KR102558127B1 - Composition comprising arylpiperazine derivative for activating stimulator of interferon gene dependent signalling - Google Patents

Abstract

The present invention relates to an interferon gene stimulant composition comprising an N-arylpiperazine derivative as an active ingredient, which can solve the problem of low disease control rate compared to the use of cyclic-di-nucleotide alone, and can have high activity even when a low concentration of cyclic-di-nucleotide is used by using it together with the compound of formula (I).

Description

Interferon gene stimulator composition containing an arylpiperazine derivative as an active ingredient

The present invention relates to an interferon gene stimulator composition comprising an arylpiperazine derivative as an active ingredient.

New insights into the mechanisms underlying immune-evasion have served as a basis for the development of vaccines or immune modulators capable of priming or boosting an effective adaptive immune response, along with combination treatment regimens that directly or indirectly enhance the efficacy of therapeutic vaccination through combination with immune checkpoint inhibitors or other therapies. These modulators consist of tumor-specific CD4+ and CD8+ T cells specific for targeted malignancies, resulting in an antitumor response and clinical benefit. How the innate immune system is engaged by targeted ligands shapes the development of adaptive responses and aids in the design of vaccines and immunomodulators (Reed et al., Trends Immunol., 30: 23-32, 2009; Dubensky and Reed, Semin. Immunol., 22: 155-61, 2010; Kastenmuller et al., J. Clin. Invest., 121: 1782-1796, 2011; Coffman et al., Immunity, 33: 492-503, 2010).

Cyclic-di-AMP (produced by Listeria monocytogenes and other bacteria), a cyclic-di-nucleotide (CDN), and its analogues, cyclic-di-GMP and cyclic-GMP-AMP, are recognized by host cells as pathogen-associated molecular patterns (PAMPs) and bind to pathogen recognition receptors (PRRs) known as stimulators of interferon genes (STING). STING is an adapter protein in the cytoplasm of host mammalian cells, which activates the TANK binding kinase (TBK1)-IRF3 and NF-κB signaling axes, resulting in the induction of IFN-β and other gene products that strongly activate innate immunity. It is now recognized that STING is a component of the host cytosolic surveillance pathway (Vance et al., 2009), which senses infection by intracellular pathogens and induces the production of IFN-β in response, triggering the development of an adaptive protective pathogen-specific immune response consisting of both antigen-specific CD4+ and CD8+ T cells as well as pathogen-specific antibodies. Examples of cyclic purine dinucleotides are described in some detail, for example in US Pat. Nos. 7,709458 and 7,592,326; Patent applications WO2007/054279, WO2014/093936 and WO2014/189805; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008)]. An uncharacterized mouse gene with significant structural homology to the catalytic domain of the human oligoadenylate synthase, cyclic GMP-AMP synthase, has been reported to be the enzyme responsible for the production of STING-binding CDNs in mammalian cells (Sun et al., Science 339(6121):786-91, 2013). This enzyme, called cyclic GMP-AMP synthase (cGAS), catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from ATP and GTP in the presence of DNA. This cGAMP then functions as a second messenger that binds to and activates STING. These cGAS-produced CDNs differed structurally from bacterially produced CDNs in that they possessed unconventional phosphodiester linkages. Thus, whereas bacterially produced CDNs contained bis-3',5' linkages between two nucleotides, mammalian CDNs contained one 2',5' linkage and one 3',5' linkage, or so-called "mixed linkage (ML) or non-canonical CDNs. These 2',5'-3',5' molecules bind STING with nM affinity, some 300-fold better than bacterial c-di-GMP. Human STING (hSTING) also has known polymorphisms including an allele encoding histidine at position 232 that is refractory to the bis-3',5' (canonical) CDN but not to the 2',5'-3',5' (non-canonical, mixed linkage) CDN (Diner et al., Cell Reports 3, 1355-61, 2013; Jin et al., Genes and Immunity , 12: 263-9, 2011) Single nucleotide polymorphisms in the hSTING gene have been reported to affect responsiveness to bacterial-derived canonical CDNs (Diner et al., 2013; Gao et al., Cell 154, 748-762, 2013; Conlon et al., J. Immunol. 190: 5216-5225, 2013). Five haplotypes of hSTING have been reported (WT, REF, HAQ, AQ and Q alleles), which differ at amino acid positions 71, 230, 232 and 293 (Jin et al., 2011; Yi et al., PLOS One 8: e77846, 2013). It responds weakly to stimulation by the DNs cGAMP, c-di-AMP and c-di-GMP, but is responsive to the endogenously produced cGAS product ML cGAMP (Diner et al., 2013).Therefore, the 2',5'-3',5' molecule has been suggested to be a much more potent physiological ligand in terms of hSTING targeting (Zhang et al., Mol. Cell. 51:226-35, 2013; Xiao and Fitzgerald, Mol. Cell 51: 135-39, 2013).

Despite these findings, in the case of CDN, mass synthesis is not easy due to the complexity of the synthesis process and the difficulty of the purification process, and there are limitations in that stability is poor and only intratumoral administration is possible. Therefore, the need for a STING antagonist based on a small molecule compound is emerging. Therefore, in this study, among ARP compounds, one species showing STING-dependent signaling and interferon-inducing effects was selected, and Type I IFN signaling was confirmed to complete the invention.

1. Reed et al., Trends Immunol., 30: 23-32, 2009

One aspect of the present invention is to provide an interferon gene stimulator composition comprising an arylpiperazine derivative as an active ingredient.

One aspect of the present invention is to provide an interferon gene stimulator composition comprising an arylpiperazine derivative compound represented by Formula 1 below.

[Formula 1]

In Formula 1,

X is sulfur (S) or oxygen (O);

Y is -NHR ₁ or R ₂ ;

R ₁ is a C ₁ -C ₈ straight or branched chain alkyl group; C ₁ -C ₈ straight-chain or branched-chain alkyl group substituted with a halogen, hydroxy group or C ₁ -C ₆ alkoxy group; C ₃ -C ₁₀ cycloalkyl group; C ₆ -C ₂₀ aryl group; C ₁ -C ₈ alkyl group, halogen-substituted C ₁ -C ₈ alkyl group, C ₁ -C ₈ alkoxy group, halogen-substituted C ₁ -C ₈ alkoxy group, nitro, nitrile, hydroxy group, or halogen-substituted C ₆ -C ₂₀ aryl group;

R ₂ is a C ₁ -C ₁₅ straight-chain or branched-chain alkyl group; C ₁ -C ₁₅ straight-chain or branched-chain alkyl group substituted with a halogen, hydroxy group or C ₁ -C ₆ alkoxy group; C ₂ -C ₈ alkenyl group; C ₃ -C ₁₀ cycloalkyl group; pyrazinyl group; or ego,

n is 0 or an integer from 1 to 5;

R ₃ , R ₄ and R ₅ are each independently hydrogen; halogen; nitro; nitrile; C ₁ -C ₈ straight or branched chain alkyl group; C ₁ -C ₈ straight-chain or branched-chain alkyl group substituted with a halogen, hydroxy group or C ₁ -C ₆ alkoxy group; C ₁ -C ₈ straight-chain or branched-chain alkoxy group; or a halogen-substituted C ₁ -C ₈ straight-chain or branched-chain alkoxy group.

Specifically, when X is sulfur in Formula 1, Y may be -NHR ₁ . At this time, R ₁ is a C ₁ -C ₈ straight-chain or branched-chain alkyl group; Or a C ₁ -C ₈ alkyl group, a halogen-substituted C ₁ -C ₈ alkyl group, a C ₁ -C ₈ alkoxy group, a halogen-substituted C ₁ -C ₈ alkoxy group, a nitro, nitrile, hydroxy group, or a halogen-substituted C ₆ -C ₂₀ aryl group; The C ₆ -C ₂₀ aryl group may be a phenyl group.

In Formula 1, when X is oxygen, Y may be -NHR ₁ or R ₂ . In this case, R ₁ may be a C ₁ -C ₈ alkyl group, a halogen-substituted C ₁ -C ₈ alkyl group, a C ₁ -C ₈ alkoxy group, a halogen-substituted C ₁ -C ₈ alkoxy group, a nitro, nitrile, hydroxy group, or a halogen-substituted C ₆ -C ₂₀ aryl group; The C ₆ -C ₂₀ aryl group may be a phenyl group.

In addition, R ₂ is a C ₁ -C ₁₅ straight-chain or branched-chain alkyl group; pyrazinyl group; or can be

That is, the arylpiperazine derivative compound represented by Chemical Formula 1 may be any one of the compounds represented by Chemical Formulas 1a to 1c.

[Formula 1a]

[Formula 1b]

[Formula 1c]

.

The definition of R ₁ in Formulas 1a and 1b is the same as the definition of R ₁ in Formula 1 above. In addition, the definition of R ₂ in Formula 1c is the same as the definition of R ₂ in Formula 1 above.

The compound represented by Chemical Formula 1a may be prepared by Reaction Scheme 1 below.

[Scheme 1]

In Reaction Scheme 1, the definition of R ₁ in Formula (a) is the same as the definition of R ₁ in Formula 1 above.

The compound represented by Formula 1b may be prepared by Reaction Scheme 2 below.

[Scheme 2]

In Reaction Scheme 2, the definition of R ₁ in Formula b is the same as the definition of R ₁ in Formula 1 above.

The compound represented by Chemical Formula 1c may be prepared by Reaction Scheme 3 below.

[Scheme 3]

In Reaction Scheme 3, the definition of R ₂ in Formula c is the same as the definition of R ₂ in Formula 1 above.

In Schemes 1 to 3, in step 1), 2,3-dichloroaniline is reacted with bis(2-chloroethyl)ethylamine to obtain 1-(2,3-dichlorophenyl)piperazine hydrochloride, and NH ₄ OH In the presence of OH, 1-(2,3-dichlorophenyl)piperazine hydrochloride can be converted to 1-(2,3-dichlorophenyl)piperazine.

In Scheme 1, step 2) may react 1-(2,3-dichlorophenyl)piperazine and the compound represented by Formula a with triethylamine (TEA) in anhydrous DCM.

In Scheme 2, step 2) may react 1-(2,3-dichlorophenyl)piperazine with the compound represented by Formula b in the presence of triphosgene.

In Scheme 3, step 2) may react 1-(2,3-dichlorophenyl)piperazine with the compound represented by Formula c in the presence of HATU and DIPEA in DMF.

The stimulator of interferon genes (STING) of the present invention is a receptor that recognizes nucleic acids different from TLRs (toll-like receptors). Examples of native ligands to be recognized include bacterial/protozoan-derived cyclic dinucleotides (CDNs), 2',3'-cGAMPs synthesized by upstream cGAS (cyclic GMP-AMP synthase), and the like (Trends in Immunology 35:88-93 (2014)). It has been reported that 2',3'-cGAMP, one of the natural ligands, is degraded by ENPP1 (ecto-nucleotide-pyrophosphatase/phosphodiesterase), a type of pyrophosphatase/phosphodiesterase, and that other CDNs are degraded by phosphodiesterase (Nat Chem Biol 10 :1043-1048 (2014); Cell Res 25:539-550 (2015); Biochemistry 55:837-849 (2016)). STING is activated by these native ligands, induces phosphorylation of TBK1 (TANK binding kinase 1) downstream, and induces type I interferon (IFN) responses by further activating IRF3 (Interferon regulatory factor 3) and NFkB signals downstream (Trends in Immunology 35:88-93 (2014)) . The importance of STING signaling in cancer has been shown by tests using knockout mice. It has been reported that in allogeneic-transplanted mice using knockout mice for STING and its downstream signal IRF3, cancer cells grow by suppression of the cancer immune system, compared to wild-type mice (Immunity 41: 830-842 (2014)). In addition, cancer cell growth in allo-tumor transplanted mice is inhibited by radiation therapy, but in knockout mice for STING and IFNAR1 (interferon (alpha and beta) receptor 1, a receptor for type I IFN produced by downstream signaling), it has been reported that the effect of radiation therapy is reduced (Immunity 41:843-852 (2014)). For these reasons, STING plays an important role in the inhibition of cancer cell growth, and activation of immune signals induced by activation of STING is believed to be associated with anticancer activity. Thus, STING agonists may also be used as anti-cancer agents to target cancer immunity. In addition, activation of STING is believed to play an important role in the immune effect of vaccines, since its activation activates innate immunity (Ther Adv Vaccines 1:131-143 (2013)). Thus, STING agonists may be used as adjuvants to various vaccines.

The arylpiperazine derivative compound represented by Formula 1 may be a compound represented by Formula 1-1 below.

[Formula 1-1]

In the above formula,

R ₆ is hydrogen; C ₁ -C ₈ Alkyl group; A halogen-substituted C ₁ -C ₈ alkyl group; A C ₁ -C ₈ alkoxy group; A halogen-substituted C ₁ -C ₈ alkoxy group; nitro; nitrile; hydroxy group; or halogen; Specifically, in Chemical Formula 1-1, R ₆ may be nitro or halogen. More specifically, R ₆ can be nitro, fluorine, chlorine, or bromine.

The compound represented by Formula 1a may include the compound represented by Formula 1-1.

The compound represented by Chemical Formula 1-1 may be a compound represented by Chemical Formula 1-1-a or Chemical Formula 1-1-b below.

[Formula 1-1-a]

[Formula 1-1-b]

In Formula 1-1-a and Formula 1-1-b, the definition of R ₆ is the same as the definition of R ₆ in Formula 1-1.

The arylpiperazine derivative compound represented by Formula 1 may be a compound represented by Formula 1-2 below.

[Formula 1-2]

In the above formula,

R ₇ is hydrogen; C ₁ -C ₈ Alkyl group; A halogen-substituted C ₁ -C ₈ alkyl group; A C ₁ -C ₈ alkoxy group; A halogen-substituted C ₁ -C ₈ alkoxy group; nitro; nitrile; hydroxy group; or halogen; Specifically, in Formula 1-2, R ₇ may be nitro, halogen, a halogen-substituted C ₁ -C ₈ alkyl group, a C ₁ -C ₈ alkoxy group, or a halogen-substituted C ₁ -C ₈ alkoxy group. More specifically, R ₇ can be nitro, fluorine, trifluoromethyl, trifluoromethoxy, or methoxy.

The compound represented by Formula 1b may include the compound represented by Formula 1-2.

The compound represented by Chemical Formula 1-2 may be a compound represented by Chemical Formula 1-2-a or Chemical Formula 1-2-b below.

[Formula 1-2-a]

[Formula 1-2-b]

In Formula 1-2-a and Formula 1-2-b, the definition of R ₇ is the same as the definition of R ₇ in Formula 1-2.

An interferon gene stimulator composition wherein the arylpiperazine derivative compound represented by Chemical Formula 1 is a compound represented by Chemical Formulas 1-3:

[Formula 1-3]

In the above formula,

n is 0 or an integer from 1 to 5;

R ₃ , R ₄ and R ₅ are each independently hydrogen; halogen; nitro; nitrile; C ₁ -C ₈ straight or branched chain alkyl group; C ₁ -C ₈ straight-chain or branched-chain alkyl group substituted with a halogen, hydroxy group or C ₁ -C ₆ alkoxy group; C ₁ -C ₈ straight-chain or branched-chain alkoxy group; or a halogen-substituted C ₁ -C ₈ straight-chain or branched-chain alkoxy group;

Specifically, in Chemical Formula 1-3, each of R ₃ , R ₄ and R ₅ may independently be hydrogen, halogen, a C ₁ -C ₈ straight or branched chain alkyl group, a halogen-substituted C ₁ -C ₈ alkyl group, a C ₁ -C ₈ alkoxy group, or nitro. More specifically, each of R ₃ , R ₄ and R ₅ may independently be trifluoromethyl, methoxy, methyl, nitro, fluorine, or chlorine.

The compound represented by Formula 1c may include the compound represented by Formula 1-3.

The compound represented by Chemical Formula 1-3 may be a compound represented by Chemical Formula 1-3-a or Chemical Formula 1-3-b below.

[Formula 1-3-a]

[Formula 1-3-b]

In Formula 1-3-a and Formula 1-3-b, the definitions of R ₃ , R ₄ and R ₅ are the same as those of R ₃ , R ₄ and R ₅ in Formula 1-3.

The arylpiperazine derivative compound represented by Chemical Formula 1 may be any one of Compounds 1 to 6 below.

[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]

.

Specifically, the compound represented by Chemical Formula 1-1 may be any one of Compound 2 to Compound 6.

The arylpiperazine derivative compound represented by Formula 1 may be any one of Compounds 7 to 14 below.

[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

[Compound 12]

[Compound 13]

[Compound 14]

.

Specifically, the compound represented by Chemical Formula 1-2 may be any one of Compound 7 to Compound 14.

The arylpiperazine derivative compound represented by Formula 1 may be any one of Compounds 15 to 25 below.

[Compound 15]

[Compound 16]

[Compound 17]

[Compound 18]

[Compound 19]

[Compound 20]

[Compound 21]

[Compound 22]

[Compound 23]

[Compound 24]

[Compound 25]

Specifically, the compound represented by Chemical Formula 1-3 may be any one of Compound 17 to Compound 25.

The composition may further include an acceptable carrier, but is not particularly limited thereto.

The acceptable carrier is one commonly used in formulation and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, etc., and may further contain other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding a suitable pharmaceutically acceptable carrier and formulation, it can be preferably formulated according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but may be formulated as an injection, an inhalant, or an external skin preparation.

The compound of Formula 1 can overcome the difficulties of preparing cyclic-di-nucleotide, a conventional interferon gene stimulating substance, low stability, and limitations of the route of administration, and exhibits STING-dependent signaling and interferon-inducing effects.

One aspect of the present invention relates to a method for preventing or treating a disease comprising administering the composition to a subject to be treated or prevented.

In one embodiment of the present invention, the subject to be treated or prevented has an autoimmune disease such as cancer, solid cancer, non-T cell invasive cancer, cancer with low immune checkpoint inhibitor reactivity, and STING-associated vasculopathy with onset in infancy (SAVI), or has a high possibility of having it.

The composition may be administered in a pharmaceutically effective amount to a subject for treatment or prevention.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to the type and severity of the subject, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrently used drugs, and other factors well known in the medical field.

The composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, single or multiple administrations may be possible. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, the composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dosage may be appropriately selected by those skilled in the art, although it varies depending on the condition and weight of the patient, the severity of the disease, the type of drug, the route of administration and time.

As a specific example, the composition may be administered in an amount of generally 0.001 to 1000 mg/kg, more specifically 0.05 to 200 mg/kg, and most specifically 0.1 to 100 mg/kg once or several times a day, but a preferred dosage may be appropriately selected by those skilled in the art depending on the condition and body weight of the subject, the severity of the disease, the form of the drug, the route of administration and the period of administration.

The composition of the present invention exhibits STING-dependent signaling and interferon-inducing effects while being able to overcome the high difficulty, low stability, and limitations of the administration route of preparing cyclic-di-nucleotides, which are conventional interferon gene stimulating substances.

Figure 1 shows the concentration-dependent ISRE reporter assay results and cytotoxicity results of the compounds of the present invention.
Figure 2 shows the results of IFNβ and IP-10 cytokine analysis induced by the compound of the present invention.
Figure 3 is a result of analyzing Interferon-stimulated gene (ISG) expression changes induced by the compound of the present invention.
Figure 4 is the result of ISRE reporter assay (RED: WT cells, BLUE: STING KO cells, Size by normalized cell viability, duplicated) of STING dependence test of the compound of the present invention.
Figure 5 is the concentration-dependent STING dependence test ISRE reporter assay results of the compounds of the present invention (RED: WT cells, BLUE: STING KO cells, Size by normalized cell viability, duplicated).

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

¹ H and ¹³ C NMR spectra were recorded using a JEOL ECZ-600R (JEOL Ltd, Japan), and chemical shifts were measured in the ppm downfield range of the internal tetramethylsilane standard.

Multiplicity is expressed as follows: s (singlet); d (doublet); t(triplet); q (quartet); m(multiplet); dd (doublet of doublet); ddd (doublet of doublet of doublet); doublet of triplet (dt); triplet of doublets (td); brs (broad singlet).

Coupling constants are reported in Hz. Routine mass analyzes were performed on an LC/MS system equipped with a reversed-phase column (C-18, 50Υ 2.1 mm, 5 μm) and a photodiode analytical detector using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI).

Molecular weight analysis of the compound was confirmed by high-resolution mass spectrometry (LRMS). LRMS analysis was performed using LCMS-2020 (Shimadzu, Japan).

Analysis of the purity and Rt (Retenion time) of the compound was confirmed by high performance liquid chromatography (HPLC). Specifically, HPLC was performed on an Alliance Waters 2695 system (column, C18, 5 mm, 4.6 mm Х 250 mm; UV wavelength, 210 nm; temperature, 25 °C; eluent: A: 0.01 M KH2PO4 in H2O; eluent: B: ACN; 1.0 mL/min).

Identification of the functional group of the compound was confirmed by infrared spectroscopy (IR). IR analysis was performed using a Compact FT-IR Spectrometer ALPHA II (Bruker, USA).

Example

Example 1: Preparation of Compound 1 (Scheme 1)

Example 1-1: Preparation of 1-(2,3-dichlorophenyl)piperazine

A mixture of 2,3-dichloroaniline (10 g, 1 equiv, 61.72 mmol) and bis(2-chloroethyl)ethylamine (8.7 g, 1 equiv, 61.72 mmol) contained p-toluenesulfonic acid (1.17 g, 0.1 equiv, 6.17 mmol) and tetrabutylammonium bromide (1.5 g, 0.1 equiv, 6.17 mmol). A solution was prepared by adding xylene (150 mL) and stirring.

Then, the prepared solution was heated at 130 to 135 °C for 48 hours. after the reaction is complete. The solution was cooled to room temperature and the pH of the solution was adjusted to 6-7 with aqueous ammonia.

Thereafter, organic compounds were extracted from the solution with ethyl acetate, dried over sodium sulfate, and dried under reduced pressure. The light brown liquid obtained was concentrated to give 1-(2,3-dichlorophenyl)piperazine (12.5 g, 88%) without further purification.

Example 1-2: Preparation of Compound 1

In an anhydrous DCM (10 mL) solution containing 1 equivalent of 1-(2,3-dichlorophenyl)piperazine, 2-isothiocyanatopropane ( ) and 3 equivalents of TEA were added and the mixture was stirred at room temperature for 20 minutes.

After the reaction was complete, water was added and the reaction mixture was extracted with EtOAc (3 x 20 mL). The extracted organic phases were combined, washed with brine, dried over anhydrous MgSO ₄ , filtered and concentrated. The concentrated organic phase was purified by silica-gel flash column chromatography (EA: Hexane = 1:5 - ethyl acetate 40%) to obtain Compound 1.

[Compound 1]

4-(2,3-dichlorophenyl)-N-isopropylpiperazine-1-carbothioamide

The physical properties of Compound 1 prepared in Example 1 are as follows.

Yield: 85%

HPLC: 97%

RT: 19.99 minutes

¹ H NMR (600 MHz, DMSO d ₆ ): δ=7.19-7.13 (m, 2H), 6.91 (dd, J = 7.9, 1.6 Hz, 1H), 5.34 (d, J = 7.3 Hz, 1H), 4.67 (dq, J = 13.1, 6.6 Hz, 1H), 3.96-3 .95 (m, 4H), 3.07-3:05 (m, 4H), 1.25 (d, J = 6.5 Hz, 6H)

¹³ C NMR (150 MHz, DMSO d ₆ ): δ=181.5, 150.4, 134.3, 127.7, 127.7, 125.3, 118.8, 50.9, 47.8, 47.5, 22.9 ppm

IR: 3925, 3752, 3679, 3152 (NH str), 2964, 2883, 2654, 2325, 2008, 1523, 1450, 1336, 1216, 1131, 949 (C=S str), 467, 702, 658 cm ^-1

LRMS (ESI) m/z: 332.2 (M + H) ⁺

Examples 2 to 6

An arylpiperazine derivative compound was synthesized in the same manner as in Example 1, except that the compound shown in Table 1 was used instead of 2-isothiocyanatopropane as the compound represented by Formula (a) in Example 1-2.

Example 2: Compound 2, 4-(2,3-dichlorophenyl)-N-(4-nitrophenyl)piperazine-1-carbothioamide

Example 3: Compound 3, 4-(2,3-dichlorophenyl)-N-(4-fluorophenyl)piperazine-1-carbothioamide

Example 4: Compound 4, N-(4-chlorophenyl)-4-(2,3-dichlorophenyl)piperazine-1-carbothioamide

Example 5: Compound 5, N-(4-bromophenyl)-4-(2,3-dichlorophenyl)piperazine-1-carbothioamide

Example 6: Compound 6, N-(3-bromophenyl)-4-(2,3-dichlorophenyl)piperazine-1-carbothioamide

number Compound represented by formula a Example 2
1-isothiocyanato-4-nitrobenzene Example 3
1-fluoro-4-isothiocyanatobenzene Example 4
1-chloro-4-isothiocyanatobenzene Example 5
1-bromo-4-isothiocyanatobenzene Example 6
1-bromo-3-isothiocyanatobenzene

number Properties Example 2 Yield: 87%
HPLC: 96%
Rt: 19.454 min
¹ H NMR (400 MHz, DMSO d ₆ ): δ = 9.95 (s, 1H), 8.18 (d, J = 9.1 Hz, 2H), 7.62 (d, J = 9.1 Hz, 2H), 7.34 (dd, J = 8.2, 5.8 Hz, 2H), 7.25-7.16 (m, 1H), 4.09 (s, 4H), 3.11–3.09 (m, 4H).
¹³ C NMR (100 MHz, DMSO d ₆ ): δ = 181.0, 150.4, 147.7, 142.1, 132.6, 128.6, 126.2, 124.9, 124.0, 122.4, 120.0, 50.6, 48.7 ppm
IR: 3856, 3814, 3706, 3612, 3388, 3277 (NH str), 3112, 3004, 2712, 2426, 2259, 2117, 1510, 1432, 1338, 1295, 1226, 1165, 1018 (C= S str), 954, 715, 675 cm ^-1
LRMS (ESI) m/z: 411.5 (M + H) ⁺ Example 3 Yield: 89%
HPLC: 94%
Rt: 18.246 min
¹ H NMR (500 MHz, DMSO d ₆ ): δ = 7.21 (d, J = 7.1 Hz, 1H), 7.16 (dd, J = 12.9, 6.2 Hz, 3H), 7.12 (s, 1H), 7.05 (t, J = 8.5 Hz, 2H), 6.93 (d, J = 7.9 Hz, 1H), 4.03 (s, 4H), 3.12-3.10 (m, 4H)
¹³ C NMR (101 MHz, CDCl ₃ + DMSO d ₆ ): δ = 181.1, 159.6, 157.2, 149.1, 135.7, 132.1, 126.6 - 126.1 (m, 1C), 125.7, 123.5, 117.8, 113.5, 1 13.3, 49.5, 47.0 ppm
IR: 3899, 3595, 3441, 3304 (NH str), 3213, 3001, 2898, 2507, 2373, 2222, 2019, 1617, 1524, 1426, 1326, 1221, 1158, 1023, 956 (C=S str), 830, 778, 721 cm ^-1
LRMS (ESI) m/z: 384.04 (M + H) ⁺ Example 4 Yield: 85%
HPLC: 91%
Rt: 20.247 min
¹ H NMR (500 MHz, CDCl ₃ ) δ = 7.33 (d, J = 8.3 Hz, 2H), 7.23-7.10 (m, 5H), 6.94 (d, J = 7.5 Hz, 1H), 4.03 (s, 4H), 3.12 (s, 4H)
13C NMR (100 MHz, DMSO d6): δ = 181.6, 150.4, 140.0, 132.6, 128.5, 128.2, 127.8, 126.6, 126.1, 124.8, 119.9, 50.6, 48.2 ppm
IR: 3948, 3838, 3758, 3665, 3576, 3096 (NH str), 2892, 2717, 1527, 1421, 1319, 1221, 1021, 955 (C=S str), 825, 779, 717 cm ^-1
LRMS (ESI) m/z: 400.06 (M + H) ⁺ Example 5 Yield: 85%
HPLC: 49%
RT: 2.764 min
¹ H NMR (400 MHz, DMSO d ₆ ): δ = 9.48 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.37 - 7.32 (m, 2H), 7.29 (d, J = 8.7 Hz, 2H), 7.19 (dd, J = 5.8, 3.7 Hz, 1H) , 4.06 (s, 4H), 3.08–3.06 (m, 4H).
¹³ C NMR (101 MHz, DMSO d ₆ ): δ = 181.5, 150.5, 140.4, 132.6, 130.8, 128.5, 127.0, 126.1, 124.8, 120.0, 116.3, 50.6, 48.2 ppm
IR: 3798, 3763, 3644, 3349 (NH str), 2823, 2477, 2091, 1981, 1580, 1527, 1420, 1318, 1221, 1018 (C=S str), 955, 821, 718 cm ^-1
LRMS (ESI) m/z: 443.9 (M + H) ⁺ Example 6 Yield: 88%
HPLC: 87%
RT: 20.850 min
^1H NMR (500 MHz, DMSO) δ = 9.49 (s, 1H), 7.49-7.48 (m, 2H), 7.47-7.34 (m, 2H), 7.30-7.28 (m, 2H), 7.19 (dd, J = 6.0, 3.6 Hz, 1H), 4.07-4.05 ( m, 4H), 3.8-3.06 (m, 4H)
¹³ C NMR (101 MHz, CDCl ₃ ) δ = 180.6, 148.9, 141.1, 131.9, 128.0, 126.4, 126.2, 125.5, 123.4, 122.3, 119.5, 117.7, 49.3, 47.1 ppm.
IR: 3943, 3849, 3698, 3613, 3378, 3323, 3174 (NH str), 2844, 2739, 2357, 2268, 1581, 1513, 1437, 1315, 1227, 958 (C=S str), 779, 725 , 647 cm ^-1
LRMS (ESI) m/z: 443.9 (M + H) ⁺

Example 7: Preparation of Compound 1 (Scheme 2)

Example 7-1: Preparation of 1-(2,3-dichlorophenyl)piperazine

1-(2,3-dichlorophenyl)piperazine was prepared in the same manner as in Example 1-1.

Example 7-2: Preparation of Compound 7

4-nitroaniline (which is a compound represented by formula b) ) To a solution of anhydrous DCM (10 mL) containing 1 equivalent and 3 equivalents of TEA, 1.5 equivalents of triphosgene was added at 0 ° C, and after 10 minutes, 1- (2,3-dichlorophenyl) piperazine diluted with DCM was added dropwise at the same temperature and stirred at room temperature for 1 hour.

After the reaction was complete, water was added and the reaction mixture was extracted with EtOAc (3 x 20 mL). The extracted organic phases were combined, washed with brine, dried over anhydrous MgSO ₄ , filtered and concentrated. The concentrated organic phase was purified by silica-gel flash column chromatography (EA: Hexane = 1:5 - ethyl acetate 40%) to obtain compound 7.

[Compound 7]

4-(2,3-dichlorophenyl)-N-(4-nitrophenyl)piperazine-1-carboxamide

The physical properties of Compound 7 prepared in Example 7 are as follows.

Yield: 95%

HPLC: 92%

RT: 19.99 minutes

¹ H NMR (600 MHz, DMSO d ₆ ) δ = 9.45 (s, 1H), 8.16 (d, J = 9.2 Hz, 2H), 7.78 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 4.3 Hz, 2H), 7.20 - 7.17 (m, 1H), 3.68 (s, 4H), 3.03 (d, J = 4.3 Hz, 4H);

¹³ C NMR (150 MHz, DMSO d ₆ ) δ = 154.1, 150.8, 147.5, 140.8, 132.6, 128.5, 126.2, 124.7, 124.6, 112.0, 118.3, 50.9, 44.1, 41.2 ppm

IR: 3833, 3599, 3361, 3176 (NH str), 2926, 2500, 2289, 2144, 1734 (C=O amide), 1557, 1494, 1299, 1246, 1178, 1109, 850, 750, 654 cm ^-1

LRMS (ESI) m/z: 395.6 (M + H) ⁺

Examples 8 to 14

An arylpiperazine derivative compound was synthesized in the same manner as in Example 7, except that the compound shown in Table 3 was used instead of 4-nitroaniline as the compound represented by Formula b in Example 7-2.

Example 8: Compound 8, 4-(2,3-dichlorophenyl)-N-(4-fluorophenyl)piperazine-1-carboxamide

Example 9: Compound 9, 4-(2,3-dichlorophenyl)-N-(4-(trifluoromethyl)phenyl)piperazine-1 carboxamide

Example 10: Compound 10, 4-(2,3-dichlorophenyl)-N-(4-(trifluoromethoxy)phenyl)piperazine-1-carboxamide

Example 11: Compound 11, 4-(2,3-dichlorophenyl)-N-(4-methoxyphenyl)piperazine-1-carboxamide

Example 12: Compound 12, 4-(2,3-dichlorophenyl)-N-(3-(trifluoromethyl)phenyl)piperazine-1-carboxamide

Example 13: Compound 13, 4-(2,3-dichlorophenyl)-N-(3-methoxyphenyl)piperazine-1-carboxamide

Example 14: Compound 14, 4-(2,3-dichlorophenyl)-N-(3-methoxyphenyl)piperazine-1-carboxamide

number Compound represented by formula b Example 8
4-fluoroaniline Example 9
4-(trifluoromethyl)aniline Example 10
4-(trifluoromethoxy)aniline Example 11
4-methoxyaniline Example 12
3-(trifluoromethyl)aniline Example 13
3-(trifluoromethoxy)aniline Example 14
3-methoxyaniline

number Properties Example 8 Yield: 89 % HPLC: 97 %
Rt: 17.452 min
¹ H NMR (600 MHz, CDCl ₃ ) δ = 7.31-7.29 (m, 2H), 7.21 -7.15 (m, 1H), 7.01 - 6.96 (m, 1H), 7.01 - 6.97 (m, 2H), 6.94 (dd, J = 7.9, 1.6 Hz, 1H), 6.38 (s, 1H), 3.67–3.65 (m, 4H), 3.08–3.06 (m, 4H).
¹³ C NMR (150 MHz, CDCl ₃ ): δ = 159.9, 158.3, 155.2, 150.7, 134.8 (d, J = 3.2 Hz), 134.3, 127.8, 127.7, 125.3, 122.6 (d, J = 8.0 Hz), 118.8 ( d, J = 3.4 Hz), 115.68 (s), 115.5, 51.2, 44.42. ppm.
IR: 3821, 3769, 3617, 3489, 3179 (NH str), 2915, 2825, 2494, 2326, 2097, 1982, 1622 (C=O amide), 1518, 1424, 1219, 1140, 944, 818, 77 8, 663 cm ^-1
LRMS (ESI) m/z: 384.5 (M + H) ⁺ Example 9 Yield: 85%
HPLC: 99%
RT: 20.003 min
¹ H NMR (600 MHz, CDCl ₃ ): δ = 7.53 (dd, J = 23.8, 8.7 Hz, 4H), 7.34 -7.10 (m, 2H), 6.96 (dd, J = 7.7, 1.8 Hz, 1H), 6.54 (s, 1H), 3.73-3.70 (m, 4H) ), 3.12–3.09 (m, 4H).
¹³ C NMR (150 MHz, CDCl ₃ ) δ = 154.5, 150.8, 144.3, 132.6, 128.5, 126.0 (d, J = 30.0 Hz), 125.9, 125.68 (t, J = 16.6 Hz), 124.7, 121.7, 121. 4, 119.9, 118.87, 50.88, 44.0. ppm
IR: 3885, 3848, 3577, 3503, 3297 (NH str), 3029, 2835, 2342, 2027, 1627 (C=O amide), 1527, 1423, 1318, 1249, 1150, 1110, 1061, 941, 8 23, 660 cm ^-1
LRMS (ESI) m/z: 418.3 (M + H) ⁺ Example 10 Yield: 91%
HPLC: 88%
Rt: 19.023 min
¹ H NMR (600 MHz, CDCl ₃ ): δ = 7.32-7.28 (m, 4H), 7.14 (dtt, J = 13.3, 8.9, 4.3 Hz, 2H), 3.78 (s, 2H), 3.66 (s, 2H), 3.33-3.31 (m, 2H), 3.0 8-3.07 (m, 2H).
¹³ C NMR (150 MHz, CDCl ₃ ): δ = 150.8 (d, J = 13.6 Hz), 147.6, 133.2 (d, J = 2.7 Hz), 129.2 - 129.0 (m), 126.8 (d, J = 7.9 Hz), 125.8 - 125.4 (m), 12 4.7, 120.6, 120.3, 51.6 (d, J = 33.9 Hz), 51.4 (d, J = 40.0 Hz), 51.0, 49.1, 49.0, 48.3, 46.8. ppm.
IR: 3867, 3775, 3730, 3641, 3537, 3447, 3336 (NH str), 3209, 2934, 2626, 2355, 2262, 1626 (C=O amide), 1540, 1505, 1208, 1148, 1051, 1003, 845, 786, 749, 644 cm ^-1
LRMS (ESI) m/z: 434.5 (M + H) ⁺ Example 11 Yield: 89%
HPLC: 91%
RT: 16.804 min
¹ H NMR (400 MHz, DMSO d ₆ ): δ = 8.45 (s, 1H), 7.34 (dd, J = 12.8, 6.7 Hz, 4H), 7.19-7.17 (m, 1H), 6.84 (t, J = 8.8 Hz, 2H), 3.70 (s, 3H), 3.60 (s , 4H), 3.17–2.85 (m, 4H).
¹³ C NMR (101 MHz, DMSO d ₆ ) δ = 155.2, 154.5, 150.9, 133.4, 132.6, 128.5, 126.2, 124.7, 121.6, 119.9, 119.9, 113.9, 113.5, 55.1, 5 0.9, 43.9 ppm
IR: 3956, 3852, 3759, 3598, 3390, 3274 (NH str), 3168, 2855, 2500, 2317, 2027, 1616 (C=O amide), 1514, 1422, 1227, 1041, 995, 941, 80 0, 656 cm ^-1
LRMS (ESI) m/z: 380.1 (M + H) ⁺ Example 12 Yield: 92%
HPLC: 81%
RT: 21.075 minutes
^1H NMR (600 MHz, CDCl ₃ ): δ = 7.21 (dd, J = 8.1, 1.5 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.92 (dd, J = 7.9, 1.5 Hz, 1H), 3.90-3.86 (m, 4H), 3.07-3.0 6 (m, 4H).
¹³ C NMR (150 MHz, CDCl ₃ ): δ = 150.2, 148.6, 134.4, 127.9, 127.81-127.64 (m), 125.74-125.16 (m), 118.9, 51.3, 50.8, 49.0, 46.5 ppm
IR: 3856, 3779, 3735, 3643, 3417, 3290 (NH str), 3213, 3007, 2437, 2146, 1711 (C=O amide), 1448, 1405, 1202, 1139, 1012, 940, 853, 78 8, 655 cm ^-1
LRMS (ESI) m/z: 418.0 (M + H) ⁺ Example 13 Yield: 91%
HPLC: 76%
Rt: 19.214 min
^1H NMR (600 MHz, CDCl ₃ ): δ = 7.21 (dd, J = 8.1, 1.5 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.92 (dd, J = 7.9, 1.6 Hz, 1H), 3.91-3.81 (m, 4H), 3.07-3.0 6 (m, 4H).
¹³ C NMR (151 MHz, CDCl ₃ ): δ = 150.2, 148.6, 134.4, 128.0, 127.7 (q, J = 1.8 Hz), 126.0-125.4 (m), 119.3-118.7 (m), 51.1 (d, J = 73.4 Hz), 49 .0, 47.0. ppm
IR: 3780, 3461, 3297 (NH str), 3019, 2914, 2389, 1639 (C=O amide), 1598, 1556, 1437, 1253, 1206, 1149, 956, 875, 782, 699, 628 cm ^-1
LRMS (ESI) m/z: 434.5 (M + H) ⁺ Example 14 Yield: 91%
HPLC: 99%
Rt: 13.113 min
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.22-7.14 (m, 4H), 6.95 (dd, J = 7.6, 1.8 Hz, 1H), 6.84 (dd, J = 8.0, 1.4 Hz, 1H), 6.61 (dd, J = 8.1, 2.2 Hz, 1H), 6.3 7 (s, 1H), 3.81 (s, 3H), 3.70–3.67 (m, 4H), 3.10–3.08 (m, 4H).
¹³ C NMR (101 MHz, CDCl ₃ ) δ = 160.2, 154.9, 150.6, 140.2, 134.2, 129.5, 127.7, 127.5, 125.2, 118.7, 112.0, 109.2, 105.6, 55.3, 51 .1, 44.4. ppm
IR: 3833, 3599, 3361 (NH str), 3176, 2926, 2500, 2289, 2144, 1734, 1557 (C=O amide), 1494, 1299, 1246, 1178, 1109, 850, 750, 654 cm ^-1
LRMS (ESI) m/z: 403.6 (M + H) ⁺

Example 15: Preparation of Compound 15 (Scheme 3)

Example 15-1: Preparation of 1-(2,3-dichlorophenyl)piperazine

1-(2,3-dichlorophenyl)piperazine was prepared in the same manner as in Example 1-1.

Example 15-2: Preparation of compound 15

Tridecanoic acid, a compound represented by formula c ( ) HATU and 3 equivalents of DIPEA were added to a DMF (3 mL) solution containing 1 equivalent, and after stirring at room temperature for 20 minutes, 1.2 equivalent of 1-(2,3-dichlorophenyl)piperazine was added. The solution was then stirred overnight at room temperature.

After the reaction was complete, water was added and the reaction mixture was extracted with EtOAc (3 x 20 mL). The extracted organic phases were combined, washed with brine, dried over anhydrous MgSO ₄ , filtered and concentrated. The concentrated organic phase was purified by silica-gel flash column chromatography (EA: Hexane = 1:5 - ethyl acetate 40%) to obtain compound 15.

[Compound 15]

1-(4-(2,3-dichlorophenyl)piperazin-1-yl)tridecan-1-one

The physical properties of Compound 15 prepared in Example 15 are as follows. Yield: 95%

HPLC: 92%

RT: 3.760 min

¹ H NMR (400 MHz, DMSO d ₆ ) δ = 7.33-7.31 (m, 2H), 7.14 (dd, J = 5.8, 3.7 Hz, 1H), 3.60 (s, 4H), 3.02-2.88 (m, 4H), 2.33 (t, J = 7.4 Hz, 2H), 1.2 4 (s, 20 H), 0.85 (t, J = 6.7 Hz, 3 H).

¹³ C NMR (101 MHz, DMSO) δ = 170.8, 150.8, 132.6, 128.5, 126.2, 124.7, 119.8, 51.3, 50.9, 45.1, 41.1, 32.2, 31.2, 29.0, 29.0, 29.0, 28.9, 28.8, 28.7, 28.7, 24.8, 22.0, 13.9. ppm

IR: 3411, 3065, 2921, 2852, 1647 (C=O str), 1576, 1449, 1428, 1232, 1145, 1034, 956, 781, 718 cm ^-1

LRMS (ESI) m/z: 427.3 (M + H) ⁺

Examples 16 to 25

An arylpiperazine derivative compound was synthesized in the same manner as in Example 15, except that the compound shown in Table 3 was used instead of tridecanoic acid as the compound represented by Formula b in Example 7-2.

Example 16: Compound 16, (4-(2,3-dichlorophenyl)piperazin-1-yl)(pyrazin-2-yl)methanone

Example 17: Compound 17, (4-(2,3-dichlorophenyl)piperazin-1-yl)(3-(trifluoromethyl)phenyl)methanone

Example 18: Compound 18, (4-(2,3-dichlorophenyl)piperazin-1-yl)(4-(trifluoromethyl)phenyl)methanone

Example 19: Compound 19, (4-(2,3-dichlorophenyl)piperazin-1-yl)(4-methoxyphenyl)methanone

Example 20: Compound 20, (4-(2,3-dichlorophenyl)piperazin-1-yl)(3,5-dimethoxyphenyl)methanone

Example 21: Compound 21, (4-(2,3-dichlorophenyl)piperazin-1-yl)(2-methyl-4-nitrophenyl)methanone

Example 22: Compound 22, (2,4-dichlorophenyl)(4-(2,3-dichlorophenyl)piperazin-1-yl)methanone

Example 23: Compound 23, 1-(4-(2,3-dichlorophenyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)ethenone

Example 24: Compound 24, 1-(4-(2,3-dichlorophenyl)piperazin-1-yl)-2-(2,4,5-trifluorophenyl)ethanone

Example 25: Compound 25, 1-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-(2-(trifluoromethyl) phenyl) propan-1-one

number Compound represented by formula c Example 16
pyrazine-2-carboxylic acid Example 17
3-(trifluoromethyl)benzoic acid Example 18
4-(trifluoromethyl)benzoic acid Example 19
4-methoxybenzoic acid Example 20
3,5-dimethoxybenzoic acid Example 21
2-methyl-4-nitrobenzoic acid Example 22
2,4-dichlorobenzoic acid Example 23
2-(3,4,5-trimethoxyphenyl)acetic acid Example 24
2-(2,4,5-trifluorophenyl)acetic acid Example 25
3-(2-(trifluoromethyl)phenyl)propanoic acid

number Properties Example 16 Yield: 90%
HPLC: 98%
Rt: 14.153 min
¹ H NMR (400 MHz, DMSO d ₆ ) δ = 8.89 (s, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.70 (s, 1H), 7.32 (dd, J = 8.1, 5.9 Hz, 2H), 7.17 (dd, J = 5.9, 3.6 Hz, 1H), 3.84 (s, 2H), 3.62–3.60 (m, 2H), 3.10–3.08 (m, 2H), 3.00 (d, J = 4.3 Hz, 2H).
¹³ C NMR (101 MHz, DMSO d ₆ ) δ = 164.8, 150.6, 149.2, 145.5, 144.5, 143.2, 132.6, 128.5, 126.2, 124.9, 120.0, 51.2, 50.8, 46.8, 41. 8. ppm
IR: 3935, 3779, 3694, 3606, 3384, 3169, 3061, 3009, 2828, 2685, 2545, 1625 (C=O str), 1435, 1235, 1140, 1010, 946, 859, 776, 71 2 cm ^-1
LRMS (ESI) m/z: 337.5 (M + H) ⁺ Example 17 Yield: 90%
HPLC: 99%
RT: 20.212 min
^1H NMR (600 MHz, CDCl ₃ ) δ = 7.71-7.68 (m, 2H), 7.61 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.19 (dd, J = 8.0, 1.6 Hz, 1H), 7.15 (t, J = 8.0 Hz) , 1H), 6.93 (dd, J = 7.9, 1.6 Hz, 1H), 3.78 (d, J = 234.6 Hz, 4H), 3.04 (d, J = 87.4 Hz, 4H).
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 169.0, 150.5, 136.5, 134.3, 131.2 (q, J = 32.9 Hz), 130.9, 130.5, 130.4, 129.2, 127.9, 127.7, 126.7 (dd, J = 7.9, 3.9 Hz), 125.4, 124.2 (dd, J = 8.2, 4.1 Hz), 122.8, 51.9, 51.2, 48.2, 42.5 ppm
IR: 3884, 3831, 3776, 3734, 3657, 3620, 3522, 3381, 3223, 2696, 2588, 2159, 1626 (C=O str), 1417, 1325, 1259, 1162, 1124, 1068, 1017, 943, 784, 706, 660 cm ^-1
LRMS (ESI) m/z: 403.6 (M + H) ⁺ Example 18 Yield: 87%
HPLC: 97%
RT: 19.933 min
¹ H NMR (400 MHz, DMSO d ₆ ) δ = 7.84 (d, J = 6.8 Hz, 2H), 7.67 (d, J = 7.1 Hz, 2H), 7.34 (s, 2H), 7.16 (s, 1H), 3.82 (s, 2H), 3.45 (s, 2H), 3.07- 2 (m, 4H).
¹³ C NMR (101 MHz, DMSO d ₆ ) δ = 167.7, 150.6, 139.9, 132.6, 129.7 (d, J =32.2 Hz), 128.5, 127.8, 126.2, 125.5, 125.5 (d, J = 3.6 Hz), 124.8, 122.5, 119.9, 51.0, 50.7, 47.2, 41.7. ppm
IR: 3875, 3832, 3650, 3607, 3478, 3363, 3260, 3156, 3022, 2851, 2711, 2534, 2260, 1630 (C=O str), 1445, 1323, 1277, 1165, 1118, 1062, 1013, 942, 848, 779, 714 cm ^-1
LRMS (ESI) m/z: 403.63 (M + H) ⁺ Example 19 Yield: 95%
HPLC: 93%
Rt: 17.203 min
¹ H NMR (400 MHz, DMSO-d ₆ ) δ = 7.42 (d, J = 8.6 Hz, 2H), 7.32 (dd, J = 6.4, 2.6 Hz, 2H), 7.20-7.11 (m, 1H), 6.99 (d, J = 8.7 Hz, 2H), 3.80 (s, 3H), 3.66 (s, 4H), 2.99 (s, 4H).
¹³ C NMR (101 MHz, DMSO-d ₆ ) δ = 169.0, 160.2, 150.7, 132.6, 131.3, 129.1, 128.4, 127.6, 126.2, 124.8, 119.9, 113.6, 56.5, 55.7, 5 5.2, 50.8, 36.1. ppm
IR: 3868, 3756, 3680, 3581, 3377, 2930, 2857, 1773 (C=O str), 1606, 1424, 1242, 1163, 1096, 1015, 945, 835, 769, 707, 658 cm ^-1
LRMS (ESI) m/z: 365.5 (M + H) ⁺ Example 20 Yield: 85%
HPLC: 99%
RT: 18.587 min
¹ H NMR (600 MHz, CDCl ₃ ) δ = 7.22-7.07 (m; 2H), 6.91 (dd, J = 7.9, 1.6 Hz, 1H), 6.53 (d, J = 2.3 Hz, 2H), 6.48 (t, J = 2.2 Hz, 1H), 3.93 (s, 2H), 3 .79 (s, 6H), 3.60 (s, 2H), 3.01 (d, J = 82.6 Hz, 4H).
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 170.2, 161.0, 150.6, 137.6, 134.3, 127.8, 127.7, 125.4, 125.3, 125.3, 118.9, 105.0, 101.7, 55.6, 52 .0, 51.4, 47.9, 42.3. ppm
IR: 3853, 3798, 3730, 3596, 3382, 3305, 2972, 2834, 2732, 2268, 3160, 2002, 1627 (C=O str), 1590, 1452, 1420, 1202, 1151, 1060, 1021, 949, 833, 782, 656 cm ^-1
LRMS (ESI) m/z : 395.7 (M + H) ⁺ Example 21 Yield: 95%
HPLC: 98%
RT: 18.762 minutes
¹ H NMR (600 MHz, CDCl ₃ ) δ = 8.13-8.07 (m, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.20 (dd, J = 8.1, 1.5 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.92 (dd, J = 7.9, 1.5 Hz, 1H), 4.01 (d, J = 25.6 Hz, 2H), 3.38 (dd, J = 9.0, 4.0 Hz, 2H), 3.12 (t, J = 4.8 Hz, 2H), 2.93 (dd, J = 10.0, 4.4 Hz, 2H), 2.45 (s, 3H).
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 170.2, 161.0, 150.6, 137.6, 134.3, 127.8, 127.7, 125.3, 125.3, 125.3, 118.9, 105.0, 101.7, 55.6, 52 .0, 51.3, 47.9, 42.3. ppm
IR: 3913, 3883, 3707, 3646, 3567, 3447, 3349, 3063, 2873, 2831, 2636, 2349, 2213, 1627 (C=O str), 1442, 1347, 1241, 1148, 1017, 960, 785, 737 cm ^-1
LRMS (ESI) m/z: 394.6 (M + H) ⁺ Example 22 Yield: 95%
HPLC: 99%
Rt: 22.020 min
^1H NMR (600 MHz, CDCl ₃ ) δ = 7.43 (d, J = 1.9 Hz, 1H), 7.31 (dd, J = 8.2, 1.9 Hz, 1H), 7.27 - 7.22 (m, 1H), 7.20 - 7.06 (m, 2H), 6.91 (dd, J = 7.9, 1.6 Hz;
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 166.2, 150.5, 135.8, 134.3, 134.2, 131.4, 129.7, 128.9, 127.8, 127.8, 127.7, 125.4, 118.9, 51.7, 51 .1, 47.1, 42.0 ppm.
IR: 3845, 3002, 3706, 3625, 3437, 3350, 3061, 2919, 2856, 2055, 1639 (C=O str), 1432, 1236, 1140, 1010, 941, 780, 710 cm ^-1
LRMS (ESI) m/z: 403.7 (M + H) ⁺ Example 23 Yield: 92%
HPLC: 97%
Rt: 16.713 min
¹ H NMR (400 MHz, CDCl ₃ ) δ = 7.21-7.11 (m, 2H), 6.86 (dd, J = 7.8, 1.5 Hz, 1H), 6.48 (s, 2H), 3.85 (d, J = 7.5 Hz, 11H), 3.72 (s, 2H), 3.66 -3.6 3 (m, 2H), 3.03-2.98 (m, 2H), 2.89 - 2.87 (m, 2H).
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 168.9, 152.7, 150.7, 136.0, 132.6, 131.3, 128.5, 126.2, 124.8, 119.8, 106.2, 60.0, 55.8, 51.2, 50.8 , 45.6, 41.4 ppm
IR: 3910, 3871, 3701, 3629, 3523, 3407, 3133, 2930, 2845, 2336, 2227, 2120, 1641 (C = O STR), 1440, 1312, 1105, 1035, 943, 777 cm ^-1
LRMS (ESI) m/z: 439.1 (M + H) ⁺ Example 24 Yield: 90%
HPLC: 97%
Rt: 19.101 min
¹ H NMR (600 MHz, DMSO-d ₆ ) δ = 7.16 (dt, J = 15.6, 7.8 Hz, 3H), 6.94–6.89 (m, 2H), 3.81 (s, 2H), 3.68 (s, 4H), 3.00 (s, 4H).
¹³ C NMR (101 MHz, CDCl ₃ ) δ = 167.8, 156.8 (d, J = 9.6 Hz), 154.3 (d, J = 9.2 Hz), 150.4, 148.0 (t, J = 13.4 Hz), 145.7 (d, J = 9.2 Hz), 134.2, 127.7 (d, J = 15.4 Hz), 125.3, 119.4–118.1 (m), 105.6, 105.4 (d, J = 7.5 Hz), 105.2, 51.6, 51.0, 46.2, 42.3, 32.6 .ppm
IR: 3875, 3827, 3730, 3636, 3501, 3407, 3332, 3106, 3052, 2725, 2595, 2301, 2062, 1627 (C=O str), 1513, 1434, 1226, 1153, 1031, 952, 900, 851, 773, 712 cm ^-1
LRMS (ESI) m/z: 403.6 (M + H) ⁺ Example 25 Yield: 87%
HPLC: 99%
RT: 21.874 min
¹ H NMR (600 MHz, CDCl ₃ ) δ = 7.62 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.22-7.10 (m, 2H), 6.88 (dd, J = 7.9, 1.4 Hz, 1H), 3.81 (s, 2H), 3.58-3.52 (m, 2H), 3.21-3.14 (m, 2H), 2.98 (t, J = 4.8 Hz, 2H), 2.93-2.86 (m, 2H), 2.69-2.61 (m, 2H).
¹³ C NMR (151 MHz, CDCl ₃ ) δ = 170.5, 150.7, 140.0, 134.3, 132.1, 131.6, 128.6 (dd, J = 59.5, 29.7 Hz), 127.8, 127.6, 126.5, 126.2 (dd, J = 12.0, 6.1 Hz), 125.6, 125.3, 123.8, 118.8, 51.6, 51.2, 45.8, 42.0, 35.2, 28.6 (d, J = 2.0 Hz) ppm.
IR: 3884, 3817, 3722, 3648, 3523, 3468, 3320, 3103, 3025, 2879, 2783, 2404, 1739 (C=O str), 1639, 1583, 1805, 1452, 1417, 1337, 1237, 1206, 1119, 1010, 957, 888, 654 cm ^-1
LRMS (ESI) m/z: 431.7 (M + H) ⁺

Experiment method

Cell culture and reagents

Human single cell lines THP-1 WT and STING KO cell lines were purchased from Invivogen. Cells were cultured in 1XRPMI 1640 containing 10% fetal bovine serum (FBS, Hyclone), 2.05mM L-glutamine (hyclone), and 1% penicillin (corning) at 37°C, 5% CO ₂ , in a humidified incubator.

Luciferase reporter assay

To confirm the immune response, THP-1 cells were placed in a 384-well plate (Grenier), treated with individual compounds and cGAMP (1 μg/ml), and ISRE activity was analyzed 24 hours later. QUANTI-Luc assay (Invivogen) was performed using the cell culture supernatant to confirm the luciferase reporter gene signal. Fluorescent signals were measured using a TECAN micro plate reader SPARK, and the results were normalized using DMSO control.

Cell viability assay

Cells were analyzed using CellTiter Clo (CTG, Promega). Absorbance was measured using a TECAN micro plate reader SPARK. Results were normalized using DMSO control.

ELISA assay

Cytokine levels were confirmed by ELISA analysis. THP-1 cells were placed in a 96-well U bottom plate and tested after 24 hours of treatment with the compound and cGAMP (1 μg/ml). After culturing, the cell supernatant was recovered and human IFNβ and IP-10 were confirmed by ELISA analysis.

Gene expression analysis by real-time quantitative PCR

For gene expression confirmation, THP-1 cells were placed in a 24-well plate (SPL) and treated with the compound of the present invention (GxF) and cGAMP (1 μg/ml) for 6 hours. After incubation, the cells were cultured using NucleoSpin RNA Plus (MN) for RNA isolation. The mRNA expression levels of IFNB, CXCL10, IFIT3, ISG15, IRF7 and OAS1 were measured by real-time qPCR (CFX96 Real-Time PCR Detection System, Bio-Rad) using IQ SYBR Green Supermix (Bio-Rad). GAPDH was used as a housekeeping gene to normalize cytokine mRNA levels.

Experimental results and analysis

Discovery results of N-arylpiperazine derivatives

When 25 N-arylpiperazine derivatives were treated, the immune response in THP-1 cells through ISRE reporter assay was confirmed through ISRE reporter gene assay, and cytotoxicity was additionally confirmed (Table 7). As a result, indolizine derivative compound 23 with the highest efficacy was used below.

compound RU ^a Cell viability (%) ^b compound 1 1.2 ± 0.4 84 ± 15 compound 2 1.1 ± 0.3 97±2 compound 3 1.3 ± 0.3 81±7 compound 4 1.2 ± 0.2 99±4 compound 5 0.4 ± 0.2 46 ± 54 compound 6 1.5 ± 0.2 85±9 compound 7 1.0 ± 0.3 90±10 compound 8 2.1 ± 1.7 95 ± 11 compound 9 3.9±1.4 61 ± 16 compound 10 4.2±1.3 96 ± 11 compound 11 3.3±2.0 98 ± 16 compound 12 3.3±1.1 76 ± 17 compound 13 3.0 ± 1.3 70 ± 26 compound 14 2.7±1.0 87 ± 15 compound 15 1.3 ± 0.7 89 ± 16 compound 16 2.5 ± 0.8 89±8 compound 17 2.8 ± 0.3 105±20 compound 18 1.8 ± 0.1 90 ± 15 compound 19 3.7 ± 0.9 81 ± 16 compound 20 1.9±1.1 83 ± 14 compound 21 1.4 ± 0.6 84 ± 16 compound 22 2.3±1.2 86 ± 18 compound 23 5.2 ± 1.5 98±22 compound 24 2.8±1.7 101 ± 15 compound 25 2.5 ± 1.4 96±12 ^a Relative unit for ISRE reporter signal. Normalized response by DMSO as a control. ^b THP-1 cell viability. RU and cell viability were measured by treatment with 40 μM of each compound and are shown as mean ± standard deviation.

Concentration-dependent ISRE reporter assay and cytotoxicity results of the compounds of the present invention

As confirmed in Figure 1, the EC ₅₀ of compound 23 of the present invention is 13.1 μM and E _max can be confirmed to increase by 4.9 times (Figure 1a). In addition, no cytotoxicity was observed even when 40 μM was used (Fig. 1b).

Confirmation of cytokine expression

As confirmed in FIG. 2, as a result of measuring the amount of immunoreactive cytokines secreted by cGAMP such as IFN-β and IP-10 using ELISA as well as ISRE reporter gene assay, it was confirmed that the secretion of immunoreactive cytokines was effectively increased through administration of the compound of the present invention.

Identification of gene expression related to Type I IFN signaling

As confirmed in Figure 3, it was confirmed that the expression of various ISGs, including IFNB, significantly increased according to the treatment with the compound of the present invention.

Through this, the function of the present invention can be confirmed as an IGN derivative efficiently without additional toxicity.

Check for STING dependencies

As confirmed in Figure 4, the immune response inducing activity of the arylpiperazine derivatives was shown only in the WT control cell line and not at all in the STING KO cell line, thereby proving that the immune response inducing activity of the arylpiperazine derivatives acts dependently on STING. As further confirmed in FIG. 5, even in a concentration-dependent experiment for compound 23, the STING KO cell line showed no immune response-inducing activity at all tested concentrations, proving the STING-dependent action.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (7)

A composition for preventing or treating cancer comprising an arylpiperazine derivative compound represented by Formula 1 as an active ingredient,
Wherein the composition stimulates the interferon gene:
[Formula 1]

In Formula 1,
X is sulfur (S) or oxygen (O);
Y is -NHR ₁ or R ₂ ;
R ₁ is isopropyl, halophenyl, nitrophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl or methoxymethyl;
R ₂ is C ₁₂ alkyl, pyrazinyl, trifluoromethylphenyl, methoxyphenyl, nitrophenyl, methylphenyl or halophenyl.
According to claim 1,
A composition for preventing or treating cancer, wherein the arylpiperazine derivative compound represented by Formula 1 is a compound represented by Formula 1-1:
[Formula 1-1]

In the above formula,
R ₆ is hydrogen; C ₁ -C ₈ Alkyl group; A halogen-substituted C ₁ -C ₈ alkyl group; A C ₁ -C ₈ alkoxy group; A halogen-substituted C ₁ -C ₈ alkoxy group; nitro; nitrile; hydroxy group; or halogen;
According to claim 1,
A composition for preventing or treating cancer, wherein the arylpiperazine derivative compound represented by Formula 1 is a compound represented by Formula 1-2:
[Formula 1-2]

In the above formula,
R ₇ is hydrogen; C ₁ -C ₈ Alkyl group; A halogen-substituted C ₁ -C ₈ alkyl group; A C ₁ -C ₈ alkoxy group; A halogen-substituted C ₁ -C ₈ alkoxy group; nitro; nitrile; hydroxy group; or halogen;
According to claim 1,
A composition for preventing or treating cancer, wherein the arylpiperazine derivative compound represented by Formula 1 is a compound represented by Formula 1-3:
[Formula 1-3]

In the above formula,
n is 0 or an integer from 1 to 5;
R ₃ , R ₄ and R ₅ are each independently hydrogen; halogen; nitro; nitrile; C ₁ -C ₈ straight or branched chain alkyl group; C ₁ -C ₈ straight-chain or branched-chain alkyl group substituted with a halogen, hydroxy group or C ₁ -C ₆ alkoxy group; C ₁ -C ₈ straight-chain or branched-chain alkoxy group; or a halogen-substituted C ₁ -C ₈ straight-chain or branched-chain alkoxy group;
According to claim 1,
An arylpiperazine derivative compound represented by Formula 1 is any one of the following compounds 1 to 6: A composition for preventing or treating cancer:
[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Compound 6]
.

According to claim 1,
An arylpiperazine derivative compound represented by Formula 1 is any one of the following compounds 7 to 14, a composition for preventing or treating cancer:
[Compound 7]

[Compound 8]

[Compound 9]

[Compound 10]

[Compound 11]

[Compound 12]

[Compound 13]

[Compound 14]
.

According to claim 1,
An arylpiperazine derivative compound represented by Formula 1 is any one of the following compounds 15 to 25: A composition for preventing or treating cancer:
[Compound 15]

[Compound 16]

[Compound 17]

[Compound 18]

[Compound 19]

[Compound 20]

[Compound 21]

[Compound 22]

[Compound 23]

[Compound 24]

[Compound 25]
.