Discovery of 7-azaindole Inhibitors of inflammasomes/IL-1β for the Treatment of Inflammatory Bowel Disease

Discovery of 7-azaindole Inhibitors of inflammasomes/IL-1β for the Treatment of Inflammatory Bowel Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery of 7-azaindole Inhibitors of inflammasomes/IL-1β for the Treatment of Inflammatory Bowel Disease Yuyun Yan, Xiuxiu Zhang, Ruiwen Wu, Xiangting Liang, Yiming Luo, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6307163/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Molecular Diversity → Version 1 posted 10 You are reading this latest preprint version Abstract Currently, a significant proportion of patients with inflammatory bowel disease (IBD) fail to respond to conventional drug therapy such as immunosuppressants and biologic agents. IL-1 signaling blockade is a promising therapeutic strategy for these unresponsive IBD patients. In this study, we identified a novel anti-NLRP3/IL-1β inhibitor, the 7-azaindole analogue Y19 , which demonstrates potent inhibitory activity with an IC 50 value of 1.26 µM. Mechanistic investigations revealed that it suppresses NLRP3 inflammasome assembly and activation by disrupting critical protein-protein interactions, including NEK7-NLRP3, NLRP3-NLRP3, NLRP3-ASC, and ASC-ASC. Furthermore, it also inhibits the AIM2 and NLRC4 inflammasome pathways. In a murine model of colitis, Y19 demonstrated anti-inflammatory efficacy comparable to that of tofacitinib, a Janus kinase inhibitor commonly prescribed for IBD patients refractory to conventional therapies. This finding highlights the potential of Y19 as a promising lead compound for the treatment of IBD. Inhibitors 7-azaindole inflammasome IL-1β Inflammatory bowel disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Inflammatory bowel disease (IBD) has gradually become a global disease that afflicts millions of people today[ 1 ]. IBD, comprising Crohn's disease (CD), ulcerative colitis (UC) and IBD unclassified, has yet no exact known cause [ 2 ], and is a kind of chronic debilitating inflammatory gastrointestinal disorder with complex pathological and clinical features, such as mucosal barrier defects, immune response dysregulation, and even autoimmunity and syndromic features in other organs[ 3 – 5 ]. Patients with IBD exhibit heterogeneity that may diversify clinical phenotypes and limit therapeutic success[ 6 – 8 ]. Currently, there is still no cure for IBD. The medical treatment for IBD aims to relieve symptoms and reduce inflammation, such as classic anti-inflammatory drugs (5-aminosalicylates and corticosteroids), immunosuppressants (thiopurines and ciclosporin-A), and biologic agents (anti-TNF-α and anti–interleukin-12/23)[ 7 , 9 ]. Novel biologics are generally indicated for patients with moderately to severely active IBD who are unresponsive to classic anti-inflammatory drugs and immunosuppressants, who are glucocorticoids-dependent, or who have unacceptable side effects from these drugs[ 10 , 11 ]. Even so, there is still a large proportion of patients who do not respond to biologic treatments[ 12 ]. For example, approximately one third of CD patients either have no initial response or stop response to anti–TNF-α biologic agents[ 13 ]. Targeting Janus kinase (JAK) may be the new approach for IBD patients with conventional treatment failure[ 14 , 15 ]. Tofacitinib, an oral pan-JAK inhibitor, was approved for patients with UC who have no response to conventional or anti–TNF-α therapy, but did not exhibit positive benefit for CD patients[ 15 ]. Upadacitinib is the selective JAK1 inhibitor currently approved for the treatment of both UC and CD[ 16 ]. However, safety concerns have led to broad regulatory restrictions on the use of JAK inhibitors. The long-term use of high doses of JAK inhibitors may increase the risk of major adverse cardiovascular events, venous thromboembolism, serious infections and cancer[ 17 , 18 ]. Recently, increasing evidence supports that inflammasome/IL-1 β pathway plays a vital role in IBD pathogenesis. Deregulated activation of inflammasome exacerbates inflammation through secretion of pro-inflammation factors IL-1β and IL-18, which is able to amplify immune responses[ 19 ]. IL-1α/IL-1β is key upstream cytokines that regulate IL-23 expression in monocytes, which is also significant mediators of intestinal inflammation[ 20 ]. The inflammatory monocytes and CX3CR1 + IL-1β + macrophages may be the primary sources of IL-23 during inflammasome activation and IL-1 production in the inflamed intestine[ 20 ]. Overactivation of inflammasomes leads to dysregulation of the neutrophil-dominated microenvironment with higher levels of TNF-α, IL-1β, IL-1α, IL-8, IL-12, IL-15, IL-17, IL-23, and IL-36[ 21 ]. IL-1β helps to prolong the survival of T cells and the proliferation of B cells to enhance the production of antibodies, to induce a potent chemokine response, and to drive the neutrophil infiltrates, inflammatory fibroblast activation and epithelial cell loss in ulcerated intestinal tissue[ 22 – 27 ]. Importantly, IL-1 has been identified as the critical signal driving stromal-neutrophil interactions, as observed in a subset of IBD patients who fail to respond to anti-TNF and other therapies[ 28 ]. These suggest that the blockade of inflammasomes/IL-1β pathway is also a promising strategy for the treatment of IBD. Herein, we describe the identification of the 7-azaindole inhibitor Y19 that can inhibit IL-1β secretion mediated by different inflammasomes. In vivo pharmacological models of colitis have shown that Y19 alleviates uncontrolled inflammation via anti-IL-1β effect. 2. Results and discussion 2.1. Discovery and synthesis of lead compounds. A cell screening model based on the activation steps of the NLRP3 pathway was established. [ 29 ]. This model initiates with lipopolysaccharide (LPS)-primed mouse macrophage line J774A.1, which activates NF-κB to produce resting NLRP3 and pro-IL-1β. Subsequently, the cells are treated with our compounds, followed by stimulation with nigericin, and finally NLRP3-mediated IL-1β levels are detected using enzyme-linked immunosorbent assay (ELISA). We identified our in-house compound 1 (see Table 1 ), which effectively inhibits NLRP3-mediated IL-1β secretion with an IC 50 value of 35.7 µM. To further investigate the structure-activity relationship (SAR) and obtain more potent leads, we designed and synthesized a diverse array of analogues. Scheme 1 briefly describes the synthesis of all compounds. In Scheme 1 A, carboxylic acids with different structures ( A1 , A2 , A4-A15 ) were coupled with the corresponding amines directly in the presence of HATU and TEA to give Y1 , 2 , 4–15 , and 29 . As shown in Scheme 1 B, the synthesis started with known compound B1 , which was readily obtained according to a reported procedure [ 30 ]. B1 was coupled with the corresponding amines in the presence of EDCI, HOBt and DIPEA to give Y3 , 16–28 [ 31 ]. In Scheme 1 C, according to the literature, α, β-unsaturated amide C1 was obtained through the acylation of phenethylamine with acryloyl chloride [ 32 ], and C2 was produced through the protection of 5-iodo-7H-pyrrolo[2,3-d]pyrimidine with 4-methylbenzenesulfonyl chloride [ 33 ]. C2 was subjected to a Heck reaction with C1 in the presence of CuI and Pd(OAc) 2 , and then Ts was removed to give Y30 [ 31 ]. 2.2. SAR Analysis. The indole analogue 1 shows anti-NLRP3/IL-1β activity with an IC 50 value of 35.7 µM. It has a molecular weight of 394.2, and its structure mainly includes the indole core, 1-methyl, 2-phenyl, and 3-α, β-unsaturated amide. Firstly, we investigated its preliminary structure − activity relationships (SAR) to determine the impact of molecular fragment on anti- NLRP3/IL-1β activation. As shown in Table 1 , the substitution of the phenyl group with a hydrogen atom ( Y1 ) exhibited a positive influence on activity. The relocation of the methyl group from the 1-position to the 2-position led to the exposure of an N-H moiety at the 1-position ( Y2 ), thereby significantly enhancing activity. Moreover, the replacement of the carbon atom at the 7-position with a nitrogen atom, along with the substitution of 2-methyl with a hydrogen atom ( Y3 ), dramatically increased the inhibitory potency with an IC 50 value of 5.67 µM. However, the removal of 3-α, β-unsaturated double bond ( Y4 and Y5 ) resulted in a complete loss in the inhibitory effect on IL-1β. These findings indicate that the α, β-unsaturated double bond is essential for inhibitory activity towards NLRP3 mediated IL-1β. On the basis of the above analysis, we firstly chose 7-azaindole moiety as a region for further modification. The azaindole was replaced with a range of different substituted aromatic rings, such as pyridine ( Y6 and Y7 ) and substituted phenyl groups ( Y8 – Y15 ). Among them, he majority of analogs exhibited a loss in potency, and only Y 8 , Y9 , and Y14 demonstrated low inhibitory activities. These results indicated that 7-azaindole core is important for anti-IL-1β activity. Thus, we directed our focus towards optimizing the α-ethylbenzylamine moiety. The shift from S -α-ethylbenzylamine to R -configuration ( Y16 ) showed a slight increase in activity. Substitution with α-methylbenzylamine ( Y17 ) or 2-methylbenzylamine ( Y18 ) to α-ethylbenzylamine exhibited the reduced activity, whereas the phenylethylamine moiety ( Y19 ) showed a remarkable improvement with an IC 50 value of 1.26 µM. Subsequently, we investigated the effect of differently substituted phenethylamines on the activity. Unfortunately, all these compounds significantly reduced the activity. For example, about 4 to 6-fold less activity was observed for chlorine- and fluorine-substituted phenethylamines ( Y20 , Y21 , Y22 , Y23 , Y24 , and Y25 ). A similar result was also observed with electron-donating group methoxy substituted phenylethylamine ( Y26 ). The introduction of the water-soluble and electron-withdrawing methylsulfone group ( Y27 ) leads to a complete loss of activity. When phenylethylamine moiety was converted to cyclohexylethylamine ( Y28 ), the inhibitory activity was also reduced by more than 10-fold. The removal of α, β-unsaturated double bond at 3-position ( Y29 ) or substitution of carbon atom by a nitrogen atom at 5-position ( Y30 ) of Y19 results in the loss of activity, further indicating the importance of α, β-unsaturated double bond and azaindole core. Therefore, we selected the most active Y 19 to further study its mechanistic studies in NLRP3 pathway. 2.3. Inhibitory effect of Y19 on inflammasomes. IL-1β is secreted in response to the activation of various inflammasomes, including NLRP3, AIM2, and NLRC4. Initially, we assessed the inhibitory effect of Y19 on the NLRP3 pathway stimulated by different agents in J774A.1 cells. The data demonstrated that treatment with Y19 effectively inhibited IL-1β release mediated by NLRP3 activation in a dose-dependent manner upon stimulation with nigericin, SiO2, and MSU (Fig. 1 A, B & C).. Subsequently, we investigated whether Y19 could inhibit IL-1β release from different inflammasomes. Bone marrow-derived macrophages (BMDMs) were primed with LPS prior to treatment with Y19 ; thereafter, the cells were stimulated with nigericin to activate the NLRP3 inflammasome (Fig. 1 D), Poly(dA:dT) to activate the AIM2 inflammasome (Fig. 1 E), and FLA-ST Ultrapure to activate the NLRC4 inflammasome (Fig. 1 F). Our results indicated that Y19 is able to inhibit the secretion of IL-1β upon activation of all the NLRP3, AIM2 and NLRC4 inflammasomes. 2.4. Biological Mechanism Study. In order to confirm the effect of Y19 on NLRP3 pathway, its cytotoxicity was firstly evaluated. Y19 did not show significant toxicity against HEK293T and J774A.1 cells after 2 h of treatment (Fig. 1 S). Of all inflammasomes, NLRP3 is the most extensively studied to date. The NLRP3 pathway consists of two stages: first, NF-κB activation upregulates the transcriptional levels of both NLRP3 and pro-interleukin-1β (pro-IL-1β); second, subsequent assembly and activation of the NLRP3 inflammasome induce caspase-1-dependent secretion of IL-1β and cell pyroptosis, ultimately leading to inflammation[ 34 ]. Therefore, we primarily investigated the mechanism of action of compound Y19 . As illustrated in Figs. 2 A & B, Y19 did not impair LPS-induced tumor necrosis factor-alpha (TNF-α) secretion as measured by ELISA or NF-κB signaling activation assessed through Western blot analysis. The levels of caspase-1 (p20, an auto-cleaved fragment of caspase-1) was dose-dependently decreased in supernatants from Y19 -treated J774A.1 cells (Fig. 2 C), indicating that Y19 suppresses the activation of caspase-1 via the NLRP3 inflammasome. Correspondingly, the formation of NT-GSDMD (N-terminal domain of GSDMD) and lactate dehydrogenase (LDH) release was inhibited by Y19 (Figs. 2 C & D). These findings enable us to speculate on the role of Y19 in the assembly of the NLRP3 inflammasome, a critical event within the NLRP3 signaling pathway. Consequently, we investigated protein-protein interactions related to NLRP3 inflammasome assembly, including NEK7-NLRP3, NLRP3-NLRP3, NLRP3-ASC, and ASC-ASC interactions. The formation of the NLRP3–NEK7 complex is essential for initiating NLRP3 inflammasome assembly. As illustrated in Fig. 3 A, Y19 inhibited the interaction between NEK7 and NLRP3. Next, we assessed whether Y19 could obstruct direct interactions between NLRP3–NLRP3 or NLRP3–ASC. Co-immunoprecipitation assays demonstrated that Y19 reduces oligomerization of NLRP3 similarly to tranilast (the positive control) as shown in Fig. 3 B and significantly alters the interaction between NLRP3 and ASC (Fig. 3 C). We also evaluated ASC-ASC interactions, and our results indicated that Y19 prevents ASC oligomerization induced by NLRP3 (Fig. 3 D). Since ASC specks are indicative of activated NLRP3 inflammasomes, we examined their formation through immunofluorescence analysis. As demonstrated by Fig. 3 E&F, treatment with Y19 exhibited the production of ASC specks. Together these results indicate that Y19 prevents the NLRP3 inflammasome activation through the disruption of NLRP3 inflammasome assembly. 2.5. In Vivo Efficacy of Y19. Dextrose sodium sulfate (DSS)-induced colitis has been demonstrated to be dependent on the NLRP3 inflammasome in murine models[ 35 ]. Given that NLRP3 pathway plays a key role in human inflammatory bowel disease (IBD) and especially in non-response or poor response to current drugs in severe IBD, we aimed to investigate the potential of Y19 as a therapeutic agent against IBD. Tofacitinib, a highly effective JAK inhibitor known for its efficacy in treating severe colitis unresponsive to current medications, was selected as the positive control. A mouse model of colitis was established by administering water containing 2.5% dextran sulfate sodium (DSS) for 7 days. As illustrated in Fig. 4 A − D, mice with colitis exhibited significant body weight loss, reduced colon length, and increased fecal occult blood levels. In our experimental models, the positive drug tofacitinib and Y19 displayed no improvement of bodyweight loss and hemafecia (Fig. 4 A&B), however, they significantly increased colon length (Figs. 4 C & D). Notably, Notably, Y19 attenuated colon shortening in a dose-dependent manner. This alleviation was also reflected by reductions in tissue-associated inflammatory factor IL-1β levels (Fig. 5 A). Surprisingly, while the positive drug tofacitinib showed suppression of IL-1β expression in vivo, our ELISA data indicated that it did not inhibit IL-1β release when assessed in vitro (Figure S2). This discrepancy suggests that the observed anti-IL-1β activity may differ between vitro and vivo conditions due to complex interactions between JAK and IL-1β pathways within an organism. Next, histological and pathological evaluations using hematoxylin and eosin staining were conducted to assess intestinal mucosal damage. Mice administering DSS showed severe mucosal damage, such as crypt architecture, goblet cell depletion and inflammatory cell infiltration. After treatment with Y19 , especially at dose of 10 mg/kg and 20 mg/kg, these damages were significantly improved (Fig. 5 C&D). Furthermore, we investigated the effect of Y19 on NLRP3 pathway in vivo . Our results indicated that Y19 reduced the levels of NLRP3 and IL-1β at both 10 mg/kg and 20 mg/kg doses (Fig. 5 B). These findings suggest that Y19 has a positive therapeutic effect on ulcerative colitis through inhibiting NLRP3 pathway in vivo . 3. Conclusions In summary, the blockage of IL-1 signaling is a promising strategy for the treatment of IBD patients without respond to current drugs. Based on the phenotypic screening model for the NLRP3/IL-1β pathway, compound 1 from our in-house compound library were identified as an inhibitor of NLRP3 pathway. A SAR-driven drug design was used to discover the more potent lead compound Y19 with good inhibition of NLRP3-dependent IL-1β release. Also, Y19 inhibited the secretion of IL-1βmediated by AIM2 and NLRC4 inflammasomes. In the animal model of colitis, Y19 demonstrated the same level of anti-IL-1β ability as tofacitinib. Taken together, these findings strongly support further chemical development as promising azaindole inhibitors of IL-1β and therapeutic investigation for IBD. 4. Experimental Section Chemistry. All reactions were performed under a nitrogen atmosphere with anhydrous solvents from commercial sources (Innochem, Alfa, and Shanghai Chemical Reagent Company) without further purification unless otherwise stated. Reactions were monitored by thin-layer chromatography carried out on Yantai silica gel plates (60F-254), and the visualization was achieved using UV light, or phosphomolybdic acid in ethanol followed by heating. Tsingdao silica gel (200–300 mesh) was used for flash column chromatography. NMR spectra were recorded with a 400 MHz ( 1 H NMR, 400 MHz; 13 C NMR, 100 MHz) spectrometer. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, and integration. All compounds are > 95% pure by HPLC analysis (HPLC traces seen in Supporting Information). The purity was determined by Waters e2695 with UV/visible detector, using a JADE-PAK ODS-AQ column (15 cm × 0.46 cm, 5 µm) eluted at 1.0 mL/min with CH 3 OH/H 2 O (85/15, v/v). General Procedure A: Amide Coupling. Carboxylic acid ( A1, A2, A4-A15, A29 ) (1 equiv.), HATU (1.1 equiv.) and triethylamine (TEA, 2 equiv.) were dissolved in CH 2 Cl 2 (0.2 − 0.3 mol/L) and stirred at 0°C for 30 min, then the corresponding amine (1 equiv.) was added, and the mixture was stirred for 6 h at room temperature. The reaction mixture was poured into water and extracted with CH 2 Cl 2 (3 × 20 mL). The organic layers were combined, washed with saturated aqueous NaHCO 3 and brine, dried with anhydrous Na 2 SO 4 , filtered and evaporated to give the crude product. Purification on silica gel column gave white powder. General Procedure B: Amide Coupling. The B1 (1 equiv.), EDCI (1 equiv) and HOBT (1.2 equiv) were dissolved in DMF and stirred at 0°C for 30 min, after which the corresponding amine (1 equiv) and DIPEA (2.5 equiv) were added, and the mixture was stirred for 6 h at room temperature. The reaction mixture was poured into water and extracted with CH 2 Cl 2 (3 × 20 mL). The organic layers were combined, washed with saturated aqueous NaHCO 3 and brine, dried with anhydrous Na 2 SO 4 , filtered and evaporated to give the crude product. Purification on silica gel column gave white powder. General Procedure C: Heck coupling. The C1 (1 equiv.) and C2 (0.6 equiv.) were dissolved in DMF, CuI (0.02 equiv.), TEA (2 equiv.), and Pd(OAc) 2 (0.02 equiv.) were added, and the mixture was stirred for 5 hours at 70°C. At the end of the reaction, saturated saline was added and extracted with EA (3 × 20 mL). The organic layers were combined, dried with anhydrous Na 2 SO 4 , filtered and evaporated to give the crude product. General Procedure D: Removal of Ts group. The crude product (1 equiv.) and LiOH (2 equiv.) were dissolved in a mixture of THF and water, 500 µL of 1,4-dioxane was added, and the reaction was stirred for 6 h at room temperature. At the end of the reaction, the organic phase was extracted with EA (3 × 20 mL). The organic layers were combined, dried with anhydrous Na 2 SO 4 , filtered and evaporated to give the crude product. The crude product was dissolved in methanol and stirred for 1 h at room temperature to obtain white crystals 30 . Synthesis of compounds Y1 , Y2 , Y4 - Y15 , Y29 . Compounds Y1 , Y2 , Y4 - Y15 , Y29 were prepared using General Procedure A. (S,E)-3-(1-methyl-1H-indol-3-yl)-N-(1-phenylpropyl)acrylamide ( Y1 ). White foam; yield 67%; 1 H NMR (400 MHz, Chloroform- d ) δ 8.34 (dd, J = 4.7, 1.5 Hz, 1H), 8.10 (dd, J = 7.9, 1.5 Hz, 1H), 7.76 (d, J = 15.6 Hz, 1H), 7.37–7.20 (m, 7H), 7.09 (dd, J = 7.9, 4.7 Hz, 1H), 6.42 (d, J = 15.5 Hz, 1H), 6.15 (d, J = 8.3 Hz, 1H), 5.06 (q, J = 7.6 Hz, 1H), 3.83 (s, 3H), 1.94–1.86 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.06, 148.83, 143.85, 142.50, 134.18, 132.38, 128.73, 128.70, 127.38, 126.87, 118.46, 116.80, 116.53, 110.63, 55.15, 31.54, 29.28, 10.97; HRMS (ESI) calcd for C 21 H 22 N 2 O [M + H] + , 319.1805, found, 319.1800. (S,E)-3-(2-methyl-1H-indol-3-yl)-N-(1-phenylpropyl)acrylamide ( Y2 ). White solid, mp 203.9℃; yield 74%; [α] D 21 +122.53 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, Chloroform- d ) δ 8.98 (s, 1H), 7.89 (d, J = 15.4 Hz, 1H), 7.83–7.77 (m, 1H), 7.38–7.31 (m, 4H), 7.31–7.25 (m, 2H), 7.17–7.10 (m, 2H), 6.43 (d, J = 15.4 Hz, 1H), 5.98 (d, J = 8.2 Hz, 1H), 5.08 (q, J = 7.6 Hz, 1H), 2.37 (s, 3H), 1.91 (q, J = 7.1 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 167.30, 142.61, 140.03, 136.00, 134.68, 128.74, 127.36, 126.84, 126.44, 122.13, 120.98, 119.82, 114.34, 111.18, 109.34, 55.25, 29.38, 12.16, 10.99; HRMS (ESI) calcd for C 21 H 22 N 2 O [M + H] + , 319.1805, found, 319.1801. (S)-N-(1-phenylpropyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide ( Y4 ). White solid, mp 226.3℃; yield 57%; [α] D 21 -60.96 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, DMSO- d 6 ) δ12.10 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.35–8.27 (m, 2H), 8.25 (s, 1H), 7.42–7.27 (m, 4H), 7.21 (d, J = 7.5 Hz, 1H), 7.17–7.11 (m, 1H), 4.93 (q, J = 7.9 Hz, 1H), 1.86–1.74 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 164.06, 148.90, 145.07, 143.97, 129.84, 128.71, 128.63, 127.09, 119.17, 117.40, 109.84, 54.50, 29.75, 12.00. HRMS (ESI) calcd for C 17 H 17 N 3 O [M + H] + , 280.1445, found, 280.1448. (R)-N-(1-phenylpropyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide ( Y5 ). White solid, mp 216.6℃; yield 68%; [α] D 21 +60.96 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.10 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.34–8.23 (m, 3H), 7.42–7.28 (m, 4H), 7.21 (d, J = 7.5 Hz, 1H), 7.17–7.11 (m, 1H), 4.93 (q, J = 7.9 Hz, 1H), 1.90–1.71 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 164.09, 148.93, 145.08, 144.00, 129.87, 128.74, 128.66, 127.12, 119.20, 117.43, 109.88, 54.53, 29.77, 12.02. HRMS (ESI) calcd for C 17 H 17 N 3 O [M + H] + , 280.1445, found, 280.1446. (S,E)-N-(1-phenylpropyl)-3-(pyridin-2-yl)acrylamide ( Y6 ). White foam; yield 58%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.60–8.55 (m, 1H), 7.69–7.64 (m, 1H), 7.59 (d, J = 15.2 Hz, 1H), 7.35–7.28 (m, 5H), 7.26–7.19 (m, 2H), 7.01 (d, J = 15.1 Hz, 1H), 6.14 (d, J = 8.4 Hz, 1H), 5.02 (q, J = 7.6 Hz, 1H), 1.91–1.82 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 164.97, 153.31, 150.01, 142.08, 139.76, 136.99, 128.76, 127.47, 126.73, 125.07, 124.74, 123.99, 56.21, 28.58, 10.82. HRMS (ESI) calcd for C 17 H 18 N 2 O [M + H] + , 267.1492, found, 267.1494. (R,E)-N-(1-phenylpropyl)-3-(pyridin-2-yl)acrylamide ( Y7 ). White foam; yield 42%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.57 (d, J = 4.6 Hz, 1H), 7.70–7.63 (m, 1H), 7.59 (d, J = 15.1 Hz, 1H), 7.35–7.21 (m, 7H), 7.01 (d, J = 15.1 Hz, 1H), 6.15 (d, J = 8.6 Hz, 1H), 5.02 (q, J = 7.6 Hz, 1H), 1.91–1.77 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 159.08, 147.40, 144.14, 136.19, 133.91, 131.12, 122.47, 121.59, 120.86, 119.15, 118.89, 118.13, 49.33, 23.40, 4.98. HRMS (ESI) calcd for C 17 H 18 N 2 O [M + H] + , 267.1492, found, 267.1494. (S,E)-3-(2-fluorophenyl)-N-(1-phenylpropyl)acrylamide ( Y8 ). White foam; yield 63%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.68 (d, J = 15.8 Hz, 1H), 7.47–7.40 (m, 1H), 7.33–7.24 (m, 6H), 7.12–7.06 (m, 1H), 7.06–6.99 (m, 1H), 6.59 (d, J = 15.8 Hz, 1H), 6.23 (d, J = 8.4 Hz, 1H), 5.02 (q, J = 7.4 Hz, 1H), 1.93–1.84 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.27, 161.44 (d, J = 253.1 Hz), 142.14, 134.39, 130.99 (d, J = 8.7 Hz), 129.87 (d, J = 3.3 Hz), 128.77, 127.48, 126.85, 124.45 (d, J = 3.6 Hz), 123.81 (d, J = 7.8 Hz), 122.94 (d, J = 11.6 Hz), 116.21 (d, J = 22.0 Hz), 55.30, 29.19, 10.96. HRMS (ESI) calcd for C 18 H 18 FNO [M + H] + , 284.1445, found, 284.1423. (S,E)-3-(2-chlorophenyl)-N-(1-phenylpropyl)acrylamide ( Y9 ). White foam; yield 75%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.98 (d, J = 15.6 Hz, 1H), 7.50 (dd, J = 7.7, 1.8 Hz, 1H), 7.37 − 7.30 (m, 5H), 7.25–7.15 (m, 3H), 6.46 (d, J = 15.6 Hz, 1H), 6.36 (d, J = 8.3 Hz, 1H), 5.00 (q, J = 7.6 Hz, 1H), 1.97–1.73 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 164.94, 142.13, 137.14, 134.85, 133.26, 130.49, 130.22, 128.77, 127.60, 127.50, 126.99, 126.87, 123.75, 55.36, 29.18, 10.99. HRMS (ESI) calcd for C 18 H 18 ClNO [M + H] + , 300.1150, found, 300.1151. (S,E)-N-(1-phenylpropyl)-3-(o-tolyl)acrylamide ( Y10 ). White solid, mp 136.5℃; yield 72%; [α] D 21 -28 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, CDCl 3 ) δ 7.91 (d, J = 15.4 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.35–7.30 (m, 4H), 7.26–7.19 (m, 2H), 7.18–7.09 (m, 2H), 6.35 (d, J = 15.4 Hz, 1H), 6.21 (d, J = 8.3 Hz, 1H), 5.01 (q, J = 7.5 Hz, 1H), 2.37 (s, 3H), 1.93–1.81 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.41, 142.25, 139.15, 137.69, 133.94, 130.83, 129.51, 128.78, 127.48, 126.87, 126.24, 126.12, 121.83, 55.30, 29.22, 19.94, 11.00. HRMS (ESI) calcd for C 19 H 21 NO [M + H] + , 280.1696, found, 280.1695. (S,E)-3-(2-methoxyphenyl)-N-(1-phenylpropyl)acrylamide ( Y11 ). White solid, mp 139.8℃; yield 68%; [α] D 21 -23 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, CDCl 3 ) δ 7.87 (d, J = 15.7 Hz, 1H), 7.44 (dd, J = 7.6, 1.7 Hz, 1H), 7.35–7.31 (m, 4H), 7.31–7.24 (m, 2H), 6.96–6.86 (m, 2H), 6.55 (d, J = 15.7 Hz, 1H), 5.92 (d, J = 8.3 Hz, 1H), 5.04 (q, J = 7.8 Hz, 1H), 3.86 (s, 3H), 1.97–1.80 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.98, 158.36, 142.40, 136.82, 130.80, 129.28, 128.73, 127.39, 126.90, 123.96, 121.83, 120.72, 111.16, 55.50, 55.14, 29.19, 10.95. HRMS (ESI) calcd for C 19 H 21 NO 2 [M + H] + , 296.1645, found, 296.1645. (S,E)-N-(1-phenylpropyl)-3-(3,4,5-trimethoxyphenyl)acrylamide ( Y12 ). White solid, mp 128.7℃; yield 56%; [α] D 21 +42.9 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, CDCl 3 ) δ 7.51 (d, J = 15.5 Hz, 1H), 7.36–7.30 (m, 5H), 6.71 (s, 2H), 6.39 (d, J = 15.5 Hz, 1H), 6.07 (d, J = 8.3 Hz, 1H), 5.01 (q, J = 7.6 Hz, 1H), 3.85 (s, 9H), 1.93–1.83 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.24, 153.44, 142.15, 141.24, 139.49, 130.55, 128.79, 127.51, 126.81, 120.26, 104.89, 61.06, 56.15, 55.21, 29.23, 10.94. HRMS (ESI) calcd for C 21 H 25 NO 4 [M + H] + , 356.1856, found, 356.1857. (S,E)-3-(2-cyanophenyl)-N-(1-phenylpropyl)acrylamide ( Y13 ). White solid, mp 122.8℃; yield 67%; [α] D 21 +14 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, CDCl 3 ) δ 7.70 (s, 1H), 7.65–7.51 (m, 3H), 7.44 (t, J = 7.8 Hz, 1H), 7.33–7.29 (m, 4H), 7.26–7.22 (m, 1H), 6.50 (d, J = 15.6 Hz, 1H), 6.37 (d, J = 8.3 Hz, 1H), 5.00 (q, J = 7.6 Hz, 1H), 1.96–1.81 (m, J = 7.0 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 164.47, 141.98, 138.62, 136.30, 132.65, 132.26, 130.76, 129.84, 128.82, 127.59, 126.81, 123.60, 118.46, 113.7, 55.48, 29.20, 10.93. HRMS (ESI) calcd for C 19 H 18 N 2 O [M + H] + , 291.1492, found, 291.1489. (S,E)-3-(2-nitrophenyl)-N-(1-phenylpropyl)acrylamide ( Y14 ). White solid, mp 172.6℃; yield 73%; 1 H NMR (400 MHz, CDCl 3 ) δ 8.05–7.92 (m, 2H), 7.64–7.44 (m, 3H), 7.37–7.30 (m, 4H), 7.29–7.26 (m, 1H), 6.33 (d, J = 15.5 Hz, 1H), 6.03 (d, J = 8.3 Hz, 1H), 5.01 (q, J = 7.6 Hz, 1H), 2.06–1.80 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 164.23, 148.39, 141.84, 136.32, 133.45, 131.20, 129.87, 129.24, 128.85, 127.63, 126.87, 126.29, 124.96, 55.40, 29.10, 10.90. HRMS (ESI) calcd for C 18 H 18 N 2 O 3 [M + H] + , 311.1390, found, 311.1389. (S,E)-3-(3-nitrophenyl)-N-(1-phenylpropyl)acrylamide ( Y15 ). White solid, mp 171.3℃; yield 54%; [α] D 21 +17.1 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.68 (dd, J = 8.5, 1.9 Hz, 1H), 8.04 (dd, J = 7.9, 1.9 Hz, 1H), 7.85–7.76 (m, 2H), 7.73–7.56 (m, 2H), 7.41–7.19 (m, 5H), 6.72 (dd, J = 15.6, 1.9 Hz, 1H), 4.82 (q, J = 8.4, 7.6 Hz, 1H), 1.84–1.68 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 164.23, 148.39, 141.84, 136.32, 133.45, 131.20, 129.87, 129.24, 128.85, 127.63, 126.87, 126.29, 124.96, 55.45, 28.10, 10.90. HRMS (ESI) calcd for C 18 H 18 N 2 O 3 [M + H] + , 311.1390, found, 311.1392. N-phenethyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide ( Y29 ). White solid, mp 203.4℃; yield 72%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.32–11.92 (m, 1H), 8.44 (dd, J = 8.0, 1.7 Hz, 1H), 8.26 (dd, J = 4.7, 1.7 Hz, 1H), 8.16 (t, J = 5.6 Hz, 1H), 8.11 (d, J = 2.7 Hz, 1H), 7.32–7.23 (m, 4H), 7.22–7.13 (m, 2H), 3.49 (t, J = 6.1 Hz, 2H), 2.84 (t, J = 7.4 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 164.47, 148.89, 143.98, 140.23, 129.78, 129.23, 128.91, 128.39, 126.62, 118.98, 117.41, 110.06, 40.82, 36.11. HRMS (ESI) calcd for C 16 H 15 N 3 O [M + H] + , 266.1288, found, 266.1291. Synthesis of compounds Y3 , Y16 - Y28 . Compounds Y3 , Y16 - Y28 were prepared using General Procedure B. (S,E)-N-(1-phenylpropyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide( Y3 ). White solid, mp 163.7℃; yield 74%; [α] D 21 +75.36 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.10 (s, 1H), 8.35–8.26 (m, 3H), 7.89 (s, 1H), 7.55 (d, J = 15.8 Hz, 1H), 7.39–7.27 (m, 4H), 7.27–7.19 (m, 2H), 6.77 (d, J = 15.8 Hz, 1H), 4.84 (q, J = 7.4 Hz, 1H), 1.80–1.68 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 174.98, 159.08, 153.33, 153.09, 142.18, 140.06, 137.79, 137.71, 136.20, 136.04, 127.05, 126.64, 126.04, 120.51, 63.53, 39.03, 20.53; HRMS (ESI) calcd for C 19 H 19 N 3 O [M + H] + , 306.1601, found, 306.1598. (R,E)-N-(1-phenylpropyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y16 ). White solid, mp 161.2℃; yield 74%; [α] D 21 -75.36 (c 0.1, CH 3 OH); 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.06 (s, 1H), 8.33–8.24 (m, 3H), 7.87 (d, J = 2.3 Hz, 1H), 7.53 (d, J = 15.8 Hz, 1H), 7.35–7.29 (m, 4H), 7.27–7.18 (m, 2H), 6.75 (d, J = 15.8 Hz, 1H), 4.83 (q, J = 7.6 Hz, 1H), 1.80–1.68 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 165.99, 150.08, 144.34, 144.10, 133.19, 131.06, 128.79, 128.72, 127.20, 127.04, 118.05, 117.64, 117.05, 111.51, 54.53, 30.04, 11.53. HRMS (ESI) calcd for C 19 H 19 N 3 O [M + H] + , 306.1601, found, 306.1597. (S,E)-N-(1-phenylethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y17 ). White foam; yield 69%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.10 (s, 1H), 8.37–8.28 (m, 3H), 7.90 (s, 1H), 7.56 (d, J = 15.9 Hz, 1H), 7.39– 7.19 (m, 6H), 6.75 (d, J = 15.9 Hz, 1H), 5.12–5.00 (m, 1H), 1.41 (d, J = 6.9 Hz, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 165.76, 149.78, 145.39, 144.15, 133.26, 131.06, 128.89, 128.74, 127.26, 126.63, 118.10, 117.72, 117.12, 111.58, 48.46, 23.25. HRMS (ESI) calcd for C 18 H 17 N 3 O [M + H] + , 292.1444, found, 291.1440. (E)-N-(2-methylbenzyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y18 ). White solid, mp 173.8℃; yield 48%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.10 (s, 1H), 8.31–8.25 (m, 2H), 8.23 (t, J = 5.7 Hz, 1H), 7.91 (s, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.29–7.24 (m, 1H), 7.22–7.19 (m, 1H), 7.19–7.16 (m, 3H), 6.78 (d, J = 15.9 Hz, 1H), 4.41 (s, 2H), 2.30 (s, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.55, 150.12, 144.12, 137.67, 136.43, 133.32, 131.07, 130.57, 128.74, 128.59, 127.59, 126.39, 117.85, 117.71, 117.10, 111.58, 39.41, 19.20. HRMS (ESI) calcd for C 18 H 17 N 3 O [M + H] + , 292.1444, found, 292.1439. (E)-N-phenethyl-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y19 ). White solid, mp 183..8℃; yield 86%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.06 (s, 1H), 8.30 (d, J = 4.7 Hz, 1H), 8.26 (d, J = 7.9 Hz, 1H), 8.01 (t, J = 5.7 Hz, 1H), 7.90 (s, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.33–7.17 (m, 6H), 6.65 (d, J = 15.8 Hz, 1H), 2.79 (t, J = 7.4 Hz, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.61, 150.06, 144.09, 140.12, 132.98, 130.96, 129.20, 128.91, 128.67, 126.65, 117.97, 117.68, 117.08, 111.48, 39.36, 35.89. HRMS (ESI) calcd for C 18 H 17 N 3 O [M + H] + , 292.1444, found, 292.1455. (E)-N-(2-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y20 ). White solid, mp 205.8℃; yield 63%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.07 (s, 1H), 8.29 (d, J = 4.6 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.88 (s, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.42 (d, J = 6.7 Hz, 1H), 7.35 (d, J = 5.3 Hz, 1H), 7.31–7.17 (m, 3H), 6.62 (d, J = 15.9 Hz, 1H), 2.92 (t, J = 7.2 Hz, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.77, 150.07, 144.14, 137.41, 133.74, 133.11, 131.59, 130.90, 129.83, 128.77, 127.87, 117.91, 117.75, 117.15, 111.50, 49.19, 33.65. HRMS (ESI) calcd for C 18 H 16 Cl [M + H] + , 326.1055, found, 326.1049. (E)-N-(3-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y21 ). White solid, mp 195.6℃; yield 52%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.09 (s, 1H), 8.32–8.23 (m, 2H), 7.98 (t, J = 5.7 Hz, 1H), 7.90 (s, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.37–7.32 (m, 2H), 7.29–7.24 (m, 2H), 7.20 (dd, J = 7.9, 4.7 Hz, 1H), 6.64 (d, J = 15.9 Hz, 1H), 3.44 (t, J = 6.6 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.64, 150.09, 144.10, 139.19, 133.04, 131.37, 131.31, 131.14, 130.98, 128.81, 128.68, 117.93, 117.71, 117.08, 111.50, 40.53, 35.15. HRMS (ESI) calcd for C 18 H 16 Cl [M + H] + , 326.1055, found, 326.1050. (E)-N-(4-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y22 ). White solid, mp 201.2℃; yield 48%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.08 (s, 1H), 8.30 (dd, J = 4.9, 1.5 Hz, 1H), 8.25 (dd, J = 8.2, 1.7 Hz, 1H), 7.98 (t, J = 5.9 Hz, 1H), 7.90 (s, 1H), 7.57 (d, J = 15.8 Hz, 1H), 7.38–7.32 (m, 2H), 7.29–7.25 (m, 2H), 7.20 (dd, J = 8.0, 4.7 Hz, 1H), 6.63 (d, J = 15.7 Hz, 1H), 3.43 (t, J = 6.5 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.64, 150.08, 144.11, 139.19, 133.05, 131.31, 131.14, 131.00, 128.81, 128.68, 117.90, 117.69, 117.09, 111.48, 40.53, 35.13. HRMS (ESI) calcd for C 18 H 16 Cl [M + H] + , 326.1055, found, 326.1049. (E)-N-(2-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y23 ). White solid, mp 223.1℃; yield 63%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.03 (s, 1H), 8.29 (d, J = 4.4 Hz, 1H), 8.25 (d, J = 7.9 Hz, 1H), 7.98 (t, J = 5.8 Hz, 1H), 7.88 (s, 1H), 7.58 (d, J = 15.8 Hz, 1H), 7.34–7.12 (m, 5H), 6.61 (d, J = 15.9 Hz, 1H), 3.44 (q, J = 6.8 Hz, 2H), 2.85 (t, J = 7.1 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.61, 161.32 (d, J = 243.4 Hz), 150.10, 144.08, 132.95, 131.67 (d, J = 4.8 Hz), 130.76, 128.81 (d, J = 8.0 Hz),128.59, 126.78, 126.63, 124.92 (d, J = 3.2 Hz), 117.90 (d, J = 28.7 Hz),117.05, 115.66 (d, J = 21.8 Hz), 111.51,39.39, 29.30. HRMS (ESI) calcd for C 18 H 16 FN 3 O [M + H] + , 310.1350, found, 310.1348. (E)-N-(3-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y24 ). White solid, mp 214.2; yield 67%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.08 (s, 1H), 8.30–8.20 (m, 2H), 7.99 (t, J = 5.7 Hz, 1H), 7.89 (s, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.38–7.29 (m, 1H), 7.20 (dd, J = 7.9, 4.7 Hz, 1H), 7.13–7.06 (m, 2H), 7.06–6.98 (m, 1H), 6.64 (d, J = 15.8 Hz, 1H), 3.46 (t, J = 6.3 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.67, 162.80 (d, J = 243.0 Hz), 150.08, 144.11, 143.15 (d, J = 7.3 Hz), 133.04, 130.94, 130.71 (d, J = 8.3 Hz), 128.67, 125.40 (d, J = 2.7 Hz), 117.93, 117.71, 117.09, 115.91 (d, J = 20.7 Hz), 113.44 (d, J = 20.8 Hz), 111.49, 40.06, 35.48. HRMS (ESI) calcd for C 18 H 16 FN 3 O [M + H] + , 310.1350, found, 310.1346. (E)-N-(4-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y25 ). White solid, mp 232.8℃; yield 72%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.00 (s, 1H), 8.26 (dd, J = 4.7, 1.5 Hz, 1H), 8.22(dd, J = 8.0, 1.6 Hz, 1H), 7.92(t, J = 5.7 Hz, 1H), 7.85 (s, 1H), 7.55 (d, J = 15.9 Hz, 1H), 7.26–7.15 (m, 3H), 7.15–7.00 (m, 2H), 6.61 (d, J = 15.9 Hz, 1H), 3.39 (m, 2H), 2.75 (t, J = 7.2 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.64, 161.43 (d, J = 242.3 Hz), 150.11, 144.13, 136.30, 133.03, 131.05, 130.98, 128.70, 117.99, 117.72, 117.12, 115.68, 115.48, 111.52, 40.80,35.02. HRMS (ESI) calcd for C 18 H 16 FN 3 O [M + H] + , 310.1350, found, 310.1346. (E)-N-(4-methoxyphenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y26 ). White solid, mp 245.1℃; yield 68%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.08 (s, 1H), 8.33–8.23 (m, 2H), 7.95 (t, J = 5.7 Hz, 1H), 7.89 (s, 1H), 7.57 (d, J = 15.9 Hz, 1H), 7.24–7.16 (m, 2H), 7.15 (s, 1H), 6.90–6.81 (m, 2H), 6.65 (d, J = 15.9 Hz, 1H), 3.71 (s, 3H), 2.72 (t, J = 7.3 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.60, 158.25, 150.08, 144.11, 132.93, 131.98, 130.89, 130.16, 128.69, 118.08, 117.72, 117.09, 114.33, 111.53, 55.52, 39.42, 35.03. HRMS (ESI) calcd for C 19 H 19 N 3 O 2 [M + H] + , 322.1550, found, 322.1544. (E)-N-(4-(methylsulfonyl)phenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y27 ). White solid, mp 245.2℃; yield 57%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.66 (s, 1H), 7.88 (dd, J = 4.7, 1.5 Hz, 1H), 7.84 (dd, J = 8.0, 1.6 Hz, 1H), 7.60 (t, J = 5.7 Hz, 1H), 7.50–7.42 (m, 3H), 7.20–7.09 (m, 3H), 6.79 (dd, J = 7.9, 4.7 Hz, 1H), 6.22 (d, J = 15.9 Hz, 1H), 3.08 (dd, J = 6.7, 1.5 Hz, 2H), 2.77 (s, 3H), 2.50 (t, J = 7.0 Hz, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.77, 150.09, 146.51, 144.14, 139.30, 133.16, 130.97, 130.23, 128.68, 127.60, 117.86, 117.73, 117.12, 111.49, 44.17, 39.43, 35.69. HRMS (ESI) calcd for C 19 H 19 N 3 O 3 S [M + H] + , 370.1220, found, 370.1217. (E)-N-(2-cyclohexylethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide ( Y28 ). White solid, mp 241.2℃; yield 56%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.05 (s, 1H), 8.32–8.23 (m, 2H), 7.90–7.79 (m, 2H), 7.54 (d, J = 15.8 Hz, 1H), 7.21 (dd, J = 8.0, 4.7 Hz, 1H), 6.63 (d, J = 15.9 Hz, 1H), 3.22–3.19 (m, 2H), 1.76–1.55 (m, 6H), 1.36 (q, J = 6.8, 2H), 1.23–1.11 (m, 3H), 0.94–0.83 (m, 2H). 13 C NMR (100 MHz, DMSO- d 6 ) δ 166.45, 150.05, 144.09, 132.75, 130.39, 128.69, 118.20, 117.71, 117.07, 111.51, 37.37, 36.76, 35.12, 33.23, 26.69, 26.33. HRMS (ESI) calcd for C 18 H 23 N 3 O [M + H] + , 298.1914, found, 298.1911. Synthesis of compound Y30 . Compound Y30 was prepared using General Procedure C and D. (E)-N-phenethyl-3-(7H-pyrrolo[2,3-d]pyrimidin-5-yl)acrylamide ( Y30 ). White foam; yield 38%; 1 H NMR (400 MHz, CD 3 OD) δ 9.23 (s, 1H), 8.80 (s, 1H), 7.80 (s, 1H), 7.68 (d, J = 15.8 Hz, 1H), 7.33–7.23 (m, 4H), 7.22–7.16 (m, 1H), 6.65 (d, J = 15.8 Hz, 1H), 3.59–3.53 (m, 2H), 2.88 (t, J = 7.3 Hz, 2H). 13 C NMR (100 MHz, CD 3 OD) δ 167.77, 152.82, 151.26, 148.32, 139.21, 132.20, 130.84, 128.50, 128.22, 126.07, 118.77, 116.24, 112.00, 40.90, 35.33. HRMS (ESI) calcd for C 17 H 16 N 4 O [M + H] + , 293.1397, found, 293.1395. Cell culture and Stimulation J774A.1 cells (Guangzhou JennioBiotechCo, China) were cultured with medium and 10% FBS (Gibco, USA) in a humidified atmosphere of 5% CO 2 at 37°C. BMDMs were obtained from the myelin sheaths of mouse tibia and femur, and were cultured with a culture medium consisting of 10% FBS, RPMI-1640 medium, 1 mM double antibody, 1 mM glutamine and 0.1 mM-CSF. For NLRP3 inflammasome activation, cells were stimulated with 1 µg/mL of LPS (Sigma-Aldrich, USA) for 5 h, treated with compounds for 1 h, followed by stimulation with Nigericin (Invitrogen, USA) for 1 h. For NLRC4 or AIM2 inflammatory activation, BMDMs were stimulated with 500 ng/mL of LPS for 3 h, treated with compounds for 1 h, followed by stimulation with 25 µg/mL FLA-ST Ultrapure (Invitrogen, USA) or a mixture of 2.5 µg/mL Poly dA:dT (Invitrogen, USA) and Lipo 3000(Invitrogen, USA) for 14 h of stimulation. LDH Release Assay J774A.1 cells were plated on 96-well plates, and the cells were stimulated with 1 µg/mL of LPS for 5 h, treated with Y19 for 1 h, and then stimulated with nigericin for 1 h. Supernatants were collected and LDH activity was measured using the LDH Assay Kit (Beyotime, China). Enzyme-linked Immunosorbent Assay IL-1β (Invitrogen, USA) or TNF-α (Invitrogen, USA) in cell cultures or animal colon homogenates supernatants were analyzed by ELISA kits. Western Blotting Analysis Cells were lysed in RIPA buffer (Beyotime, China) containing protease inhibitors for 0.5 h at 4°C, after which the lysate was collected and the protein concentration was determined by the BCA Protein Assay Kit (23227, Thermo) at 562 nm. The Protein samples were separated by electrophoresis on sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, which were closed with 5% skimmed milk solution for 1 h at room temperature and incubated with primary antibody for 12 h. Subsequently, the membranes were lubricated by TBST and then treated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Finally, the membranes were incubated with ECL protein blotting reagent (ThermoFisher Scientific, USA) and visualized by a gel imaging system. The above primary antibodies were anti-mouse IL-1β (AF-401-NA, R&D Systems), anti-mouse caspase-1 (AG-20B0042, Adipogen), anti-NLRP3 (15100S, Cell Signaling Technology), anti-ASC (67824S, Cell Signaling Technolology), anti-NEK7 (ab133514, Abcam), anti-phosphorylated NF-κB (3033T, Cell Signaling Technologies), anti-NF-κB (8242T, Cell Signaling Technologies), anti-phospho-IκBα (2859T, Cell Signaling Technologies) NLRP3 Oligomerization Assay J774A.1 cells plated on 6-well plates were grouped, modelled and given drug administration according to the LPS-induced NLRP3 inflammasome activation step. Cells were lysed with cell lysate for 30 min and then the lysate was collected and centrifuged at 12,000 rpm for 5 min at 4°C, after which the lysate was quantified by BCA protein quantification. The proteins were processed by agarose gel electrophoresis and protein immunoblotting. After the proteins had been fully transferred to the PVDF membrane, the membrane was closed with skimmed milk for 1 h. The whole membrane was incubated with NLRP3 primary antibody at 4°C overnight, and the secondary antibody was applied and developed the next day. ASC Oligomerization Assay J774A.1 cells were plated on six large plates and treated following the steps for LPS-induced NLRP3 inflammasome activation. Cells were lysed with cell lysate for 30 min and then centrifuged at 12,000 rpm for 5 min at 4°C to collect the lysate, which was subsequently quantified by BCA protein quantification. The cell pellet was washed twice with pre-cooled PBS and 4 mM of disuccinimidyl suberate dissolved in DMSO was added to the cell pellet and the mixture was crosslinked for 30 min at room temperature before centrifugation at 8,000 rpm for 5 min at 4°C. The supernatant was discarded and the residual disuccinimidyl suberate was neutralized by the addition of 4 mM Tris and left at room temperature for 20 min. Centrifuge at 8,000 rpm for 10 min at 4°C, discard the supernatant, add 20 µL of 2X Sample Buffer directly to the tube, and boil for 10 min at 100°C on a metal bath before analyzing the samples by immunoblotting. Detection of ASC specks BMDMs were treated with 500 ng/mL LPS (Sigma-Aldrich, USA) for 3 h, treated with Y19 for 1 h and stimulated with 5 µM nigericin (Invitrogen, USA) for 30 min. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After blocking with 1% BSA for 1 h, the cells were stained with cells were stained with ASC antibodies overnight at 4°C. After washed three times with PBS, the cells were incubated with the rhodamine-labelled anti-rabbit IgG antibody (A11008, Invitrogen) for 90 min at room temperature. Cell nuclei were stained with DAPI (2 µM) for 30 min and washed three times with PBS. Images were captured using a fluorescence microscope (DM4000 BLED, Leica, Germany). Co-immunoprecipitation Assay J774A.1 cells were plated on six large plates and treated according to the steps of LPS-induced NLRP3 inflammasome activation. The cells were lysed with IP lysis buffer containing protease inhibitors and collected, centrifuged at 4°C for 10 min at 10,000 g. The supernatant was collected and 20 µL of protein A/G PLUS-beads (sc-2003, Santa Cruz) were added and incubated for 12 h at 4°C in a shaker. The supernatant was collected by centrifugation at 2800 rpm for 10 min at 4°C. The primary antibody (an equal amount of normal IgG as negative control) was added and incubated at 4°C for 8 h. Afterwards, 20 µL of protein A/G PLUS-beads were added and the mixture was incubated overnight at 4°C in a shaker. The immune complexes were washed with PBS and protein-containing additives were added. The mixture was boiled for 10 min and then subjected to immunoblotting. Animal Experiments. All animal experiments were conducted in accordance with the Guidelines for the Keeping and Use of Laboratory Animals and approved by the Ethics Committee of Guangzhou Medical University (GY2022-045). The animal studies were reported in accordance with the ARRIVE guidelines. All male C57BL/6 mice (8 weeks, 6 mice per group) were kept at room temperature with free access to food and water. The blank group received water treatment for 7 days. Ulcerative colitis was induced by the addition of 2.5% DSS to distilled water for 7 consecutive days. DSS was added to distilled water for 7 consecutive days to induce ulcerative colitis. Y19 (5, 10 and 20 mg/kg) and Tofacitinib (10 mg/kg) were administered by gavage on days 1, 3, 5 and 7 respectively. Equal volume of solution was given by gavage in blank and DSS treated groups. Mice were executed on day 8 after induction of colitis. Body weight and fecal occult blood were monitored daily. The colon was scored microscopically and biochemical tests were performed on colon specimens. Statistical Analysis. The data are expressed as mean ± s.e.m. Statistical analysis preformed was an unpaired, two-tailed Student’s t test using GraphPad Prism software. The accepted levels of significance were *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: not significant. Abbreviations AIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a CARD protein; DCM, dichloromethane; DMF, N, N-dimethylformamide; DSS, dextran sulfate sodium; NLRC4, NOD-like receptor family CARD-containing 4; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; Tofa., Tofacitinib. Declarations Conflicts of interest The authors declare no competing financial interests. Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC) (NO. 81803364 to Z. Yang). 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Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformation.docx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 20 Aug, 2025 Read the published version in Molecular Diversity → Version 1 posted Editorial decision: Revision requested 22 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviews received at journal 11 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 26 Mar, 2025 Submission checks completed at journal 26 Mar, 2025 First submitted to journal 25 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6307163","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442159115,"identity":"03a731e4-fb7a-49e4-9a85-666f20202cf2","order_by":0,"name":"Yuyun Yan","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuyun","middleName":"","lastName":"Yan","suffix":""},{"id":442159116,"identity":"97634218-1da9-4572-a06c-5c83b4207bb2","order_by":1,"name":"Xiuxiu Zhang","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiuxiu","middleName":"","lastName":"Zhang","suffix":""},{"id":442159117,"identity":"f0417990-7fb4-4263-9062-3799c246dd32","order_by":2,"name":"Ruiwen Wu","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruiwen","middleName":"","lastName":"Wu","suffix":""},{"id":442159118,"identity":"64b9b2aa-7351-4f48-a0c1-42e7488840ec","order_by":3,"name":"Xiangting Liang","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiangting","middleName":"","lastName":"Liang","suffix":""},{"id":442159119,"identity":"a3e9c465-6116-4986-963f-af9ce779ce21","order_by":4,"name":"Yiming Luo","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiming","middleName":"","lastName":"Luo","suffix":""},{"id":442159120,"identity":"e17b0246-e187-4148-9a44-58b85e1f05ab","order_by":5,"name":"Jie Yang","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yang","suffix":""},{"id":442159121,"identity":"a2789eb0-b8d6-4102-afca-18937dad25b7","order_by":6,"name":"Dan Wu","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wu","suffix":""},{"id":442159122,"identity":"e63399db-94dd-478f-a28e-50c8a8e22b35","order_by":7,"name":"Geng Lin","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Geng","middleName":"","lastName":"Lin","suffix":""},{"id":442159123,"identity":"dcb7a83d-6575-45de-bb0a-5cb3e0a25646","order_by":8,"name":"Ping Sun","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Sun","suffix":""},{"id":442159124,"identity":"d27c251c-2846-4118-a242-5868089c4402","order_by":9,"name":"Wenhui Hu","email":"","orcid":"","institution":"Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenhui","middleName":"","lastName":"Hu","suffix":""},{"id":442159125,"identity":"322223f3-9d24-4569-8418-f9e654846891","order_by":10,"name":"Zhongjin Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYHCCxIcfKiTk5EnRkmwsccbC2LCBBC1sErxtFYkMB4hVb3D+wAMJyXkSCYwNzA8f3SBKy4EDCQaF2yTy2BnYjI1ziNJysCEhQXKbRDFjAw+bNHFaDjMkHOCdI5HYcIBoLccYEht4G0jRInmGIZlZ4piEsWEzsX7hO38m/eeHmjo5efbmh4+J0qJwgCcBwmImRjkIyDewHyBW7SgYBaNgFIxUAAC65jC5pJ3guAAAAABJRU5ErkJggg==","orcid":"","institution":"Tianjin University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhongjin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-03-25 23:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6307163/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6307163/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11030-025-11316-1","type":"published","date":"2025-08-20T16:30:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81695173,"identity":"4d926cb3-e8bd-4377-a50c-a14daa305b77","added_by":"auto","created_at":"2025-04-30 11:56:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":159008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eY19 inhibits the activation of inflammasomes.\u003c/strong\u003e (A-C) LPS-primed J774A.1 cells were treated with various doses of Y19 for 1 h and stimulated with nigericin (A) or SiO\u003csub\u003e2 \u003c/sub\u003e(B) or MSU(C) for another 1 h, and then supernatants were analyzed by ELISA for IL-1β; (D-F) ELISA of IL-1β in supernatants from LPS-primed BMDMs treated with Y19 for 1 h and then stimulated with nigericin (D) or poly (dA/dT) (E) or FLA-ST Ultrapure (F). The p-values referred to the comparison between the negative control and indicated compound treatments and were analyzed using an unpaired, two-tailed Student’s t-test, ns: not significant, *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage66.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/ec9a49b5967bdce05b1e4064.png"},{"id":81695214,"identity":"3e4f2709-e2c7-4b1f-825a-dbba5d71305d","added_by":"auto","created_at":"2025-04-30 11:56:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":144082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eY19 inhibits NLRP3-mediated apoptosis. \u003c/strong\u003e(A, B) LPS-primed J774A.1 cells were treated with various doses of Y19 for 1 h, and then supernatants were analyzed by ELISA for TNF-α (A) or Protein levels of p65, phosphorylated p65, IκBα, phosphorylated IκBα were analyzed by western blotting (B); (C, D) LPS-primed J774A.1 cells were pretreated for 1 h with Y19 (3 and 9 μM), followed by stimulation with nigericin.(C) Immunoblot analysis of mature IL-1β and activated caspase-1 in the supernatant and pro-IL-1β, ASC, NLRP3 and GSDMD expression in lysates; (D) Cell death was evaluated by detecting LDH release in the cell supernatants (n=5).\u003c/p\u003e","description":"","filename":"floatimage67.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/8cd939e091d7915b3b7583ab.png"},{"id":81695186,"identity":"75f6638e-cf25-4d92-9668-4e41043c2388","added_by":"auto","created_at":"2025-04-30 11:56:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eY19 blocks the assembly of NLRP3 inflammasome. \u003c/strong\u003e(A-D) LPS-primed J774A.1 cells were pretreated for 1 h with Y19 (3 and 9 μM), followed by stimulation with nigericin. (A) Co-IP and immunoblot analysis of the NEK7-NLRP3 interaction; (B) Immunoblot analysis by the native PAGE or SDS-PAGE; (C) Co-IP and western blotting of ASC-NLRP3 interaction; (D) Immunoblot analysis of ASC oligomerization; (E, F) Y19 inhibits the formation of ASC specks. (E) ASC-pyroptosome immunofluorescence analysis (Merge: green for ASC and blue for DAPI) of BMDM cells stimulated with LPS/nigericin, with or without Y19 (3 and 9 μM) pretreatment for 1 h. (F) Statistical plot of the proportion of cells with ASC specks.\u003c/p\u003e","description":"","filename":"floatimage68.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/3e9d0dd061363d804c5bc6a8.png"},{"id":81695169,"identity":"4f710818-430c-411d-9d0d-7a9933e87f94","added_by":"auto","created_at":"2025-04-30 11:56:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":131256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eY19 ameliorates DSS-induced colon shortening in mice. \u003c/strong\u003eC57BL/6 mice were fed with drinking water for 3 days and administrated with 2.5% DSS for another 7 days. Y19 (5, 10, and 20 mg/kg) and Tofacitinib (10 mg/kg) was administrated orally on days 1, 3, 5 and 7. (A) Body weight measurements were performed daily on different groups of mice and expressed as a percentage change in body weight (n = 6 per group); (B) The feces of mice were collected daily, and the occult blood of mice was measured by using the fecal occult blood kit to express the blood degree score (n = 6 per group); (C) Representative photographs of the colon lengths; (D) Colon length was measured (n = 6 per group);\u003c/p\u003e","description":"","filename":"floatimage69.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/67f67bf276947aaa5d7eed94.png"},{"id":81695183,"identity":"7208be5a-ae76-467e-b27d-fe1d2bcab7e2","added_by":"auto","created_at":"2025-04-30 11:56:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":240202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Y19 shows protective effects on the mice with DSS-induced ulcerative colitis.\u003c/strong\u003e (A) The protein levels of IL-1β in colon homogenates were determined by ELISA; (B) Western blotting analysis of NLRP3, IL-1β, and in colon; (C) Colitis score of each mouse in different groups; (D) Serial sections of paraffin-embedded colon tissue were stained with hematoxylin and eosin. Results shown are from 1 of 6 representative paraffin specimens studied (n = 6). Magnification: × 200. Statistics were analyzed by unpaired, two-tailed Student’s t-test, ns: not significant, *p \u0026lt; 0.05, and **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage70.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/e83b9f9d0210dca9a65647ea.png"},{"id":81695179,"identity":"7bb75f40-20da-4505-806a-c94b5efbdf0c","added_by":"auto","created_at":"2025-04-30 11:56:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":316515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Chemical Synthesis of the Designed Analogues\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eReagents and conditions: (a) HATU, DCM, TEA 0℃ to r.t; (b) EDCI-HCl, HOBT, DMF, DIPEA, 0℃ to r.t; (c) Pd(OAc)\u003csub\u003e2\u003c/sub\u003e, CuI, DMF, TEA, 70℃; (d) \u0026nbsp;LiOH, THF, H\u003csub\u003e2\u003c/sub\u003eO, 1,4-Dioxane, r.t.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/22371271d794e27df53532bb.png"},{"id":89847575,"identity":"6b19a8d6-fe7e-4287-aef8-ec6e96d7488e","added_by":"auto","created_at":"2025-08-25 16:43:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2331478,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/68517624-a147-473d-b624-dfc36bcc288d.pdf"},{"id":81695761,"identity":"bb1a0a17-8a38-459e-a5fc-b634e857b792","added_by":"auto","created_at":"2025-04-30 12:04:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1840965,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/41cf1c4a2d6d1b3b0d3466bf.docx"},{"id":81695198,"identity":"483862f0-a4a1-4e4e-a6c7-ef0a26932cce","added_by":"auto","created_at":"2025-04-30 11:56:25","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":113252,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6307163/v1/1124a6c224264dccb73a6515.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of 7-azaindole Inhibitors of inflammasomes/IL-1β for the Treatment of Inflammatory Bowel Disease","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammatory bowel disease (IBD) has gradually become a global disease that afflicts millions of people today[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. IBD, comprising Crohn's disease (CD), ulcerative colitis (UC) and IBD unclassified, has yet no exact known cause [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and is a kind of chronic debilitating inflammatory gastrointestinal disorder with complex pathological and clinical features, such as mucosal barrier defects, immune response dysregulation, and even autoimmunity and syndromic features in other organs[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Patients with IBD exhibit heterogeneity that may diversify clinical phenotypes and limit therapeutic success[\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, there is still no cure for IBD. The medical treatment for IBD aims to relieve symptoms and reduce inflammation, such as classic anti-inflammatory drugs (5-aminosalicylates and corticosteroids), immunosuppressants (thiopurines and ciclosporin-A), and biologic agents (anti-TNF-α and anti\u0026ndash;interleukin-12/23)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Novel biologics are generally indicated for patients with moderately to severely active IBD who are unresponsive to classic anti-inflammatory drugs and immunosuppressants, who are glucocorticoids-dependent, or who have unacceptable side effects from these drugs[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Even so, there is still a large proportion of patients who do not respond to biologic treatments[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For example, approximately one third of CD patients either have no initial response or stop response to anti\u0026ndash;TNF-α biologic agents[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Targeting Janus kinase (JAK) may be the new approach for IBD patients with conventional treatment failure[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Tofacitinib, an oral pan-JAK inhibitor, was approved for patients with UC who have no response to conventional or anti\u0026ndash;TNF-α therapy, but did not exhibit positive benefit for CD patients[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Upadacitinib is the selective JAK1 inhibitor currently approved for the treatment of both UC and CD[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, safety concerns have led to broad regulatory restrictions on the use of JAK inhibitors. The long-term use of high doses of JAK inhibitors may increase the risk of major adverse cardiovascular events, venous thromboembolism, serious infections and cancer[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, increasing evidence supports that inflammasome/IL-1\u003cem\u003eβ\u003c/em\u003e pathway plays a vital role in IBD pathogenesis. Deregulated activation of inflammasome exacerbates inflammation through secretion of pro-inflammation factors IL-1β and IL-18, which is able to amplify immune responses[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. IL-1α/IL-1β is key upstream cytokines that regulate IL-23 expression in monocytes, which is also significant mediators of intestinal inflammation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The inflammatory monocytes and CX3CR1\u003csup\u003e+\u003c/sup\u003eIL-1β\u003csup\u003e+\u003c/sup\u003e macrophages may be the primary sources of IL-23 during inflammasome activation and IL-1 production in the inflamed intestine[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Overactivation of inflammasomes leads to dysregulation of the neutrophil-dominated microenvironment with higher levels of TNF-α, IL-1β, IL-1α, IL-8, IL-12, IL-15, IL-17, IL-23, and IL-36[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. IL-1β helps to prolong the survival of T cells and the proliferation of B cells to enhance the production of antibodies, to induce a potent chemokine response, and to drive the neutrophil infiltrates, inflammatory fibroblast activation and epithelial cell loss in ulcerated intestinal tissue[\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Importantly, IL-1 has been identified as the critical signal driving stromal-neutrophil interactions, as observed in a subset of IBD patients who fail to respond to anti-TNF and other therapies[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These suggest that the blockade of inflammasomes/IL-1β pathway is also a promising strategy for the treatment of IBD.\u003c/p\u003e \u003cp\u003eHerein, we describe the identification of the 7-azaindole inhibitor \u003cb\u003eY19\u003c/b\u003e that can inhibit IL-1β secretion mediated by different inflammasomes. In vivo pharmacological models of colitis have shown that \u003cb\u003eY19\u003c/b\u003e alleviates uncontrolled inflammation via anti-IL-1β effect.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Discovery and synthesis of lead compounds.\u003c/h2\u003e\n \u003cp\u003eA cell screening model based on the activation steps of the NLRP3 pathway was established. [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. This model initiates with lipopolysaccharide (LPS)-primed mouse macrophage line J774A.1, which activates NF-\u0026kappa;B to produce resting NLRP3 and pro-IL-1\u0026beta;. Subsequently, the cells are treated with our compounds, followed by stimulation with nigericin, and finally NLRP3-mediated IL-1\u0026beta; levels are detected using enzyme-linked immunosorbent assay (ELISA). We identified our in-house compound \u003cstrong\u003e1\u003c/strong\u003e (see Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), which effectively inhibits NLRP3-mediated IL-1\u0026beta; secretion with an IC\u003csub\u003e50\u003c/sub\u003e value of 35.7 \u0026micro;M. To further investigate the structure-activity relationship (SAR) and obtain more potent leads, we designed and synthesized a diverse array of analogues.\u003c/p\u003e\n \u003cp\u003eScheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e briefly describes the synthesis of all compounds. In Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, carboxylic acids with different structures (\u003cstrong\u003eA1\u003c/strong\u003e, \u003cstrong\u003eA2\u003c/strong\u003e, \u003cstrong\u003eA4-A15\u003c/strong\u003e) were coupled with the corresponding amines directly in the presence of HATU and TEA to give \u003cstrong\u003eY1\u003c/strong\u003e, \u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e4\u0026ndash;15\u003c/strong\u003e, and \u003cstrong\u003e29\u003c/strong\u003e. As shown in Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the synthesis started with known compound \u003cstrong\u003eB1\u003c/strong\u003e, which was readily obtained according to a reported procedure [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cstrong\u003eB1\u003c/strong\u003e was coupled with the corresponding amines in the presence of EDCI, HOBt and DIPEA to give \u003cstrong\u003eY3\u003c/strong\u003e, \u003cstrong\u003e16\u0026ndash;28\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. In Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, according to the literature, \u0026alpha;, \u0026beta;-unsaturated amide \u003cstrong\u003eC1\u003c/strong\u003e was obtained through the acylation of phenethylamine with acryloyl chloride [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], and \u003cstrong\u003eC2\u003c/strong\u003e was produced through the protection of 5-iodo-7H-pyrrolo[2,3-d]pyrimidine with 4-methylbenzenesulfonyl chloride [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. \u003cstrong\u003eC2\u003c/strong\u003e was subjected to a Heck reaction with \u003cstrong\u003eC1\u003c/strong\u003e in the presence of CuI and Pd(OAc)\u003csub\u003e2\u003c/sub\u003e, and then Ts was removed to give \u003cstrong\u003eY30\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. SAR Analysis.\u003c/h2\u003e\n \u003cp\u003eThe indole analogue \u003cstrong\u003e1\u003c/strong\u003e shows anti-NLRP3/IL-1\u0026beta; activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 35.7 \u0026micro;M. It has a molecular weight of 394.2, and its structure mainly includes the indole core, 1-methyl, 2-phenyl, and 3-\u0026alpha;, \u0026beta;-unsaturated amide. Firstly, we investigated its preliminary structure\u0026thinsp;\u0026minus;\u0026thinsp;activity relationships (SAR) to determine the impact of molecular fragment on anti- NLRP3/IL-1\u0026beta; activation. As shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the substitution of the phenyl group with a hydrogen atom (\u003cstrong\u003eY1\u003c/strong\u003e) exhibited a positive influence on activity. The relocation of the methyl group from the 1-position to the 2-position led to the exposure of an N-H moiety at the 1-position (\u003cstrong\u003eY2\u003c/strong\u003e), thereby significantly enhancing activity. Moreover, the replacement of the carbon atom at the 7-position with a nitrogen atom, along with the substitution of 2-methyl with a hydrogen atom (\u003cstrong\u003eY3\u003c/strong\u003e), dramatically increased the inhibitory potency with an IC\u003csub\u003e50\u003c/sub\u003e value of 5.67 \u0026micro;M. However, the removal of 3-\u0026alpha;, \u0026beta;-unsaturated double bond (\u003cstrong\u003eY4\u003c/strong\u003e and \u003cstrong\u003eY5\u003c/strong\u003e) resulted in a complete loss in the inhibitory effect on IL-1\u0026beta;. These findings indicate that the \u0026alpha;, \u0026beta;-unsaturated double bond is essential for inhibitory activity towards NLRP3 mediated IL-1\u0026beta;.\u003c/p\u003e\n \u003cp\u003eOn the basis of the above analysis, we firstly chose 7-azaindole moiety as a region for further modification. The azaindole was replaced with a range of different substituted aromatic rings, such as pyridine (\u003cstrong\u003eY6\u003c/strong\u003e and \u003cstrong\u003eY7\u003c/strong\u003e) and substituted phenyl groups (\u003cstrong\u003eY8\u003c/strong\u003e\u0026ndash;\u003cstrong\u003eY15\u003c/strong\u003e). Among them, he majority of analogs exhibited a loss in potency, and only \u003cstrong\u003eY 8\u003c/strong\u003e, \u003cstrong\u003eY9\u003c/strong\u003e, and \u003cstrong\u003eY14\u003c/strong\u003e demonstrated low inhibitory activities. These results indicated that 7-azaindole core is important for anti-IL-1\u0026beta; activity. Thus, we directed our focus towards optimizing the \u0026alpha;-ethylbenzylamine moiety. The shift from \u003cem\u003eS\u003c/em\u003e-\u0026alpha;-ethylbenzylamine to \u003cem\u003eR\u003c/em\u003e-configuration (\u003cstrong\u003eY16\u003c/strong\u003e) showed a slight increase in activity. Substitution with \u0026alpha;-methylbenzylamine (\u003cstrong\u003eY17\u003c/strong\u003e) or 2-methylbenzylamine (\u003cstrong\u003eY18\u003c/strong\u003e) to \u0026alpha;-ethylbenzylamine exhibited the reduced activity, whereas the phenylethylamine moiety (\u003cstrong\u003eY19\u003c/strong\u003e) showed a remarkable improvement with an IC\u003csub\u003e50\u003c/sub\u003e value of 1.26 \u0026micro;M. Subsequently, we investigated the effect of differently substituted phenethylamines on the activity. Unfortunately, all these compounds significantly reduced the activity. For example, about 4 to 6-fold less activity was observed for chlorine- and fluorine-substituted phenethylamines (\u003cstrong\u003eY20\u003c/strong\u003e, \u003cstrong\u003eY21\u003c/strong\u003e, \u003cstrong\u003eY22\u003c/strong\u003e, \u003cstrong\u003eY23\u003c/strong\u003e, \u003cstrong\u003eY24\u003c/strong\u003e, and \u003cstrong\u003eY25\u003c/strong\u003e). A similar result was also observed with electron-donating group methoxy substituted phenylethylamine (\u003cstrong\u003eY26\u003c/strong\u003e). The introduction of the water-soluble and electron-withdrawing methylsulfone group (\u003cstrong\u003eY27\u003c/strong\u003e) leads to a complete loss of activity. When phenylethylamine moiety was converted to cyclohexylethylamine (\u003cstrong\u003eY28\u003c/strong\u003e), the inhibitory activity was also reduced by more than 10-fold. The removal of \u0026alpha;, \u0026beta;-unsaturated double bond at 3-position (\u003cstrong\u003eY29\u003c/strong\u003e) or substitution of carbon atom by a nitrogen atom at 5-position (\u003cstrong\u003eY30\u003c/strong\u003e) of \u003cstrong\u003eY19\u003c/strong\u003e results in the loss of activity, further indicating the importance of \u0026alpha;, \u0026beta;-unsaturated double bond and azaindole core. Therefore, we selected the most active \u003cstrong\u003eY 19\u003c/strong\u003e to further study its mechanistic studies in NLRP3 pathway.\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e2.3. Inhibitory effect of Y19 on inflammasomes.\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003cp\u003eIL-1\u0026beta; is secreted in response to the activation of various inflammasomes, including NLRP3, AIM2, and NLRC4. Initially, we assessed the inhibitory effect of \u003cstrong\u003eY19\u003c/strong\u003e on the NLRP3 pathway stimulated by different agents in J774A.1 cells. The data demonstrated that treatment with \u003cstrong\u003eY19\u003c/strong\u003e effectively inhibited IL-1\u0026beta; release mediated by NLRP3 activation in a dose-dependent manner upon stimulation with nigericin, SiO2, and MSU (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, B \u0026amp; C).. Subsequently, we investigated whether \u003cstrong\u003eY19\u003c/strong\u003e could inhibit IL-1\u0026beta; release from different inflammasomes. Bone marrow-derived macrophages (BMDMs) were primed with LPS prior to treatment with \u003cstrong\u003eY19\u003c/strong\u003e; thereafter, the cells were stimulated with nigericin to activate the NLRP3 inflammasome (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD), Poly(dA:dT) to activate the AIM2 inflammasome (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE), and FLA-ST Ultrapure to activate the NLRC4 inflammasome (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Our results indicated that \u003cstrong\u003eY19\u003c/strong\u003e is able to inhibit the secretion of IL-1\u0026beta; upon activation of all the NLRP3, AIM2 and NLRC4 inflammasomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Biological Mechanism Study.\u003c/h2\u003e\n \u003cp\u003eIn order to confirm the effect of \u003cstrong\u003eY19\u003c/strong\u003e on NLRP3 pathway, its cytotoxicity was firstly evaluated. Y19 did not show significant toxicity against HEK293T and J774A.1 cells after 2 h of treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eS).\u003c/p\u003e\n \u003cp\u003eOf all inflammasomes, NLRP3 is the most extensively studied to date. The NLRP3 pathway consists of two stages: first, NF-\u0026kappa;B activation upregulates the transcriptional levels of both NLRP3 and pro-interleukin-1\u0026beta; (pro-IL-1\u0026beta;); second, subsequent assembly and activation of the NLRP3 inflammasome induce caspase-1-dependent secretion of IL-1\u0026beta; and cell pyroptosis, ultimately leading to inflammation[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we primarily investigated the mechanism of action of compound \u003cstrong\u003eY19\u003c/strong\u003e. As illustrated in Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; B, \u003cstrong\u003eY19\u003c/strong\u003e did not impair LPS-induced tumor necrosis factor-alpha (TNF-\u0026alpha;) secretion as measured by ELISA or NF-\u0026kappa;B signaling activation assessed through Western blot analysis. The levels of caspase-1 (p20, an auto-cleaved fragment of caspase-1) was dose-dependently decreased in supernatants from \u003cstrong\u003eY19\u003c/strong\u003e-treated J774A.1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating that \u003cstrong\u003eY19\u003c/strong\u003e suppresses the activation of caspase-1 via the NLRP3 inflammasome. Correspondingly, the formation of NT-GSDMD (N-terminal domain of GSDMD) and lactate dehydrogenase (LDH) release was inhibited by \u003cstrong\u003eY19\u003c/strong\u003e (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC \u0026amp; D). These findings enable us to speculate on the role of \u003cstrong\u003eY19\u003c/strong\u003e in the assembly of the NLRP3 inflammasome, a critical event within the NLRP3 signaling pathway. Consequently, we investigated protein-protein interactions related to NLRP3 inflammasome assembly, including NEK7-NLRP3, NLRP3-NLRP3, NLRP3-ASC, and ASC-ASC interactions. The formation of the NLRP3\u0026ndash;NEK7 complex is essential for initiating NLRP3 inflammasome assembly. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, Y19 inhibited the interaction between NEK7 and NLRP3. Next, we assessed whether \u003cstrong\u003eY19\u003c/strong\u003e could obstruct direct interactions between NLRP3\u0026ndash;NLRP3 or NLRP3\u0026ndash;ASC. Co-immunoprecipitation assays demonstrated that \u003cstrong\u003eY19\u003c/strong\u003e reduces oligomerization of NLRP3 similarly to tranilast (the positive control) as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and significantly alters the interaction between NLRP3 and ASC (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). We also evaluated ASC-ASC interactions, and our results indicated that \u003cstrong\u003eY19\u003c/strong\u003e prevents ASC oligomerization induced by NLRP3 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Since ASC specks are indicative of activated NLRP3 inflammasomes, we examined their formation through immunofluorescence analysis. As demonstrated by Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026amp;F, treatment with \u003cstrong\u003eY19\u003c/strong\u003e exhibited the production of ASC specks. Together these results indicate that \u003cstrong\u003eY19\u003c/strong\u003e prevents the NLRP3 inflammasome activation through the disruption of NLRP3 inflammasome assembly.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. In Vivo Efficacy of Y19.\u003c/h2\u003e\n \u003cp\u003eDextrose sodium sulfate (DSS)-induced colitis has been demonstrated to be dependent on the NLRP3 inflammasome in murine models[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Given that NLRP3 pathway plays a key role in human inflammatory bowel disease (IBD) and especially in non-response or poor response to current drugs in severe IBD, we aimed to investigate the potential of \u003cstrong\u003eY19\u003c/strong\u003e as a therapeutic agent against IBD. Tofacitinib, a highly effective JAK inhibitor known for its efficacy in treating severe colitis unresponsive to current medications, was selected as the positive control.\u003c/p\u003e\n \u003cp\u003eA mouse model of colitis was established by administering water containing 2.5% dextran sulfate sodium (DSS) for 7 days. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026thinsp;\u0026minus;\u0026thinsp;D, mice with colitis exhibited significant body weight loss, reduced colon length, and increased fecal occult blood levels. In our experimental models, the positive drug tofacitinib and \u003cstrong\u003eY19\u003c/strong\u003e displayed no improvement of bodyweight loss and hemafecia (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026amp;B), however, they significantly increased colon length (Figs. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC \u0026amp; D). Notably, Notably, \u003cstrong\u003eY19\u003c/strong\u003e attenuated colon shortening in a dose-dependent manner. This alleviation was also reflected by reductions in tissue-associated inflammatory factor IL-1\u0026beta; levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Surprisingly, while the positive drug tofacitinib showed suppression of IL-1\u0026beta; expression in vivo, our ELISA data indicated that it did not inhibit IL-1\u0026beta; release when assessed in vitro (Figure S2). This discrepancy suggests that the observed anti-IL-1\u0026beta; activity may differ between vitro and vivo conditions due to complex interactions between JAK and IL-1\u0026beta; pathways within an organism. Next, histological and pathological evaluations using hematoxylin and eosin staining were conducted to assess intestinal mucosal damage. Mice administering DSS showed severe mucosal damage, such as crypt architecture, goblet cell depletion and inflammatory cell infiltration. After treatment with \u003cstrong\u003eY19\u003c/strong\u003e, especially at dose of 10 mg/kg and 20 mg/kg, these damages were significantly improved (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026amp;D). Furthermore, we investigated the effect of \u003cstrong\u003eY19\u003c/strong\u003e on NLRP3 pathway \u003cem\u003ein vivo\u003c/em\u003e. Our results indicated that \u003cstrong\u003eY19\u003c/strong\u003e reduced the levels of NLRP3 and IL-1\u0026beta; at both 10 mg/kg and 20 mg/kg doses (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings suggest that \u003cstrong\u003eY19\u003c/strong\u003e has a positive therapeutic effect on ulcerative colitis through inhibiting NLRP3 pathway \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Conclusions","content":"\u003cp\u003eIn summary, the blockage of IL-1 signaling is a promising strategy for the treatment of IBD patients without respond to current drugs. Based on the phenotypic screening model for the NLRP3/IL-1β pathway, compound \u003cb\u003e1\u003c/b\u003e from our in-house compound library were identified as an inhibitor of NLRP3 pathway. A SAR-driven drug design was used to discover the more potent lead compound \u003cb\u003eY19\u003c/b\u003e with good inhibition of NLRP3-dependent IL-1β release. Also, \u003cb\u003eY19\u003c/b\u003e inhibited the secretion of IL-1βmediated by AIM2 and NLRC4 inflammasomes. In the animal model of colitis, \u003cb\u003eY19\u003c/b\u003e demonstrated the same level of anti-IL-1β ability as tofacitinib. Taken together, these findings strongly support further chemical development as promising azaindole inhibitors of IL-1β and therapeutic investigation for IBD.\u003c/p\u003e"},{"header":"4. Experimental Section","content":"\u003cp\u003eChemistry. All reactions were performed under a nitrogen atmosphere with anhydrous solvents from commercial sources (Innochem, Alfa, and Shanghai Chemical Reagent Company) without further purification unless otherwise stated. Reactions were monitored by thin-layer chromatography carried out on Yantai silica gel plates (60F-254), and the visualization was achieved using UV light, or phosphomolybdic acid in ethanol followed by heating. Tsingdao silica gel (200\u0026ndash;300 mesh) was used for flash column chromatography. NMR spectra were recorded with a 400 MHz (\u003csup\u003e1\u003c/sup\u003eH NMR, 400 MHz; \u003csup\u003e13\u003c/sup\u003eC NMR, 100 MHz) spectrometer. Data are reported as follows: chemical shift, multiplicity (s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, t\u0026thinsp;=\u0026thinsp;triplet, q\u0026thinsp;=\u0026thinsp;quartet, br\u0026thinsp;=\u0026thinsp;broad, m\u0026thinsp;=\u0026thinsp;multiplet), coupling constants, and integration. All compounds are \u0026gt;\u0026thinsp;95% pure by HPLC analysis (HPLC traces seen in Supporting Information). The purity was determined by Waters e2695 with UV/visible detector, using a JADE-PAK ODS-AQ column (15 cm \u0026times; 0.46 cm, 5 \u0026micro;m) eluted at 1.0 mL/min with CH\u003csub\u003e3\u003c/sub\u003eOH/H\u003csub\u003e2\u003c/sub\u003eO (85/15, v/v).\u003c/p\u003e \u003cp\u003eGeneral Procedure A: Amide Coupling. Carboxylic acid (\u003cb\u003eA1, A2, A4-A15, A29\u003c/b\u003e) (1 equiv.), HATU (1.1 equiv.) and triethylamine (TEA, 2 equiv.) were dissolved in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (0.2\u0026thinsp;\u0026minus;\u0026thinsp;0.3 mol/L) and stirred at 0\u0026deg;C for 30 min, then the corresponding amine (1 equiv.) was added, and the mixture was stirred for 6 h at room temperature. The reaction mixture was poured into water and extracted with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (3 \u0026times; 20 mL). The organic layers were combined, washed with saturated aqueous NaHCO\u003csub\u003e3\u003c/sub\u003e and brine, dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered and evaporated to give the crude product. Purification on silica gel column gave white powder.\u003c/p\u003e \u003cp\u003eGeneral Procedure B: Amide Coupling. The \u003cb\u003eB1\u003c/b\u003e (1 equiv.), EDCI (1 equiv) and HOBT (1.2 equiv) were dissolved in DMF and stirred at 0\u0026deg;C for 30 min, after which the corresponding amine (1 equiv) and DIPEA (2.5 equiv) were added, and the mixture was stirred for 6 h at room temperature. The reaction mixture was poured into water and extracted with CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (3 \u0026times; 20 mL). The organic layers were combined, washed with saturated aqueous NaHCO\u003csub\u003e3\u003c/sub\u003e and brine, dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered and evaporated to give the crude product. Purification on silica gel column gave white powder.\u003c/p\u003e \u003cp\u003eGeneral Procedure C: Heck coupling. The \u003cb\u003eC1\u003c/b\u003e (1 equiv.) and \u003cb\u003eC2\u003c/b\u003e (0.6 equiv.) were dissolved in DMF, CuI (0.02 equiv.), TEA (2 equiv.), and Pd(OAc)\u003csub\u003e2\u003c/sub\u003e (0.02 equiv.) were added, and the mixture was stirred for 5 hours at 70\u0026deg;C. At the end of the reaction, saturated saline was added and extracted with EA (3 \u0026times; 20 mL). The organic layers were combined, dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered and evaporated to give the crude product.\u003c/p\u003e \u003cp\u003eGeneral Procedure D: Removal of Ts group. The crude product (1 equiv.) and LiOH (2 equiv.) were dissolved in a mixture of THF and water, 500 \u0026micro;L of 1,4-dioxane was added, and the reaction was stirred for 6 h at room temperature. At the end of the reaction, the organic phase was extracted with EA (3 \u0026times; 20 mL). The organic layers were combined, dried with anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered and evaporated to give the crude product. The crude product was dissolved in methanol and stirred for 1 h at room temperature to obtain white crystals \u003cb\u003e30\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSynthesis of compounds \u003cb\u003eY1\u003c/b\u003e, \u003cb\u003eY2\u003c/b\u003e, \u003cb\u003eY4\u003c/b\u003e- \u003cb\u003eY15\u003c/b\u003e, \u003cb\u003eY29\u003c/b\u003e. Compounds \u003cb\u003eY1\u003c/b\u003e, \u003cb\u003eY2\u003c/b\u003e, \u003cb\u003eY4\u003c/b\u003e- \u003cb\u003eY15\u003c/b\u003e, \u003cb\u003eY29\u003c/b\u003e were prepared using \u003cem\u003eGeneral Procedure A.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(1-methyl-1H-indol-3-yl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY1\u003c/b\u003e). White foam; yield 67%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) δ 8.34 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.5 Hz, 1H), 8.10 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.5 Hz, 1H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H), 7.37\u0026ndash;7.20 (m, 7H), 7.09 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.42 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H), 6.15 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.06 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 3.83 (s, 3H), 1.94\u0026ndash;1.86 (m, 2H), 0.93 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.06, 148.83, 143.85, 142.50, 134.18, 132.38, 128.73, 128.70, 127.38, 126.87, 118.46, 116.80, 116.53, 110.63, 55.15, 31.54, 29.28, 10.97; HRMS (ESI) calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 319.1805, found, 319.1800.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-methyl-1H-indol-3-yl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY2\u003c/b\u003e). White solid, mp 203.9℃; yield 74%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +122.53 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Chloroform-\u003cem\u003ed\u003c/em\u003e) δ 8.98 (s, 1H), 7.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4 Hz, 1H), 7.83\u0026ndash;7.77 (m, 1H), 7.38\u0026ndash;7.31 (m, 4H), 7.31\u0026ndash;7.25 (m, 2H), 7.17\u0026ndash;7.10 (m, 2H), 6.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4 Hz, 1H), 5.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 5.08 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 2.37 (s, 3H), 1.91 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H), 0.95 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 167.30, 142.61, 140.03, 136.00, 134.68, 128.74, 127.36, 126.84, 126.44, 122.13, 120.98, 119.82, 114.34, 111.18, 109.34, 55.25, 29.38, 12.16, 10.99; HRMS (ESI) calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 319.1805, found, 319.1801.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S)-N-(1-phenylpropyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide\u003c/em\u003e(\u003cb\u003eY4\u003c/b\u003e). White solid, mp 226.3℃; yield 57%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e -60.96 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ12.10 (s, 1H), 8.40 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 8.35\u0026ndash;8.27 (m, 2H), 8.25 (s, 1H), 7.42\u0026ndash;7.27 (m, 4H), 7.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 7.17\u0026ndash;7.11 (m, 1H), 4.93 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 1.86\u0026ndash;1.74 (m, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.06, 148.90, 145.07, 143.97, 129.84, 128.71, 128.63, 127.09, 119.17, 117.40, 109.84, 54.50, 29.75, 12.00. HRMS (ESI) calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 280.1445, found, 280.1448.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(R)-N-(1-phenylpropyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide\u003c/em\u003e(\u003cb\u003eY5\u003c/b\u003e\u003cem\u003e).\u003c/em\u003e White solid, mp 216.6℃; yield 68%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +60.96 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.10 (s, 1H), 8.40 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H), 8.34\u0026ndash;8.23 (m, 3H), 7.42\u0026ndash;7.28 (m, 4H), 7.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 7.17\u0026ndash;7.11 (m, 1H), 4.93 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 1.90\u0026ndash;1.71 (m, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.09, 148.93, 145.08, 144.00, 129.87, 128.74, 128.66, 127.12, 119.20, 117.43, 109.88, 54.53, 29.77, 12.02. HRMS (ESI) calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 280.1445, found, 280.1446.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-N-(1-phenylpropyl)-3-(pyridin-2-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY6\u003c/b\u003e). White foam; yield 58%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 8.60\u0026ndash;8.55 (m, 1H), 7.69\u0026ndash;7.64 (m, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.2 Hz, 1H), 7.35\u0026ndash;7.28 (m, 5H), 7.26\u0026ndash;7.19 (m, 2H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.1 Hz, 1H), 6.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 5.02 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.91\u0026ndash;1.82 (m, 2H), 0.91 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.97, 153.31, 150.01, 142.08, 139.76, 136.99, 128.76, 127.47, 126.73, 125.07, 124.74, 123.99, 56.21, 28.58, 10.82. HRMS (ESI) calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 267.1492, found, 267.1494.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(R,E)-N-(1-phenylpropyl)-3-(pyridin-2-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY7\u003c/b\u003e). White foam; yield 42%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 8.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.6 Hz, 1H), 7.70\u0026ndash;7.63 (m, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.1 Hz, 1H), 7.35\u0026ndash;7.21 (m, 7H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.1 Hz, 1H), 6.15 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 1H), 5.02 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.91\u0026ndash;1.77 (m, 2H), 0.91 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 159.08, 147.40, 144.14, 136.19, 133.91, 131.12, 122.47, 121.59, 120.86, 119.15, 118.89, 118.13, 49.33, 23.40, 4.98. HRMS (ESI) calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 267.1492, found, 267.1494.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-fluorophenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY8\u003c/b\u003e). White foam; yield 63%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.47\u0026ndash;7.40 (m, 1H), 7.33\u0026ndash;7.24 (m, 6H), 7.12\u0026ndash;7.06 (m, 1H), 7.06\u0026ndash;6.99 (m, 1H), 6.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 6.23 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 5.02 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 1H), 1.93\u0026ndash;1.84 (m, 2H), 0.91 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.27, 161.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;253.1 Hz), 142.14, 134.39, 130.99 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz), 129.87 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.3 Hz), 128.77, 127.48, 126.85, 124.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.6 Hz), 123.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz), 122.94 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.6 Hz), 116.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22.0 Hz), 55.30, 29.19, 10.96. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eFNO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 284.1445, found, 284.1423.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-chlorophenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY9\u003c/b\u003e). White foam; yield 75%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H), 7.50 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1.8 Hz, 1H), 7.37 \u0026minus;\u0026thinsp;7.30 (m, 5H), 7.25\u0026ndash;7.15 (m, 3H), 6.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H), 6.36 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.00 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.97\u0026ndash;1.73 (m, 2H), 0.90 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.94, 142.13, 137.14, 134.85, 133.26, 130.49, 130.22, 128.77, 127.60, 127.50, 126.99, 126.87, 123.75, 55.36, 29.18, 10.99. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eClNO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 300.1150, found, 300.1151.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-N-(1-phenylpropyl)-3-(o-tolyl)acrylamide\u003c/em\u003e(\u003cb\u003eY10\u003c/b\u003e). White solid, mp 136.5℃; yield 72%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e -28 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4 Hz, 1H), 7.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 7.35\u0026ndash;7.30 (m, 4H), 7.26\u0026ndash;7.19 (m, 2H), 7.18\u0026ndash;7.09 (m, 2H), 6.35 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4 Hz, 1H), 6.21 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.01 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 2.37 (s, 3H), 1.93\u0026ndash;1.81 (m, 2H), 0.91 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.41, 142.25, 139.15, 137.69, 133.94, 130.83, 129.51, 128.78, 127.48, 126.87, 126.24, 126.12, 121.83, 55.30, 29.22, 19.94, 11.00. HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eNO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 280.1696, found, 280.1695.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-methoxyphenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY11\u003c/b\u003e). White solid, mp 139.8℃; yield 68%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e -23 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.87 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.7 Hz, 1H), 7.44 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6, 1.7 Hz, 1H), 7.35\u0026ndash;7.31 (m, 4H), 7.31\u0026ndash;7.24 (m, 2H), 6.96\u0026ndash;6.86 (m, 2H), 6.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.7 Hz, 1H), 5.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.04 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 3.86 (s, 3H), 1.97\u0026ndash;1.80 (m, 2H), 0.93 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.98, 158.36, 142.40, 136.82, 130.80, 129.28, 128.73, 127.39, 126.90, 123.96, 121.83, 120.72, 111.16, 55.50, 55.14, 29.19, 10.95. HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 296.1645, found, 296.1645.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-N-(1-phenylpropyl)-3-(3,4,5-trimethoxyphenyl)acrylamide\u003c/em\u003e(\u003cb\u003eY12\u003c/b\u003e). White solid, mp 128.7℃; yield 56%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +42.9 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H), 7.36\u0026ndash;7.30 (m, 5H), 6.71 (s, 2H), 6.39 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H), 6.07 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.01 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 3.85 (s, 9H), 1.93\u0026ndash;1.83 (m, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.24, 153.44, 142.15, 141.24, 139.49, 130.55, 128.79, 127.51, 126.81, 120.26, 104.89, 61.06, 56.15, 55.21, 29.23, 10.94. HRMS (ESI) calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 356.1856, found, 356.1857.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-cyanophenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY13\u003c/b\u003e). White solid, mp 122.8℃; yield 67%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +14 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 7.70 (s, 1H), 7.65\u0026ndash;7.51 (m, 3H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.33\u0026ndash;7.29 (m, 4H), 7.26\u0026ndash;7.22 (m, 1H), 6.50 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H), 6.37 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.00 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.96\u0026ndash;1.81 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.47, 141.98, 138.62, 136.30, 132.65, 132.26, 130.76, 129.84, 128.82, 127.59, 126.81, 123.60, 118.46, 113.7, 55.48, 29.20, 10.93. HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 291.1492, found, 291.1489.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(2-nitrophenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY14\u003c/b\u003e). White solid, mp 172.6℃; yield 73%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 8.05\u0026ndash;7.92 (m, 2H), 7.64\u0026ndash;7.44 (m, 3H), 7.37\u0026ndash;7.30 (m, 4H), 7.29\u0026ndash;7.26 (m, 1H), 6.33 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H), 6.03 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H), 5.01 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 2.06\u0026ndash;1.80 (m, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e164.23, 148.39, 141.84, 136.32, 133.45, 131.20, 129.87, 129.24, 128.85, 127.63, 126.87, 126.29, 124.96, 55.40, 29.10, 10.90. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 311.1390, found, 311.1389.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-3-(3-nitrophenyl)-N-(1-phenylpropyl)acrylamide\u003c/em\u003e(\u003cb\u003eY15\u003c/b\u003e). White solid, mp 171.3℃; yield 54%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +17.1 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 8.68 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 1.9 Hz, 1H), 8.04 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.9 Hz, 1H), 7.85\u0026ndash;7.76 (m, 2H), 7.73\u0026ndash;7.56 (m, 2H), 7.41\u0026ndash;7.19 (m, 5H), 6.72 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6, 1.9 Hz, 1H), 4.82 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 7.6 Hz, 1H), 1.84\u0026ndash;1.68 (m, 2H), 0.86 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e164.23, 148.39, 141.84, 136.32, 133.45, 131.20, 129.87, 129.24, 128.85, 127.63, 126.87, 126.29, 124.96, 55.45, 28.10, 10.90. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 311.1390, found, 311.1392.\u003c/p\u003e \u003cp\u003e \u003cem\u003eN-phenethyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide\u003c/em\u003e(\u003cb\u003eY29\u003c/b\u003e). White solid, mp 203.4℃; yield 72%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 12.32\u0026ndash;11.92 (m, 1H), 8.44 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.7 Hz, 1H), 8.26 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.7 Hz, 1H), 8.16 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 1H), 8.11 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.7 Hz, 1H), 7.32\u0026ndash;7.23 (m, 4H), 7.22\u0026ndash;7.13 (m, 2H), 3.49 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz, 2H), 2.84 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 164.47, 148.89, 143.98, 140.23, 129.78, 129.23, 128.91, 128.39, 126.62, 118.98, 117.41, 110.06, 40.82, 36.11. HRMS (ESI) calcd for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 266.1288, found, 266.1291.\u003c/p\u003e \u003cp\u003eSynthesis of compounds \u003cb\u003eY3\u003c/b\u003e, \u003cb\u003eY16\u003c/b\u003e- \u003cb\u003eY28\u003c/b\u003e. Compounds \u003cb\u003eY3\u003c/b\u003e, \u003cb\u003eY16\u003c/b\u003e- \u003cb\u003eY28\u003c/b\u003e were prepared using \u003cem\u003eGeneral Procedure B.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-N-(1-phenylpropyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide(\u003c/em\u003e \u003cb\u003eY3\u003c/b\u003e \u003cem\u003e).\u003c/em\u003e White solid, mp 163.7℃; yield 74%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e +75.36 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.10 (s, 1H), 8.35\u0026ndash;8.26 (m, 3H), 7.89 (s, 1H), 7.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.39\u0026ndash;7.27 (m, 4H), 7.27\u0026ndash;7.19 (m, 2H), 6.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 4.84 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 1H), 1.80\u0026ndash;1.68 (m, 2H), 0.87 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO- \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 174.98, 159.08, 153.33, 153.09, 142.18, 140.06, 137.79, 137.71, 136.20, 136.04, 127.05, 126.64, 126.04, 120.51, 63.53, 39.03, 20.53; HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 306.1601, found, 306.1598.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(R,E)-N-(1-phenylpropyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY16\u003c/b\u003e). White solid, mp 161.2℃; yield 74%; [α]\u003csup\u003eD\u003c/sup\u003e \u003csub\u003e21\u003c/sub\u003e -75.36 (c 0.1, CH\u003csub\u003e3\u003c/sub\u003eOH); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.06 (s, 1H), 8.33\u0026ndash;8.24 (m, 3H), 7.87 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.3 Hz, 1H), 7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.35\u0026ndash;7.29 (m, 4H), 7.27\u0026ndash;7.18 (m, 2H), 6.75 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 4.83 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.80\u0026ndash;1.68 (m, 2H), 0.86 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.99, 150.08, 144.34, 144.10, 133.19, 131.06, 128.79, 128.72, 127.20, 127.04, 118.05, 117.64, 117.05, 111.51, 54.53, 30.04, 11.53. HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 306.1601, found, 306.1597.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(S,E)-N-(1-phenylethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY17\u003c/b\u003e). White foam; yield 69%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.10 (s, 1H), 8.37\u0026ndash;8.28 (m, 3H), 7.90 (s, 1H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.39\u0026ndash; 7.19 (m, 6H), 6.75 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 5.12\u0026ndash;5.00 (m, 1H), 1.41 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.9 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 165.76, 149.78, 145.39, 144.15, 133.26, 131.06, 128.89, 128.74, 127.26, 126.63, 118.10, 117.72, 117.12, 111.58, 48.46, 23.25. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 292.1444, found, 291.1440.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(2-methylbenzyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY18\u003c/b\u003e). White solid, mp 173.8℃; yield 48%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.10 (s, 1H), 8.31\u0026ndash;8.25 (m, 2H), 8.23 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.91 (s, 1H), 7.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.29\u0026ndash;7.24 (m, 1H), 7.22\u0026ndash;7.19 (m, 1H), 7.19\u0026ndash;7.16 (m, 3H), 6.78 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 4.41 (s, 2H), 2.30 (s, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.55, 150.12, 144.12, 137.67, 136.43, 133.32, 131.07, 130.57, 128.74, 128.59, 127.59, 126.39, 117.85, 117.71, 117.10, 111.58, 39.41, 19.20. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 292.1444, found, 292.1439.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-phenethyl-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY19\u003c/b\u003e). White solid, mp 183..8℃; yield 86%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.06 (s, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7 Hz, 1H), 8.26 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 8.01 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.90 (s, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.33\u0026ndash;7.17 (m, 6H), 6.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 2.79 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.61, 150.06, 144.09, 140.12, 132.98, 130.96, 129.20, 128.91, 128.67, 126.65, 117.97, 117.68, 117.08, 111.48, 39.36, 35.89. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 292.1444, found, 292.1455.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(2-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY20\u003c/b\u003e). White solid, mp 205.8℃; yield 63%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 12.07 (s, 1H), 8.29 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.6 Hz, 1H), 8.25 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 8.05 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.88 (s, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.42 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7 Hz, 1H), 7.35 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.3 Hz, 1H), 7.31\u0026ndash;7.17 (m, 3H), 6.62 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 2.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) δ 166.77, 150.07, 144.14, 137.41, 133.74, 133.11, 131.59, 130.90, 129.83, 128.77, 127.87, 117.91, 117.75, 117.15, 111.50, 49.19, 33.65. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 326.1055, found, 326.1049.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(3-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY21\u003c/b\u003e). White solid, mp 195.6℃; yield 52%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.09 (s, 1H), 8.32\u0026ndash;8.23 (m, 2H), 7.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.90 (s, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.37\u0026ndash;7.32 (m, 2H), 7.29\u0026ndash;7.24 (m, 2H), 7.20 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.44 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6 Hz, 2H), 2.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.64, 150.09, 144.10, 139.19, 133.04, 131.37, 131.31, 131.14, 130.98, 128.81, 128.68, 117.93, 117.71, 117.08, 111.50, 40.53, 35.15. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 326.1055, found, 326.1050.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(4-chlorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY22\u003c/b\u003e). White solid, mp 201.2℃; yield 48%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.08 (s, 1H), 8.30 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.9, 1.5 Hz, 1H), 8.25 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 1.7 Hz, 1H), 7.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 1H), 7.90 (s, 1H), 7.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.38\u0026ndash;7.32 (m, 2H), 7.29\u0026ndash;7.25 (m, 2H), 7.20 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 4.7 Hz, 1H), 6.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.7 Hz, 1H), 3.43 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 2H), 2.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.64, 150.08, 144.11, 139.19, 133.05, 131.31, 131.14, 131.00, 128.81, 128.68, 117.90, 117.69, 117.09, 111.48, 40.53, 35.13. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eCl [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 326.1055, found, 326.1049.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(2-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY23\u003c/b\u003e). White solid, mp 223.1℃; yield 63%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.03 (s, 1H), 8.29 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.4 Hz, 1H), 8.25 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H), 7.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 1H), 7.88 (s, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.34\u0026ndash;7.12 (m, 5H), 6.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.44 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, 2H), 2.85 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.61, 161.32 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;243.4 Hz), 150.10, 144.08, 132.95, 131.67 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8 Hz), 130.76, 128.81 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz),128.59, 126.78, 126.63, 124.92 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.2 Hz), 117.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;28.7 Hz),117.05, 115.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21.8 Hz), 111.51,39.39, 29.30. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 310.1350, found, 310.1348.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(3-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY24\u003c/b\u003e). White solid, mp 214.2; yield 67%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.08 (s, 1H), 8.30\u0026ndash;8.20 (m, 2H), 7.99 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.89 (s, 1H), 7.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.38\u0026ndash;7.29 (m, 1H), 7.20 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 7.13\u0026ndash;7.06 (m, 2H), 7.06\u0026ndash;6.98 (m, 1H), 6.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 3.46 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 2H), 2.82 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.67, 162.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;243.0 Hz), 150.08, 144.11, 143.15 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz), 133.04, 130.94, 130.71 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz), 128.67, 125.40 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.7 Hz), 117.93, 117.71, 117.09, 115.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20.7 Hz), 113.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20.8 Hz), 111.49, 40.06, 35.48. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 310.1350, found, 310.1346.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(4-fluorophenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY25\u003c/b\u003e). White solid, mp 232.8℃; yield 72%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.00 (s, 1H), 8.26 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.5 Hz, 1H), 8.22(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.6 Hz, 1H), 7.92(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.85 (s, 1H), 7.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.26\u0026ndash;7.15 (m, 3H), 7.15\u0026ndash;7.00 (m, 2H), 6.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.39 (m, 2H), 2.75 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.64, 161.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;242.3 Hz), 150.11, 144.13, 136.30, 133.03, 131.05, 130.98, 128.70, 117.99, 117.72, 117.12, 115.68, 115.48, 111.52, 40.80,35.02. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eFN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 310.1350, found, 310.1346.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(4-methoxyphenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY26\u003c/b\u003e). White solid, mp 245.1℃; yield 68%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.08 (s, 1H), 8.33\u0026ndash;8.23 (m, 2H), 7.95 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.89 (s, 1H), 7.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 7.24\u0026ndash;7.16 (m, 2H), 7.15 (s, 1H), 6.90\u0026ndash;6.81 (m, 2H), 6.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.71 (s, 3H), 2.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.60, 158.25, 150.08, 144.11, 132.93, 131.98, 130.89, 130.16, 128.69, 118.08, 117.72, 117.09, 114.33, 111.53, 55.52, 39.42, 35.03. HRMS (ESI) calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 322.1550, found, 322.1544.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(4-(methylsulfonyl)phenethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY27\u003c/b\u003e). White solid, mp 245.2℃; yield 57%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 11.66 (s, 1H), 7.88 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.7, 1.5 Hz, 1H), 7.84 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.6 Hz, 1H), 7.60 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H), 7.50\u0026ndash;7.42 (m, 3H), 7.20\u0026ndash;7.09 (m, 3H), 6.79 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 4.7 Hz, 1H), 6.22 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.08 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7, 1.5 Hz, 2H), 2.77 (s, 3H), 2.50 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.77, 150.09, 146.51, 144.14, 139.30, 133.16, 130.97, 130.23, 128.68, 127.60, 117.86, 117.73, 117.12, 111.49, 44.17, 39.43, 35.69. \u003cem\u003eHRMS (ESI) calcd for\u003c/em\u003e C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 370.1220, found, 370.1217.\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-(2-cyclohexylethyl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)acrylamide\u003c/em\u003e(\u003cb\u003eY28\u003c/b\u003e). White solid, mp 241.2℃; yield 56%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 12.05 (s, 1H), 8.32\u0026ndash;8.23 (m, 2H), 7.90\u0026ndash;7.79 (m, 2H), 7.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.21 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 4.7 Hz, 1H), 6.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz, 1H), 3.22\u0026ndash;3.19 (m, 2H), 1.76\u0026ndash;1.55 (m, 6H), 1.36 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8, 2H), 1.23\u0026ndash;1.11 (m, 3H), 0.94\u0026ndash;0.83 (m, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e 166.45, 150.05, 144.09, 132.75, 130.39, 128.69, 118.20, 117.71, 117.07, 111.51, 37.37, 36.76, 35.12, 33.23, 26.69, 26.33. HRMS (ESI) calcd for C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 298.1914, found, 298.1911.\u003c/p\u003e \u003cp\u003eSynthesis of compound \u003cb\u003eY30\u003c/b\u003e. Compound \u003cb\u003eY30\u003c/b\u003e was prepared using \u003cem\u003eGeneral Procedure C and D.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003e(E)-N-phenethyl-3-(7H-pyrrolo[2,3-d]pyrimidin-5-yl)acrylamide\u003c/em\u003e (\u003cb\u003eY30\u003c/b\u003e). White foam; yield 38%; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e 9.23 (s, 1H), 8.80 (s, 1H), 7.80 (s, 1H), 7.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 7.33\u0026ndash;7.23 (m, 4H), 7.22\u0026ndash;7.16 (m, 1H), 6.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz, 1H), 3.59\u0026ndash;3.53 (m, 2H), 2.88 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD) \u003cem\u003eδ\u003c/em\u003e 167.77, 152.82, 151.26, 148.32, 139.21, 132.20, 130.84, 128.50, 128.22, 126.07, 118.77, 116.24, 112.00, 40.90, 35.33. HRMS (ESI) calcd for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e, 293.1397, found, 293.1395.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell culture and Stimulation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eJ774A.1 cells (Guangzhou JennioBiotechCo, China) were cultured with medium and 10% FBS (Gibco, USA) in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. BMDMs were obtained from the myelin sheaths of mouse tibia and femur, and were cultured with a culture medium consisting of 10% FBS, RPMI-1640 medium, 1 mM double antibody, 1 mM glutamine and 0.1 mM-CSF.\u003c/p\u003e \u003cp\u003eFor NLRP3 inflammasome activation, cells were stimulated with 1 \u0026micro;g/mL of LPS (Sigma-Aldrich, USA) for 5 h, treated with compounds for 1 h, followed by stimulation with Nigericin (Invitrogen, USA) for 1 h. For NLRC4 or AIM2 inflammatory activation, BMDMs were stimulated with 500 ng/mL of LPS for 3 h, treated with compounds for 1 h, followed by stimulation with 25 \u0026micro;g/mL FLA-ST Ultrapure (Invitrogen, USA) or a mixture of 2.5 \u0026micro;g/mL Poly dA:dT (Invitrogen, USA) and Lipo 3000(Invitrogen, USA) for 14 h of stimulation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLDH Release Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eJ774A.1 cells were plated on 96-well plates, and the cells were stimulated with 1 \u0026micro;g/mL of LPS for 5 h, treated with Y19 for 1 h, and then stimulated with nigericin for 1 h. Supernatants were collected and LDH activity was measured using the LDH Assay Kit (Beyotime, China).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnzyme-linked Immunosorbent Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIL-1β (Invitrogen, USA) or TNF-α (Invitrogen, USA) in cell cultures or animal colon homogenates supernatants were analyzed by ELISA kits.\u003c/p\u003e \u003cp\u003e \u003cem\u003eWestern Blotting Analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eCells were lysed in RIPA buffer (Beyotime, China) containing protease inhibitors for 0.5 h at 4\u0026deg;C, after which the lysate was collected and the protein concentration was determined by the BCA Protein Assay Kit (23227, Thermo) at 562 nm. The Protein samples were separated by electrophoresis on sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, which were closed with 5% skimmed milk solution for 1 h at room temperature and incubated with primary antibody for 12 h. Subsequently, the membranes were lubricated by TBST and then treated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Finally, the membranes were incubated with ECL protein blotting reagent (ThermoFisher Scientific, USA) and visualized by a gel imaging system. The above primary antibodies were anti-mouse IL-1β (AF-401-NA, R\u0026amp;D Systems), anti-mouse caspase-1 (AG-20B0042, Adipogen), anti-NLRP3 (15100S, Cell Signaling Technology), anti-ASC (67824S, Cell Signaling Technolology), anti-NEK7 (ab133514, Abcam), anti-phosphorylated NF-κB (3033T, Cell Signaling Technologies), anti-NF-κB (8242T, Cell Signaling Technologies), anti-phospho-IκBα (2859T, Cell Signaling Technologies)\u003c/p\u003e \u003cp\u003e \u003cem\u003eNLRP3 Oligomerization Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eJ774A.1 cells plated on 6-well plates were grouped, modelled and given drug administration according to the LPS-induced NLRP3 inflammasome activation step. Cells were lysed with cell lysate for 30 min and then the lysate was collected and centrifuged at 12,000 rpm for 5 min at 4\u0026deg;C, after which the lysate was quantified by BCA protein quantification. The proteins were processed by agarose gel electrophoresis and protein immunoblotting. After the proteins had been fully transferred to the PVDF membrane, the membrane was closed with skimmed milk for 1 h. The whole membrane was incubated with NLRP3 primary antibody at 4\u0026deg;C overnight, and the secondary antibody was applied and developed the next day.\u003c/p\u003e \u003cp\u003e \u003cem\u003eASC Oligomerization Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eJ774A.1 cells were plated on six large plates and treated following the steps for LPS-induced NLRP3 inflammasome activation. Cells were lysed with cell lysate for 30 min and then centrifuged at 12,000 rpm for 5 min at 4\u0026deg;C to collect the lysate, which was subsequently quantified by BCA protein quantification. The cell pellet was washed twice with pre-cooled PBS and 4 mM of disuccinimidyl suberate dissolved in DMSO was added to the cell pellet and the mixture was crosslinked for 30 min at room temperature before centrifugation at 8,000 rpm for 5 min at 4\u0026deg;C. The supernatant was discarded and the residual disuccinimidyl suberate was neutralized by the addition of 4 mM Tris and left at room temperature for 20 min. Centrifuge at 8,000 rpm for 10 min at 4\u0026deg;C, discard the supernatant, add 20 \u0026micro;L of 2X Sample Buffer directly to the tube, and boil for 10 min at 100\u0026deg;C on a metal bath before analyzing the samples by immunoblotting.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDetection of ASC specks\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBMDMs were treated with 500 ng/mL LPS (Sigma-Aldrich, USA) for 3 h, treated with Y19 for 1 h and stimulated with 5 \u0026micro;M nigericin (Invitrogen, USA) for 30 min. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. After blocking with 1% BSA for 1 h, the cells were stained with cells were stained with ASC antibodies overnight at 4\u0026deg;C. After washed three times with PBS, the cells were incubated with the rhodamine-labelled anti-rabbit IgG antibody (A11008, Invitrogen) for 90 min at room temperature. Cell nuclei were stained with DAPI (2 \u0026micro;M) for 30 min and washed three times with PBS. Images were captured using a fluorescence microscope (DM4000 BLED, Leica, Germany).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCo-immunoprecipitation Assay\u003c/em\u003e \u003c/p\u003e \u003cp\u003eJ774A.1 cells were plated on six large plates and treated according to the steps of LPS-induced NLRP3 inflammasome activation. The cells were lysed with IP lysis buffer containing protease inhibitors and collected, centrifuged at 4\u0026deg;C for 10 min at 10,000 g. The supernatant was collected and 20 \u0026micro;L of protein A/G PLUS-beads (sc-2003, Santa Cruz) were added and incubated for 12 h at 4\u0026deg;C in a shaker. The supernatant was collected by centrifugation at 2800 rpm for 10 min at 4\u0026deg;C. The primary antibody (an equal amount of normal IgG as negative control) was added and incubated at 4\u0026deg;C for 8 h. Afterwards, 20 \u0026micro;L of protein A/G PLUS-beads were added and the mixture was incubated overnight at 4\u0026deg;C in a shaker. The immune complexes were washed with PBS and protein-containing additives were added. The mixture was boiled for 10 min and then subjected to immunoblotting.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimal Experiments.\u003c/em\u003e \u003c/p\u003e \u003cp\u003e All animal experiments were conducted in accordance with the Guidelines for the Keeping and Use of Laboratory Animals and approved by the Ethics Committee of Guangzhou Medical University (GY2022-045). The animal studies were reported in accordance with the ARRIVE guidelines. All male C57BL/6 mice (8 weeks, 6 mice per group) were kept at room temperature with free access to food and water. The blank group received water treatment for 7 days. Ulcerative colitis was induced by the addition of 2.5% DSS to distilled water for 7 consecutive days. DSS was added to distilled water for 7 consecutive days to induce ulcerative colitis. Y19 (5, 10 and 20 mg/kg) and Tofacitinib (10 mg/kg) were administered by gavage on days 1, 3, 5 and 7 respectively. Equal volume of solution was given by gavage in blank and DSS treated groups. Mice were executed on day 8 after induction of colitis. Body weight and fecal occult blood were monitored daily. The colon was scored microscopically and biochemical tests were performed on colon specimens.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analysis.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;s.e.m. Statistical analysis preformed was an unpaired, two-tailed Student\u0026rsquo;s t test using GraphPad Prism software. The accepted levels of significance were *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, and ns: not significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAIM2, absent in melanoma 2; ASC, apoptosis-associated speck-like protein containing a CARD protein; DCM, dichloromethane; DMF, N, N-dimethylformamide; DSS, dextran sulfate sodium; NLRC4, NOD-like receptor family CARD-containing 4; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; Tofa., Tofacitinib.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (NSFC) (NO. 81803364 to Z. Yang).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edata\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR and\u003csup\u003e\u0026nbsp;13\u003c/sup\u003eC NMR of all target compounds are available free of charge\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCaron B, Honap S, Peyrin-Biroulet L (2024) Epidemiology of Inflammatory Bowel Disease across the Ages in the Era of Advanced Therapies. J Crohns Colitis 18(Supplement_2):ii3\u0026ndash;ii15. https://doi.org/10.1093/ecco-jcc/jjae082 .\u003c/li\u003e\n\u003cli\u003ePenagini F, Lonoce L, Abbattista L, Silvera V, Rendo G, Cococcioni L, Dilillo D, Calcaterra V, Zuccotti GV (2023) Dual biological therapy and small molecules in pediatric inflammatory bowel disease. 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Bioorg Med Chem Lett 30:127396. https://doi.org/10.1016/j.bmcl.2020.127396. \u003c/li\u003e\n\u003cli\u003eMeyer A, Vasseur J, Morvan F (2013) Synthesis of Monoconjugated and Multiply Conjugated Oligonucleotides by \u0026ldquo;Click Thiol\u0026rdquo; Thiol-Michael-Type Additions and by Combination with CuAAC \u0026ldquo;Click Huisgen\u0026rdquo;. Eur J Org Chem 2013:465-473. https://doi.org/10.1002/ejoc.201390003.\u003c/li\u003e\n\u003cli\u003eBilledeau RJ, Kondru RK, Lopez-Tapia FJ, Lou Y, Owens TD, Qian Y, So SS, Thakkar KC, Wanner J, Phthalazinone derivatives as inhibitors of Bruton\u0026apos;s tyrosine kinase and their preparation and use in the treatment of inflammatory and autoimmune diseases, 2012, WO2012156334A1.\u003c/li\u003e\n\u003cli\u003eBaldwin AG, Brough D, Freeman S (2016) Inhibiting the Inflammasome: A Chemical Perspective. 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Gut, 59:1192\u0026ndash;1199. https://doi.org/10.1136/gut.2009.197822.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Inhibitors, 7-azaindole, inflammasome, IL-1β, Inflammatory bowel disease","lastPublishedDoi":"10.21203/rs.3.rs-6307163/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6307163/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrently, a significant proportion of patients with inflammatory bowel disease (IBD) fail to respond to conventional drug therapy such as immunosuppressants and biologic agents. IL-1 signaling blockade is a promising therapeutic strategy for these unresponsive IBD patients. In this study, we identified a novel anti-NLRP3/IL-1β inhibitor, the 7-azaindole analogue \u003cb\u003eY19\u003c/b\u003e, which demonstrates potent inhibitory activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 1.26 \u0026micro;M. Mechanistic investigations revealed that it suppresses NLRP3 inflammasome assembly and activation by disrupting critical protein-protein interactions, including NEK7-NLRP3, NLRP3-NLRP3, NLRP3-ASC, and ASC-ASC. Furthermore, it also inhibits the AIM2 and NLRC4 inflammasome pathways. In a murine model of colitis, \u003cb\u003eY19\u003c/b\u003e demonstrated anti-inflammatory efficacy comparable to that of tofacitinib, a Janus kinase inhibitor commonly prescribed for IBD patients refractory to conventional therapies. This finding highlights the potential of \u003cb\u003eY19\u003c/b\u003e as a promising lead compound for the treatment of IBD.\u003c/p\u003e","manuscriptTitle":"Discovery of 7-azaindole Inhibitors of inflammasomes/IL-1β for the Treatment of Inflammatory Bowel Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-30 11:56:18","doi":"10.21203/rs.3.rs-6307163/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-22T13:37:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-08T13:14:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"222901755638089524745904795871516625291","date":"2025-04-29T03:37:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-11T19:18:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173284566110939915655351342235505244245","date":"2025-04-03T05:49:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167899749742370610872052868269062270575","date":"2025-04-02T12:24:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T17:43:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T11:17:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-26T09:54:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Diversity","date":"2025-03-25T23:35:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-diversity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"modi","sideBox":"Learn more about [Molecular Diversity](http://link.springer.com/journal/11030)","snPcode":"11030","submissionUrl":"https://submission.nature.com/new-submission/11030/3","title":"Molecular Diversity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8a16ce2b-3f99-4aff-ae5d-2b6830f9f35f","owner":[],"postedDate":"April 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T16:38:55+00:00","versionOfRecord":{"articleIdentity":"rs-6307163","link":"https://doi.org/10.1007/s11030-025-11316-1","journal":{"identity":"molecular-diversity","isVorOnly":false,"title":"Molecular Diversity"},"publishedOn":"2025-08-20 16:30:14","publishedOnDateReadable":"August 20th, 2025"},"versionCreatedAt":"2025-04-30 11:56:18","video":"","vorDoi":"10.1007/s11030-025-11316-1","vorDoiUrl":"https://doi.org/10.1007/s11030-025-11316-1","workflowStages":[]},"version":"v1","identity":"rs-6307163","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6307163","identity":"rs-6307163","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}