Type I Interferon (IFN) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus IFNs are widely used as anti-viral agents and treatment for immune disorder or cancer. However, there is a growing demand for novel small-molecule IFN inducer due to tolerance, toxicity, or short duration of action following direct administration of IFNs.
In this study, we assessed arylpiperazine (ARP) as a new core skeleton of IFN inducer. To investigate structure-activity relationship, we designed and synthesized a series of ARP analogues and evaluated the ability to stimulate IFN response in THP-1 human monocyte cells. Compound 5i was identified as a potent type I IFN inducer as it significantly increased cytokine secretion and increased expression of various IFN-stimulating genes which are representative biomarkers of type I IFN pathway. Our results suggested a beneficial therapeutic potential of 5i as an anti-viral agent.
Keyword: interferon inducer, type I Interferon, arylpiperazine, anti-viral agent, innate immunity
We designed and synthesized a series of arylpiperazine derivatives as interferon inducers  We tested different linker system and carbonyl moiety resulted in increase of potency  5i exhibited the activity of interferon inducer with EC 50 of 13.1 μM and no cytotoxicity  5i initiated immune responses by mediating type I IFN signaling
Interferons (IFNs) are essential signaling peptides which act as the first line of surveillant in defense system and modulate innate and adaptive immune activation [1, 2] . In response to virus infection, IFNs are released by host cells, after which they bind to their specific receptors, thereby stimulating JAK/STAT signaling pathway and initiating transcription of various interferon-stimulated genes (ISGs) such as CXCL10, IRF7, IFIT3, and OAS etc [3] [4] [5] [6] [7] . Activation of IFN signaling pathway regulates not only anti-viral response but also induces cellular immunity by stimulating macrophage or monocyte [8] .
Based on the class of binding receptor and signaling cascade, IFNs are categorized into three major group: type I, type II and type III. Type I IFNs including IFNα and IFNβ are considered as therapeutic targets for treatment of hepatitis B and C infections and multiple sclerosis [9, 10] . In addition, IFNs are used in cancer treatments of hematological malignancy such as leukemia and lymphomas [11, 12] .
Despite their therapeutic potential, there are several limitations of IFNs treatments including toleration, dose-related toxicity and side-effects such as dizziness, headache, muscle pain, and depression [13] .
Moreover, IFNs are typically administrated by intramuscular injection for therapeutic use. PEGylated
IFNs with enhancing stability are generally used in clinical treatment to overcome the short duration of effect. However, PEGylated IFNs are not able to be used on patients with hyperbilirubinemia [14, 15] .
For these reasons, continuous efforts have been made to develop small-molecule modulators so as to stimulate IFNs. For example, tilorone is the first synthetic small-molecule drugs used as orally active IFN inducer which showed efficient anti-viral activity against broad spectrum of viruses including ebola virus and middle east respiratory syndrome-related coronavirus (MERS-Cov), especially [16] .
Anti-malarial agent Anti-bacterial agent Therefore, we aimed to develop new small-molecule regulators to stimulate type I IFN effect and identified that particular arylpiperazine (ARP) derivatives exhibited the desired biological activity using phenotypic screening. Remarkably, the ARP core structure was reported to exert anti-malarial, antimicrobial, anti-cancer activity or selective 5-HT 1A antagonistic effect ( Figure 1a ) [17] [18] [19] [20] . In addition, 2,3-di-chloro substituent in ARP pharmacophore plays important role in particular drugs such as cariprazine, aripiprazol or bioactive DRD3 antagonist ( Figure 1a ) [21] [22] [23] . Based on these important characteristics of the ARP skeleton, we designed and synthesized a series of 2,3-dichloro ARP derivatives by further N-modification on piperazine part with different linker moiety such as thiourea, urea, and carbonyl functional group in order to assess structure-activity relationship (SAR; Figure 1b All the synthesized compounds were evaluated using ISRE reporter assay on THP-1 human monocyte cells for monitoring immune response. In this system, ISG54 minimal promoter in conjunction with five ISRE elicits transcription of secreted luciferase reporter gene upon stimulation of the type I IFN pathway, and IFN-related immune activation was measured based on luminescence [24] . As a result of the SAR study, all thiourea derivatives revealed negligible or very weak potency, regardless of alkyl and aryl moiety at R 1 (3a to 3f). No significant effects of p-nitro, fluoro, chloro substituents were observed on the aryl ring, however, the p-bromo substitution revealed undesired cellular toxicity (3c to 3e). In the aryl part, meta substitution lead to slightly increased activity compared to para substitution (3e and 3f).
To enhance activity, we introduced a urea moiety as a linker with diverse substitution on the aryl ring, which increased potency of ARP analogues for immune stimulation. Compared with the thiourea linker, para substitution on the phenyl ring exhibited stronger potency than meta substitution in most urea linker cases (4a to 4h). In para-substituted derivatives, no significant activity was observed in the nitro moiety (4a) which corresponded to thiourea linker, whereas the fluoro and trifluoromethyl moieties showed increased activity (4b and 4c). However, compound 4c showed undesired cellular toxicity, thereby a trifluoromethyl substituent was replaced by a trifluoromethoxy moiety which substantially enhanced potency (4.2-fold change) and generated no toxicity (4d). Considering the interesting results of thiourea and urea linker, we next incorporated carbonyl moiety as a linker for ARP derivatives. Somewhat weaker or stronger potency was observed in case of trifluoromethyl and methoxy substituents, compared to individual functional group in urea linker (5c to 5e). For the further investigation, aliphatic chain or heteroaryl group were introduced at R 1 position which produced no significant difference and only marginally increased activity (5a and 5b) .
Furthermore, disubstituted ARP analogues were considered to increase activity (5f to 5h) that resulted in weak activity which was below that of p-methoxy analogue (5e).
To increase potency of ARP derivatives, we examined the effects on carbon element by incorporating a benzyl group with a trisubstituted functional group. For this purpose, trimethoxy benzyl (5i) or tri-fluoro benzyl (5j) were introduced at the phenyl ring. As a result, immune response was considerably improved due to tri-methoxy substitution on the aryl ring (5.2-fold change), whereas tri-fluoro benzyl substitution showed no drastic change on ISRE result with a bit increase of activity (2.8-fold change). In addition, we confirmed no effect on cell viability by benzyl group. For the further modification, we inserted a phenethyl moiety at R 1 position (5k) to assess effects of carbon chain which showed activity similar to that of dichlorophenyl (5h) or trifluorobenzyl (5j) moiety (2.5, 2.3 and 2.8, respectively) Based on these SAR results, we identified the compound 5i as an effective type I IFN inducer and conducted further biological evaluation. Corresponding to the cytokine level, mRNA expression of IFNB and CXCL10 was clearly increased by 5i treatment (Figure 4 ). In addition to IFNB and CXCL10, 5i significantly elicited transcription of various ISGs such as IRF7, IFIT3, and OAS1 which are considered biomarkers of type I IFN signaling pathway ( Figure 4 ). In conclusion, all these results indicated the potential ability of 5i as an efficient type I IFN inducer without undesired toxicity. Although, it is hard to conclude that 5i showed superior potency compared to tilorone, an efficient small molecule anti-viral agent inducing type I IFN, due to the different evaluating system. Considering 50 mg/kg required to treat Evola virus by tilorone [25] , the fact that 5i induced significant increased IFNβ secretion at 20 μM suggested structure insight for developing new anti-viral therapy.
In conclusion, we confirmed ARP motif as a new core skeleton of type I IFN regulation. Moreover, we synthesized various ARP analogues and investigated their biological activity for anti-viral state and innate immunity. Based on the SAR analysis, we verified the importance of the carbonyl linker and the benzyl moiety with a trimethoxy attachment on adjacent aryl ring in the ARP pharmacophore. As a result, compound 5i was identified as a potent type I IFN inducer. Further investigation about cytokine secretion and IFN-mediated ISG expression by 5i indicated effective stimulation of type I IFN pathway.
All these results suggest a beneficial therapeutic potential of 5i for anti-viral agent.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.