WO2017115914A1 - PPARγ PHOSPHORYLATION INHIBITOR AND PHARMACEUTICAL COMPOSITION COMPRISING SAME - Google Patents

Abstract

The present invention relates to a PPARγ phosphorylation inhibitor and a pharmaceutical composition comprising the same. The PPARγ phosphorylation inhibitor of the present invention is characterized by effectively inhibiting only the phosphorylation of Ser273 selectively without showing transactivation, unlike conventional drugs targeting PPARγ, and by strongly binding to PPARγ through a covalent bond. Therefore, the PPARγ phosphorylation inhibitor is expected to be useful in related fields such as drugs for PPARγ related diseases.

Description

PPARγ phosphorylation inhibitor and pharmaceutical composition comprising the same

The present invention relates to a PPARγ phosphorylation inhibitor and a pharmaceutical composition comprising the same.

As the organism loses its homeostasis due to various internal and external factors, the homeostasis of cells is lost, and the metabolic efficiency of sugar and fat decreases. In addition, overnutrition due to changes in eating habits increases metabolic dysfunctions such as obesity, hyperlipidemia and diabetes, and cardiovascular diseases such as hypertension and atherosclerosis, and these diseases are complicated by genetic and nutritional factors. I have causes.

Peroxysomes are one of the organelles that cause these metabolic disorders, and have been considered to play a minor role in cellular function for many years. It has been found to play an important role in metabolism. In addition, peroxysomes have been reported to have a wide range of effects on regulation of cell proliferation / differentiation and regulation of inflammatory mediators.

Ongoing research in this area suggests that nuclear hormone receptors called peroxisome proliferator-activated receptors (also called "PPARs") will be good targets for the pharmacological approach of these diseases. I put them out.

There are three types of PPARs, PPARα, PPARβ / δ, and PPARγ, of which PPARγ is most frequently found in adipose tissue and is found in vascular endothelial cells, macrophages, and pancreatic β-cells. PPARγ regulates the differentiation of fat cells and plays a critical role in systemic lipid homeostasis. Furthermore, since PPARγ is closely related to insulin sensitivity, PPARγ has been a major target in the development of therapeutics such as obesity and diabetes.

Recently, it has been suggested that the prevention of phosphorylation of Ser273 residues of PPARγ by Cdk5 (Cyclin dependent kinase 5) reduces the insulin resistance and may be a possibility of treating diabetes (Non-Patent Documents 1 and 2). In relation to the relationship between PPARγ and obese diabetes, the study found that obesity increased the inflammatory factors TNF-α, IL-6 and free fatty acids (FFAs), resulting in phosphorylation of Ser273 of PPARγ in adipocytes. The mechanism for reducing the expression of genes that can be activated and improve insulin resistance, such as adiponectin and adipsin, is being actively researched for PPARγ phosphorylation activation.

Meanwhile, as a drug targeting PPARγ, a thiazolidinediones (TDZ) -based drug has been developed as a diabetic agent, but problems such as increased weight, edema, and decreased bone density have been reported. Troglitazone, approved by the US FDA in January 1997, has been recovered from the market for hepatotoxicity. There is a report from the academic society indicating that these side effects are caused by the transactivation of PPARγ, the need for research on the development of therapeutic agents that selectively inhibit the phosphorylation of PPARγ without causing the transactivation of PPARγ It is becoming.

The present invention provides a PPARγ phosphorylation inhibitor that can effectively inhibit PPARγ phosphorylation (specifically, inhibits Ser273 phosphorylation of PPARγ) without causing transactivation of PPARγ, and a pharmaceutical composition containing the same as an active ingredient. It aims to provide.

In order to solve the above problems, the present invention provides a PPARγ phosphorylation inhibitor comprising at least one selected from the group consisting of compounds represented by the following formula (1) to compound represented by 3.

[Formula 1]

[Formula 2]

[Formula 3]

In Chemical Formulas 1 to 3,

R ₁ is-(CH ₂ ) nA ₁ -X- (CH ₂ ) mA ₂ -R ₆ ,

A ₁ is a cycloalkyl compound or an aromatic compound having 5 to 6 carbon atoms,

X is an oxygen atom, a sulfur atom or a nitrogen atom,

A ₂ is an aromatic compound, or a heteroaromatic compound,

N is an integer of 0 or 1,

M is an integer of 0 to 10,

R ₆ is a hydrogen atom, or — (CH ₂ ) oA ₃ — (CH- ₂ ) pR ₇ ,

A ₃ is an oxygen atom, —NH—, —NH—C (═O) —, or —C (═O) —NH—,

R ₇ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

O and p are each an integer of 0 to 5,

In Formula 2, R ₂ is hydrogen, an alkyl group having 1 to 3 carbon atoms, a substituted or unsubstituted phenyl group, or -NR ₈ R ₉ , wherein R ₈ and R ₉ may be the same as or different from each other, and each hydrogen An atom or an alkyl group having 1 to 3 carbon atoms,

R ₃ and R ₄ may be the same as or different from each other, and each is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a substituted or unsubstituted phenyl group,

In Formula 3, R ₅ may be a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group.

In some embodiments, A ₁ and A ₂ are an aromatic compound, X is an oxygen atom, n is O, and m may be 1.

Another embodiment is that R ₁ is

or

It may be

In another embodiment, R ₆ is a hydrogen atom,

,

,

,

.

,

,

,

,,

,

, or

Is,

R ₇ is -A ₄ -R ₁₀ , wherein A ₄ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

R ₁₀ is a hydrogen atom, a methyl group,

,

, or

ego,

R ₁₁ may be a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

Another embodiment may include one or more selected from the group consisting of a compound represented by Formula 4 to a compound represented by 19.

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity, metabolic disease or cardiovascular disease, characterized in that it contains the PPARγ phosphorylation inhibitor as an active ingredient.

In one embodiment, the metabolic disease is diabetes, hyperlipidemia, hyperinsulinemia, hyperuricemia, hypercholesterolemia, hypertriglyceridemia, metabolic syndrome (syndrome X) or endothelial dysfunction, wherein the cardiovascular disease is a cardiovascular disease Hypertension, precoagulant state, dyslipidemia or atherosclerosis disease.

Another embodiment may further comprise a pharmacologically acceptable medicament, diluent or excipient.

Unlike PPARγ-phosphorylation inhibitors of the present invention, the PPARγ-phosphorylation inhibitor effectively inhibits only phosphorylation of Ser273 without transactivation, and is characterized by strong binding with PPARγ.

1 shows the structures of SB1405, SB1406, GW9662 and SB1404 according to Example 1. FIG.

Figure 2 shows the results of confirming the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 1.

3 shows X-ray crystal structures of SB1405 and SB1406 according to Experimental Example 1. FIG.

4 shows the chemical structures of A to F according to Example 2. FIG.

5 shows the results of confirming the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 2.

6 shows a design process of SB1405, SB1451 and SB1453 according to the third embodiment.

Figure 7 shows the results confirm the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 3.

8 shows the results of confirming the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 4.

Figure 9 shows the results of confirming the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 5.

Figure 10 shows the results confirming the phosphorylation inhibitory effect of the PPARγ Ser273 residue according to Experimental Example 6.

Figure 11 shows the results of confirming the phosphorylation inhibitory effect of PPARγ Ser273 residue on the cell according to Experimental Example 7.

12 shows the results of confirming the transcriptional activity effect according to Experimental Example 8.

Figure 13 shows the result of confirming the lipid accumulated through Oil Red O staining according to Experimental Example 9.

14 shows the results of confirming the effect of inhibiting phosphorylation of PPARγ Ser273 residues in vivo according to Experimental Example 10. FIG.

15 shows expression evaluation results of 17 genes regulated by PPARγ phosphorylation according to Experimental Example 11. FIG.

16 shows expression evaluation results of 17 genes regulated by transcriptional activity according to Experimental Example 12.

Figure 17 shows the sugar load test results according to Experimental Example 13.

The present invention relates to a PPARγ phosphorylation inhibitor and a pharmaceutical composition comprising the same.

Hereinafter, the present invention will be described in detail.

The present invention provides a PPARγ phosphorylation inhibitor comprising at least one member selected from the group consisting of compounds represented by Formula 1 to compounds represented by 3.

In Chemical Formulas 1 to 3,

R ₁ is-(CH ₂ ) nA ₁ -X- (CH ₂ ) mA ₂ -R ₆ ,

A ₁ is a cycloalkyl compound or an aromatic compound having 5 to 6 carbon atoms,

X is an oxygen atom, a sulfur atom or a nitrogen atom,

A ₂ is an aromatic compound, or a heteroaromatic compound,

N is an integer of 0 or 1,

M is an integer of 0 to 10,

R ₆ is a hydrogen atom or — (CH ₂ ) oA ₃ — (CH ₂ ) pR ₇ ,

A ₃ is an oxygen atom, —NH—, —NH—C (═O) —, or —C (═O) —NH—,

R ₇ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

O and p are each an integer of 0 to 5,

In Formula 2, R ₂ is hydrogen, an alkyl group having 1 to 3 carbon atoms, a substituted or unsubstituted phenyl group, or -NR ₈ R ₉ , wherein R ₈ and R ₉ may be the same as or different from each other, and each hydrogen An atom or an alkyl group having 1 to 3 carbon atoms,

R ₃ and R ₄ may be the same as or different from each other, and each is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a substituted or unsubstituted phenyl group,

In Formula 3, R ₅ is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group.

In Formula 1, the part except R ₁ is a structure for covalently binding PPARγ, and the part except R ₆ in R ₁ occupies a hydrophobic specific binding site to inhibit phosphorylation of PPARγ Ser273 residue. In the case where R ₆ is-(CH ₂ ) oA _3- (CH ₂ ) pR ₇ instead of a hydrogen atom, R ₆ is a structure having in order to further increase the PPARγ phosphorylation inhibitory effect.

Formula 2 is a modification of the structure for covalently binding PPARγ in Formula 1 to improve solubility, Formula 3 is a non-specific binding while maintaining the high potency of the covalent inhibitor (Covalent inhibitor) In order to prevent this from happening, the structure for covalent bonding is modified in Chemical Formula 1. In particular, the formula (3) is characterized in that it can act as a reversible inhibitor.

In the present specification, cycloalkyl compounds include, but are not limited to, cyclopentyl, cyclohexyl, and the like.

The heteroaromatic group or heteroaromatic compound includes at least one oxygen atom, sulfur atom or nitrogen atom, and is not limited to the position of the oxygen atom, sulfur atom or nitrogen atom. For example, the heteroaromatic compound includes, but is not limited to, furan, thiophene, pyrrole, imidazole, and the like.

An alkyl group having 1 to 3 carbon atoms means a straight or branched alkyl group and includes, for example, methyl, ethyl, n-propyl, i-propyl.

An alkyl group having 1 to 5 carbon atoms means a straight or branched alkyl group, and examples include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, and the like. It doesn't work.

The position of X linked to A ₁ is not limited, but when A ₁ is an aromatic compound, ortho or meta is preferable, and most preferably, ortho may be ortho.

The position of R ₆ connected to A ₂ is not limited, but when A ₂ is an aromatic compound, ortho or meta is preferable.

In R ₁ of Formulas 1 to 3, A ₁ and A ₂ are an aromatic compound, X is an oxygen atom, n is O, and m may be 1.

For example, R ₁ is

or

It may be

In order to further increase the PPARγ phosphorylation inhibitory effect, R ₆ may have a structure of forming a part having hydrophobicity and a part having hydrophilicity on the basis of A ₂ .

For example, R ₆ is a hydrogen atom,

,

,

,

. ,

,

,

,,

,

, or

Is,

R ₇ is -A ₄ -R ₁₀ , wherein A ₄ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

R ₁₀ is a hydrogen atom, a methyl group,

,

, or

ego,

R ₁₁ may be a hydrogen atom or an alkyl group having 1 to 3 carbon atoms.

More specifically, the inhibitor may include one or more selected from the group consisting of compounds represented by Formula 4 to compounds represented by 19.

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

[Formula 17]

[Formula 18]

[Formula 19]

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity, metabolic diseases or cardiovascular diseases, characterized in that it contains a PPARγ phosphorylation inhibitor as an active ingredient.

Since it is well known in the art to affect obesity, metabolic diseases, cardiovascular diseases, etc. according to whether PPARγ is activated, the composition inhibits PPARγ phosphorylation to prevent or treat diabetes, obesity, metabolic diseases or cardiovascular diseases. It is characterized in that, more specifically inhibits the phosphorylation of PPARγ Ser273 exhibits the prevention or treatment of the diabetes, obesity, metabolic disease or cardiovascular disease.

Specifically, hypercholesterolemia among the metabolic diseases is low HDL-cholesterolemia, high LDL-cholesterolemia or hypertriglyceridemia, and the cardiovascular disease is hypertension, precoagulant state, dyslipidemia or atherosclerosis. It may be a sclerotic disease, but is not particularly limited thereto.

As an active ingredient in the pharmaceutical composition according to the present invention, the amount of the inhibitor including at least one selected from the group consisting of the compound represented by Formula 1 to the compound represented by Formula 3 is in the form and purpose of use, patient condition It may be appropriately adjusted according to the kind and severity of symptoms, and may be 0.001 to 99.9% by weight, 0.1 to 99% by weight, or 1 to 50% by weight based on the weight of the composition, but is not limited thereto.

The dosage of the pharmaceutical composition according to the present invention can be determined in consideration of the method of administration, the age, sex of the patient, the severity, the condition of the patient, the absorption of the active ingredient in the body, the inactivation rate and the drug used in combination, 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg (body weight) or 1 mg / kg (body weight) to 300 mg / kg based on ingredients It may be administered in (body weight), it may be administered once or divided into several, but is not limited thereto.

The pharmaceutical composition of the present invention can be administered to a mammal including a human by various routes. The mode of administration can be any of the routinely used methods and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebroventricular injection. The pharmaceutical compositions of the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, transdermals, suppositories, and sterile injectable solutions in accordance with conventional methods. Can be formulated and used.

The pharmaceutical composition of the present invention may further contain an adjuvant such as a pharmaceutically suitable and physiologically acceptable carrier, excipient and diluent in addition to the active ingredient. Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form forms at least one excipient such as starch, calcium carbonate, sucrose or It may be prepared by mixing lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, transdermal agents and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, Whitepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention, the present invention is not limited by the following examples can be variously modified and changed.

Example 1 Preparation of Inhibitors (Irreversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 1

It was confirmed that ligands that inhibit phosphorylation of PPARγ Ser273 residues commonly occupy specific hydrophobic specific binding sites. SB1405 (Formula 4) and SB1406 (Formula 5) are selected from the covalent inhibitors expected to occupy the specific binding sites as described in Scheme 1 below based on the GW9662 and PPARγ crystal structure, which are known covalent ligands. SB1404 (Formula 21), which is expected to not be occupied at all, was also manufactured, and the structure thereof is shown in FIG. 1.

Scheme 1

Formula 20] (GW9662)

Formula 21] (SB1404, 2-chloro-N-methyl-5-nitrobenzamide)

(SB1405, N- (2- (benzyloxy) phenyl) -2-chloro-5-nitrobenzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.78 (brs, 1H), 8.64 (d, J = 2.8 Hz, 1H), 8.48 (dd, J = 7.6, 1.2 Hz, 1H), 8.16 (dd, J = 8.8, 2.4 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.41-7.34 (m, 5H), 7.10 (d, J = 7.6, 1.6 Hz, 1H), 7.05-7.00 (m, 2H ), 5.11 (s, 2 H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 161.5, 147.8, 146.7, 137.5, 136.2, 136.0, 131.7, 128.8, 128.6, 128.0, 127.3, 126.1, 125.9, 125.0, 121.6, 120.6, 111.8, 71.2; LRMS (ESI) m / z calcd for C ₂₀ H ₁₆ ClN ₂ O ₄ [M + H] ⁺ : 383.08; Found: 382.91.

(SB1406, N- (3- (benzyloxy) phenyl) -2-chloro-5-nitrobenzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.52 (s, 1H), 8.20 (dd, J = 8.8, 2.4 Hz, 1H), 7.99 (s, 1H), 7.60 (d, J = 9.2 Hz, 1H) , 7.45-7.24 (m, 7H), 7.09 (d, J = 8.4 Hz, 1H), 6.81 (dd, J = 8.4, 2.0 Hz, 1H), 5.07 (s, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 162.4, 159.5, 146.7, 138.2, 137.7, 136.8, 136.6, 131.7, 130.1, 128.7, 128.2, 127.7, 126.1, 125.3, 112.8, 112.2, 107.2 70.2; LRMS (ESI) m / z calcd for C ₂₀ H ₁₆ ClN ₂ O ₄ [M + H] ⁺ : 383.08; Found: 382.87.

Experimental Example 1. Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residues 1

The phosphorylation inhibitory effect of the PPARγ Ser273 residue of the inhibitor prepared in Example 1 was confirmed using an in vitro Cdk5 assay (Cell Signaling Technology, USA) according to the manufacturer's instructions.

Specifically, phosphorylation was performed by reacting 0.5 μg of purified PPARγ LBD (Ligand Binding Domain) at 30 ° C. for 30 minutes with active Cdk5 / p35 (Millipore, USA), and the assay was performed under the following conditions:

25 uM ATP, 25 mM Tris-HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na ₃ VO ₄ , 10 mM MgCl ₂ .

In order to confirm the inhibitory effect of phosphorylation, PPARγ LBD and the inhibitor were first reacted at 30 ° C. 30 minutes before the assay, and the substrate was Rb peptide (residues 773) to confirm that the inhibitor did not directly act on Cdk5. -928, Millipore) followed by the same assay (Rb peptide is one of the well known substrates of Cdk5). The degree of phosphorylation of the substrate was confirmed by Western blotting using aniti-phospho-Ser Cdk substrate antibody (Cell Signaling Technology, USA), and the results are shown in FIG. 2. At this time, Rosiglitazone (Rosi) and SR1664, which are known as antidiabetic PPARγ ligands, were assayed in the same manner.

[Formula 22] (Rosiglitazone)

[Formula 23] (SR1664)

As shown in FIG. 2, only SB1405 among the covalent inhibitors showed the same activity as Rosiglitazone and SR1664, which are antidiabetic PPARγ ligands.

In addition, X-ray crystallography confirmed the binding site, unlike SB1406 SB1405 occupies the specific binding site, it was confirmed that effectively inhibits the phosphorylation of PPARγ Ser273 residues (see Figure 3).

Example 2 Construction of Inhibitors (Irreversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 2

According to the results of Experimental Example 1, based on the structure of SB1405, various inhibitors were prepared as described in Scheme 2 below, and the prepared inhibitors were represented by A (Formula 6), C (Formula 7), It is represented by D (formula 8), E (formula 9) and F (formula 10).

Scheme 2

(A, 2-chloro-5-nitro-N- (2-phenoxyphenyl) benzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.63 (s, 1H), 8.61 (s, 1H), 8.59 (d, J = 7.2 Hz, 1H), 8.23 (dd, J = 7.2, 2.0 Hz, 1H) , 7.60 (d, J = 6.8 Hz, 1H), 7.36 (t, J = 6.4 Hz, 2H), 7.17-7.10 (m, 3H), 7.02 (d, J = 6.4 Hz, 2H), 6.92 (d, J = 6.0 Hz, 1H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 161.8, 156.3, 146.7, 146.1, 137.4, 136.3, 131.6, 130.0, 129.0, 125.9, 125.7, 125.2, 124.2, 124.1, 121.3, 118.5, 118.0; LRMS (ESI) m / z calcd for C ₁₉ H ₁₄ ClN ₂ O ₄ [M + H] ⁺ : 369.06; Found: 369.05.

(C, 2-chloro-5-nitro-N- (2-phenethoxyphenyl) benzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.54 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.36 (s, 1H), 8.26 (dd, J = 8.4, 2.8 Hz, 1H) , 7.64 (d, J = 8.8 Hz, 1H), 7.18-7.10 (m, 6H), 7.04 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.30 (t, J = 6.6 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 161.7, 147.4, 146.8, 137.8, 137.7, 136.9, 131.7, 128.8, 128.6, 127.3, 126.7, 125.6, 125.5, 125.0, 121.6, 120.4, 111.6, 69.1, 35.6; LRMS (ESI) m / z calcd for C ₂₁ H ₁₈ ClN ₂ O ₄ [M + H] ⁺ : 397.10; Found: 397.15.

(D, 2-chloro-N- (2-ethoxyphenyl) -5-nitrobenzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.77 (brs, 1H), 8.72 (d, J = 2.8 Hz, 1H), 8.50 (dd, J = 8.0, 1.2 Hz, 1H), 8.25 (dd, J = 8.8, 2.8 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.13 (dd, J = 8.0, 1.2 Hz, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.14 (q, J = 6.8 Hz, 2H), 1.45 (t, J = 6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 161.5, 147.7, 146.9, 137.5, 136.5, 131.9, 127.1, 126.2, 126.0, 125.0, 121.2, 120.3, 111.1, 64.4, 15.0; LRMS (ESI) m / z calcd for C ₁₅ H ₁₄ ClN ₂ O ₄ [M + H] ⁺ : 321.06; Found: 321.15.

(E, 2-chloro-N- (2- (hexyloxy) phenyl) -5-nitrobenzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.76 (brs, 1H), 8.72 (d, J = 2.8 Hz, 1H), 8.50 (dd, J = 8.0, 1.2 Hz, 1H), 8.25 (dd, J = 8.8, 2.8 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.13 (t, J = 8.0, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.06 (t, J = 6.8 Hz, 2H), 1.81 (m, 2H), 1.45 (m, 2H), 1.32 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 161.5, 147.9, 146.9, 137.5, 136.5, 131.8, 127.1, 126.2, 126.0, 125.0, 121.2, 120.3, 111.1, 68.9, 31.6, 29.3, 25.9, 22.7, 14.1; LRMS (ESI) m / z calcd for C ₁₉ H ₂₂ ClN ₂ O ₄ [M + H] ⁺ : 377.13; Found: 377.20.

(F, 2-chloro-N- (2- (2- (4-methylpiperazin-1-yl) ethoxy) phenyl) -5-nitrobenzamide)

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.31 (brs, 1H), 8.64 (d, J = 2.8 Hz, 1H), 8.45 (dd, J = 8.0, 1.6 Hz, 1H), 8.26 (dd, J = 8.4, 2.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.13 (dd, J = 8.0, 1.6 Hz, 1H), 7.08 (dd, J = 8.0, 1.2 Hz, 1H), 6.98 ( dd, J = 8.0, 1.2 Hz, 1H), 4.19 (t, J = 5.6 Hz, 2H), 2.72 (t, J = 5.6 Hz, 2H), 2.50-2.43 (m, 8H), 2.16 (s, 3H ); ^{13 C NMR (100 MHz, CDCl} 3) δ 162.1, 148.1, 146.7, 138.0, 137.2, 131.7, 128.2, 125.9, 125.7, 125.3, 122.1, 121.2, 113.4, 66.9, 57.2, 54.8, 53.3, 46.0; LRMS (ESI) m / z calcd for C ₂₀ H ₂₄ ClN ₄ O ₄ [M + H] ⁺ : 419.15; Found: 419.06.

Experimental Example 2. Confirmation of the phosphorylation inhibitory effect of the PPARγ Ser273 residue 2

Assays A, B, C, D, E and F prepared in Examples 1 and 2 were carried out in the same manner as in Experimental Example 1, and the results are shown in FIG. 5.

As shown in FIG. 5, in the case of F having a hydrophilic functional group in the R group, the phosphorylation of PPARγ Ser273 residues was not inhibited at all, but the compounds having a hydrophobic functional group in the R group generally inhibited phosphorylation well. In particular, it was confirmed that B (SB1405) having a benzyl group as the R group is the most effective structure to inhibit phosphorylation.

Example 3 Construction of Inhibitors (Irreversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 3

In order to improve physical properties as inhibitors, SB1451 (Scheme 11) and SB1453 (Scheme 12) were prepared as shown in Scheme 3 based on the crystal structure as described in FIG. 6.

 Scheme 3

(SB1451, 2-chloro-N- (2-((2-((4-((4-methylpiperazin-1-yl) methyl) benzamido) methyl) benzyl) oxy) phenyl) -5-nitrobenzamide )

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.09 (s, 1H), 8,99 (t, J = 5.6 Hz, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.26 (dd, J = 8.4, 2.8 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 6.8 Hz, 1H), 7.39-7.24 (m, 6H), 7.20 (t, J = 8.4 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 5.34 (s, 2H), 4.58 ( d, J = 5.6 Hz, 2H), 3.48 (s, 2H), 2.34 (brs, 8H), 2.15 (s, 3H); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 166.1, 163.1, 150.5, 146.0, 141.8, 137.9, 137.7, 137.2, 134.2, 132.8, 131.3, 128.5, 128.2, 128.1, 127.1, 126.8, 126.29, 126.26, 125.5 , 124.7, 124.0, 120.5, 113.1, 67.9, 61.6, 54.7, 52.5, 45.7; LRMS (ESI) m / z calcd for C ₃₄ H ₃₅ ClN ₅ O ₅ [M + H] ⁺ : 628.23; Found: 628.30.

(SB1453, 2-chloro-N- (2-((3-((4-((4-((4-methylpiperazin-1-yl) methyl) benzamido) methyl) benzyl) oxy) phenyl) -5-nitrobenzamide )

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 10.07 (s, 1H), 8.98 (t, J = 6.0 Hz, 1H), 8.37 (d, J = 2.4 Hz, 1H), 8.30 (dd, J = 8.8, 2.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 4H), 7.43-7.31 (m, 5H), 7.26 (d, J = 7.6 Hz, 1H), 7.17-7.14 (m, 2H), 7.01-6.97 (m, 1H), 5.19 (s, 2H), 4.46 (d, J = 6.0 Hz, 2H), 3.49 (s, 2H), 2.34 (brs, 8H), 2.14 (s, 3H); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 166.0, 163.0, 150.6, 146.0, 141.8, 139.9, 137.7, 137.2, 137.0, 133.0, 131.3, 128.6, 128.4, 127.2, 126.6. 126.33, 126.28, 125.9, 125.6, 124.7, 124.1, 120.5, 113.2, 69.8, 61.6, 54.7, 52.6, 45.7, 42.5; LRMS (ESI) m / z calcd for C ₃₄ H ₃₅ ClN ₅ O ₅ [M + H] ⁺ : 628.23; Found: 628.41.

Experimental Example 3. Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residue 3

As SB1451 and SB1453 prepared in Example 3, the assay was carried out in the same manner as in Experiment 1 with different concentrations (0.1 uM, 1 uM, 10 uM), the results are shown in FIG.

As shown in Figure 7, SB1451 and SB1453 was found to effectively inhibit the phosphorylation of PPARγ Ser273 residues in a concentration-dependent manner.

Example 4 Construction of Inhibitors (Irreversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 4

SB1450 and SB1452 were prepared in a similar manner as in Scheme 3.

Formula 13] (SB1450)

(SB1452)

Experimental Example 4. Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residue 4

Assays were performed in the same manner as Experimental Example 1 for comparing the inhibitors prepared in Examples 1 to 4, and the results are shown in FIG. 8.

As shown in FIG. 8, SB1450 and SB1452 effectively inhibited PPARγ phosphorylation, but the degree of inhibition was slightly lower than that of SB1451 and SB1453.

Example 5 Construction of Inhibitors (Irreversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 5

In order to improve solubility, Compound A (Formula 15), Compound B (Formula 16), Compound C (Formula 17), and Compound D (Formula 18), which modify the structure of the covalently bonded site, are represented as in Scheme 4. Produced.

Scheme 4

[Formula 15] (Compound A)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31 (s, 1H), 7.87 (brs, 1H), 6.76 (brs, 1H), 3.02 (d, J = 6.0 Hz, 3H), 3,01 (d, J = 5.6 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 165.3, 162.3, 157.8, 155.0, 108.1, 28.2, 26.6; LRMS (ESI) m / z calcd for C ₇ H ₉ ClN ₄ O [M + H] ⁺ : 201.05; Found: 201.10.

[Compound B]

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.45 (brs, 1H), 8.34 (s, 1H), 7.62 (brs, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.39 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 7.6 Hz, 1H), 3.04 (d, J = 4.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 162.9, 162.2, 158.0, 155.0, 136.7, 129.2, 125.5, 120.6, 108.8, 28.3; LRMS (ESI) m / z calcd for C ₁₂ H ₁₁ ClN ₄ O [M + H] ⁺ : 263.06; Found: 263.10.

Formula 17] (Compound C)

^{1 H NMR (400 MHz, CDCl} 3) δ 9.12 (brs, 1H), 8.43 (d, J = 7.6 Hz, 1H), 8.32 (s, 1H), 8.09 (brs, 1H), 7.40-7.34 (m, 5H), 7.12 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 7.6 Hz, 2H), 5.12 (s, 2H), 3.03 (d, J = 4.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 162.8, 162.7, 157.9, 155.3, 147.8, 135.7, 128.8, 128.6, 128.0, 127.1, 124.9, 121.3, 120.5, 111.6, 108.3, 71.2, 28.3; LRMS (ESI) m / z calcd for C ₁₉ H ₁₇ ClN ₄ O ₂ [M + H] ⁺ : 369.10; Found: 369.20.

[Compound D]

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (s, 1H), 8.32 (brs, 1H), 7.63 (brs, 1H), 7.45-7.28 (m, 7H), 7.09 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.08 (s, 2H), 3.04 (d, J = 4.8 Hz, 3H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 162.9, 162.3, 159.4, 158.1, 155.1, 137.9, 136.6, 129.9, 128.6, 128.0, 127.5, 112.9, 111.9, 108.6, 107.3, 70.1, 28.3; LRMS (ESI) m / z calcd for C ₁₉ H ₁₇ ClN ₄ O ₂ [M + H] ⁺ : 369.10; Found: 369.10.

Experimental Example 5. Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residues 5

Assay was carried out in the same manner as in Experiment 1 with Compound A, Compound B, Compound C and Compound prepared in Example 4 to a concentration of 10 uM, the results are shown in FIG.

As shown in Figure 9, it was confirmed that compound C (Formula 17) effectively inhibits PPARγ phosphorylation. These results show that Formula 1 and Formula 2 having the same R ₁ exhibit the same activity in inhibiting PPARγ phosphorylation.

Example 6 Construction of Inhibitors (Reversible) Inhibiting Phosphorylation of PPARγ Ser273 Residues 6

As a reversible inhibitor in which nonspecific binding does not occur while maintaining high potency, which is an advantage of a covalent inhibitor, BH273 (Formula 19) was modified in which the structure of the covalent binding site was modified.

Formula 19] (BH273)

Experimental Example 6. Confirmation of phosphorylation inhibitory effect of PPARγ Ser273 residue 6

The assay was performed in the same manner as in Experiment 1 with different concentrations (0.1 uM, 1 uM, 10 uM) of BH273 prepared in Example 5, and the results are shown in FIG. 10.

As shown in FIG. 10, BH273 showed Ser273 phosphorylation inhibition similar to that of rosiglitazone, an antidiabetic PPARγ ligand.

Experimental Example 7. Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residue on the cell

The phosphorylation inhibitory effect of PPARγ Ser273 residues of SB1451 and SB1453 prepared in Example 3 was confirmed on HEK-293 cells.

Specifically, HEK-293 cells expressing PPARγ were treated with PMA (phorbol 12-myristate 13-acetate) for 30 minutes and then incubated in FLAG M2 agarose (Sigma Aldrich, USA) at 4 ° C. Immunoprecipitate was analyzed using phosphor-specific or anti-PPARγ antibodies of PPARγ Ser273 and the results are shown in FIG. 11.

 As shown in FIG. 11, it was confirmed that both SB1451 and SB1453 effectively inhibited phosphorylation of PPARγ even on cells.

Experimental Example 8. Confirmation of transcriptional activity effect

It was tested whether SB1451 and SB1453 prepared in Example 3 cause transactivation.

Specifically, HEK-293T cells were seeded in 96-well plates at a density of 7000 cells / well before transfection. Then, the cells were transfected with pDR-1 luciferase reporter plasmid, PPARγ RXRα and pRL-renillin using calcium phosphate transfection. After transfection, rosiglitazone, SR1664, SB1451, and SB1453 were treated for 24 hours, and after culturing the cells, a reporter gene assay (Promega, USA) was performed using the Dual-luciferase kit. Luciferase activity was measured using a Bio-Tek microplate reader (ELx800TM, Bio-Tek Instruments Inc., USA) and normalized to Renilla activity, resulting in Fold based on DMSO-treated cells. The change is shown in FIG. 12.

As shown in FIG. 12, SB1451 and SB1453 had little effect on the transcriptional activity of PPARγ, unlike Rosiglitazone.

Experimental Example 9. 3T3L1 Adipocyte Differentiation Promotion Test

3T3L1 adipocytes were grown to 100% confluency in 6-well plates, followed by 1 uM dexamethasone, 850 nM insulin and 10 uM inhibitor (in DMEM containing 10% FBS) for 2 days. Treated. Since then, 10% FBS containing DMEM and 850 nM insulin was continuously cultured while treating and replacing. On day 8 of the culture, lipids accumulated in 3T3L1 cells were confirmed by Oil Red O staining, and the results are shown in FIG. 13.

Figure 13 shows the result of confirming the lipid accumulated through the Oil Red O staining.

As shown in FIG. 13, SB1451 and SB1453 did not show the result of promoting lipid formation, whereas Rosiglitazone promoted adiogenesis and differentiated 3T3L1 cells into mature adipocytes.

Experimental Example 10 Confirmation of the phosphorylation inhibitory effect of PPARγ Ser273 residues (In vivo)

SB1451 and SB14533 were administered at 10 mg / kg / day for 7 days in obese-induced obese mice (DIO mice) to confirm the effect of inhibiting the phosphorylation of PPARγ Ser273 residues in the same manner as in Experiment 1, and the results 14 is shown.

As shown in Figure 14, SB1451 and SB1453 was confirmed to inhibit PPARγ phosphorylation in DIO mice. In particular, SB1453 was found to inhibit PPARγ phosphorylation at a level similar to Rosiglitazone in DIO mice.

Experimental Example 11. Evaluation of expression of genes regulated by PPARγ phosphorylation

Using the DIO mice proceeded in Experiment 10, the expression was evaluated for 17 genes significantly affected by the conventional PPARγ phosphorylation:

<Target gene>

Adipsin, Adiponectin, Cd24a, Txnip, Nr1d1, Peg10, Acyl, Cidec, Rarres2, Car3, Selenbp1, Aplp2, Nr3c1, Cyp2f2, Nr1d2, Ddx17, Rybp

 Specifically, RNA was separated from adipose tissue and qPCR was performed, and the expression level of the genes according to the inhibitor treatment is shown in FIG. 15.

As shown in FIG. 15, it was confirmed that the expression of target genes was significantly increased in the DIO mice treated with SB1453.

Experimental Example 12. Evaluation of expression of genes regulated by transcriptional activity

Using DIO mice proceeded in Experiment 10, the expression of 17 genes significantly affected by the transcriptional activity by the conventional PPARγ activation was evaluated:

<Target gene>

aP2, Cycs, Idh3a, Ppcs, Fdx1, Fgfrl1, Pdk4, Cib2, Fmr1, Pim3, Las1l, Abhd12, Nadk, Arhgap5, Lass4, Plin2, Phospho1

Specific experimental method was carried out in the same manner as in Experiment 12, the results are shown in FIG.

As shown in FIG. 16, the expression level of 17 target genes increased significantly in DIO mice treated with Rosiglitazone, but when treated with SB1453, there was no statistically significant difference when compared to Control.

Experimental Example 13. Glucose Tolerance Test

For glucose tolerance testing, rosiglitazone, SB1453, and vehicle were intraperitoneally injected at 10 mg / kg daily in DIO mice. Thereafter, 1.5 g / kg of D-glucose was administered to fasting mice, and blood was drawn at regular intervals from the tail vein to measure glucose concentration using a Truetrack glucometer (Nipro Diagnostics, Japan). Shown in

As shown in FIG. 17, it was confirmed that mice administered SB1453 or rosiglitazone effectively reduced blood glucose levels as compared to vehicle control.

It can be usefully used in related fields, such as drugs of PPARγ-related diseases.

Claims (8)

1. A PPARγ phosphorylation inhibitor comprising at least one selected from the group consisting of compounds represented by Formula 1 to compounds represented by 3 below:

   [Formula 1]

   

   [Formula 2]

   

   [Formula 3]

   

   In Chemical Formulas 1 to 3,

   R ₁ is-(CH ₂ ) nA ₁ -X- (CH ₂ ) mA ₂ -R ₆ ,

   A ₁ is a cycloalkyl compound or an aromatic compound having 5 to 6 carbon atoms,

   X is an oxygen atom, a sulfur atom or a nitrogen atom,

   A ₂ is an aromatic compound, or a heteroaromatic compound,

   N is an integer of 0 or 1,

   M is an integer of 0 to 10,

   R ₆ is a hydrogen atom, or — (CH ₂ ) oA ₃ — (CH- ₂ ) pR ₇ ,

   A ₃ is an oxygen atom, —NH—, —NH—C (═O) —, or —C (═O) —NH—,

   R ₇ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

   O and p are each an integer of 0 to 5,

   In Formula 2, R ₂ is hydrogen, an alkyl group having 1 to 3 carbon atoms, a substituted or unsubstituted phenyl group, or -NR ₈ R ₉ , wherein R ₈ and R ₉ may be the same as or different from each other, and each hydrogen An atom or an alkyl group having 1 to 3 carbon atoms,

   R ₃ and R ₄ may be the same as or different from each other, and each is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a substituted or unsubstituted phenyl group,

   In Formula 3, R ₅ is a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group.
2. The method according to claim 1,

   A ₁ and A ₂ are an aromatic compound,

   X is an oxygen atom,

   N is O,

   M is 1, PPARγ phosphorylation inhibitor.
3. The method according to claim 1,

   R ₁ is

   

   or

   

   It is a PPARγ phosphorylation inhibitor.
4. The method according to claim 1,

   R ₆ is a hydrogen atom,

   

   ,

   

   ,

   

   ,

   

   .

   

   ,

   

   ,

   

   ,

   

   ,,

   

   ,

   

   , or

   

   Is,

   R ₇ is -A ₄ -R ₁₀ , wherein A ₄ is a substituted or unsubstituted aromatic group, or a substituted or unsubstituted heteroaromatic group,

   R ₁₀ is a hydrogen atom, a methyl group,

   

   ,

   

   , or

   

   ego,

   R ₁₁ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, PPARγ phosphorylation inhibitor.
5. The method according to claim 1,

   PPARγ phosphorylation inhibitor comprising at least one selected from the group consisting of compounds represented by the following formula (4) to compound represented by 19.

   [Formula 4]

   

   [Formula 5]

   

   [Formula 6]

   

   [Formula 7]

   

   [Formula 8]

   

   [Formula 9]

   

   [Formula 10]

   

   [Formula 11]

   

   [Formula 12]

   

   [Formula 13]

   

   [Formula 14]

   

   [Formula 15]

   

   [Formula 16]

   

   [Formula 17]

   

   [Formula 18]

   

   [Formula 19]

   
6. A pharmaceutical composition for the prevention or treatment of obesity, metabolic disease or cardiovascular disease, comprising the PPARγ phosphorylation inhibitor of claim 1 as an active ingredient.
7. The method according to claim 6,

   The metabolic disease is diabetes, hyperlipidemia, hyperinsulinemia, hyperuricemia, hypercholesterolemia, hypertriglyceridemia, metabolic syndrome (syndrome X) or endothelial dysfunction,

   The cardiovascular disease is a cardiovascular disease is a hypertension, a precoagulant state, dyslipidemia or atherosclerosis disease, pharmaceutical composition.
8. The method according to claim 6,

   A pharmaceutical composition further comprising a pharmacologically acceptable agent, diluent or excipient.

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