Hz, 2H), 7.60 (d, J =9.0 Hz, 2H), 6.93-6.91 ( m, 2H), 5.29 (s 2H), 4.67 (s, 2H), 3.11 (s, 3H), 2.27 (s, 3H). 111.38 N/A 9-13 CO ₂ CH ₃ H CF ₃ 1H-NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 7.59-7.55 (m, 4H), 7.48 (d, J = 7.5 Hz, 1H), 6.89-6.87 (m, 2H) 5.25 (s, 2H), 4.65 (s, 2H), 3.80 (s, 3H), 2.25 (s, 3H). 107.89 N/A

Experimental Example One. CLOCK:BMAL1 Dimer Efficacy evaluation for transcriptional activity

To evaluate the effect of oxyiminomethylbenzene derivatives on the transcriptional activity of CLOCK:BMAL1 dimer mediated by E-box, an NIH-3T3 fibroblast line expressing E-box-LUC reporter (Non-Patent Document 6) was used. Did. Reporter-expressing fibroblast lines were prepared using Dulbecco's modified eagles medium (Invitrogen) with 10% serum, 1% penicillin/streptomycin (Invitrogen), and 1% L-glutamine. It was maintained and cultured in an incubator where a humidified state of 5% carbon dioxide and 37 degrees Celsius was maintained.

The reporter expressing cell line was cultured by dispensing in a 48-well culture plate 24 hours before compound treatment. The cultured cells were finally treated with a concentration of 0.1, 0.3, 1.0, 3.0, 10, 30 or 100 μM. As a control (0 μM), cells treated with the same amount of dimethyl sulfoxide (DMSO) were used. After 36 hours of treatment with the compound, the cells were washed twice with 1X D-PBS and lysed for 15 minutes with 100 µl per cell lysis buffer (Possive Lysis buffer, Promega). After reacting a sample of 20 μl in the lysate with the same amount of Luciferase assay reagent (Promega), the luciferase activity is measured using a luminometer (TD-20/20 Luminometer, Turner systems). Was measured. The measured activity of luciferase was corrected by the protein content of the lysate, and the protein content was measured using Bio-rad's Bradford dyeing reagent.

The concentration-response correlation for each derivative was analyzed by converting it to a standard curve through Four Parameter Logistic Equation, and the calculation process utilized the statistical analysis function provided by SigmaPlot 10.0 (SigmaPlot 8.0, Systat Software Inc.). Based on the concentration-response curve, the maximum activity (Max) and EC ₅₀ values of each derivative were calculated and recorded in [Table 1] above. In the case of cell death among the compounds, it was indicated that cytotoxicity (Tox.) occurred in the EC ₅₀ column. As a representative example, concentration-response correlation curves of compounds of Formulas 8-9, 9-9, 8-10, and 9-10 are shown in FIG. 1.

1 and Table 1, among the compounds of Example 1, the compound of Formula 8-9 had a maximum activity of 193.04% compared to the control group, an EC ₅₀ value of 6.44 μM, and no significant cytotoxicity was observed until 100 μM. It was found to be the best in terms of maximum activity, EC ₅₀ and cytotoxicity. The compound of Formula 8-10, which has a structure similar to Formula 8-9, also showed a significant level of maximum activity (161.26%) and EC ₅₀ value (10.17 μM).

Experimental Example 2. CRY dependency verification according to compound treatment

The following experiments were performed to verify whether the compounds (8-9 and 8-10) prepared in Example 1 actually modulate E-box mediated transcriptional activity via CRY.

The HEK293 cell line used in this experiment was used in Dulbecco's modified Eagle's medium containing 10% serum, 1% penicillin/streptomycin and 1% L-glutamine, in an incubator maintaining a humidified state of 5% carbon dioxide and 37 degrees Celsius. It was maintained and cultured. After 24 hours prior to transduction, HEK293 cells were dispensed into a 48-well culture plate, followed by mouse CLOCK expression vector (with E-box-LUC luciferase reporter (50 ng/well), pRL-mTK reporter (100 ng/well)) A mixture of 600 ng/well) and a mouse BMAL1 expression vector (200 ng/well) or equivalent pcDNA3.1 plasmid and Flag-CRY1 expression vector (20 ng/well) or equivalent pcDNA3.1 plasmid were introduced into cells. For transfection, Lipofectamine 3000 (Invitrogen) was used, and the procedure was performed according to the manufacturer's manual.

After 48 hours from transduction, compounds of formulas 9-9 or 8-10 were finally treated to a concentration of 1, 3 or 10 μM. As a control (0 μM), the same amount of dimethyl sulfoxide treated was used. After 36 hours of treatment with the compound, the cells were washed twice with 1X D-PBS and lysed with 100 μl of cell lysis buffer per well for 15 minutes. After reacting a 20 μl sample in the lysate with an equal amount of luciferase activity evaluation reagent, the activity of luciferase was measured using a luminometer. The measured activity of E-box-LUC luciferase was corrected using pRL-mTK ranilla luciferase activity.

The control group (E-box-LUC luciferase reporter and pRL-mTK reporter and pcDNA3.1 only) were introduced and the dimethyl sulfoxide group was treated with the degree of change in E-box-LUC reporter activity in each experimental group for each derivative treatment. , Expressed in CTL) as a ratio with respect to the average value, and illustrated in FIG. 2. As a result, the expression of CRY1, as known, inhibited the E-box mediated transcriptional activity of CLOCK:BMAL1 to a significant level, and the action of CRY was concentration-dependently inhibited for the treatment of the compounds of Formulas 8-9 and 8-10. .

As shown in Fig. 2, among the compounds of Example 1, the compounds of Formulas 8-9 and 8-10 enhance the transcriptional activity of CLOCK:BMAL1 in a CRY-dependent manner, and thus the oxyiminomethylbenzene derivatives of the present invention are added to the CRY protein. It shows that it acts as an inhibitor for Korea.

Experimental Example 3. PER2 -Of LUC fusion proteins Circadian Derivative efficacy evaluation for expression

The effects of the compounds (8-9 and 8-10) prepared in Example 1 on the circulatory expression of molecular bioclocks expressed by cells through inhibition of CRY and enhancement of transcriptional activity of CLOCK:BMAL1 were evaluated. To this end, a mutated fibroblast line (PER2-LUC knock-in fibroblast) expressing a PER2-LUC fusion protein was used by linking the luciferase gene from the position where the stop codon of the PER2 gene was located. This cell line was established from lung tissue of PER2-LUC knock-in mutant mice (non-patent document 31), one week after birth. PER2-LUC knock-in (KI) fibroblasts are maintained in a humidified state of 5% carbon dioxide and 37 degrees Celsius using a Dulbecco's modified Eagle's medium containing 10% serum, 1% penicillin/streptomycin and 1% L-glutamine. It was maintained and cultured in an incubator.

PER2-LUC KI cells were cultured by dispensing in a 35 mm culture dish 24 hours before compound treatment. After incubating the cultured cells with 200 nM dexamethasone (DEX) for 2 hours to synchronize, 20 μM of the compound of Formula 8-9 or 8-10, and luciferase substrate luciferin (final concentration 0.1 mM) It was replaced with a medium containing. As a control group, cells treated with the same amount of dimethyl sulfoxide (VEH) instead of the compound were used, and as another control group, KS15, a 2-ethoxypropionic acid-based CRY inhibitor (non-patent document 6) (final concentration 20 μM) Was used. Control and KS15 treated cells were also replaced with media containing the same concentration of luciferin.

Biophosphorescence signals generated from cells cultured in a medium containing a compound and luciferin were continuously measured for up to 5 days at 10 minute intervals using real-time biophosphorescence measurement equipment (Kronos-DIO, ATTO), and the results were statistically trended. It is shown in FIG. 3 through detrending. Amplitude, period, and stability of periodicity are analyzed by analyzing the cyclic expression pattern of PER2-LUC fusion protein using Cosinor analysis program (free distribution from http://www.circadian.org ). Based on the results obtained through 4 replicates, the statistical significance of the changes due to compound treatment was verified.

As a result, as shown in FIGS. 3A and 3B, the treatment of the compound of Chemical Formulas 8-9 or 8-10 increases the amplitude of periodic expression of the PER2-LUC fusion protein to a significant level, and significantly affects the cycle or stability. Did not cause This is in contrast to KS15, which reduces the amplitude of the circadian molecular rhythm and reduces its stability to a significant level, although the treatment of KS15 enhances the transcriptional activity of the CLOCK:BMAL1 dimer. That is, unlike 2-ethoxypropionic acid derivatives such as KS15, the oxyiminomethylbenzene derivatives of the present invention have anti-efficacy against the activity of the circadian molecular bioclock.

Experimental Example 4. High lipid On a diet Derivative efficacy evaluation for induced obesity

The effect of the compound (8-9) prepared in Example 1 on obesity caused by a high lipid diet was verified. A high-lipid diet comprising lipids corresponding to 60% of the total calories is known to induce obesity and various metabolic abnormalities in mice, and is the most widely used animal model of metabolic disease (Non-Patent Document 15).

After the start of the experiment, mice (28 heads in total) were divided into two experimental groups, each with a normal diet (including normal chow, NC, 15.2% protein and 2.9% lipid), or a high-fat diet (HFD, 20.3%). Protein and 27.0% lipids) were fed for 2 weeks. At the beginning of week 3, each diet group was divided into 2 experimental groups and used as a compound administration group or a control group of formula 8-9. In the case of the administration group for each diet group, 8-9 compounds (10 mg/kg body weight, 200 μl administration per time) were administered once every 2 days for 8 weeks in total by intraperitoneal injection, and the control group was treated with solvent (VEH, 0.5 in the same way). % Dimethylsulfoxide in equal volume of physiological saline). The weight of the mice was measured twice a week (1st and 4th days of the week) and ZT06-07 of the day, and the average of the 2 measurements was recorded as the weight of the corresponding parking lot and shown in FIG. 4.

4A is a representative photograph showing the appearance of mice (7 heads) for each experimental group immediately after the experiment for a total of 10 weeks, and FIG. 4B shows a change in body weight at weekly intervals. Figure 4c is a result showing the change in body weight during the 8-9 or 8-week administration period.

As can be seen in Figure 4, administration of the compound of Formula 8-9 showed an anti-obesity effect, such as reducing the weight gain caused by a high-lipid diet by about 40% compared to the control group. In contrast, in the mice receiving the normal diet, there was no statistically significant difference between the treatment group and the control group, which appears in a specific situation in which the effect of administering the compound of Formula 8-9 is obese, and in particular of the compound of the Formula 8-9. This means that the weight loss effect is not due to the toxicity of the potential compounds.

Experimental Example 5. High lipid On a diet Derivative efficacy evaluation for induced circadian bioclock anomalies

As described above, metabolic diseases including obesity are frequently accompanied by abnormalities of the circadian biorhythm, and the compound (8-9) prepared in Example 1 above is caused by a high-lipid diet. The effect of the nucleus on the circadian function of the central biological clock was verified.

To this end, the day after the diet and compound administration described in Experimental Example 4 were completed, the mice were sacrificed at the time of ZT02 (3 heads per experimental group) and ZT12 (4 heads per experimental group), and then the brain was excised to slice 1 mm thick brain tissue. Prepared from this, the optic nerve crossing nucleus region located in the hypothalamus was extracted to a certain size (2 mm horizontal, 1 mm vertical and 1 mm thick). RNA was isolated from the optic nerve cross-phase nucleus using a commercialized RNA isolation kit (microRNeasy, Qiagen), and cDNA was synthesized using a commercially available cDNA synthesis kit (cDNA synthesis kit, Takara) for 500 ng of RNA samples. . For this cDNA, the expression level of the target gene mRNA was measured by quantitative real-time PCR using the SYBR Green I PCR kit (Invitrogen). The gene expression levels of the biological clock genes Bmal1, Npas2, Rev-erbα, and Per2 were corrected by the expression level of TATA-box binding protein (TBP) mRNA, one of the house-keeping genes, and the overall result was standard RNA. It was expressed as a ratio to the gene level measured in the sample. The measurement results are shown in FIG. 5.

As shown in FIG. 5, unlike the control mice (NC and VEH) fed with a normal diet and administered with solvents, cyclic expression of Bmal1 and Rev-erbα when obesity was induced (HFD and VEH) by a high lipid diet. Disappeared, and the expression pattern of Npas2 mRNA was reversed. On the other hand, despite supplying a high-lipid diet, the expression of these bio-clock genes was normally maintained in mice (HFD and 8-9) administered the compound of Example 1. As in Experimental Example 4, the normal expression pattern of the bio-clock genes was normal in the mouse group (NC and 8-9) receiving the normal diet and administered the 8-9 compound.

The above results are consistent with the results of the cultured cell model of Experimental Example 3, and the administration of the derivative (8-9) of Example 1 may contribute to the enhancement of the circadian molecular bioclock in vivo, resulting in circadian disturbance. It is a result that shows that the obese animal model can maintain the circulatory function of the central biological clock as well as weight loss.

Experimental Example 6. Efficacy and Intoxication Control Derivative Efficacy Evaluation for Brain Function

Compound (8-9) prepared in Example 1 according to the present invention on the basis of the fact that emotional and addictive disorders are frequently accompanied by abnormalities of the circadian rhythm as described above, affect the emotional and addictive behaviors of mice. The impact was verified.

To this end, after the compound (8-9) or solvent was repeatedly administered for 6 weeks at the same dose as described in Experimental Example 4 (once per 2 days, 10 mg/kg body weight per dose, 200 μl administration), the following 3 animals Behavioral tests were performed sequentially, and the results are shown in FIG. 6. Compounds and solvents were administered in the same way during behavioral testing.

Sucrose preference test (hereinafter referred to as SPT) is a behavioral test method for measuring pleasure response related to the risk of addiction and depression (non-patent document 20). The normal drinking water and 2% sugar water were each placed in the same drinking water bottle and supplied to the subject mice for 48 hours. After the positions of the two drinking water bottles were changed on the third day, the weight of the normal drinking water and 2% sugar water consumed for 24 hours was measured. The preference for sugar water is the ratio of the amount of sugar water consumed to the total consumption (the sum of general drinking water and sugar water consumption) in %, and there is no significant difference between the two mouse groups (FIG. 6A ). While many substances showing metabolic and emotional improvement efficacy have a high risk of poisoning, the results of this experimental example mean that treatment of the compound of Formula 8-9 does not cause a major problem in the pleasure response.

Tail suspension test (hereinafter referred to as TST) is a behavioral test that measures the risk of depression (Non-Patent Document 20). After fixing the tail of the subject mouse to a pre-installed stand, the motion of the mouse was photographed for 6 minutes. The absence of movement other than breathing is defined as'Immobility', and the time of immobilization reaction in 6 minutes is expressed as %. The higher the degree of depression, the longer the time of immobilization reaction and the antidepressant effect. Drugs are known to reduce the time of immobilization reactions. In mice administered with the compound of Formula 8-9, the immobility time was reduced compared to the control group at a statistically significant level, which means that the compound of Formula 8-9 has an antidepressant effect (FIG. 6B).

The open field test (hereinafter referred to as OFT) is a behavioral test for measuring anxiety in mice (non-patent document 20). When the subject mice are left alone in an open chamber (45 cm each in horizontal, vertical, and depth), they tend to stay at the edge of the wall rather than the central area, which is an instinctively open space. Therefore, after photographing the behavior of the mouse for 15 minutes, the time to stay in the central area corresponding to 25% of the total area was expressed as %. The higher the anxiety, the less the time to stay in the central area, and the anti-anxiety effect. It is known that administration of drugs increases the time to stay in the central region. In the mice administered with the compound of Formula 8-9, the residence time in the central region was increased at a statistically significant level compared to the control group, which means that the compound of Formula 8-9 has an anti-anxiety effect (FIG. 6C). . The total distance traveled in the test chamber for 15 minutes did not show a significant difference between the administration group and the control group, which is not due to the modulation of motor function due to the difference in the TST immobility time or the OFT central area occupancy time. And the fact that it is a characteristic caused by a difference in anxiety (FIG. 6D).

Meanwhile, the oxyiminomethylbenzene derivative represented by Chemical Formula 1 according to the present invention can be formulated in various forms depending on the purpose. The following are examples of several formulation methods in which the compound represented by Formula 1 according to the present invention is included as an active ingredient.

Formulation example 1. Preparation of tablets (pressurized)

As an active ingredient, after sieving 5.0 mg of the compound represented by Chemical Formula 1 of the present invention, 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressurized to form a tablet.

Formulation example 2. Preparation of tablets (wet granulation)

As an active ingredient, after sieving 5.0 mg of the compound represented by Formula 1 of the present invention, 16.0 mg of lactose and 4.0 mg of starch were mixed. After dissolving 0.3 mg of polysorbate 80 in pure water, an appropriate amount of this solution was added, followed by atomization. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were compressed into tablets.

Formulation example 3. Preparation of powders and capsules

As an active ingredient, after sieving 5.0 mg of the compound represented by Formula 1 of the present invention, it was mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. Mix the mixture using a suitable device. Filled in 5 gelatin capsules.

Formulation example 4. Preparation of Injection

As an active ingredient, 100 mg of the compound represented by Chemical Formula 1 of the present invention was added, and 180 mg of mannitol, 26 mg of Na2HPO4, 26 mg of 12H2O, and 2,974 mg of distilled water were prepared.

As described above, although the embodiments have been described by the limited drawings, those skilled in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order than the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (2)

Oxyiminomethylbenzene derivative represented by the following formula (1).
[Formula 1]

(In the above formula 1, R ¹ is a substituted or unsubstituted phenyl group connected by C1-C3 alkyl, wherein the substituent is hydrogen, halogen, cyano, hydroxy, NO ₂ , C1-6 haloalkyl, C1-6 alkoxy, Represents 1 to 4 substituents selected from the group consisting of C 1-6 haloalkoxy; R ² is a methyl group; A, E is hydrogen; B, C, D are each independently COOH, COOC 1-6 alkyl, COO (pyridine- NHC1-6alkyl) or C1 alkoxy substituted with COO (histamine), or hydrogen.)
According to claim 1,
The oxyiminomethylbenzene derivative enhances the amplitude of the circadian biorhythm and stabilizes the cycle by inhibiting the activity of CRYs, a transcriptional regulator of the circadian bioclock molecular network, and enhancing the activity of the CLOCK:BMAL1 dimer. Oxyiminomethylbenzene derivative.

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