KR101999899B1 - Novel benzopyranyl tetracycle compound and pharmaceutical composition having excellent anti-inflammatory effect comprising the same - Google Patents

Abstract

[화학식 5]

The present invention relates to novel compounds of the following formula (3j) or (5) (2l), and pharmaceutical compositions having excellent antiinflammatory effects comprising the same. These compounds inhibit the migration of the HMGB1 protein, which is an inflammation-inducing factor, from the nucleus to the cytoplasm, thereby having an excellent inflammation-inhibiting effect, and particularly, an excellent therapeutic or preventive effect against sepsis.
(3)

[Chemical Formula 5]

Description

TECHNICAL FIELD The present invention relates to novel benzopyranyl tetracycline compounds, and pharmaceutical compositions having excellent anti-inflammatory effects,

The present invention relates to a novel compound having benzopyran as a central structure, its use and a pharmaceutical composition containing the same. Specifically, the present invention relates to a novel benzopyranyl tetracycline compound, and a pharmaceutical composition having excellent antiinflammatory effect containing the same.

Inflammation refers to the pathological condition of abscesses formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various allergens). Specifically, when foreign bacteria invade a specific tissue and proliferate, the leukocyte of the living body recognizes it and actively attacks the propagated external bacteria. The death of leukocyte during this process is accumulated in the invaded tissue At the same time, the cell debris of invading microorganisms killed by leukocytes melts into the invading tissues and abscess forms. The treatment of abscess due to inflammation can be promoted by the anti-inflammatory action. The anti-inflammatory action is to inhibit the growth of invading microorganisms by using an antibacterial agent or to activate the macrophage that digests foreign substances accumulated in the abscess, And enhancing the function of excretory macrophages. Generally, an inflammatory reaction is a biological defense reaction process for repairing and regenerating damage caused by an invasion that causes a fundamental change in a cell or tissue of a living body. In the reaction process, localized blood vessels, various tissue cells of body fluids, Lt; / RTI > Normally, the inflammatory response induced by external invading bacteria is essential for survival as this defensive mechanism to protect the living body. However, temporally or spatially inadequate inflammatory responses may be caused by a wide range of diseases, including diseases that are apparently not associated with leukocytes such as arthritis or Alzheimer ' s disease, as well as by obvious leukocyte components such as autoimmune diseases, asthma or atherosclerosis . In these inflammatory diseases, leukocytes can be formed into tissues by an autoimmune response in which antibodies inadvertently recognize host proteins, or by improper triggering, such as persistent cell killing, extracellular cholesterol deposits, or accumulated tissue damage such as particulate matter in the lung Gathered. Such diseases are not able to treat the astringent white blood cells, for example, white blood cells can not remove or kill all the host cells expressing the autoantigen, or can not contain excessively large particles for the cells. Thus, it is often chronic and continues to release inflammatory cytokines, leading to additional leukocytes in unnecessary locations, leading to chronic inflammation. Such inflammatory reactions have been reported to cause chronic progressive diseases such as arteriosclerosis, obesity, insulin resistance, rheumatoid arthritis, glomerulonephritis, and cancer progression and play a major role in the progress of aging.

Sepsis, on the other hand, is a systemic inflammatory response syndrome due to microbial infection, which can progress to severe sepsis or septic shock. It is also referred to as severe sepsis when it is associated with low perfusion, hypotension, and organ dysfunction in the pathophysiology of sepsis, which occurs in about 10% of patients in the US intensive care unit.

Although many therapies for sepsis have been developed, the mortality rate for severe sepsis is nearly 20%, and annual hospital health care costs for patients with severe sepsis in the United States are the highest of all diseases and reach about $ 20 billion.

The major obstacle to the discovery of sepsis treatments is the diverse pathogenesis among patients. Because the heterogeneous forms of sepsis depend on pathogenic organisms and sites of infection, due to the diverse nature of the pathogenesis of sepsis, Mechanism.

Previous methods for treating sepsis using antibiotics, mediators such as TNF-α, IL-6, and IL-1, which mediate or promote the inflammatory response for the treatment of sepsis based on the inflammatory system and activation of early clinical sepsis There are many attempts to suppress the survival rate of the patients until now, but the situation is not much.

Sepsis occurs when the immune response becomes overactive and involves a refractory inflammatory response associated with unbalanced cytokine production, and an inflammatory cytokine cascade results in systemic shock.

In addition, tumor necrosis factor (TNF) -α and interleukin (IL) -1β are two major inflammatory cytokines associated with sepsis, and their secretion is regulated by positive reactions during systemic inflammatory responses.

Significant amounts of cytokines are produced when secreted cytokines circulate throughout the body, resulting in septic shock and secondary multiple organ dysfunction. Thus, a therapeutic approach to antagonize the production of these cytokines has been used in the treatment of sepsis.

For example, large-scale clinical trials of anti-TNF- [alpha] antibodies have been performed, but the production of TNF- [alpha] and IL-1 [beta] is present in the early stages of systemic inflammation, And persistent inflammatory stimuli were uneventfully restored after treatment with anti-TNF- [alpha] antibodies and development was discontinued.

On the other hand, HMGB1 (High Mobility Group Box 1) is a nuclear protein that is constantly expressed and secreted from ganglion cells and peripheral cells in which tissue damage occurs. There is more than one receptor for extracellular HMGB1, and receptor signaling induces cell division and cell migration, activates inflammation, and initiates an immune response. HMGB1-secreting cells include monocytes, macrophages, and human umbilical vein endothelial cells (HUVECs). When HMGB1 is activated and secreted, it causes severe vascular inflammation and sepsis , Leading to death.

HMGB1 induces the expression of adhesion molecules (VCAM-1, ICAM-1, E-selectin) on endothelial cells by binding to RAGE (receptor for advanced glycation end products) or pattern recognition receptors (TLR2 and TLR4). The inflammatory response in endothelial cells is initiated by HMGB1, which increases the secretion of TNF, IL-6 and induces phosphorylation of NF-kB, ERK-1 and ERK-2.

It is known that HMGB1 is detected in the serum of sepsis patients and the serum level of HMGB1 is greatly increased in patients with bad prognosis. It is also known that increased plasma HMGB1 levels in patients with severe sepsis are likely to die. Thus, HMGB1 is the target molecule for the prevention or treatment of sepsis or other vascular inflammatory diseases.

HMGB1 has been shown to mediate the inflammatory response, and when the damaged cells are destroyed by necrosis, HMGB1 is released into the extracellular environment and warns of damage to adjacent cells. In immune cells such as macrophages, HMGB1 is actively secreted as a pro-inflammatory cytokine during the systemic inflammatory response.

In previous reports, secreted HMGB1 plays an important role in regulating the production of other proinflammatory cytokines, and inhibition of HMGB1 secretion has been shown to be successful in the pathogenesis of sepsis in cecal ligation and puncture (CLP) mouse models used in the field of sepsis ( Proc. Natl. Acad. Sci . USA 2004, 101, 296-301).

In the CLP mouse model, it is established by performing surgical operations on the cecum, an organ in which the bacteria reside. The cecum is stuck and the tip is pierced with a needle to induce multinuclear contamination to the intraperitoneal system. This action subsequently leads to systemic inflammation, ie, sepsis. Previous researchers on the role of HMGB1 in sepsis have reported that the anti-HMGB1 antibody reduced the mortality of CLP mice by inhibiting the release of HMGB1.

In addition, the known self-modulating (-) - epigallocatechin-3-gallate (EGCG) modulator has been investigated to induce the degradation of cytosolic HMGB1, thereby promoting autophagic processes by preventing lipopolysaccharide (LPS) come.

The present inventors have found a novel compound, inflachromene (ICM, 1d), which inhibits the activation of BV-2 microglia-like cells by screening a pDOS (Diversity-Oriented Synthesis) -1645942).

(Compound ICM (1d))

HMM1 and HMGB2 were identified as the target proteins of the compound ICM (1d). As a result of biochemical and biophysical studies, the compounds ICM (1d ) Inhibited the secretion of HMGB1 and HMGB2 in microglia.

Therefore, the present inventors modified the substituent of the compound ICM (1d) to synthesize a number of candidate compounds, which are similar compounds, and then, based on the structure-activity correlation study, ≪ / RTI > to the present invention.

The present inventors examined the inflammation inhibitory effect using the candidate compounds, and confirmed the migration and secretion inhibitory action of HMGB1 protein which induces inflammation in particular. Specifically, the present invention has been accomplished by measuring the survival rate and the like after in vivo administration of the compound ICM (1d) and the candidate compounds to the CLP mouse model.

In addition, the present inventors have found that controlling HMGB1 release is a promising strategy for prevention or treatment of sepsis, thereby providing novel compounds having an excellent therapeutic effect on sepsis.

The present invention provides novel compounds which show excellent effects in the body based on systematic structure-activity correlation studies.

The novel compounds provide novel uses for the anti-inflammatory effect.

In particular, the novel compounds provide an application for treating or preventing sepsis.

Particularly, the novel compounds inhibit the HMGB1 protein acting in the latter half of the inflammatory reaction, so that the anti-inflammatory effect, especially the sepsis treatment effect, is superior to the existing therapeutic agents.

First, the present inventors provide a compound of the formula (I), or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

R is optionally substituted C _1-8 alkyl; Substituted or unsubstituted C _1-8 alkoxy; Substituted or unsubstituted C _1-8 dialkoxy; Substituted or unsubstituted C _1-8 alkylcarbonyl; Lt; RTI ID = 0.0 &

Preferably, the compound represented by Formula 1 may be a compound represented by Formula 2 (2i) to 5 (Formula 2l).

Most preferably, the compound represented by Formula 1 is a compound represented by Formula 3 (compound 2j) or Formula 5 (compound 2l).

Further, the present inventors provide a pharmaceutical composition having an inflammation-inhibiting effect comprising a compound represented by the general formula (3) or (5).

The present invention also provides a pharmaceutical composition for treating and preventing sepsis comprising the compound represented by the general formula (3) or (5).

The novel compounds of the present invention can block the inflammatory response by inhibiting the HMGB1 protein from being transferred from the nucleus to the cytoplasm and secreted out of the cell. In particular, since the HMGB1 protein acts in the latter half of the inflammatory reaction, the novel compounds of the present invention that inhibit the migration and secretion of HMGB1 protein can be used as an excellent therapeutic agent that can be effective even in patients who have not undergone early treatment.

In addition, the novel compounds of the present invention can be used as therapeutic agents for excellent inflammation-related diseases, by decreasing the secretion of IL-6 and increasing the survival rate of the patient.

The novel compounds of the present invention have pharmacokinetic properties such as excellent solubility, stability and PK data, and thus have advantages of being easily formulated into pharmaceuticals in the future.

Figure 1 shows the inflammatory response inhibitory pathway of compound 1d.
Figure 2 shows the survival rate of the CLP mouse model in the control and compound 1d treated groups.
Figure 3 shows mouse serum IL-6 levels of control (A) and compound 1d treated group (B) in a CLP mouse model.
Fig. 4 shows the inhibition rate of NO emission of the compound 1d (A), the compound (B) in which the double bond of the tetrahydropyridazine ring is removed, and the compound (C) in which the double bond is transferred.
Figure 5 shows the cytopathological fluorescence image of HMGB1 after pretreatment with compound 1d and compounds 2i to 2l.
Figure 6 shows Western bloc analysis results to confirm the inhibitory effect of IL-6 release after pretreatment with compound 1d and compounds 2i to 2l.
Figure 7 shows the binding energy (A) of compounds 1d, 2j and 2l as a ligand of HMGB1, the binding mode (B) and the interaction (C) with adjacent amino acids in the binding cavity of HMGB1.
8 shows the result of Western blot analysis (A) on the phosphorylation (p) of p38, JNK and ERK after pre-treatment with compounds 1d, 2j and 2l in Raw264.7 cells, (B). ≪ / RTI >
Fig. 9 shows a cell NF-κB p65 immunofluorescence image obtained by pretreating compounds 1d and 2j and 2l in Raw264.7 cells, respectively, and then treating with 500 ng / ml LPS.
Fig. 10 shows the survival rate of CLP mice treated with the control group and compounds 1d, 2j and 2l, respectively.
Figure 11 shows the IL-6 levels of mice treated with serum IL-6 (A), compound 1d (B), compound 2j (C) and compound 21 (D), respectively.

The present inventors have found that Compound 1d, a compound that inhibits the HMGB1 protein in the CLP mouse model, effectively improves the pathogenesis of sepsis, and synthesized candidate compounds similar to Compound 1d.

Among these compounds, compounds having a superior anti-inflammatory effect were selected. As a result, the present inventors have invented novel compounds having the best anti-inflammatory effect, based on structure-activity correlation studies and biological evaluation experiments.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

R is optionally substituted C _1-8 alkyl; Substituted or unsubstituted C _1-8 alkoxy; Substituted or unsubstituted C _1-8 dialkoxy; Substituted or unsubstituted C _1-8 alkylcarbonyl; Lt; RTI ID = 0.0 &

Most preferably, the present invention provides a compound represented by the following formula (3) or a compound represented by the following formula (5), or a pharmaceutically acceptable salt thereof.

(3)

[Chemical Formula 5]

There is provided a pharmaceutical composition having an anti-inflammatory effect comprising the novel compound represented by the above-mentioned general formula (3) or (5).

The inflammation-inhibiting effect is characterized by exhibiting an anti-inflammatory effect by acting on HMGB1 protein, an inflammation-inducing factor.

Specifically, the compound inhibits the phosphorylation or acetylation of the HMGB1 protein, thereby inhibiting the migration of the HMGB1 protein from the nucleus to the cytoplasm and consequently inhibiting the secretion of the HMGB1 protein into the cell.

The inflammatory reaction is a biological defense reaction process for restoring and regenerating damage caused by invasion that causes a fundamental change in the cell or tissue of a living body. In this reaction process, localized blood vessels, various tissue cells of body fluids, do. Normally, the inflammatory reaction induced by external invading bacteria is a defense system to protect the living body. On the other hand, when abnormally excessive inflammatory reaction is induced, various diseases appear. Such diseases include gastritis, colitis, rheumatoid arthritis, Chronic diseases such as pancreatitis, septicemia, seizures, cancer, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease and neuroinflammatory diseases such as acute brain injury such as stroke and brain trauma are known. Especially, sepsis, seizure, HMGB is known to affect.

Accordingly, the pharmaceutical composition comprising the novel compound represented by the general formula (3) or (5) is useful as a pharmaceutical composition for preventing or treating sepsis, gastritis, colitis, rheumatoid arthritis, nephritis, hepatitis, pancreatitis, sepsis, seizure, multiple sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Related diseases selected from the group consisting of amyotrophic lateral sclerosis (ALS), stroke, trauma, spinal cord injury, and cancer.

In particular, the pharmaceutical composition comprising the novel compound represented by the general formula (3) or (5) has an effect of treating or preventing sepsis.

The present invention relates to a novel pharmaceutical composition having an anti-inflammatory effect comprising a compound represented by the general formula (3) or (5).

The present invention also relates to a pharmaceutical composition for treating or preventing sepsis comprising the novel compound represented by the general formula (3) or (5).

Preparation of compounds 1a to 1j

Compounds 1a to 1j were synthesized according to Reaction Scheme 1 below.

[Reaction Scheme 1]

In step a), hydroxyacetophenone having appropriate R ₁ , R ₂ , R ₃ functional groups was added with ketone and pyrrolidine in ethanol (EtOH) solvent and refluxed.

b) In step it was refluxing and the addition of CuBr2 under EtOAc / CHCl ₃ / MeOH solvent mixture.

In step c), NaBH ₄ was added in EtOH solvent and reacted at 40 ° C.

In step d), p-TsOH was added in a toluene solvent, and the reaction was carried out at 70 ° C.

In step e), dibutyl vinylboronic acid ester, Na ₂ CO ₃ , and Pd (PPh ₃ ) ₄ were added in a mixed solvent of EtOH / toluene / H ₂ O and the reaction was carried out at 70 ° C.

In step f), phenyltriazolindione was added in a toluene solvent, and the reaction was allowed to proceed at room temperature.

In the above production method, it is preferable to protect the R ₂ or R ₃ functional group with an OH group (1d, 1h, 1i, 1j) with a triisopropylsilyl protecting group before the step d) The title compound was deprotected with a mixture of hydrofluoric acid and pyridine in THF solvent to obtain the final compound.

When R ₂ functional group is an N-methylamino group (1b), it is preferable to protect with a Boc protecting group before step e), and after f), the compound is deprotected with trifluoroacetic acid in a dichloromethane solvent to obtain the final compound.

When the R ₄ functional group is N-propyl-4'-piperidyl or (1i) N-acetyl-4'-piperidyl (1j), N-Boc-piperidinone protected with a Boc protecting group After the boc protecting group is first deblocked with trifluoroacetic acid in a dichloromethane solvent prior to deprotonation with respect to the R ₂ functional group, the biphenyltetracarboxylic acid is deprotected with propioldehyde (1i) was subjected to a reductive amination reaction using triacetoxyborohydride or an acetylation reaction (1j) using acetic anhydride and diisopropylethylamine base in a dichloromethane solvent to give a final compound.

Table 1 below shows the structures, molecular weights, MS analysis values and NMR of the compounds 1a to 1j prepared through the above reaction schemes.

compound rescue Molecular Weight MS analysis value
(Molecular weight + H ⁺ ) NMR 1a

361.40 361.1429 [M] ^< ^{+ & 1 H-NMR (500 MHz,} CDCl 3): d 7.64 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 5.80 (t , J = 2.5 Hz, 1H) , 5.73 (s, 1H), 4.30 (dd, J = 16.5 and 5.0 Hz, 1H), 4.11 (dt, J = 16.5 and 2.5 Hz, 1H), 1.62 (s, 3H) , ≪ / RTI > 1.57 (s, 3H); ^{13 C-NMR (125 MHz,} CDCl 3): d153.8, 153.4, 151.6, 138.2, 131.4, 129.4, 129.3, 128.5, 125.6, 124.3, 123.5, 121.9, 118.2, 112.9, 79.4, 50.7, 44.3, 28.2, 27.2 1b

390.44 391.09 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.64 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.85 (dd, J = 8.5 and 2.0 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 5.85 (t, J = 2.5 Hz, 1H), 5.73 (s, 1H), 4.30 (dd, J = 14.0 and 3.5 Hz, 1H), 4.10 (dt, J = 14.0 and 2.5 Hz, 1H) s, 3H), 1.46 (s, 9H) 1c

379.39 380.1409 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.62 (d, J = 7.5 Hz, 2H), 7.52 (t, J = 7.5 Hz, 2H), 7.45 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 8.0 Hz, 1H), 6.66 (td, J = 8.5 and 2.5 Hz, 1H), 6.60 (dd, J = 8.5 and 2.5 Hz, 1H), 5.86 (t, J = 2.5 Hz, 1H) , 5.70 (s, 1H), 4.30 (dd, J = 16.5 and 5.0 Hz, 1H), 4.11 (dt, J = 16.5 and 2.5 Hz, 1H), 1.60 (s, 3H), 1.59 ^{13 C-NMR (125 MHz,} CDCl 3): d 164.5, 162.5, 154.7, 153.9, 151.7, 137.2, 131.4, 129.5, 128.6, 125.6, 125.2 (d), 119.6 (d), 113.7, 108.8 (d), 105.6 (d), 79.7, 50.3, 44.4, 27.9, 27.0 1d

377.40 377.1380 [M] ^< ^{+ & 1 H-NMR (500 MHz,} acetone- d 6): d 8.47 (br.s., 1H), 7.65 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.44 (dd, J = 8.5 and 2.5 Hz, 1H), 6.35 (d, J = 2.5 Hz, 1H), 6.02 (t, J = 2.5 Hz, 1H), 5.64 (s, 1 H), 4.25 (dd, J = 16.5 and 5.0 Hz, 1 H), 4.14 (dt, J = 16.5 and 2.5 Hz, 1 H), 1.61 (s, 3 H), 1.57 (s, 3 H); ^{13 C-NMR (125 MHz,} acetone- d 6): d 159.2, 155.3, 155.0, 152.2, 138.0, 133.0, 129.6, 128.7, 126.7, 125.6, 116.3, 114.6, 109.4, 105.3, 79.8, 50.6, 45.2, 27.8 , 27.2 1e

391.43 391.16
[M] ^{+ 1 H-NMR (500 MHz,} CDCl 3): d 7.63 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.52 (dd, J = 8.5 and 3.0 Hz, 1H), 6.45 (d, J = 3.0 Hz, 1H), 5.86 (t, J = 2.0 Hz, 1H), 5.74 (d, J = 16.0 and 5.0 Hz, 1H), 4.12 (dt, J = 16.0 and 2.5 Hz, 1H), 3.77 (s, 3H), 1.62 s, 3H); ¹³ C-NMR (125 MHz, CDCl ₃ ): d 161.0, 154.5, 154.1, 151.7, 137.5, 131.5, 129.5, 128.5, 125.7, 124.9, 115.7, 113.1, 108.0, 103.3, 79.0, 55.6, 50.3, 27.7, 27.0 1f

391.43 392.10 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.64 (d, J = 7.0 Hz, 2H), 7.53 (t, J = 7.0 Hz, 2H), 7.43 (t, J = 7.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.61 (s, 1H), 5.76 (q, J = 2.5 Hz, 1H), 5.67 (s, 1H), 4.30 (dd, J = 16.5 and 5.0 Hz, 1H), 4.13 (dd, J = 16.5 and 2.5 Hz, 1H), 3.74 (s, 3H), 1.53 (s, 3H), 1.42 (s, 3H); ^{13 C-NMR (125 MHz,} CDCl 3): d154.9, 153.6, 151.6, 147.1, 139.3, 131.4, 129.4, 128.5, 126.1, 125.6, 119.0, 114.1, 112.6, 109.1, 79.6, 55.9, 51.2, 44.1, 28.4, 27.3 1g

421.45 422.11 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.60 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.43 (t, J = 7.5 Hz, 1H), 6.76 (s, 1H), 6.46 ( s, 1H), 5.88 (t, J = 2.5 Hz, 1H), 5.74 (s, 1H), 4.32 (dd, J = 16.5 and 5.0 Hz, 1H), 4.13 (dt, J = 16.5 and 2.5 Hz, 1H ), 3.83 (s, 3H), 3.78 (s, 3H), 1.61 (s, 3H), 1.59 (s, 3H) 1h

407.43 408.11 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.62 (d, J = 7.5 Hz, 2H), 7.52 (t, J = 8.0 Hz, 2H), 7.41 (t, J = 7.5 Hz, 1H), 6.58 (d, J = 9.0 Hz, 1H), 6.53 (d, J = 8.5 Hz, 1H), 5.84 (t, J = 2.5 Hz, 1H) , 4.31 (dd, J = 16.5 and 5.0 Hz, 1H), 4.09 (dt, J = 16.5 and 2.5 Hz, 1H), 3.86 (s, 3H), 1.65 (s, 3H), 1.63 (s, 3H); ^{13 C-NMR (125 MHz,} CDCl 3): d154.0, 151.7, 147.5, 140.7, 138.0, 135.3, 131.4, 129.5, 128.6, 125.7, 117.9, 113.6, 113.4, 105.0, 80.4, 56.5, 50.4, 44.5, 28.0, 27.3 1i

460.53 460.91 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.62 (d, J = 8.5 Hz, 2H), 7.52 (t, J = 7.5 Hz, 2H), 7.44 (t, J = 7.5 Hz, 1H), 6.93 (t, J = 9.0 Hz, 1H), 6.53 (dd, J = 8.5 and 2.0 Hz, 1H), 6.46 (d, J = 2.0 Hz, 1H), 5.92 (t, J = 2.5 Hz, 1H), 5.61 (s, 1H), 4.30 ( dd, J = 16.5 and 5.0 Hz, 1H), 4.11 (dt, J = 16.5 and 2.5 Hz, 1H), 3.12 (q, J = 7.5 Hz, 2H), 2.92-2.89 ( 2H), 1.23 (q, J = 7.5 Hz, 2H), 2.34-3.39 (m, 2H), 2.18-2.03 (m, 2H), 1.87-1.78 3H), 1.08 (d, J = 7.5Hz, 18H), 1.01 (t, J = 7.5 Hz, 3H) 1j

460.49 460.94 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.63 (d, J = 7.5 Hz, 2H), 7.53 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.51 (dd, J = 8.5 and 2.0 Hz, 1H), 6.48 (d, J = 2.0 Hz, 1H), 5.80 (t, J = 2.0 Hz, 1H), 5.68 (d, J = 16.5 and 5.0 Hz, 1H), 4.12 (d, J = 16.5 Hz, 1H), 3.46-3.37 (m, 2H), 3.35-3.18 2.17-2.13 (m, 2H), 1.92-1.78 (m, 2H), 1.50 (s, 9H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5Hz, 18H)

Preparation of compounds 2a to 2o

Compounds 2a to 2o were synthesized according to Reaction Scheme 2 below.

[Reaction Scheme 2]

Starting from the diene compound prepared in step a) through e) of Scheme 1 in step 1), in step g), a triazole dione derivative having various R-functional groups was added in a toluene / THF mixed solvent In step h), the OH functional group is deblocked with a fluoric acid-pyridine mixture in a THF solvent to obtain the final compound 2a to 2o.

Table 2 shows the structure, molecular weight, MS analysis value and NMR of the compounds 2a to 2o prepared through the above reaction formula.

compound rescue Molecular Weight MS analysis value
(Molecular weight + H ⁺ ) NMR 2a

315.33 315.91 ^{1 H-NMR (500 MHz,} CDCl 3): d 6.82 (d, J = 8.5 Hz, 1H), 6.46 (dd, J = 8.0 and 2.5 Hz, 1H), 6.41 (d, J = 2.5 Hz, 1H) , 5.77 (t, J = 2.5 Hz, 1H), 5.61 (s, 1H), 4.21 (dd, J = 16.5 and 5.0 Hz, 1H), 3.96 (dt, J = 16.5 and 2.5 Hz, 1H), 3.23 ( (s, 3H), 1.57 (s, 3H), 1.56 (s, 3H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5 Hz, 18H); ¹³ C-NMR (125 MHz, CDCl ₃ ): d 157.3, 155.7, 154.2, 153.0, 137.9, 124.3, 116.6, 113.5, 112.9, 109.5, 79.0, 50.2, 44.3, 27.8, 27.1, 25.6, 18.1, 12.8. 2b

391.43 391.01 [M] ^< ⁺ & gt ^{; 1 H-NMR (500 MHz,} CDCl 3): d 7.48 (d, J = 8.0 Hz, 2H), 7.37-7.30 (m, 3H), 6.68 (d, J = 8.5 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 6.34 (dd, J = 8.5 and 2.0 Hz, 1H), 5.74 (t, J = 2.5 Hz, 1H), 5.56 (s, 1H), 4.82 (d, J = 4.5 Hz, 2H), 4.19 (dd, J = 16.5 and 5.0 Hz, 1H), 3.95 (dt, J = 16.5 and 2.5 Hz, 1H), 1.55 (s, 3H), 1.54 (s, 3H), 1.23 (q, J = 7.5 Hz, 3H), 1.10 (d, J = 7.5 Hz, 18H). 2c

383.45 384.01 ^{1 H-NMR (500 MHz,} CDCl 3): d 6.75 (d, J = 8.5 Hz, 1H), 6.45 (dd, J = 8.5 and 2.5 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H) , 5.73 (t, J = 2.5 Hz, 1H), 5.55 (s, 1H), 4.15 (dd, J = 16.0 and 5.0 Hz, 1H), 4.02 (tt, J = 10.0 and 3.0 Hz, 1H), 3.92 ( (d, J = 16.0 and 2.5 Hz, 1H), 2.23 (t, J = 2.5 Hz, 2H), 1.90-1.83 (m, 6H), 1.71-1.67 (s, 3H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5 Hz, 18H). 2d

503.30 503.86 ^{1 H-NMR (500 MHz,} CDCl 3): d 8.02 (t, J = 8.0 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.49 (dd, J = 8.5 and 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 5.83 (t, J = 2.5 Hz, 1H), 5.73 (s, 1H), 4.32 (dd, J = 16.5 and 5.0 Hz, 1H), 4.12 (dt, J = 16.5 and 2.5 Hz, 1H), 1.61 (s, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5 Hz, 18H) 2e

407.43 408.2 ^{1 H NMR (400 MHz, DMSO-} d 6): d 9.47 (s, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.38-6.42 , 1.58 (s, 3H), 1.58 (s, 3H) (more stable conformal); 5.58 (s, 1H), 4.02-4.24 (m, 2H), 3.88 ^{13 C NMR (100 MHz, DMSO-} d 6): δ 158.2, 158.2, 155.4, 154.0, 153.9, 151.4, 136.5, 131.2, 130.4, 123.7, 120.7, 119.4, 114.8, 112.5, 104.3, 104.2, 79.2, 56.0, 49.3, 44.1, 27.3, 26.8 2f

407.43 407.98 ^{1 H-NMR (500 MHz,} CDCl 3): d7.43 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 7.20 (t, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.5 and 2.5 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.48 (dd, J = 8.5 and 2.5 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H J = 16.5 and 2.5 Hz, 1H), 3.81 (dd, J = 16.5 and 5.0 Hz, 1H), 5.81 (t, J = 2.5 Hz, 1H) (s, 3H), 1.60 ( s, 3H), 1.59 (s, 3H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5Hz, 18H) 2g

407.43 407.92 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.51 (d, J = 10.0 Hz, 2H), 7.03 (d, J = 10.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.47 (dd, J = 8.5 and 2.0 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 5.80 (t, J = 2.5 Hz, 1H), 5.68 (s, 1H), 4.30 (dd, J = (S, 3H), 1.57 (s, 3H), 1.23 (q, J = 7.5 Hz, 1H), 4.08 (dt, J = 16.5 and 2.5 Hz, Hz, 3H), 1.08 (d, J = 7.5 Hz, 18H). 2h

395.39 393.82
[MH] ^{- 1 H-NMR (500 MHz,} CDCl 3): d 7.62 (dt, J = 7.0 and 2.5 Hz, 2H), 7.20 (t, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 1H) , 6.47 (dd, J = 8.5 and 2.0 Hz, 1H), 6.43 (d, J = 2.0 Hz, 1H), 5.80 (t, J = 2.5 Hz, 1H), 5.68 (s, 1H), 4.29 (dd, J = 16.5 and 5.0 Hz, 1H ), 4.09 (dt, J = 16.5 and 2.5 Hz, 1H), 1.59 (s, 3H), 1.57 (s, 3H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5 Hz, 18 H); ^{13 C-NMR (125 MHz,} CDCl 3): d163.2, 161.2, 157.4, 154.2, 153.9, 151.6, 137.9, 127.6 (d), 124.3, 116.6, 116.4, 113.6, 112.7, 109.6, 79.1, 50.5, 44.4 , 27.9, 27.1, 18.1, 12.8. 2i

391.43 391.1535 [M] ^< ⁺ & gt ^{; 1 H-NMR (500 MHz,} CDCl 3): d 7.46 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 1H), 6.36 (dd, J = 8.5 and 2.5 Hz, 1H), 6.33 (d, J = 2.5 Hz, 1H), 5.81 (t, J = 2.5 Hz, 1H), 5.68 (s, 1H), 5.44 (br.s, 1H), 4.29 (dd, J = 16.5 and 5.0 Hz, 1H), 4.08 (dt, J = 16.5 and 2.5 Hz, 1H), 2.40 (s, 3H), 1.57 (s, 6H); ¹³ C-NMR (125 MHz, CDCl ₃ ): d 157.3 154.2, 154.1, 151.8, 138.8 137.4, 130.0, 128.3, 125.7, 124.5, 115.3, 112.6, 109.1, 105.0, 78.9, 50.2, 44.3, 27.6, 2j

419.44 420.1 ^{1 H NMR (400 MHz, DMSO-} d 6): δ 9.47 (s, 1H), 8.13 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.4 Hz, 1H), 6.37 (dd, J = 8.8 Hz, 2.0 Hz, 1H), 6.25 (d, J = 1.6 Hz, 1H), 5.95 (s, 1H), 5.61 (s, 1H), 4.11- 4.25 (m, 2H), 2.64 (s, 3H), 1.54 (s, 3H), 1.51 (s, 3H); ^{13 C NMR (100 MHz, DMSO-} d 6): δ 197.2, 158.2, 153.8, 153.3, 150.5, 136.0, 135.8, 135.6, 128.9, 125.7, 124.5, 114.6, 113.6, 108.6, 104.1, 79.0, 49.3, 44.0, 27.4, 26.9, 26.8 2k

421.41 422.1 ^{1 H NMR (400 MHz, CDCl} 3): δ 7.00-7.04 (m, 2H), 6.83-6.92 (m, 2H), 6.31-6.36 (m, 2H), 6.07 (s, 1H), 6.02 (s, 2H), 5.78-5.81 (m, IH), 5.66 (s, IH), 4.24-4.30 (m, IH), 4.04-4.10 (m, IH), 1.78 (s, 6H); ^{13 C NMR (100 MHz, CDCl} 3): δ 157.2, 154.3, 154.1, 151.8, 148.3, 147.9, 137.4, 124.6, 124.4, 120.1, 115.4, 112.7, 109.0, 108.6, 107.4, 105.0, 102.0, 78.9, 50.2, 44.3, 27.6, 26.9 2l

437.45 438.1 ^{1 H NMR (400 MHz, DMSO-} d 6): δ 9.46 (s, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 2.4 Hz, 2H), 6.614 (s, 1H ), 6.38 (dd, J = 8.0 Hz, 2.2 Hz, 1H), 6.25 (d, J = 2.4 Hz, 1H), 5.94 ), 3.79 (s, 6 H), 1.53 (s, 3 H), 1.50 (s, 3 H); ^{13 C NMR (100 MHz, DMSO-} d 6): δ 160.4, 158.2, 153.8, 153.7, 150.8, 136.1, 133.0, 124.4, 114.6, 113.6, 108.6, 105.0, 79.0, 55.6, 55.5, 49.2, 44.0, 27.3, 26.7 2m

445.12 446.0 ^{1 H NMR (400 MHz, DMSO-} d 6): δ 9.47 (brs, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 1H), 6.37 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 6.25 (d, J = 2.0 Hz, 1H), 5.95 (s, 1H), 5.61 (s, 1H), 4.16- 4.23 (m, 2 H), 1.54 (s, 3 H), 1.51 (s, 3 H); ^{13 C NMR (100 MHz, DMSO-} d 6): δ 158.2, 153.8, 153.2, 150.4, 130.9, 135.2, 126.4, 126.1, 124.5, 114.5, 113.5, 108.6, 104.1, 79.2, 79.0, 49.3, 44.0, 27.4, 26.8 2n

433.51 433.96 ^{1 H-NMR (500 MHz,} CDCl 3): d 7.53 (s, 4H), 6.91 (d, J = 8.5 Hz, 1H), 6.46 (dd, J = 8.5 and 2.5 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 5.80 (t, J = 2.5 Hz, 1H), 5.69 (s, 1H), 4.31 (dd, J = 16.5 and 5.0 Hz, 1H), 4.08 (dt, J = 16.5 and 2.5 Hz, 1H), 1.59 (s, 3H), 1.57 (s, 3H), 1.35 (s, 9H), 1.23 (q, J = 7.5 Hz, 3H), 1.08 (d, J = 7.5 Hz, 18H); ¹ H NMR (125 MHz, CDCl ₃ ): d 157 7.3, 154.2, 151.9, 151.6, 147.9, 128.7, 126.5, 125.3, 124.4, 116.5, 113.6, 112.8, 109.6, 79.1, 50.5, 44.5, 35.0, 31.5, 29.9, 27.9, 27.1, 18.1, 12.9 2o

433.51 434.2 ^{1 H NMR (400 MHz, CDCl} 3): δ 7.48 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.0 Hz, 1H), 6.32- (M, 2H), 5.68 (s, 1H), 4.28 (dd, J = 16.4 Hz, 4.4 Hz, 1H), 4.05-4.10 J = 7.6 Hz, 2H), 1.55-1.64 (m, 2H), 1.56 (s, 6H), 1.37 (sextet, J = 7.5 Hz, 2H), 0.94 (t, J = 7.2 Hz, 2H); ^13.1 C NMR (100 MHz, CDCl ₃ ):? 157.1, 154.3, 154.1, 151.8, 143.6, 137.5, 129.4, 128.6, 125.5, 124.7, 115.6, 112.8, 109.0, 105.0, 79.0, 50.2, 44.3, 35.5, 33.6, 27.6, 26.9, 22.4, 14.1

[Test Example 1]

Inhibitory Effect of Compound 1d on CLP Mouse Model

When pathogens infiltrate into host organisms, acute inflammatory reactions begin in the body. Macrophages, one of the major immune cells mediating the inflammatory response, are recognized to be activated by pathogens, and various inflammatory cytokines such as HMGB, TNF-α and IL- Causes the secretion of cine.

As shown in FIG. 1, after recognizing LPS (lipopolysaccharides) and activating macrophages, HMGB migrates from the nucleus to the cytoplasm and is released into the extracellular environment. Proinflammatory cytokines activate other nearby macrophages and initiate a positive feedback mechanism of the immune response.

On the other hand, when Compound 1d is treated, it can be seen that the inhibited immune response is exhibited.

The survival rate of CLP mice after intraperitoneal administration of compound 1d at a daily dose of 10 mg / kg for 9 days in the CLP mouse model is shown in Fig. It was confirmed that the survival rate of the compound 1d-treated group was significantly increased compared with the survival rate of the control group.

In addition, the survival rate and the IL-6 level of compound 1d administered at a daily dosage of 10 mg / kg for 9 days starting from 1 hour before CLP induction in the CLP mouse model are shown in FIG. As shown in FIG. 3, IL-6 levels in surviving mice were significantly reduced in the compound 1d-treated group as compared with the control group.

In conclusion, IL-6 levels are highly correlated with survival rates.

[Test Example 2]

The inflammation-inhibiting effect of the compounds 1a to 1j

When inflammation is induced, the secretion of nitric oxide increases, and the treatment of inflammation inhibitor suppresses nitric oxide secretion. Therefore, the Griess assay was used to measure nitric oxide release in Raw264.7 cells for evaluation of inhibition of inflammation.

Raw264.7 cell culture

Raw 264.7 macrophages were cultured in DMEM (Dulbecco Modified Eagle Medium) medium containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) antibiotic and antimycotic solution Lt; / RTI > The cells were cultured in 100 mm cell culture dishes under standard cell culture conditions (37 ° C, 5% CO2, humidified atmosphere).

Griess assay

0.1% (m / v), sulfanilamide 1% (m / v), phosphoric acid (85%), N- (1-naphthyl) ethylenediamine dihydrocholoride, ) And deionized water 5% (v / v).

Raw 264.7 cells were seeded on a clear 96-well plate and cultured for 1 day. After the medium was absorbed, each well was washed twice with serum-free DMEM medium and the serum-free DMEM medium was replenished. Thereafter, cells were treated with 100 ng / mL LPS and each compound and incubated for 24 hours.

 After incubation, the supernatant culture medium was transferred to a new 96-well plate and mixed with 50 μL of Griess assay reagent. The wells treated with the medium and reagent mixture were incubated at room temperature for several minutes and absorbance was measured at 548 nm.

As a control group, two wells treated with LPS and DMSO and two wells treated with DMSO were prepared and blank values were measured. Blank values were determined by mixing 50 μL of DMEM medium containing 10% FBS and 1% antibiotic and antifungal agent and Griess analysis reagent.

The experiment was performed at least three times independently.

When the compounds 1a to 1j were each treated, the inhibition rate (%) of nitric oxide release was confirmed as compared with the control group, and the results are shown in Table 3 below.

As shown in Table 3, it was confirmed that Compound 1d inhibited NO release by 87% in Raw264.7 cells.

Compounds 1a to 1h, in which the substituents R1 to R3 of the benzopyranyl core structure are modified, show that the inhibitory effect on NO release is reduced compared to compound 1d, which has an inhibition rate of 87% against NO release.

It can also be seen that when the methyl group at the R4 position of the compound 1d is modified like the compounds 1i and 1j, the inhibition of NO release is significantly reduced.

Therefore, it was confirmed that the substituents R1 to R4 of Compound 1d are most appropriate for the inflammation-inhibiting effect.

[Test Example 3]

Inflammation-inhibiting effect of a compound in which a double bond of a tetrahydropyridazine ring is moved or removed

The inhibition rate of NO release was measured in the same manner as in Test Example 2 for the compounds in which the double bond of the tetrahydropyridazine ring was removed or removed from the compound 1d and is shown in FIG.

(B, 17%) and double bond migration (C, 56%) compared to compound 1d (A, 87%). Therefore, it can be seen that the inhibitory effect of NO release due to the migration or elimination of the double bonds is rather reduced.

[Test Example 4]

The anti-inflammatory effect (rate of inhibition of NO release)

In order to confirm the anti-inflammatory effect of the compounds 2a to 2o prepared in Example 2, the inhibition rate of NO release was measured in the same manner as in Test Example 2 and is shown in Table 4. [

compound rescue Nitric oxide release
% Inhibition 1d

87 2a

45 2b

70 2c

63 2d

48 2e

66 2f

96 2g

89 2h

87 2i

94 2j

93 2k

92 2l

94 2m

85 2n

99 2o

99

As shown in Table 4, Compounds 2d (63%) and 2e (48%) in which a substituent was introduced at the Ortho position did not have an inhibitory effect on NO release as compared with Compound 1d.

Compounds 2f (96%) with a methoxy substituent at the meta position and 2g (89%) with a methoxy substituent at the para position have NO release And the inhibitory effect of < RTI ID = 0.0 >

As a result, it can be seen that the compounds 2f, 2g to 2l, 2n and 2o are superior to the compound 1d in the suppression effect of NO release.

[Test Example 5]

Comparative cell activity of 2a to 2o compounds

Soluble tetrazolium (WST) assay in order to determine relative cell viability for the compounds 2a to 2o prepared in Example 2.

WST (water-soluble tetrazolium) analysis

Raw 264.7 cells were seeded on a clear 96-well plate and maintained for 1 day. After the medium was absorbed, each well was washed twice with serum-free DMEM and refilled with serum-free DMEM. After treatment with 100 ng / mL LPS and each compound and incubation for 24 hours, the supernatant culture medium was transferred to a new 96-well plate.

Immediately after transferring the medium, 100 μL of DMEN medium containing 10% FBS and 1% antibiotic and antimicrobial agent was replenished in the seeded wells of the cells. Each well was then treated according to the manufacturer's protocol using 10 μL of Ez-cytox WST assay reagent.

After incubation for 20 min in standard cell culture conditions (37 ° C, 5% CO2, humidified atmosphere), the absorbance was measured at 450 nm.

The blank was determined by measuring the absorbance of the well treated with DMEM medium containing 10% FBS and 1% antibiotic and antimicrobial agent. The experiment was performed at least three times.

The results of measuring relative cell viability through the above experiment are shown in Table 5 below.

compound relative cell viability (%) 1d 105 2a 114 2b 106 2c 117 2d 109 2e 115 2f 118 2g 127 2h 109 2i 130 2j 111 2k 121 2l 121 2m 106 2n 105 2o 50

As shown in Table 5, the comparative cell activity of Compound 1d was 105, and the compounds 2a to 2l showed superior comparative cell activity as compared with Compound 1d.

[Test Example 6]

The inhibitory effect of the 2a-2l compound on inflammation (TNF-a secretion inhibiting rate)

From the results of Test Example 5, the results of Test Example 4 are shown to show similar or inferior NO release inhibitory effects as compared with Compound 1d, except for compounds 2m to 2o which exhibit similar or inferior comparative cell activity as Compound 1d Compounds 2a to 2e were excluded.

Therefore, in order to further confirm the anti-inflammatory effect of the compounds 2f to 2l, the TNF-a secretion inhibition rate was measured using an ELISA.

Cells were treated with each compound id and 2f to 2l, and LPS for 24 hours, followed by ELISA for TNF-a present in the culture.

ELISA

To determine the amount of secreted TNF- [alpha], Raw 264.7 cells were seeded in clear 96-well plates and cultured for 1 day. After 1 day, the absorbed medium was replaced with serum-free DMEM medium containing 1% (v / v) antibiotic and antifungal agent solution, and treated with 100 ng / mL LPS and 10 μM each compound.

After 4 hours, the medium was transferred to a new clear 96-well plate, diluted with PBS (phosphate buffer) containing 1% BSA, and subjected to the following ELISA analysis according to the manufacturer's protocol.

Blood was collected from mice 24 hours after surgery to quantify serum IL-6 levels in CLP (Cecal Ligation and Puncture) induced C57BL / 6 mice.

ELISA plates (Costar, Cambridge, Mass.) Were coated overnight with anti-IL-6 monoclonal antibody (eBioscience, San Diego, Calif.) At 4 ° C. Wells were blocked with PBS containing 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature (RT) and incubated overnight at 4 ° C with serum.

After washing, the plates were incubated at room temperature for 1 hour with biotinylated anti-IL-6 antibody (eBioscience). After streptavidin-HRP (horseradish peroxidase) of eBioscience company was added for 1 hour at room temperature, the solution was developed with TMB solution (eBioscience). Absorbance was measured at 450 nm using Multiskan Ascent (Labsystems, Kennett Square, PA). All analyzes were performed in triplicate.

The blank value of Test Example 6 was determined by ELISA analysis of a solution lacking TNF- ?, and a standard curve was drawn according to the dosage of the control TNF-alpha protein supplied with the ELISA kit.

When Compound 1d and Compound 2f to 2l were treated, the inhibitory rate (%) of TNF-α secretion was compared with that of the control, and the results are shown in Table 6 below.

compound TNF-α secretion inhibition rate (%) 1d 43 2f 38 2g 44 2h 44 2i 45 2j 68 2k 46 2l 60

As shown in Table 6, it was confirmed that Compound 1d and Compound 2f to 2l inhibited TNF-a release in Raw264.7 cells. In particular, Compounds 2j (68%) and 2l (60%) exhibit superior TNF-a release-inhibiting effects compared to Compound 1d (43%).

[Test Example 7]

HMGB1 migration inhibition

From the results of Test Example 6, the compounds 2i to 2l, which exhibit an excellent TNF-a secretion inhibitory effect as compared with the compound 1d, were tested for the ability of these compounds to inhibit the migration of HMGB1 from the nucleus to the cytoplasm, (immunofluorescence).

Immunofluorescence

Raw 264.7 cells were seeded in a "chambered coverglass" of "Nunc Lab-Tek II" and cultured for one day.

Treated with each compound at a concentration of 20 μM for 1 hour and then treated with 500 ng / mL of LPS for 2 hours.

After the medium was absorbed and washed with PBS, 200 μl of 4% paraformaldehyde solution was treated and cultured at room temperature for 20 minutes.

When the 4% paraformaldehyde solution was absorbed, the sample was washed three times with PBS. 200 [mu] l of methanol was treated and incubated at -20 [deg.] C for 20 minutes for permeability conferring antibody binding.

Then, the methanol was removed and the sample was washed three times with PBS. Samples were blocked with 2% BSA in PBS solution for 1 hour at room temperature. Then, 2% BSA in PBS solution was removed and the primary antibody containing 1% BSA in PBS was treated overnight at 4 ° C. Antibody concentrations were optimized according to the manufacturer's protocol.

The primary antibody solution was removed and the samples were washed three times with PBS. The secondary antibody of the PBS solution containing 1% BSA was treated at room temperature for 1 hour. The secondary antibody used was Abcam's goat anti-rabbit IgG (TRITC). After 1 hour, the antibody solution was absorbed and the sample was washed 3 times with PBS.

To stain the nuclei, each well was treated with Hoechst 33342 diluted in PBS and incubated at room temperature for 1 hour. Hoechst 33342 was removed, the chamber was filled with PBS and imaged using a DeltaVision microscope.

The results of the above analysis are shown in FIG. 5, and it was confirmed that the compound 1d and the compounds 2i to 2l inhibited the migration of HMGB1 from the nucleus to the cytoplasm.

[Test Example 8]

Decreased IL-6 secretion

From the results of Test Example 6, in order to further confirm the inflammation-inhibiting effect of the compound 2i to 2l, which exhibits the superior suppression effect of TNF-α secretion compared with the compound 1d, the IL-6 secretion inhibitory effect was evaluated by Western blotting blot analysis.

Western blot

Protein sampling was performed at 4 ° C. Protease inhibitor cocktail and phosphatase inhibitor were dissolved in RIPA lysis buffer. After centrifugation at 15000 rpm for 20 minutes, the supernatant was transferred to a new 1.5 mL tube. Protein concentrations were normalized by Micro BCA protein assay kit.

The protein samples were analyzed by SDS-PAGE and western blotting. Protein was transferred to PVDF membrane after SDS-PAGE. The cells were blocked with TBST (tris-buffered saline Tween 20) containing 2% BSA at room temperature for 1 hour and then the PVDF membrane was blocked.

The membrane was treated with primary antibody overnight at 4 ° C, treated at room temperature for 1 hour, and then washed with TBST. HRP (horseradish peroxidase) conjugated secondary antibody was treated at room temperature for 1 hour. The antibody was diluted to the indicated concentration in the antibody manufacturer's protocol using TBST solution containing 1% BSA. After washing with TBST, the membrane was developed with Amersham's ECL (enhanced chemiluminescence) prime solution.

Chemiluminescent signals were measured with a ChemiDoc MP imaging system and relative quantification was performed using ImageLab 4.0 software. Quantification results are shown using GraphPad Prism 5.0 software.

The results of the above analysis are shown in FIG. 6, and it was confirmed that IL-6 levels secreted from Raw 264.7 cells were significantly decreased upon treatment with Compound 1d and Compound 2i to 2l.

In particular, it can be seen that the chemicals 2j and 2l exhibit an excellent reduction effect with respect to IL-6 secretion.

[Test Example 9]

Relative affinity for HMGB1

The binding energies of the compounds 1d, 2j and 2l were measured with a ligand of HMGB1, and the results are shown in Fig. As shown in FIG. 7, compounds 2j and 21 show better binding to HMGB1 as compared to compound 1d.

[Test Example 10]

Inflammatory signal suppression

The intracellular inflammatory signaling pathway plays an important role in increasing the immune response in activated macrophages.

Mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain enhancer in B cell (NF-κB) complexes are two of the most important inflammatory signaling systems that regulate the release of HMGB and other cytokines in macrophages. There are three types of MAPKs, p38, JNK, and ERK. After macrophage activation, MAPK is phosphorylated and induces inflammatory signaling in the cells.

Western blot data are shown in FIG. 8, and as shown in FIG. 8, it was confirmed that compounds 1d, 2j and 2l apparently inhibited the phosphorylation of MAPK including p38, JNK and ERK.

The NF-κB pathway is another important signaling pathway that regulates the inflammatory response, and NF-κB is a complex of two different protein domains, p50 and p65. The activity of the NF-κB complex is inhibited by the nuclear factor of the kappa-light polypeptide gene enhancer in the B cell inhibitor (IκB).

When the activation signal reaches the macrophage, IκB kinase (IKK) phosphorylates IκB and induces activation of the NF-κB complex. Activated NF-κB acts as a transcription factor that moves to the nucleus and produces an inflammatory cytokine. In this signal transduction pathway, the intracellular transport of the NF-κB complex plays an important role in the activation of macrophages.

9, it can be seen that the compounds 1d, 2j and 2l inhibit NF-κB from migrating to the nucleus.

Finally, it can be seen that compounds 2j and 2l regulate or block inflammatory signaling pathways in activated macrophages.

[Test Example 11]

Pharmacokinetic properties of compounds

The water solubility, microsomal stability and PK profile of compounds 1d, 2j and 2l in the CLP mouse model were measured and are shown in Table 7.

Compared with compound 1d, compounds 2j and 2l have improved solubility in aqueous media and have better liver microsomal stability in both mice and humans. In addition, compounds 1d, 2j, and 2l were all easily administered by intravenous injection and maintained their activity for an extended period of time.

[Test Example 12]

Effects on the CLP mouse model

The therapeutic effects of compounds 1d, 2j and 2l in the CLP mouse model were analyzed.

Compound 1d, 2j, and 2l were administered intraperitoneally at a daily dose of 10 mg / kg for 21 days after the start of administration of CLP 1 hour before induction, and the survival rate was measured and shown in FIG.

As shown in Fig. 10, the survival rate of the control group was about 10%, but the survival rate of the compound 1d-treated group was 45%. In addition, the survival rates of the compound 2j and 2l administration groups were 74% and 54%, respectively, and the survival rate was remarkably superior to that of the control and compound 1d administration groups.

In addition, serum IL-6 levels in mice treated with a daily dose of 10 mg / kg for 21 days after starting the administration of compounds 1d, 2j and 2l 1 hour before CLP induction in the CLP mouse model were measured, Respectively.

As shown in Fig. 11, it was confirmed that serum IL-6 levels were remarkably decreased in surviving mice.

As can be seen from the test results, compounds 2j and 2l have excellent inflammation-inhibiting effect, survival rate and pharmacokinetic properties in comparison with compounds having Compound 1d and other similar structures. Also, it can be seen that compounds 2j and 2l block the inflammatory response through various pathways of the inflammatory response, such as inhibiting the migration of HMGB1 from the nucleus to the cytoplasm and reducing IL-6 release.

As a result, it has been confirmed that the novel compounds 2j and 2l have an excellent inflammation-inhibiting effect and can be used particularly as an excellent sepsis treatment and prevention drug.

Claims (5)

A compound represented by the following formula (5), or a pharmaceutically acceptable salt thereof:
[Chemical Formula 5]

.
A pharmaceutical composition having an anti-inflammatory effect comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for treating or preventing sepsis comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition having an anti-inflammatory effect comprising a compound represented by the following formula (3), or a pharmaceutically acceptable salt thereof:
(3)

.
A pharmaceutical composition for the treatment or prevention of sepsis comprising a compound represented by the following formula (3), or a pharmaceutically acceptable salt thereof:
(3)

.

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