Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors

Abstract In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found 18p possessed high potency (IC50 = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN-β and IP-10 were induced by 18p in a concentration dependent manner. Finally, we discovered that 18p causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.


Introduction
Cancer immunotherapy has recently emerged as a new paradigm in treating cancer patients in that patient's immune system components are involved in cancer therapies 1 . Although several adaptive immune checkpoint inhibitors such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 have received FDA approval, limitations of the immune checkpoint inhibitors have been reported including low response rate in many cases 2 . The reason of initial resistance to immune checkpoint inhibitors is that most tumours lack sufficient T cell infiltration (i.e. "cold" tumour). Thus, activating innate immune system, which is upstream of adaptive immune tumour-infiltrating lymphocytes (TILs), to turn "cold" tumour into "hot" tumour could be a novel strategy in cancer immunotherapy 3 . The innate immune system is the first responder against microbial infection. When the foreign DNAs from pathogens or dead cells are detected by cGAS (cyclic GMP-AMP synthase), cGAS-STING pathway is activated 4 . Activated cGAS by DNA binding synthesises cGAMP (cyclic GMP-AMP), which activates STING protein (stimulator of interferon genes) on the endoplasmic reticulum. Activated STING then transmit a signal that leads to production of interferons (IFNs) and ultimately recruits tumour-infiltrated lymphocytes (TIL) around tumour. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been discovered as the principal hydrolase for cGAMP and thus, ENPP1 restrains innate immune responses to cancer. It has been reported that both genetic knockdown and inhibition of ENPP1 by small molecule could increase tumour-infiltrating DCs and decrease tumour growth 5 .
Although ENPP1 have recently drawn much attention in the fields of medicinal chemistry 5,6 and efforts to synthesise small molecule ENPP1 inhibitor have been reported, development of novel and potent drug-like ENPP1 inhibitor has proven challenging ( Figure 1a). Therefore, discovery of a novel scaffold for synthesis of non-nucleotide derived ENPP1 inhibitor is highly important. Inspired by the reported ENPP1 inhibitors, QS1(2) 6h,i and 4 5,6a , we designed and synthesised a series of new sulfamide derivatives possessing the pyrrolopyrimidine and pyrrolopyridine core scaffolds and performed SAR study in an effort to discover novel and potent ENPP1 inhibitors (Figure 1b).
Nucleophilic aromatic substitution of pyrrolopyrimidines or pyrrolopyridines 12 with piperidines 13 afforded carbamates 14 in 45-97% yields 10 . Removal of the Boc group under acidic conditions provided amines 15 in good yields. Reaction between amines 15 and Burgess-type reagent 16, which was developed by Winum et al. provided 17 11 . Cleavage of the Boc group in compounds 17 with 4 N HCl in dioxane formed sulfamides 18 (Scheme 2a). Sulfone amides 20 were also synthesised by N-sulfonylation of amines 15 (Scheme 2b) 12 .
In addition, we attempted to prepare 4-phenyl substituted pyrrolopyrimidines or pyrrolopyridines 25 (Scheme 3). The Suzuki coupling of 4-chloropyrrolopyrimidines or 4-chloropyrrolopyridines 12 and boronic acids 21 generated Boc-protected amines 22 in moderate to good yields (Scheme 3). 10a Then, cleavage of the Boc group under acidic conditions and subsequent addition of sulfamide moieties to the free amines 23 followed by the removal of the Boc group afforded 25.

Structure-activity relationships
With the compounds in hand, % inhibition and IC 50 values for all synthetic 4-piperidine and 4-aryl substituted pyrrolopyridines or pyrrolopyrimidines (i.e. 18, 20, and 25) were determined using  in vitro recombinant human ENPP1 enzymatic assay with AMP-Glo assay (Table 1) 13 . Since MV658, discovered by Mavupharma, was reported to possess less than 100 nM K i value, We used MV658 as a positive control in enzymatic assay (  to 18p, we decided to evaluate cell-based assay with 18p, which possessed larger cLogP values. Moreover, percent inhibition and IC 50 values against ENPP1 with 4-phenyl substituted sulfamides 25 were evaluated ( Table 2). The percent inhibitory activities of 4-phenyl substituted sulfamides 25 was quite low compared to those with 4-piperidine substituted sulfamides 18. Both 25a and 25b displayed low percent inhibitory activities (entries 2 and 3). N-benzyl sulfamides 25c and 25d were found to have weak potency to ENPP1 (entries 4 and 5). A similar inhibitory potency was observed with N-methylated pyrrolopyrimidine 25e (entry 6). Pyrrolopyridine 25f was more potent than pyrrolopyrimidine 25d (entries 5 and 7). Addition of the sulfamide functional group at the meta position of phenyl has no beneficial effect on potency (entry 8). Interestingly, compound 25h, containing gen-dimethyl group exhibited enhanced potency (entry 9). However, N-pyridyl compound 25i showed no activity (entry 10). Surprisingly, addition of mono-methyl substitution at the benzylic position resulted in remarkable potency against ENPP1 (25j, IC 50 ¼ 28.3 nM, entry 11).

Docking studies
The three-dimensional structures of ENPP1 in complex with 18p were predicted by protein-ligand docking study. Compound 18p occupies the known cGAMP binding site of ENPP1. The pyrrololpyrimidine core forms p-p interactions with Y322 and Y353. In addition, nitrogens on the pyrrolopyrimidine forms hydrogen bond interactions with W304 and F303. The sulfamide functional group interacts with the zinc ion and the NH 2 of the sulfamide forms hydrogen bonds with T238 and N259. The piperidine linker forms hydrophobic interactions with L272 ( Figure 2).

Inhibition of CYPs, microsomal stabilities, and PK profiling
Encouraged by the great inhibitory activities of 18p against ENPP1, we evaluated the inhibitory activities of 18p against cytochrome P450s (CYPs) ( Table 3). We observed that five cytochrome P450 enzymes, CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, were only slightly inhibited by 18p. We also found that 18p possesses good microsomal stabilities (>60%) against human, rat, and mouse liver microsomes (Table 4). Moreover, the mouse pharmacokinetic profile of compound 18p showed great oral bioavailability, moderately long half-life, and a plasma exposure of 1698 ngh/mL following an oral dose of 10 mg/kg (Table 5).

Biological evaluation
To verify the cellular activity of 18p with cGAMP, we tested whether the compound could stimulate type I IFN response using THP-1 human monocyte cells bearing interferon sensitive response elements (ISRE) luciferase reporter system because THP-1 is wellknown monocyte cell line and widely used to evaluate cGAS-STING mediated innate immune activity. We used MV658 as a positive control for the cell-based assay 10a . While the low concentration of cGAMP (1 lg/mL) only induce luciferase signal slightly, co-treatment of 18p clearly increased the cGAMP-induced ISRE activation in dose-dependent manner ( Figure 3a). Treatment of 18p dramatically increased ISRE signal compared to the result with MV658. Furthermore, no obvious toxicity was observed in 18p-treatment THP-1 cells in all concentrations tested (Figure 3b).
To elucidate the endogenous immune activation by 18p, we investigated the secretion of innate immune-related cytokines such as IFN-b and IP-10 which have been mainly considered as biomarkers for STING activation. Extracellular secretion of IFN-b Scheme 3. Synthesis of phenyl substituted sulfamide 25. Table 1. Enzymatic inhibitory activities of 4-piperidine substituted sulfamides 18 and sulfone amides 20 against ENPP1. a Average ± SD from two or three independent repeats. and IP-10 was measured by enzyme-linked immunosorbent assay (ELISA). We discovered that co-treatment of 18p with cGAMP secreted more cytokine than with cGAMP alone in both IFN-b and IP-10 analysis ( Figure 4).
Next, we further explored type I IFN response induced by activation of STING signalling. The gene expression for various interferon stimulated genes (ISGs) such as CXCL10, OAS1, and IFITM1 was analysed by real-time PCR. Interestingly, co-treatment of 18p with cGAMP induced expression of all ISG genes we tested, and triggered transcription of the key activation markers like CXCL10. The result was corresponded to ELISA result shown in Figure 5.
To evaluate whether 18p induced desired immune response via activation of STING pathway, we investigated the activation of downstream effector proteins, IRF3 and STAT1 that were known as major regulatory proteins of STING and IFN-b, respectively. By western blot analysis, the increased phosphorylation of IRF3 and STAT1 was observed in dose-dependent manner by co-treatment of cGAMP and 18p in THP-1 cells ( Figure 6). Based on all these results, we concluded that potent ENPP1 inhibitor 18p obviously stimulated cGAMP-mediated STING activation by resulting in the synergy effect with cGAMP for innate immunity.

In vivo efficacy
The Bakhoum group showed that Enpp1-KO 4T1 mouse model led to significant longer overall survival rate 14 . Thus, we evaluated the anti-cancer efficacy in the 4T1 syngeneic mouse tumour model to test the therapeutic potential of 18p. We postulated the marginal Table 2. Enzymatic inhibitory activities of sulfamides 25 against ENPP1. a Average ± SD from two or three independent repeats. amount of cGAMP released by tumour could initiate the synergy effect of 18p, then administrated the compound by single-treatment 5 . After 4T1 tumour cells were implanted and grown in the flank of mice, 18p was orally administered (40 mg/kg) once daily and monitored the tumour volume and body weight of each mouse. After two weeks of treatment, 18p significantly suppressed tumour growth (TGI ¼ 39% on day 14, Figure 7(a,b) and no weight loss occurred upon treatment of 18p ( Figure 7c).

Conclusion
In conclusion, we discovered the novel small molecular ENPP1 inhibitor, 18p possessing the pyrrolopyrimidine core. Pyrrolopyrimidine 18p showed high in vitro potency (IC 50 ¼ 25.0 nM) against ENPP1. Also, 18p increased the cGAMP-induced ISRE activation and induced cytokine secretion in a concentration dependent manner in THP-1 dual cells. These results showed that 18p obviously stimulated cGAMP-mediated STING activation. Finally, we discovered that 18p causes suppression of the tumour growth in 4T1 syngeneic mouse model. This study provides insightful guidelines for design and development of novel ENPP1 inhibitors.

General informations
Unless otherwise stated, reactions were performed in flame-dried glassware under a nitrogen atmosphere using dry solvents.
Reaction progress was monitored by thin-layer chromatography (TLC). Purified water was obtained using a Barnstead NANOpure Infinity UV/UF system. Brine solutions are saturated aqueous solutions of sodium chloride. Commercially available reagents were purchased from Sigma-Aldrich, Acros Organics, Combi-Blocks, TCI or Alfa Aesar, and BLDpharm used as received unless otherwise stated. Reaction temperatures were controlled by an IKAmag temperature modulator unless otherwise indicated. TLC was performed using E. Merck silica gel 60 F254 precoated glass plates (0.25 mm) and visualised by UV fluorescence quenching, KMnO 4 staining. Silicycle SiliaFlash P60 Academic Silica gel (particle size 0.040-0.064 mm) was used for flash column chromatography. 1 H NMR spectra were recorded on Bruker 400 MHz, 600 MHz, and 800 MHz spectrometer and are reported relative to residual CDCl 3 (d 7.26 ppm), CD 3 OD (d 3.31 ppm), D 2 O (d 4.79 ppm) or (CD 3 ) 2 SO (d 2.50 ppm). 13 13 C are reported in terms of chemical shifts (d ppm). High resolution mass spectra (HRMS) were obtained from Bruker ESI Q-TOF Mass Spectrometer with electrospray ionisation (ESIþ), atmospheric pressure chemical ionisation (APCIþ), or mixed ionisation mode (MM: ESI-APCIþ). HPLC was performed on an Agilent 1100 system with an Agilent EC-C18 column (4.6 Â 150 mm, 4 mm).

4-Chloro
To a stirred solution of pyrrolopyrimidine 8 (250 mg, 1.63 mmol, 1.00 equiv) and Cs 2 CO 3 (800 mg, 2.45 mmol, 1.50 equiv) in DMF (5.40 mL) was added MeI (0.200 mL, 3.26 mmol, 2.00 equiv). The reaction mixture was stirred for 1 h at 25 C. The solution was filtered through celite and H 2 O was added. The aqueous phase was extracted with EtOAc (3 Â 7.00 mL). The combined organic phase was washed with brine, dried over MgSO 4 and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc:hexanes ¼ 1:4) to give methylated

4-Chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (9j)
n-BuLi (2.5 M in hexanes; 3.8 mL, 9.5 mmol, 2.2 equiv) was added dropwise to a solution 5-bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine S1 (1.00 g, 4.30 mmol, 1.00 equiv) in THF (24 mL) at À78 C over a period of 5 min under nitrogen atmosphere. After the reaction mixture was stirred for 30 min at À78 C, methyl iodide (0.428 mL, 6.88 mmol, 1.60 equiv) was added. The reaction mixture was stirred for 1 h at 25 C then diluted with water (25 mL), and extracted with EtOAc (3 Â 20 mL). The organic layer was washed with brine (30 mL) then dried over MgSO 4 , and concentrated in vacuo.  Step 1: S N Ar reaction. To a solution of pyrrolopyrimidine 12 (1.00 equiv) and amine 13 (1.30 equiv) in n-butanol (0.100 M) was added N,N-diisopropylethylamine (3.00 equiv) under nitrogen atmosphere. After the reaction mixture was stirred for 16 h at 100 C, the reaction mixture was cooled down to 25 C. The solid was filtered to obtain intermediate 14, or water was poured into the mixture and extracted with CH 2 Cl 2 . The combined organic phases were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash column chromatography (1:20 MeOH:CH 2 Cl 2 ) on silica gel. The collected fraction was then used in the next step (45 À 97% yield).

4.1.13.2.
Step 2: Boc deprotection. To a solution of collected fraction (1.00 equiv) in CH 2 Cl 2 (0.041 M) was added hydrochloric acid (4 M in 1,4-dioxane, 0.200 M) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C. The reaction mixture was stirred for 16 h and concentrated in vacuo. The crude mixture was then filtered and washed with Et 2 O and CH 2 Cl 2 to afford intermediate 15 (72 À 100% yield).

4.1.13.3.
Step 3: Sulfamoylation reaction. To a solution of intermediate 15 (1.00 equiv) and (tert-butoxycarbonyl)((4-(dimethylamino)pyridin-1-ium-1-yl)sulfonyl)amide 16 (1.10 equiv) in CH 2 Cl 2 (0.0873 M) was added Et 3 N (3.00 equiv) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C and the mixture was stirred for 16 h. The solution was then quenched with water and extracted with CH 2 Cl 2 . The combined organic phases were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash column chromatography (1:20 MeOH:CH 2 Cl 2 ) or Prep TLC (1:20 MeOH:CH 2 Cl 2 ). The obtained compound was then used in the next step (8 À 88% yield).  (4 M in 1,4-dioxane, 0.200 M) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C. The reaction mixture was stirred for 16 h and concentrated in vacuo. The crude mixture was then filtered and washed with Et 2 O and CH 2 Cl 2 to afford title compound 18 (21 À 98% yield).

General procedure B2 for synthesis of sulfone amides 20
To a flame dried two-neck 10-mL round-bottom flask was added amine 15 (1.00 equiv). The flask was covered with a septum, and then placed under vacuum and refilled with N 2 (three cycles). After the addition of anhydrous CH 2 Cl 2 (0.0800 M) to the flask, Et 3 N (4.50 equiv) and RSO 2 Cl (1.05 equiv) were added. The reaction mixture was stirred for 16 h at 25 C. The reaction mixture was stirred until no starting material remained by TLC. After completion of the reaction, a water (10.0 mL) was added. The aqueous phase was extracted with CH 2 Cl 2 (3 Â 10 mL). The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (1:20 MeOH:CH 2 Cl 2 ) to afford sulfone amides 20 (22 À 55% yield).
Note: This procedure is the same as step1 and step 2 of General procedure B1. Step 1: Suzuki reaction. Procedure 1) To a solution of pyrrolopyrimidine 12 (1.00 equiv) in 1,4-dioxane (0.090 M) and water (0.90 M) was added boronic acid 21 (1.05 equiv) and potassium carbonate (3.75 equiv) under nitrogen atmosphere. Tetrakis(triphenylphosphine) palladium(0) (Pd(PPh 3 ) 4 , 0.060 equiv) was added into reaction mixture and purged by nitrogen for 5 min. After the reaction mixture was stirred for 24 h at 110 C, the reaction mixture was cooled down to 25 C and quenched with water (5.00 mL). The reaction mixture was then filtered through a pad of Celite and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (40 À 89% yield).
Procedure 2) To a solution of pyrrolopyrimidine 12 (1.00 equiv) and boronic acid 21 (1.05 equiv) in 1,4-dioxane (0.300 M) was added 1,1 0 -bis(diphenylphosphino)ferrocene-palladium(II) dichloride (PdCl 2 (dppf), 0.100 equiv), and potassium phosphate (1.50 equiv) under nitrogen atmosphere. After the reaction mixture was stirred for 24 h at 110 C, the reaction mixture was cooled down to 25 C and quenched with water (5.00 mL). The reaction mixture was then filtered through a pad of Celite and extracted with CH 2 Cl 2 . The combined organic phases were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (74% yield).

4.1.15.2.
Step 2: Boc deprotection. To a collected fraction (1.00 equiv) in CH 2 Cl 2 (0.0410 M) was added hydrochloric acid (4 M in 1,4-dioxane, 0.200 M) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C. The reaction mixture was stirred for 16 h and concentrated in vacuo. The crude mixture was then filtered and washed with diethyl ether to afford intermediate 23 (76 À 100% yield).

4.1.15.3.
Step 3: Sulfamoylation reaction. To a solution of intermediate 23 (1.00 equiv) and (tert-butoxycarbonyl)((4-(dimethylamino)pyridin-1-ium-1-yl)sulfonyl)amide 16 (1.10 equiv) in CH 2 Cl 2 (0.0873 M) was added Et 3 N (3.00 equiv) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C and the mixture was stirred for 16 h. This solution was then quenched with water and extracted with CH 2 Cl 2 . The combined organic phases were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel or Prep TLC (1:20 MeOH:CH 2 Cl 2 ). The collected fraction was then used directly in the next step (15 À 52% yield).

4.1.15.4.
Step 4: Boc deprotection. To a solution of collected fraction (1.00 equiv) in CH 2 Cl 2 (0.040 M) was added hydrochloric acid (4 M in 1,4-dioxane, 0.200 M) at 0 C. The temperature of the reaction mixture was gradually increased to 25 C. The reaction mixture was stirred for 16 h and concentrated in vacuo. The crude mixture was then filtered and washed with diethyl ether to afford title compound 25 (40-100% yield).

Docking
The examined 18p was drawn using ChemDraw and converted to 3D-structure via openbabel 3.1.0 15 . Molecular docking was conducted through ICM version 3.9-2d based on Monte Carlo simulations to search the globally minimum binding pose between our compound and a target protein 16 . The co-crystal complex of ENPP1 and IJE (PDB-ID: 6 Â kd)6a was downloaded from the Protein Data Bank 17 . The centre of IJE with the coordinates of À4.04(x), 27.29(y), 40.16(z) was defined as the centre of grid box with its width of 20 Å in each x, y, z direction respectively. Then, IJE, cofactor, and water molecules were extracted from the complex. Grid maps for five different energy terms were generated with a spacing of 0.5 Å between the grid points. Docking simulation was performed with an effort parameter of 10 during 10 iterative runs, and all the conformations with different binding affinity were collected and ranked from the dock score of ICM-Pro. The pose with the best binding energy was analysed about its intermolecular interactions by virtue of LigPlot þ and PLIP18 and visualised using PYMOL 19 .

Biology
4.3.1. In vitro enzyme assay protocol ENPP1 hydrolyses nucleotides or nucleotide derivatives and produces nucleoside-5 0 -monophosphates and pyrophosphates. ENPP1 also hydrolyses 2 0 3 0 -cGAMP and produces 5 0 -adenosine monophosphate (AMP) and 5 0 guanosine monophosphate (GMP). The produced AMP from the reaction is detected using the AMP-Glo V R kit (Promega). The assay kit consists of two reagents; one to terminate the AMP-generating enzymatic reaction, remove ATP and convert AMP produced into ADP, and a second reagent to convert ADP to ATP, which is used to generate a luminescence in a luciferase reaction. The amount of light measured is proportional to the amount of AMP produced by ENPP1.
The final assay reaction mixture contains a buffer of 50 mM Tris (pH 8.5), 250 mM NaCl, 0.5 mM CaCl 2 , 1 lM ZnCl 2 , 5% glycerol and 1% DMSO. Serially diluted ENPP1 inhibitors (usually range from 10 lM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5-10 min at room temperature (RT). The reaction is initiated by adding cGAMP (at final concentration of 5 lM) and incubating for 90 min at 37 C. At the end of incubation, the reaction is stopped by adding 10 lL of AMP-Glo reagent I and followed by incubation at RT for one hour. After incubation, 20 lL of AMP detection solution (mixture of AMP-Glo II reagent and Kinase-Glo one at 1:100 ratio) is added and incubated at RT for one hour. The luminescence signal generated is measured using the ClarioStar plate reader (BMG Labtech).
Maximal activity control (containing enzyme and substrate in presence of 1% DMSO only; MAX) and background control (containing substrate and 1% DMSO; MIN) are evaluated simultaneously. In each experiment, serially diluted reference ENPP1 inhibitor is tested together. The IC 50 values for % remaining activity versus compound concentration are determined by fitting the inhibition curves using a three-parameter variable method in GraphPad Prism V R software. Triplicate wells are run at each inhibitor concentration and averaged IC 50 value for each compound is calculated.
4.3.2. Cell based assay for IRF activation in THP-1 dual cells THP-1 dual cells were purchased from Invivogen and IRF-Lucia luciferase reporter assay was performed by manufacturer's instruction. Briefly, cells were plated in medium in 384-well plate. Individual compounds were pre-treated with an indicated concentration before cGAMP activation, then cGAMP was added at an indicated concentration. After 24 h of incubation, supernatant was analysed using QUANTI-Luc TM assay kit by measuring the relative light units of luciferase signal (RU) in a microplate reader (TECAN).

Cell viability assay
Cell viability assay was performed using residual cells after luciferase assay. Cells were analysed using CellTiter 96 Aqueous One Solution Proliferation Assay (MTS, Promega) according to the manufactural protocols. Absorbance signal were measured using microplate reader (TECAN). Results were normalized using DMSO as a control.

Gene expression analysis by rtPCR
Cells were harvest and RNA were isolated using NucleoSpin RNA plus (MN) according to the manufacturer's instructions. Genomic DNA was removed and cDNA was synthesised using an iScript gDNA clear cDNA synthesis kit (Bio-Rad). Expression levels were assessed by real-time PCR (CFX96 Real-Time PCR detection system, Bio-rad) using IQ SYBR green Supermix (Bio-Rad). The mRNA levels were normalized by GAPDH and calculated by comparative Ct method. 4.3.5. Enzyme-linked immunosorbent assay (ELISA) IFN-b cytokine secretion were quantified by using ELISA kits from R&D Systems. THP-1 cells were seeded in 96 well plate and treated with the compounds for 8 h incubation. 100 lL of cell culture supernatant was harvested and analysed using manufactural protocols.

Western Blot analysis
Cells were washed by PBS and harvested with RIPA buffer (Bioseasang). After centrifugation with 20,000 Â g for 20 min at 4 C. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred on PVDF membranes through the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked in 5% BSA in TBST for 1 h at room temperature and followed by incubation with primary antibody solution for overnight. All the antibodies were used in TBST and dilution for each antibody as follows: anti-pIRF3 (Cell Signalling/no. 4947, 1:1000), anti-pSTAT (Cell Signalling/no. 9167, 1:1000), and anti-b-tubulin (Cell signalling/no. 2146, 1:1000). After washing with TBST, all antibodies were detected by anti-rabbit-HRP-linked secondary antibody (Cell signalling/no. 7074, 1:3000). b-Tubulin was used as a loading control for the amount of total protein. Luminescence signal was detected using ECL reagent and immunoreactive bands were captured with the ChemiDoc imaging systems (Bio-Rad).

In vivo efficacy
The study for murine 4T1 colon carcinoma was approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology (KIST-2021-04-048). Female BALB/c mice were 8 weeks obtained from DBL (Korea). Mice were subcutaneously inoculated with 4T1 cells (4 Â 10 5 ) suspended in PBS into the right flank. The individual group was classified based on the tumour size when each tumour of each mouse reached 30-100 mm 3 . Compound or vehicle were administrated by QD per oral injection in 0.5% MC/Saline (v/v) solution. Tumour volume was calculated by following formula: Tumour volume (mm 3 ) ¼ 0.5 Â [length, (mm) Â width, (mm)2].

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was supported by KIST Institutional Program [2E31624,