KR102673131B1 - Novel Toll-like Receptor 3/7/9 Inhibitory Small Molecule Compounds - Google Patents

Abstract

The present invention relates to an antagonistic small molecule compound with a Toll-like receptor (TLR) 3/7/9 inhibitory function, and more specifically, to inhibit the Toll-like receptor 3/7/9 signaling pathway. It relates to a novel small molecule compound and a composition containing the same for preventing or treating autoimmune diseases or inflammatory diseases. The novel compound according to the present invention not only blocks the secretion of TNF-α induced by Poly I:C (TLR3 agonist), Imiquimod (TLR7 agonist) or ODN2395 (TLR9 agonist), but also blocks the production of inflammatory cytokines. In particular, it is useful for preventing or treating TLR3/7/9-related autoimmune diseases and inflammatory diseases, including systemic lupus erythematosus and psoriasis.

Description

Novel Toll-like Receptor 3/7/9 Inhibitory Small Molecule Compounds}

The present invention relates to an antagonistic small molecule compound with a Toll-like receptor (TLR) 3/7/9 inhibitory function, and more specifically, to inhibit the Toll-like receptor 3/7/9 signaling pathway. It relates to a novel small molecule compound and a composition containing the same for preventing or treating autoimmune diseases or inflammatory diseases.

Innate (or innate) immunity is the first defense against bacterial infection in the mammalian immune system, in which pattern recognition receptors such as Toll-like receptors (TLRs) identify pathogen-associated molecular patterns. It is activated by recognizing a pattern (PAMP) or damage-associated molecular pattern (DAMP). For example, triacyl lipoproteins (eg Pam ₃ CSK ₄ ) recognized by TLR1/2 (Takeuchi, O. , et al. J Immunol 169: 10-14 (2002)), diacyl lipoproteins recognized by TLR2/6 Lipoprotein (eg Pam ₂ CSK ₄ ) (Takeuchi, O. , et al. Int Immunol 13: 933-940 (2001)), lipopolysaccharide (LPS) by TLR4 (Poltorak, A. , et al., Science 282: 2085-2088 (1998)), bacterial flagellin by TLR5 (Poltorak, A. , et al. Science 282 : 2085-2088 (1998)), viral double-stranded RNA (dsRNA) by TLR3 ( Poltorak, A. , et al., Science 282 : 2085-2088 (1998)), viral single-stranded RNA (ssRNA) by TLR7 and TLR8 (Diebold, SS et al., Science 303 : 1529-1531 (2004); Heil, F. , et al., Science 303 : 1526-1529 (2004)) and unmethylated CpG-containing oligodeoxy nucleotides (ODN) by TLR9 (Hemmi, H. , et al. Nature 408: 740-745) (2000)), etc.

TLRs play an important role in innate immune responses, with extracellular TLRs acting at the plasma membrane, including TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11, and intracellular TLRs, such as endosomes, containing TLR3, TLR7, TLR8, and TLR9. It can be divided into intracellular TLRs that act in Structurally, TLRs have a leucine-rich repeat (LRR) region at the N-terminus of the extracellular domain that is recognized by a ligand or accessory molecule, and a signal signal region at the C-terminus of the intracellular portion. It has a Toll/interleukin 1 receptor (TIR) domain that transmits .

In particular, TLR7, TLR8, TLR9, and TLR3 recognize exogenous ssRNA (single stranded RNA), dsRNA (double stranded RNA), and CpG DNA exposed to endosomes from invaders outside the cell, or cause cell necrosis inside the tissue due to abnormal reactions. Alternatively, endogenous ssRNA or DNA fragments exposed from tissues damaged by apoptosis are recognized as ligands, and inflammatory cytokines are amplified through a signal transduction process. In general, TLR7 and TLR8 recognize ssRNA from influenza or damaged cells, while TLR9 detects CpG DNA fragments generated from the genomes of bacteria and viruses or damaged tissues, and TLR3 recognizes dsRNA, an intermediate product of viral proliferation. Activates the innate immune response. Due to the role of TLR, research to utilize TLR as a target for treating immune-related diseases is actively underway around the world.

MyD88 (myeloid differentiation primary response 88)-dependent signaling of TLR7/8/9 forms a dimer by the corresponding ligand, and the TIR domain of TLR and the TIR domain of MyD88 bind to form a complex, thereby establishing a signal transduction pathway. Activates (Hemmi, H. et al. Nat Immunol 3, 196-200, (2002)). Activated TLR signals induce the activation and movement of NF-κB to the nucleus, activation of MAPK, and expression of interferon α (IFNα) and IFN-inducible genes. Activation of the NF-κB and MAPK secretes inflammatory cytokines such as TNF-α, interleukin 1β (IL-1β), and IL-6. The MyD88-independent signaling process of TLR3 is initiated by binding between the TIR domain of TLR3 and the TIR domain of TRIF (TIR domain-containing adapter-inducing interferon-β), which induces type 1 interferon by activation of IRF (interferon-regulatory factor). secrete. Additionally, TLR activity generates oxidative stress substances such as NO and ROS in macrophages.

TLRs located in the endosomal membrane (TLR3, 7, 8, and 9) are important in protecting the host from various viral and bacterial infections. In particular, expression of TLRs 7, 8 and 9 is essential for sustained defense against pathogenic components or self-antigens released from damaged or stressed tissues/cells (Demaria, O. , et al ., J Clin Invest 120: 3651 -3662 (2010)). Malfunction of these nucleic acid-sensing TLRs has been implicated in several autoimmune pathologies, such as psoriasis and systemic lupus erythematosus (SLE) (Vincent, FB et al., Nat Rev Rheumatol 10: 365-373 (2014)). there is. However, the etiology of these diseases remains unclear (Krieg, AM & Vollmer, J., Immunol Rev 220: 251-269 (2007); Terhorst, D. , et al. J Immunol 195: 4953-4961 (2015)). Therefore, there is an increasing need for the development of novel antagonists that reduce endosomal TLR-mediated disease progression.

In this way, TLR can be a target for treating various diseases such as autoimmune diseases, inflammatory diseases, and cancer, so research is being actively conducted on substances targeting TLR and medical compositions for treating diseases related to TLR. .

Accordingly, the present inventors have made diligent efforts to develop substances targeting TLR and medical compositions for treating TLR-related diseases. As a result, new compounds, including compounds defined as IM (imiquimod mimic), have been found to activate TLR3, TLR7 or TLR9. It was confirmed that the secretion of cytokines was suppressed by inhibiting the TLR signaling pathway induced by , and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. It may not be possible.

The purpose of the present invention is to provide an antagonistic small molecule compound with a toll-like receptor 3/7/9 inhibitory function and a TLR3, TLR7 and TLR9 inhibitor comprising the same.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases or inflammatory diseases containing the above compounds.

Another object of the present invention is a method for preventing or treating autoimmune diseases/inflammatory diseases using the compounds, the use of the compounds for preventing or treating autoimmune diseases/inflammatory diseases, and the production of a drug for preventing or treating autoimmune diseases/inflammatory diseases. To provide use of the compound for.

In order to achieve the above object, the present invention provides a compound or a pharmaceutically acceptable salt represented by any one formula selected from the group consisting of the following formulas 1 to 4.

[Formula 1]

;

;

[Formula 2]

;

;

[Formula 3]

;

;

[Formula 4]

,

,

In Formula 1, R ₁ to R ₁₇ are the same or different from each other, and each independently represents a hydrogen atom, a halogen atom, straight or branched alkyl, amino, alkylamine, nitrile group, nitro group, nitroso group, hydroxy, cyclo. Alkyl, benzyl, haloalkyl, allyl, alkoxy, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, alkylarylcarbonyl, alkoxycarbonyl, cycloalkoxy, aryl, heteroaryl, heterocycloalkyl, aryloxy, alkoxyhetero Aryl, heteroaryloxyalkyl, alkylheteroaryl, alkylaryl, arylalkyl, alkylheteroaryl, alkyl ester, alkylamide, or acrylic,

or R ₁ and R ₂ are cycloalkyl or heterocycloalkyl linked to each other,

Here, alkyl, alkylamine or alkoxy is C _1-30 , cycloalkyl is C _3-30 , allyl is C _2-30, aryl is C _6-30 , heteroaryl and heterocycloalkyl are fluorine, oxygen, sulfur and Contains heteroatoms selected from nitrogen.

The present invention also provides a composition for inhibiting TLR (Toll-like receptor) containing the above compound or a pharmaceutically acceptable salt.

The present invention also provides a pharmaceutical composition for preventing or treating autoimmune diseases or inflammatory diseases, comprising a compound represented by any one of the formulas selected from the group consisting of Formulas 1 to 4, or a pharmaceutically acceptable salt. .

The present invention also provides a method for preventing or treating an autoimmune disease/inflammatory disease comprising administering a compound represented by Formula 1 to Formula 4, the use of the compound for preventing or treating an autoimmune disease/inflammatory disease, and the use of the compound for autoimmune disease/inflammatory disease prevention or treatment. Provided is the use of said compound for manufacturing a medicament for preventing or treating immune/inflammatory diseases.

The novel compound according to the present invention not only blocks the secretion of TNF-α induced by Poly I:C (TLR3 agonist), Imiquimod (TLR7 agonist) or ODN2395 (TLR9 agonist), but also blocks the production of inflammatory cytokines. In particular, it is useful for preventing or treating TLR3/7/9-related autoimmune diseases and inflammatory diseases, including systemic lupus erythematosus and psoriasis.

Figure 1 is a schematic diagram showing the overall virtual screening workflow.
Figure 2 is a graph showing the inhibition of (A) TLR7 (B) TLR9 by three initial hit candidates (IM1, IM5, and IM9).
Figure 3 shows IC50 curves of three initial hits against (A) TLR7 and (B) TLR9.
Figure 4 is a graph comparing the inhibitory activity of IM9 and three active derivatives (IM30, IM34, and IM38).
Figure 5 shows the chemical details of the identified hit molecules, showing (A) the structure of the active molecule and (B) the SMILES, IUPAC name and molecular weight of the active molecule.
Figure 6 is a graph showing the inhibitory effect of IM34 on TLR1/2, TLR2/6, and TLR4.
Figure 7 is a graph showing the inhibitory effect of IM34 on PolyI:C, a TLR3 ligand.
Figure 8 is a diagram showing a representative interaction model between TLR7 (PDB ID: 5GMH) and IM9. (A) Surface view of IM9-bound TLR7, and (B) atomic view of the interaction between IM9 and the small molecule binding site of TLR. (C) An overall view of the IM9 position on the TLR7 homodimer, and (D) a two-dimensional diagram showing the interaction between IM9 and the TLR7 ligand binding site.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, novel compounds, including IM9 (imiquimod mimic 9) and compounds defined as IM1, IM5, IM30, IM34, and IM38, inhibit the TLR signaling pathway induced by TLR3, TLR7, or TLR9 activation to secrete cytokines. was confirmed to inhibit , which suggests that it may have a therapeutic effect on autoimmune or inflammatory diseases such as systemic lupus erythematosus and psoriasis caused by activation of TLR3, TLR7, or TLR9.

Accordingly, in one aspect, the present invention relates to a compound or a pharmaceutically acceptable salt represented by any one formula selected from the group consisting of Formulas 1 to 4.

[Formula 1]

;

[Formula 2]

;

[Formula 3]

;

[Formula 4]

,

In Formula 1, R ₁ to R ₁₇ are the same or different from each other, and each independently represents a hydrogen atom, a halogen atom, straight or branched alkyl, amino, alkylamine, nitrile group, nitro group, nitroso group, hydroxy, cyclo. Alkyl, benzyl, haloalkyl, allyl, alkoxy, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, alkylarylcarbonyl, alkoxycarbonyl, cycloalkoxy, aryl, heteroaryl, heterocycloalkyl, aryloxy, alkoxyhetero Aryl, heteroaryloxyalkyl, alkylheteroaryl, alkylaryl, arylalkyl, alkylheteroaryl, alkyl ester, alkylamide, or acrylic,

or R ₁ and R ₂ are cycloalkyl or heterocycloalkyl linked to each other,

Here, alkyl, alkylamine or alkoxy is C _1-30 , cycloalkyl is C _3-30 , allyl is C _2-30, aryl is C _6-30 , heteroaryl and heterocycloalkyl are fluorine, oxygen, sulfur and Contains heteroatoms selected from nitrogen.

As used herein, the term “C _1-30 alkyl” refers to a monovalent linear or branched saturated hydrocarbon moiety consisting only of carbon and hydrogen atoms and having 1 to 30 carbon atoms. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary-butyl, etc. Examples of “branched alkyl” include isopropyl, isobutyl, and tert-butyl.

The term “C _1-30 alkoxy” refers to alkyl of the formula -OC _1-30 , including but not limited to methoxy, ethoxy, isopropoxy, tert-butoxy, etc.

Specific examples of the term “halogen (or halo)” include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

The term “C _6-30 aryl” includes at least one ring with a shared pi electron chain, for example monocyclic or fused ring polycyclic (i.e. rings dividing adjacent pairs of carbon atoms). Includes groups. That is, in this specification, aryl may include phenyl, naphthyl, etc., and biaryl, unless otherwise defined. In one embodiment of the present invention, aryl refers to an aromatic ring having 6 to 30 carbon atoms.

The term “C _3-30 cyclic alkyl” refers to a cyclic, saturated hydrocarbon moiety consisting only of carbon and hydrogen atoms, having 5 to 6 carbon atoms. Examples of such cyclic alkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, etc.

The term “heteroaryl”, unless otherwise defined, is an aromatic ring with 5 or 6 ring atoms containing 1 to 4 heteroatoms selected from the group consisting of N, O and S, or wherein the heteroaryl ring is a benzene ring or Refers to a bicyclic ring fused to another heteroaryl ring. Examples of monocyclic heteroaryls include thiazolyl, oxazolyl, thiophenyl, furanyl, pyrrolyl, imidazolyl, isoxazolyl, isothiazolyl, pyrazolyl, triazolyl, triazinyl, thiadiazolyl, and tetrazolyl. , oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl and similar groups, but are not limited thereto. Examples of bicyclic heteroaryls include indolyl, azaindolyl, indolinyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, benzthiadiazolyl, and benztria. Examples include, but are not limited to, zolyl, quinolinyl, isoquinolinyl, purinyl, furopyridinyl, and similar groups.

The term “heterocycloalkyl” refers to a saturated or partially unsaturated carbocyclic ring having 5 to 9 ring atoms containing, in addition to carbon atoms, 1 to 3 heteroatoms selected from N, O and S. For example, heterocyclyl includes azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, pipe Ridinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanil, diazepanyl, Furiferazinyl, oxazepanyl, dihydroindolyl, dihydrofuryl, dihydroimidazolinyl, dihydroxazolyl, tetrahydropyridinyl, dihydropyranyl, dihydrobenzofuranyl, benzodioxolyl, or It is benzodioxanil.

The term “amino” refers to a functional group in which one hydrogen atom has been removed from ammonia, and includes amine groups in which one or more hydrogens are replaced with residues such as hydrocarbons, and amine groups are primary, secondary, or primary depending on the number of substituted alkyls. It may contain tertiary alkylamines.

In the present invention, the alkyl, alkylamine or alkoxy is preferably C _1-20 , more preferably C _1-10 , and most preferably C _1-3 .

In the present invention, R ₁ to R ₁₇ may be a substituent selected from the group consisting of a hydrogen atom, straight-chain or branched-chain alkyl, amino, alkylamine, benzyl, and aryl, but are not limited thereto. .

Preferably, the compound represented by Formula 1 may be characterized as a compound of the following Formula 1-1 or Formula 1-2, but is not limited thereto.

[Formula 1-1]

;

;

[Formula 1-2]

.

.

Preferably, the compound represented by Formula 2 may be characterized as a compound of Formula 2-1 or Formula 2-2, but is not limited thereto.

[Formula 2-1]

;

;

[Formula 2-2]

.

.

Preferably, the compound represented by Formula 3 may be characterized as a compound of the following Formula 3-1, but is not limited thereto.

[Formula 3-1]

.

.

Preferably, the compound represented by Formula 4 may be characterized as a compound of the following Formula 4-1, but is not limited thereto.

[Formula 4-1]

.

.

In the present specification, the compound of Formula 1-1 is named IM9 (imiquimod mimic 9), the compound of Formula 1-2 is IM34, the compound of Formula 2-1 is IM1, the compound of Formula 2-2 is IM5, and the compound of Formula 2-2 is IM5. The compound of Formula 3-1 was named IMI30, and the compound of Formula 4-1 was named IM38 (Table 1).

SMILES, IUPAC name and molecular weight of the compound Name SMILES IUPAC Name MW (g/ mol ) IM1 CO[C@H]1CN(C(C)C)C[C@@H]1Nc1nc(-c2ccccn2)nc2c1c(C)nn2C (3S,4S)-N-[1,3-dimethyl-6-(pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-4-methoxy-1-(propan -2-yl)pyrrolidin-3-amine 381.5 IM5 Cn1ncc2c(NC3CCc4ccccc4C3)nc(N)nc21 1-methyl-N4-(1,2,3,4-tetrahydronaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine 294.362 IM9 COc1ccc(Cc2c(C)nc3c(c(C)c(C)n3Cc3ccccn3)c2N)cc1 5-[(4-methoxyphenyl)methyl]-2,3,6-trimethyl-1-[(pyridin-2-yl)methyl]-1H-pyrrolo[2,3-b]pyridin-4-amine 386.5 IM30 COc1ccc(-n2c(C)c(C)c3c(NCCN(C)C)ncnc32)cc1 N-[2-(dimethylamino)ethyl]-7-(4-methoxyphenyl)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine 339.443 IM34 Cc1c(C)n(Cc2ccncc2)c2nc3c(c(N)c12)CC(c1ccccc1)CC3 2,3-dimethyl-6-phenyl-1-[(pyridin-4-yl)methyl]-1H,5H,6H,7H,8H-pyrrolo[2,3-b]quinolin-4-amine 382.511 IM38 Cc1c(C)n(Cc2ccccn2)c2ncn(Cc3ccccc3)c(=N)c12 3-benzyl-5,6-dimethyl-7-[(pyridin-2-yl)methyl]-3H,4H,7H-pyrrolo[2,3-d]pyrimidin-4-imine 343.434

The compound according to the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Inorganic acids and organic acids can be used as free acids. Inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, and methane sulfur. Fonic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, etc. can be used.

The compounds according to the present invention include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates that can be prepared by conventional methods.

Additionally, the compounds according to the present invention may be prepared in crystalline or amorphous form, and when the compounds of Formulas 1 to 4 are prepared in crystalline form, they may be optionally hydrated or solvated.

In the present invention, the compounds represented by Formulas 1 to 4 exhibit the effect of suppressing the secretion of inflammatory cytokines such as TNF-α by inhibiting the TLR3, TLR7 or TLR9 signaling pathway.

Therefore, from another point of view, the present invention provides a composition for inhibiting a TLR (Toll-like receptor) comprising a compound represented by any one of the formulas selected from the group consisting of Formulas 1 to 4, or a pharmaceutically acceptable salt. It's about.

In the present invention, the compound may be characterized by inhibiting the signaling pathway of any one or more TLRs selected from the group consisting of endosomal TLRs, TLR7, TLR9, TLR3, and TLR8. Preferably, it may be characterized by inhibiting the signaling pathway of TLR3, TLR7 or TLR9, but is not limited thereto.

In the present invention, the term “inhibition” refers to a phenomenon in which biological activity or signaling activity is reduced due to deficiency, incompatibility, or many other causes, and partially or completely blocks, reduces, prevents, or activates the activity of TLR. It may be delaying, inactivating, or downregulating.

In the present invention, the term “inhibitor” refers to a molecule that partially or completely inhibits the effects of other molecules, such as receptors or intracellular mediators, by any mechanism. In this specification, a composition for inhibiting TLR is used in the same sense as a TLR inhibitor.

As used herein, the term "TLR3, TLR7 or TLR9 inhibitor" refers to a substance that can directly or indirectly, or substantially, interfere with, reduce or inhibit the biological activity of TLR3, TLR7 or TLR9, preferably TLR3, TLR7 or TLR9. It refers to a substance that can block the TLR3, TLR7 or TLR9 signaling pathway by binding to the receptor and neutralizing their activity, thereby reducing the secretion of NF-κB, MAPK, inflammatory cytokines, NO and ROS.

According to one embodiment of the present invention, IM1, IM5, IM9, IM30, IM34 or IM38 inhibits hyperactivation of cytokines such as TNF-α by inhibiting the TLR signaling pathway induced by TLR3, TLR7 or TLR9 activation. It was confirmed that Therefore, since it has an excellent effect on reducing inflammatory cytokines (alleviating inflammatory responses), it can be usefully used as a composition for preventing or treating autoimmune diseases and inflammatory diseases caused by activation of TLR3, TLR7, or TLR9.

In the present invention, the term "TLR3" refers to a protein belonging to the TLRs, a family of transmembrane proteins that function as a sentinel for pathogen infection. It refers to a protein encoded by the TLR3 gene, and is also named CD283 or IIAE2. The TLR3 recognizes double-stranded RNA or Poly I:C and activates the innate immune system.

In the present invention, the term "TLR7" refers to a protein belonging to TLRs, a family of transmembrane proteins that function as a sentinel for pathogen infection, and is encoded by the TLR7 gene, and is also named UNQ248/PRO285. The TLR7 activates the innate immune system by recognizing ssRNA (single-stranded RNA) of RNA viruses or synthetic small molecules such as imidazoquinoline, loxoribine, and bropirimine.

In the present invention, the term "TLR9" refers to a protein belonging to the TLR, a family of transmembrane proteins that functions as a sentinel for pathogen infection. It refers to a protein encoded by the TLR9 gene, and is also named CD289 or UNQ5798/PRO19605. . The TLR9 recognizes unmethylated CpG oligodeoxynucleotide DNA from bacteria or DNA viruses and activates the innate immune system.

In the present invention, the term "TLR signaling pathway" refers to a signaling pathway through TLR, and a complex formed by TLR and adapter protein MyD88 (for TLR7/8/9) or TLR and adapter protein TRIF (for TLR3). It may be a reaction dependent on , through which signals are transmitted. Activated TLR7/8/9 activates NF-κB through Myd88-dependent signaling process, moves it to the nucleus, and induces the activation of MAPK. Due to the activation of NF-κB and MAPK, inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are secreted, and nitric oxide (hereinafter referred to as NO) and reactive oxygen species (hereinafter referred to as ROS) are released from macrophages. produces oxidative stress substances such as In addition, TLR3 induces a MyD88-independent signaling process by activating TRIF, interferon-regulator (IRF), and NF-κB, resulting in the secretion of type 1 interferon.

From another aspect, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases or inflammatory diseases, comprising a compound represented by any one of the chemical formulas selected from the group consisting of Formulas 1 to 4, or a pharmaceutically acceptable salt. It's about.

From another aspect, the present invention provides a method for preventing autoimmune diseases/inflammatory diseases, comprising the step of administering a compound or a pharmaceutically acceptable salt represented by any one of the formulas selected from the group consisting of Formulas 1 to 4. It's about treatment methods.

In another aspect, the present invention relates to the use of said compounds for the prevention or treatment of autoimmune diseases/inflammatory diseases.

In another aspect, the present invention relates to the use of the above compounds for the preparation of medicaments for the prevention or treatment of autoimmune diseases/inflammatory diseases.

In the present invention, the compound represented by Formula 1 is a compound of Formula 1-1 or Formula 1-2, and the compound represented by Formula 2 is a compound of Formula 2-1 or Formula 2-2, wherein The compound represented by 3 is a compound of the following formula 3-1, and the compound represented by the formula 4 may be characterized as a compound of the following formula 4-1, but is not limited thereto.

In the present invention, the autoimmune disease or inflammatory disease includes psoriasis, systemic lupus erythematosus (SLE), skin rash, photosensitivity, arthritis, oral ulcer, nephritis, cytopenia, vasculitis, serositis, inflammatory bowel disease (IBD), and diabetes. , multiple sclerosis, scleroderma, pemphigus, atopic dermatitis, urethritis, cystitis, arteriosclerosis, allergic disease, rhinitis, asthma, acute pain, chronic pain, periodontitis, gingivitis, gout, myocardial infarction, congestive heart failure, hypertension, It may be characterized as a disease selected from the group consisting of angina, gastric ulcer, cerebral infarction, Down syndrome, obesity, dementia, depression, schizophrenia, tuberculosis, sleep disorders, sepsis, burns, pancreatitis, Parkinson's disease, and stroke, but is limited thereto. That is not the case.

In the present invention, the term "autoimmune disease" refers to a disease that is caused by a problem in inducing or maintaining self-tolerance, resulting in an immune response to self-antigens, which causes an attack on one's own tissues. It means disease. The self-tolerance refers to immunological unresponsiveness that does not react harmfully to antigenic substances constituting the self. Autoimmune diseases include diseases resulting from the breakdown of self-resistance, which allows the adaptive immune system to respond to self-antigens and mediate cell and tissue damage. In certain embodiments, an autoimmune disease is characterized as being at least in part the result of a humoral immune response.

The autoimmune diseases of the present invention include systemic lupus erythematosus, insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, autoimmune arthritis, myasthenia gravis, thyroiditis, experimental form of uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, Pernicious anemia, autoimmune atrophic gastritis, Addison's disease, early menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmitis, phakic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary tract Includes, but is not limited to, cirrhosis, chronic active hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis/dermatomyositis, and discoid LE.

Additionally, examples of autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, and ankylosing spondylitis. , antibody-mediated transplant rejection, anti-GBM/anti-TBM nephritis, antiphospholipid antibody syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune Autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune diabetic retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy. , Balo disease, Behcet's disease, pemphigoid, cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO) ), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia (essential mixed cryoglobulinemia), demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devick's disease, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, nodular Erythema, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain -Barré syndrome, Hashimoto's encephalitis, Hashimono's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestation, hypogammaglobuminemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related Sclerosing disease, immunomodulatory lipoprotein, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Eaton-Lambert syndrome, leukocytic vasculitis, squamous Lichen sclerosus, lignoconjunctivitis, glandular IgA disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of unknown significance (MGUS), Encroaching corneal ulcer, Mucha-Habermann's disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis perplexus (Devick's disease), neutropenia, ocular cicatricial pemphigoid, optic neuritis, recurrent rheumatism, PANDAS (Streptococcal infection-related childhood disease) autoimmune neuropsychiatric disease), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), hemifacial atrophy, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral nerves disease, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, type II, and type III autoimmune polyglandular syndrome, polymyalgia rheumatism, polymyositis, myocardial infarction Post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmitt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmitis, Takayasu's arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, Waldenström's macroglobulinemia (WM), and Wegener's granulomatosis (polyangiitis granulomatosis). (GPA)).

In the present invention, the term "inflammatory disease" refers to TNF-α, IL-1, IL-6, and prostaglandins secreted by immune cells such as macrophages due to excessive stimulation of the immune system due to harmful stimuli such as inflammatory factors or radiation. It refers to a disease caused by inflammatory substances (inflammatory cytokines) such as prostaglandin, leukotriene, or NO. An “inflammatory disease” may be an acute or chronic inflammatory condition and may arise from infectious or non-infectious causes.

Inflammatory diseases of the present invention include psoriasis, asthma, eczema, allergies, rheumatoid arthritis, psoriatic arthritis, contact dermatitis, atopic dermatitis, acne, atopic rhinitis, allergic dermatitis, chronic sinusitis, and seborrheic diseases. Seborrheic dermatitis, gastritis, gout, gout arthritis, ulcers, chronic bronchitis, pulmonary inflammation, Crohn's disease, ulcerative colitis, ankylosing spondylitis, sepsis, vasculitis, bursitis, lupus, polymyalgia rheumatica, temporal arteritis , multiple sclerosis, solid tumors, Alzheimer's disease, arteriosclerosis, obesity, and viral infections.

Additionally, examples of inflammatory diseases include, but are not limited to, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatism (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, Cystic fibrosis, osteoarthritis, rheumatoid arthritis, inflammatory arthritis, Sjögren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g. type I) , myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory skin disease, common interstitial pneumonia (UIP), asbestos disease. , silicosis, bronchiectasis, beryllium poisoning, talcosis, pneumoconiosis, sarcoidosis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and vasculature. Salt-related forms (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed hypersensitivity reactions (e.g. lacquer poisoning), pneumonia, airway inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, Hay fever, allergy, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic damage), allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, Bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacritis, dermatomyositis, endocarditis, endometritis, enteritis, panenteritis, epicondylitis, epididymitis, fasciitis, connective tissueitis, gastritis, gastroenteritis, gingivitis, ileitis, iritis , laryngitis, myelitis, myocarditis, nephritis, laminitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, rhinitis, phlebitis, interstitial pneumonia, proctositis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis , synovitis, orchitis, tonsillitis, urethritis, bladder infection (urocystitis), uveitis, vaginitis, vasculitis, vulvovaginitis, and vulvovaginitis, vasculitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, depression, cerebrospinal fasciitis, and brain-dead enteritis. In certain embodiments, the inflammatory disease is selected from the group consisting of atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory arthritis, and myocarditis.

The term “prevention” used in the present invention refers to all actions that suppress or delay autoimmune diseases and/or inflammatory diseases by administering a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof. In addition, the term “treatment” used in the present invention refers to the treatment of symptoms of autoimmune disease and/or inflammatory disease by administration of a pharmaceutical composition containing a compound represented by Formula 1 to Formula 4, or a pharmaceutically acceptable salt thereof. refers to any action that improves or completely cures.

The composition for preventing or treating autoimmune diseases and/or inflammatory diseases according to the present invention contains a pharmaceutically effective amount of the compounds represented by Formulas 1 to 4 alone, or one or more pharmaceutically acceptable carriers and excipients. Alternatively, it may include a diluent. In the above, the pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat symptoms of autoimmune disease and/or inflammatory disease.

The term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal upset, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Examples include polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

The term “carrier” refers to a substance that facilitates the addition of a compound into cells or tissues.

The term “diluent” is defined as a substance diluted in water that not only stabilizes the biologically active form of the compound of interest, but also dissolves the compound.

In addition, the composition of the present invention may include the compounds represented by Formulas 1 to 4, as well as one or more known active ingredients that have a therapeutic effect on autoimmune diseases and/or inflammatory diseases.

Compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal other than a human. Dosage forms may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

The composition of the present invention can be administered through several routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, and the dosage of the active ingredient depends on various factors such as the route of administration, the patient's age, gender, weight, and patient's severity. It can be appropriately selected, and the composition according to the present invention can be administered in combination with known compounds that have the effect of preventing, improving, or treating symptoms of autoimmune diseases and/or inflammatory diseases.

Other terms and abbreviations used in this specification, unless otherwise defined, may be interpreted as commonly understood by those skilled in the art to which the present invention pertains.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

Example 1: Method

Example 1-1: endosome TLR's for inhibitory molecules in silico (Computer Programming in Virtual Experiments) Screening

The overall methodology of the virtual screening workflow is shown in Figure 1. First, a multiconformational library of screening compounds was prepared using molecules from ChemBridge, Chemspace, Mcule, MOE Leadlike, MolPort, ZINC Drug-Like, and ZINC Lead-Like chemical databases. Ligand structures were washed using the MOE wash protocol. Briefly, salts and broken fragments were removed, protonation states were calculated at pH 6.5, and energy was minimized. After removing duplicate conformations, a BIT:MACCS fingerprint similarity search (80% similarity cutoff) was performed for a series of known TLR7/8/9 inhibitors reported in the literature. Based on the pharmacophore models of selected highly active antagonists, the generated ligands were screened. Next, the ligand satisfying the pharmacophore constraints was selected from MOE software (Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2017 (2013) ) was docked into the R848 binding site of TLR7 (PDB ID: 5GMH (Zhang, Z. et al. Immunity 45 , 737-748 (2016))). Docking poses were ranked according to their S score (binding affinity) implemented in the London DG scoring function. The resulting docking poses were rescored using the guided-fit method and 'GBVI/WSA dG' force field refinement. Finally, a set of 15 top ligands was obtained to test TLR7/9 inhibitory activity in cell-based assays.

Example 1-2: of TLR7 Molecular docking of IM9 to the ligand-binding site.

To construct a representative interaction model with the extracellular domain of TLR7, the hit with the highest activity, IM9 (imiquimod mimic 9), was selected. The receptor structure was prepared by fixing broken residues, adjusting the protonation state at pH 6.5, and minimizing energy using the Amber12:EHT force field in the MOE program. By specifying the R848 binding site as the interaction site (Zhang, Z. et al. Immunity 45 , 737-748 (2016)), the energy-minimized structure of the ligand was docked into the receptor. Triangle matcher placement and London DG scoring schemes were applied to rank the docking poses. The receptor remained rigid and the ligand was flexible during the docking process.

Example 1-3: Identification of structural analogs of IM5 and IM9

Based on the initial activity data of IM5 and IM9, a series of similar structural analogs to IM5 or IM9 were prepared by searching in the MolPort database (URL: https://www.molport.com/shop/index) with a Tanimoto coefficient of 0.8. did. The obtained set of 100 analogs was washed using MOE's cleaning protocol. These ligands were docked into the ligand binding site of TLR7 using the same protocol as described in Examples 1-2. A set of 25 ligands with scores higher than IM9 were selected for another round of activity assays to determine whether they showed improvement in inhibition of TLR3/7/9 signaling.

Example 1-4: Cell culture and seeding

RAW 264.7 cells were purchased from the Korean Cell Line Bank and cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS (Gibco) and 1% penicillin and streptomycin solution (Hyclone).

For the ligand screening work, cells were seeded in 96-well cell culture plates at a density of 2 × 10 ⁴ cells per well and cultured overnight.

Example 1-5: Enzyme-linked immunosorbent assay (ELISA)

To confirm the inhibitory effect of the test-ligand, RAW264.7 cells were pretreated with each compound for 1 hour and then incubated with Pam ₃ CSK ₄ (TLR1/2, 100 nM), FSL-1 (TLR2/6, 100 ng/ml ), lipopolysaccharide (TLR4, 100ng/ml), imiquimod (Imiquimod; TLR7, 1μg/ml), and ODN2395 (TLR9, 0.5μM) ligands for 4 hours, and poly I showing delayed reactivity. :C (TLR3, 1μg/ml) was treated for 24 hours. After activation of TLRs, the supernatant was transferred to a pre-coated 96-well assay plate, and the level of mouse TNF-alpha secretion was measured with a mouse-TNF-alpha ELISA-kit (Invitrogen) according to the manufacturer's instructions.

Example 1-6: IC50 analysis

To plot concentration-dependent IC50 curves, three active ligands (IM1, IM5 and IM9) were pretreated at different concentrations up to 50 μM (twice the initial concentration). Cellular responses mediated by TLR7 and TLR9 (TNF-α secretion levels) were analyzed by ELISA as described in Examples 1-5. Cytokine levels in each plot were normalized from negative control (untreated) to positive control (ligand treated) and IC50 values were determined by nonlinear regression (Graph Pad Prism 7.0).

Example 2: endosome TLR's Identification of potential inhibitors

To select potential inhibitory molecules for TLR7/TLR9 activation, a virtual screening workflow (Figure 1) was used. First, a set of 15 ligands was used in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay and ELISA assay to monitor TNF-α secretion levels in RAW264.7 cells. were evaluated for their inhibitory activity. Among the tested ligands, IM1 and IM9 showed dose-dependent inhibition of TLR7/TLR9 (Figure 2), while the ligand IM5 significantly inhibited only the activation of TLR7.

Based on the preliminary activities of IM1, IM5 and IM9, the IC50 values were determined to identify the most potent of the three ligands (Figure 3). The analysis showed that IM5 was the most active ligand for TLR7 activation (IC50 = 5.48 μM), while IM9 was the most potent against TLR9 (IC50 = 3.85 μM) but was moderately active against TLR7 (IC50 = 8.58 μM). . Taken together, IM9 was collectively found to be the best inhibitory molecule for TLR7 and TLR9 signaling.

Then, a set of 25 ligands with at least 80% similarity to the IM1, IM5 or IM9 structures were obtained from commercial sources to determine whether the initial inhibitory activity of these ligands could be further improved. Among the tested derivatives, IM30, IM34 and IM38 were found to inhibit TLR7/TLR9 activation in a dose-dependent manner. IM34 (IM9-derivative) showed a better effect on activation of both receptors compared to IM9 (Figure 4). The structures of all active ligands and some of their chemical information are shown in Figure 5. The specificity of IM34 for endosomal TLRs was confirmed by analyzing the activity of IM34 on TLR1/2, TLR2/6, TRL3 and TLR4 activation profiles. As a result, IM34 ligand did not have any inhibitory effect on stimulation of Pam ₃ CSK ₄ , FSL-1 or LPS at a concentration of 10 μM (Figure 6). On the other hand, for TLR3, which has structural similarity to TLR7 and TLR9 and is active in endosomes, the IM34 ligand showed an inhibitory effect upon stimulation by poly I:C, a TLR3 ligand, at a concentration of 20 μM (FIG. 7).

Example 3: With IM9 TLR7 Interaction analysis between binding sites

To model a representative interaction between the lead molecule IM9 and the TLR7 extracellular domain, the ligand was docked into the R848-binding site of the TLR7 crystal structure (5GMH) (Figure 5A, C). Detailed analysis revealed that the binding mode of IM9 is similar to that of the agonist R848 (Zhang, Z. et al. Immunity 45 , 737-748 (2016)). The 4-amino-pyrrolopyridine group was fixed between the side chains of F408 and L557* (Figure 8B). Meanwhile, 2-pyridine or 4-methoxyphenyl groups were stabilized by several hydrophobic contacts, including residues L557*, V381, Y356, V355, F351, I585*, F466, F349, and Y264. The 2-pyridine ring of the ligand was partially exposed to the solvent, whereas the 4-methoxyphenyl was deeply buried in the binding site (Figure 8D). Van der Waals and nonpolar interactions are important for the stability of the ligand, as no hydrogen bonding has been observed to date in the docked conformation. However, the negatively charged residues E352, E583* and D555* can create strong electrostatic interactions with the amino group or nitrogen atom of the pyridine ring. Taken together, the IM9 ligand exhibited stable intermolecular interactions with the TLR7 ligand-binding pocket with partial similarity to the agonist ligand R848.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

delete delete delete delete delete delete delete delete A pharmaceutical composition for preventing or treating an autoimmune disease or inflammatory disease caused by TLR (Toll-like receptor) activation comprising a compound represented by the following Formula 1-1 or Formula 1-2 or a pharmaceutically acceptable salt:
[Formula 1-1]
;
[Formula 1-2]
.
delete delete delete delete delete The method of claim 9, wherein the autoimmune disease or inflammatory disease caused by TLR (Toll-like receptor) activation includes psoriasis, systemic lupus erythematosus (SLE), skin rash, arthritis, nephritis, cytopenia, diabetes, scleroderma, A pharmaceutical composition characterized in that the disease is selected from the group consisting of arteriosclerosis, periodontitis, gingivitis, myocardial infarction, congestive heart failure, hypertension, angina, sepsis, pancreatitis and stroke.

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