Discovery and cytotoxic activity of N-CF3-containing pyrroloindolines

Discovery and cytotoxic activity of N-CF3-containing pyrroloindolines | 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 and cytotoxic activity of N-CF3-containing pyrroloindolines Qigang Sun, Jun-Yunzi Wu, Yu Zhang, Qingjiang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6126144/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 May, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 6 You are reading this latest preprint version Abstract Pyrroloindoline scaffolds are widely found in numerous biologically active and medicinally significant natural products. Meanwhile, the potential applications of N -CF 3 compounds in medicinal chemistry have garnered increasing attention due to their unique properties. However, the pharmacological activities and therapeutic potential of N -CF 3 -containing pyrroloindolines remain underexplored. Building on our previously developed method for constructing N -CF 3 -containing pyrroloindoline scaffolds, this study systematically evaluated the antitumor activities of a diverse series of N -CF₃-containing pyrroloindoline derivatives. Cell-based assays demonstrated that these derivatives exhibited significant antiproliferative activities against human cancer cell lines, including 786-O, A498, and HepG2. Further investigations revealed that N -CF 3 -containing pyrroloindolines not only induce apoptosis in cancer cells but also inhibit colony formation. Moreover, they showed potential to suppress cancer cell metastasis and proliferation, primarily by inducing apoptosis and cell cycle arrest. These findings suggest that N -CF 3 -containing pyrroloindolines hold great promise as potential anticancer agents and merit further development. N-CF3 moiety pyrroloindoline natural product cytotoxic activity Figures Figure 1 Figure 2 INTRODUCTION To date, the SciFinder database search has revealed that approximately three thousand pyrroloindoline-containing compounds have been investigated for their diverse biological activities. Pyrroloindoline-containing natural products have garnered significant attention from the synthetic and medicinal chemistry communities due to their widespread prevalence in structurally complex natural indole alkaloids and their diverse range of biological activities, such as antibacterial (Lam et al. 1990 ; Leet et al. 1990 ; Ding et al. 1994 ; Umezawa et al. 1999 ; Umezawa et al. 2000 ; Broberg et al. 2006 ; Pohanka et al. 2006 ; Kumar and Goodfellow. 2008; Guo et al. 2009 ; Guo et al. 2010 ; Ji et al. 2012 ), antitumor (Alley et al. 1988 ; Feng et al. 2004 ), and insecticidal properties (Shiono et al. 2000 ; Furutani et al. 2017 ; Furutani et al. 2014 ). A notable example is (-)-physostigmine and its analogs, which have been clinically employed for treating myasthenia gravis and glaucoma, while also emerging as promising candidates for alleviating symptoms of Alzheimer's disease (Greig et al. 1995 ). The trifluoromethyl (CF 3 ) group is widely employed to modulate the biophysical properties of small molecules, such as lipophilicity, permeability, and metabolic stability (Purse et al.2008; Zhou et al. 2016 ; Meanwel 2018; Inoue et al. 2020 ), and has played a pivotal role in the structural optimization of pharmaceuticals. However, the lack of efficient methods to synthesize N -CF 3 -containing pyrroloindolines remains a significant limitation for exploring their potential applications in medicinal chemistry. Current research has largely focused on the synthesis of various complex natural product derivatives (Mei et al. 2020 ; Mei et al. 2021 ; Shu et al. 2023 ; Jiang et al. 2024 ), with limited exploration of the pharmacological properties and application potential of N -CF 3 -containing pyrroloindolines. To date, in-depth studies on their biological activities and potential therapeutic value remain scarce. Building on a robust and operationally straightforward approach previously developed by our research group (Wu et al. 2024 ), this intriguing structural framework was synthesized from tryptamine-derived isocyanates through a cascade sequence involving oxidative trifluoromethylation followed by halogenative cyclization. Thereby, a diverse array of N -CF 3 -containing pyrroloindoline derivatives were readily accessed. In this study, we report the discovery of N -CF 3 -containing pyrroloindolines with significant cytotoxic activity, laying a foundation for future exploration and development of these compounds as potential anticancer therapeutics. Materials and methods General Experiments. All synthetic reagents were obtained from domestic chemical vendors and employed as received unless specifically noted. Reaction progress monitoring was conducted using Thin-layer chromatography analysis with silica gel GF 254 plates. Nuclear magnetic resonance spectra were recorded on Bruker Avance III spectrometers (400/500 MHz, Germany). High-resolution mass spectrometry (HRMS) data were acquired using a SHIMADZU LCMS-IT-TOF system equipped with ion trap-time-of-flight technology. General procedure for synthesis of N -CF 3 -containing pyrroloindolines. In a 10 ml polyethylene reaction vessel maintained at ambient conditions, a dichloromethane solution (0.5 ml) containing N-halosuccinimide (2.5 molar equivalents) was subjected to sequential addition of Et 3 N∙HF (62 µl, 12.0 equiv hydrogen fluoride) and the respective tryptamine-derived isocyanide substrate (0.1 mmol, 1.0 equiv) under vigorous stirring. The heterogeneous mixture was agitated at 25°C for 5–10 min until Thin-layer chromatography analysis confirmed complete substrate conversion. Subsequent purification through wet-loaded flash chromatography on activated silica gel yielded the target compounds. General procedure for the Ag(I)-mediated nucleophilic substitution reaction. 1 (0.1 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (0.1 M) and thermally equilibrated in an ice-water bath (0°C). The nucleophilic reagent and 2,6-di-tert-butylpyridine (0.2 mmol, 2.0 equiv) were sequentially introduced into the agitated mixture. Following a 20-minute agitation period at maintained temperature, AgNTf₂ (1.2 equiv) was portion-wise incorporated into the heterogeneous system. Post 15-minute cryogenic reaction, the resultant slurry was filtered through a celite-packed filtration assembly, with subsequent washing of the solid residue using dichloromethane (3×5 ml). The combined organic phases were subjected to liquid-liquid extraction with saturated sodium bicarbonate solution (5 ml) supplemented with dichloromethane (5 ml), followed by mechanical agitation (30 min, 25°C). Phase separation was achieved through triplicate dichloromethane extractions (3×5 ml). The organic fractions were successively treated with brine (10 ml), dehydrated over anhydrous sodium sulfate, and solvent-stripped under vacuum. Final isolation of the target compound was accomplished via gradient elution flash chromatography using silica gel stationary phase. Cell culture and MTT assay. The cytotoxic effects of synthetic compounds were evaluated using the CCK-8 method against three human cancer cell models: renal clear cell adenocarcinoma (786-O), renal carcinoma (A498), and hepatocellular carcinoma (HepG2). Cells were maintained under standard conditions in RPMI 1640 medium supplemented with 10% fetal bovine serum, cultured at 37°C in a 5% CO₂ humidified incubator. For cytotoxicity assessment, cells were plated in 96-well microplates at a density of 5 × 10³ cells/well with 100 µl culture medium. After 24-hour exposure to test compounds (prepared in DMSO stock solutions), which were administered either as a single concentration (10 µM) or a nine-point dilution series (20 µM to 0.391 µM, two-fold gradient), 10 µl CCK-8 reagent (Beyotime, China) was added to each well. Following incubation, optical density (OD) at 450 nm was recorded using a microplate reader. Growth inhibition values (GI₅₀) were derived through nonlinear curve fitting with GraphPad Prism software (Version 10). All assays were conducted in triplicate with biological replicates. Colony Formation Assay. 786-O cells were plated in 6-well plates (1000 cells/well) and pre-cultured for 24 hours at 37°C. Subsequently, cells were exposed to compound ‌ 31 ‌ at varying concentrations for 14 days. Following treatment, monolayers were rinsed with PBS, fixed with chilled methanol (10 min), and stained with 0.5% crystal violet (30 min) to visualize colonies. Plates were air-dried after thorough water washing, and colony images were acquired using a digital imaging system. Biological triplicates were performed for statistical rigor. Cell Scrape Assay. 786-O cells (3 × 10⁵ cells/well) were cultured in 6-well plates until confluent. A linear wound was generated across confluent monolayers using a sterile pipette tip. Cells were then incubated with gradient concentrations of ‌ 31 ‌ for 48 hours post-scratching. Migration progression was documented via phase-contrast microscopy at 0 h (initial wound) and 48 h, with cell boundaries annotated for quantitative analysis. Three independent replicates ensured data reproducibility. Apoptosis Detection via FITC Annexin V/PI Staining Apoptotic rates were quantified using a FITC Annexin V/PI Kit (Multi Sciences). Briefly, ‌ 31 ‌-treated 786-O cells were harvested, resuspended in 500 µl binding buffer, and dual-stained with FITC Annexin V (5 µl) and PI (5 µl). After 15 min dark incubation at 25°C, samples were subjected to flow cytometric analysis (Beckman Coulter Epics Elite). Fluorescence signals from 15,000 cellular events per sample were recorded, with apoptotic populations differentiated as early (Annexin V+/PI−) or late (Annexin V+/PI+) stages. Cell cycle arrest detection. Cell cycle progression was assessed using a Cell Cycle Staining Kit (Multi Sciences). ‌ 31 ‌-exposed 786-O cells were fixed in 75% ethanol overnight at − 20°C, pelleted by centrifugation, and resuspended in PI/RNase A staining solution (50 µg/ml PI, 75 KU/mL RNase A). After 30 min dark incubation at 25°C, DNA content was measured via flow cytometry (Beckman Coulter Epics Elite XL). Cell cycle phases were quantified from 15,000 events using EXPO32 ADC software, with triplicate experiments ensuring analytical validity. Results and Discussion Hit Discovery for Potential Anti-Cancer Agents . In our quest for novel anti-cancer drugs, we screened our in-house library of natural products and synthetic compounds using the CCK-8 assay. The results revealed that several chemotypes exhibited notable cytotoxic activities, with a common structural feature: the presence of pyrroloindoline scaffolds. Given our extensive prior work on the synthesis of N -CF 3 -containing pyrroloindolines, we decided to systematically evaluate their cytotoxic activities and conduct an in-depth investigation into their pharmacological properties and potential applications as therapeutic agents. Chemistry. Previously, we developed an efficient method for synthesizing N -CF 3 -containing pyrroloindolines via a cascade oxidative trifluorination and halogenative cyclization reaction of tryptamine-derived isocyanides (Wu et al. 2024 ). In this study, all tested compounds were selected from our established N -CF 3 -containing pyrroloindoline library, with their structures depicted in Scheme 1 . A total of 38 N -CF 3 -containing pyrroloindolines with diverse functional group substitutions were investigated, including 6-Cl, Br, F, Me ( 2 – 5 ); 5-Cl, OMe, CN, Me ( 6 – 9 ); 4-Cl, Br ( 10 and 11 ); and 7-Me ( 12 ). Additionally, compounds with 2-aryl ( 13 – 15 ) and 2-alkyl ( 16 – 18 ) substitutions were included. Two phenylene-bridged bipyrroloindolines ( 19 and 20 ) were also examined. Furthermore, the influence of different 1- N -protecting groups was investigated, including sulfonyl groups ( 21 − 14 ), tert-butyloxycarbonyl (Boc, 25 ), carbobenzyloxy (Cbz, 26 ), 2,2,2-trichloroethoxycarbonyl (Troc, 27 ), and allyloxycarbonyl (Alloc, 28 ). Substituents such as 3-chloro ( 29 ) and 3-iodo ( 30 ) on the pyrroloindoline core, as well as an N -CF 3 -containing pyridoindole ( 31 ), were also included in the study. Lastly, a series of C3-substituted N -CF 3 -containing pyrroloindoline derivatives ( 32 – 38 ) were also tested. Cytotoxicity. The cytotoxicity of the synthesized compounds was evaluated using the CCK-8 assay against three human cancer cell lines: renal carcinoma 786-O, A498 and hepatoma HepG2, with staurosporine (STS) serving as the positive control. Initially, we carried out an assessment to evaluate the growth inhibition rate of N -CF 3 -containing pyrroloindolines derivatives against cancer cells at a concentration of 10 µM (Table 1 ). The results of the present study demonstrated that these compounds effectively reduced the viability of renal carcinoma and hepatoma cells. Based on these findings, the GI 50 values of several preferred compounds ( 6 , 7 , 8 , 19 , 20 , and 31 ) against tumor cells were further investigated. GI 50 values, defined as the concentrations required to inhibit 50% of cell growth, were calculated and summarized in Table 2 . The results indicated that N -CF 3 -containing pyrroloindolines analogues exhibit moderate cytotoxicity against 786-O, A498, and HepG2 cell lines at micromolar concentrations. Table 1 The cell viability of the N -CF 3 -containing pyrroloindolines Cpd. Cell Viability (100%) a Cpd. Cell Viability (100%) a 786-O A498 HepG2 786-O A498 HepG2 STS 0.34 0.13 0.44 20 0.48 0.63 0.75 1 0.93 1.00 0.86 21 1.04 0.89 0.92 2 0.92 0.93 0.99 22 1.06 0.88 0.93 3 1.00 0.90 0.87 23 1.01 0.84 0.84 4 1.00 0.92 1.00 24 1.15 0.84 0.80 5 1.01 1.04 0.92 25 0.96 0.98 1.00 6 0.95 0.94 0.63 26 1.07 0.93 0.88 7 0.91 0.97 0.63 27 1.02 0.90 0.89 8 0.98 0.78 0.59 28 0.90 0.93 0.92 9 0.99 0.83 0.94 29 1.02 0.76 0.74 10 1.08 0.69 1.06 30 0.90 0.81 0.93 11 0.97 0.68 0.98 31 0.42 0.50 0.75 12 1.01 0.81 0.93 32 0.90 0.66 0.88 13 0.99 0.82 1.06 33 1.08 0.99 0.96 14 1.01 0.76 0.93 34 1.09 0.95 0.92 15 1.01 0.83 0.91 35 1.15 0.80 0.96 16 1.02 0.88 0.94 36 1.01 0.97 0.92 17 1.04 1.11 1.10 37 1.15 0.99 0.93 18 1.00 0.96 1.03 38 1.00 0.81 0.87 19 0.43 0.63 0.78 a N -CF 3 -containing pyrroloindolines derivatives (10 µM) against 786-O, A498 and HepG2 cells cells. Staurosporine (1 µM) was used as a positive control. Every experiment was repeated independently at least three times. Table 2 Cytotoxicity of the synthesized compounds. Cpd. GI 50 (µM) a 786-O A498 HepG2 6 > 20 > 20 13.36 7 > 20 > 20 15.60 8 > 20 > 20 13.52 19 7.933 5.744 > 20 20 7.127 8.690 > 20 31 5.627 6.184 > 20 a GI 50 values were defined as the compound concentrations that resulted in 50% cell growth inhibition. Every experiment was repeated independently at least three times. Colony Formation and Cell Scrape Assay. Given that cancer metastasis significantly contributes to the high mortality rate and poor prognosis of cancer patients, the potential inhibitory effect of compound 31 on metastasis provides promising avenues for the development of more effective cytotoxic therapies. To validate the cytotoxic efficacy of 31 , a colony formation assay was carried out using 786-O cells. As depicted in Fig. 1 A, after a 14-day incubation period, prominent disparities in the growth of colony formation were observed. At a concentration of 5.0 µM, 31 manifested a pronounced inhibitory effect on colony growth, with the inhibition displaying a clear dose-dependent trend. These results were consistent with those obtained from the CCK-8 assay. Furthermore, scrape assays were performed to evaluate the potential of 31 in suppressing the metastasis of cancer cells. As presented in Fig. 1 B, the migration of 786-O cells was significantly impeded by 31 in a dose-dependent manner. These findings suggest that 31 possesses the capability to effectively suppress cancer cell metastasis, underscoring its potential significance in advancing cancer treatment strategies. Cell Apoptosis and Cell Cycle Arrest. To evaluate the induction of apoptosis by 31 , flow cytometry assays were performed on 786-O cells. After being incubated with 31 for 24 h, the apoptotic cells were measured by using Annexin–FITC/PI double-staining. As shown in Fig. 2 A, 31 could significantly induce cell apoptosis in 786-O cell lines in a dose-dependent manner. The apoptotic cells were obviously observed even after being incubated at low concentration (0.625 µM). Upon incubation at a concentration of 1.25 µM, the total apoptotic cells (including both early and late apoptotic cells) accounted for 27.91% in 786-O cells. Subsequently, when the concentration was elevated to 5 µM, the proportion of total apoptotic cells in 786-O cells surged to 62.60%. This evidently indicates that 31 can markedly induce apoptosis in 786-O cells. To examine the effect of 31 on cellular events, we applied a flow cytometry assay on 786-O cells. Firstly, the percentage of cells was determined in each phase of the cell cycle. As shown in Fig. 2 B, after 48 h treatment with increasing concentrations of 31 , 786-O cells in S phase showed an observable increase from 3.42–17.29%, and an obvious decrease of cells in G0/G1 phase from 57.72–47.50%. These results suggested that 31 induced a cell cycle arrest in S phase, and its effect on cell cycle was found to be concentration dependent. This is basically consistent with our previous research. 31 causes cell cycle arrest in the S phase, which may further lead to the obstruction of intracellular DNA synthesis, changes in metabolic activities and then induce apoptosis, thus further exerting cytotoxic effects. Conclusions To deeply investigate the pharmacological activities and potential applications of N -CF₃-containing pyrroloindolines, a diverse range of their derivatives was synthesized, and their cytotoxic activities were rigorously evaluated. CCK-8 assays demonstrated that N -CF₃-containing pyrroloindolines exhibited potent antiproliferative effects against three human cancer cell lines (786-O, A498, and HepG2). Among them, compound 31 demonstrated significantly enhanced efficacy against the renal clear cell adenocarcinoma cell line (786-O). The cytotoxic effects of these compounds were further validated through colony formation assay, cell scrape assay, flow cytometric analysis, and studies on apoptosis induction and cell cycle arrest in 786-O cells. These findings highlight the potential of N -CF₃-containing pyrroloindoline derivatives as promising candidates for anti-cancer drug development, warranting further exploration and advancement. Declarations Author Contributions : Data curation, Yu Zhang; Formal analysis, Qigang Sun, Jun-Yunzi Wu and Yu Zhang; Funding acquisition, Yu Zhang and Qingjiang Li; Investigation, Qigang Sun and Jun-Yunzi Wu; Project administration, Qingjiang Li; Supervision, Qingjiang Li; Writing – original draft, Yu Zhang; Writing – review & editing, Qingjiang Li. All authors have read and agreed to the published version of the manuscript. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] . ORCID Qigang Sun: 0009-0001-4708-0613 Jun-Yunzi Wu: 0009-0007-4436-8615 Yu Zhang: 0000-0003-3179-0538 Qingjiang Li: 0000-0001-5535-6993 Declaration Ethics approval Not applicable. Competing interests The authors declare no competing financial interest. Authors' contributions These authors contributed equally to this work. Funding This work was financially supported by the National Natural Science Foundation of China (22171293), the Guangdong Basic and Applied Basic Research Foundation (2024A1515012178), the Hunan Provincial Natural Science Foundation of China (2024JJ6397), and the Hunan Provincial Department of Education Project (23B0414). Availability of data and material The data that support the findings of this study are available from the corresponding author upon reasonable request. References Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. 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Supplementary Files Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 24 May, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Reviewers agreed at journal 06 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor invited by journal 26 Mar, 2025 Editor assigned by journal 23 Mar, 2025 First submitted to journal 23 Mar, 2025 Editorial decision: Major revisions 14 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-6126144","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436884807,"identity":"848fe9e2-88c4-4280-a444-4b6495aafd7c","order_by":0,"name":"Qigang Sun","email":"","orcid":"","institution":"Hainan Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qigang","middleName":"","lastName":"Sun","suffix":""},{"id":436884808,"identity":"a6c7835a-d446-4d67-a608-5fd830bd8ad5","order_by":1,"name":"Jun-Yunzi Wu","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Jun-Yunzi","middleName":"","lastName":"Wu","suffix":""},{"id":436884809,"identity":"fea356e7-dc2a-4344-bf89-dba75af3f7d0","order_by":2,"name":"Yu Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYJAC5j8GNnLsDSCmgQWRengq0ox5DoC1SBCr5czhxB6wFgYitMjPyDFgkGxLS+9h7zHd8KNAgoG/vTsBrxZGkBbDNpvcHp5jaTd7gA6TOHN2A14tzBI55j8S29Jy90skH7vBA9RiIJGLXwubBNCWg22H03kkEttu/iFGCw9QC2PDmcMJPEBbbhNliwTPswJmhoo0Q5BfbssYSPAQ9It8e/IGZgYDG3ke9h6zm2/+2Mjxt/fi18IgkIDmUvzKQYD/AGE1o2AUjIJRMMIBAOdRQFCA9W/1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3179-0538","institution":"University of South China","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":436884810,"identity":"6c14de10-bb41-40e2-8089-973bc41dd860","order_by":3,"name":"Qingjiang Li","email":"","orcid":"","institution":"Sun Yat-Sen University","correspondingAuthor":false,"prefix":"","firstName":"Qingjiang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-02-28 07:17:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6126144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6126144/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-025-00653-9","type":"published","date":"2025-05-24T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81112824,"identity":"086b684a-a492-4baf-ac64-13e32ef3adea","added_by":"auto","created_at":"2025-04-22 11:00:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4609801,"visible":true,"origin":"","legend":"\u003cp\u003eThe colony formation effect of \u003cstrong\u003e31\u003c/strong\u003e on 780-O cells (A). The cells were treated with the indicated concentration of \u003cstrong\u003e31\u003c/strong\u003e (0.625, 1.25, 2.5 and 5.0 μM) for 14 days, and photographed. Effect of \u003cstrong\u003e31\u003c/strong\u003e on metastasis in 780-O cells (B). The cells were treated with the indicated concentration of \u003cstrong\u003e31\u003c/strong\u003e for 48 h. The wounded areas were observed and photographed using microscopy.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6126144/v1/e5354927d65de7b7d8be7a93.png"},{"id":81112817,"identity":"312b3d42-75ac-435c-ae8f-9b781e00adfa","added_by":"auto","created_at":"2025-04-22 11:00:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2426667,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry histograms of apoptosis induced by \u003cstrong\u003e31 \u003c/strong\u003e(A). The cells were incubated with \u003cstrong\u003e31\u003c/strong\u003e (0.625, 1.25, 2.5 and 5.0 μM) for 24 h. Cell cycle analysis upon propidium iodide (PI) staining after 48 h treatment with \u003cstrong\u003e31 \u003c/strong\u003e(B).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6126144/v1/58ecaea084f04bd171c9a61c.png"},{"id":83460108,"identity":"30594224-e2fb-4ddf-8367-0b130f2c81c8","added_by":"auto","created_at":"2025-05-26 16:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7861123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6126144/v1/bee30f2d-8ae6-4cd7-9a7f-369b2f9a26e4.pdf"},{"id":81112833,"identity":"338985ac-92da-4d80-b217-5622d9876357","added_by":"auto","created_at":"2025-04-22 11:00:31","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":39416680,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6126144/v1/8ef1c656f9543dd84e897366.tif"}],"financialInterests":"","formattedTitle":"Discovery and cytotoxic activity of N-CF3-containing pyrroloindolines","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTo date, the SciFinder database search has revealed that approximately three thousand pyrroloindoline-containing compounds have been investigated for their diverse biological activities. Pyrroloindoline-containing natural products have garnered significant attention from the synthetic and medicinal chemistry communities due to their widespread prevalence in structurally complex natural indole alkaloids and their diverse range of biological activities, such as antibacterial (Lam et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Leet et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Umezawa et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Umezawa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Broberg et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pohanka et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kumar and Goodfellow. 2008; Guo et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), antitumor (Alley et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and insecticidal properties (Shiono et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Furutani et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Furutani et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A notable example is (-)-physostigmine and its analogs, which have been clinically employed for treating myasthenia gravis and glaucoma, while also emerging as promising candidates for alleviating symptoms of Alzheimer's disease (Greig et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe trifluoromethyl (CF\u003csub\u003e3\u003c/sub\u003e) group is widely employed to modulate the biophysical properties of small molecules, such as lipophilicity, permeability, and metabolic stability (Purse et al.2008; Zhou et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Meanwel 2018; Inoue et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and has played a pivotal role in the structural optimization of pharmaceuticals. However, the lack of efficient methods to synthesize \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines remains a significant limitation for exploring their potential applications in medicinal chemistry. Current research has largely focused on the synthesis of various complex natural product derivatives (Mei et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mei et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), with limited exploration of the pharmacological properties and application potential of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines. To date, in-depth studies on their biological activities and potential therapeutic value remain scarce.\u003c/p\u003e \u003cp\u003eBuilding on a robust and operationally straightforward approach previously developed by our research group (Wu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), this intriguing structural framework was synthesized from tryptamine-derived isocyanates through a cascade sequence involving oxidative trifluoromethylation followed by halogenative cyclization. Thereby, a diverse array of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindoline derivatives were readily accessed. In this study, we report the discovery of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines with significant cytotoxic activity, laying a foundation for future exploration and development of these compounds as potential anticancer therapeutics.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eGeneral Experiments.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll synthetic reagents were obtained from domestic chemical vendors and employed as received unless specifically noted. Reaction progress monitoring was conducted using Thin-layer chromatography analysis with silica gel GF\u003csub\u003e254\u003c/sub\u003e plates. Nuclear magnetic resonance spectra were recorded on Bruker Avance III spectrometers (400/500 MHz, Germany). High-resolution mass spectrometry (HRMS) data were acquired using a SHIMADZU LCMS-IT-TOF system equipped with ion trap-time-of-flight technology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral procedure for synthesis of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e\u003cb\u003e-CF\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e-containing pyrroloindolines.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn a 10 ml polyethylene reaction vessel maintained at ambient conditions, a dichloromethane solution (0.5 ml) containing N-halosuccinimide (2.5 molar equivalents) was subjected to sequential addition of Et\u003csub\u003e3\u003c/sub\u003eN∙HF (62 \u0026micro;l, 12.0 equiv hydrogen fluoride) and the respective tryptamine-derived isocyanide substrate (0.1 mmol, 1.0 equiv) under vigorous stirring. The heterogeneous mixture was agitated at 25\u0026deg;C for 5\u0026ndash;10 min until Thin-layer chromatography analysis confirmed complete substrate conversion. Subsequent purification through wet-loaded flash chromatography on activated silica gel yielded the target compounds.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral procedure for the Ag(I)-mediated nucleophilic substitution reaction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e1\u003c/b\u003e (0.1 mmol, 1.0 equiv) was dissolved in anhydrous dichloromethane (0.1 M) and thermally equilibrated in an ice-water bath (0\u0026deg;C). The nucleophilic reagent and 2,6-di-tert-butylpyridine (0.2 mmol, 2.0 equiv) were sequentially introduced into the agitated mixture. Following a 20-minute agitation period at maintained temperature, AgNTf₂ (1.2 equiv) was portion-wise incorporated into the heterogeneous system. Post 15-minute cryogenic reaction, the resultant slurry was filtered through a celite-packed filtration assembly, with subsequent washing of the solid residue using dichloromethane (3\u0026times;5 ml). The combined organic phases were subjected to liquid-liquid extraction with saturated sodium bicarbonate solution (5 ml) supplemented with dichloromethane (5 ml), followed by mechanical agitation (30 min, 25\u0026deg;C). Phase separation was achieved through triplicate dichloromethane extractions (3\u0026times;5 ml). The organic fractions were successively treated with brine (10 ml), dehydrated over anhydrous sodium sulfate, and solvent-stripped under vacuum. Final isolation of the target compound was accomplished via gradient elution flash chromatography using silica gel stationary phase.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell culture and MTT assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cytotoxic effects of synthetic compounds were evaluated using the CCK-8 method against three human cancer cell models: renal clear cell adenocarcinoma (786-O), renal carcinoma (A498), and hepatocellular carcinoma (HepG2). Cells were maintained under standard conditions in RPMI 1640 medium supplemented with 10% fetal bovine serum, cultured at 37\u0026deg;C in a 5% CO₂ humidified incubator.\u003c/p\u003e \u003cp\u003eFor cytotoxicity assessment, cells were plated in 96-well microplates at a density of 5 \u0026times; 10\u0026sup3; cells/well with 100 \u0026micro;l culture medium. After 24-hour exposure to test compounds (prepared in DMSO stock solutions), which were administered either as a single concentration (10 \u0026micro;M) or a nine-point dilution series (20 \u0026micro;M to 0.391 \u0026micro;M, two-fold gradient), 10 \u0026micro;l CCK-8 reagent (Beyotime, China) was added to each well. Following incubation, optical density (OD) at 450 nm was recorded using a microplate reader. Growth inhibition values (GI₅₀) were derived through nonlinear curve fitting with GraphPad Prism software (Version 10). All assays were conducted in triplicate with biological replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eColony Formation Assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e786-O cells were plated in 6-well plates (1000 cells/well) and pre-cultured for 24 hours at 37\u0026deg;C. Subsequently, cells were exposed to compound \u0026zwnj;\u003cb\u003e31\u003c/b\u003e\u0026zwnj; at varying concentrations for 14 days. Following treatment, monolayers were rinsed with PBS, fixed with chilled methanol (10 min), and stained with 0.5% crystal violet (30 min) to visualize colonies. Plates were air-dried after thorough water washing, and colony images were acquired using a digital imaging system. Biological triplicates were performed for statistical rigor.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Scrape Assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e786-O cells (3 \u0026times; 10⁵ cells/well) were cultured in 6-well plates until confluent. A linear wound was generated across confluent monolayers using a sterile pipette tip. Cells were then incubated with gradient concentrations of \u0026zwnj;\u003cb\u003e31\u003c/b\u003e\u0026zwnj; for 48 hours post-scratching. Migration progression was documented via phase-contrast microscopy at 0 h (initial wound) and 48 h, with cell boundaries annotated for quantitative analysis. Three independent replicates ensured data reproducibility.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis Detection via FITC Annexin V/PI Staining\u003c/h2\u003e \u003cp\u003eApoptotic rates were quantified using a FITC Annexin V/PI Kit (Multi Sciences). Briefly, \u0026zwnj;\u003cb\u003e31\u003c/b\u003e\u0026zwnj;-treated 786-O cells were harvested, resuspended in 500 \u0026micro;l binding buffer, and dual-stained with FITC Annexin V (5 \u0026micro;l) and PI (5 \u0026micro;l). After 15 min dark incubation at 25\u0026deg;C, samples were subjected to flow cytometric analysis (Beckman Coulter Epics Elite). Fluorescence signals from 15,000 cellular events per sample were recorded, with apoptotic populations differentiated as early (Annexin V+/PI\u0026minus;) or late (Annexin V+/PI+) stages.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell cycle arrest detection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCell cycle progression was assessed using a Cell Cycle Staining Kit (Multi Sciences). \u0026zwnj;\u003cb\u003e31\u003c/b\u003e\u0026zwnj;-exposed 786-O cells were fixed in 75% ethanol overnight at \u0026minus;\u0026thinsp;20\u0026deg;C, pelleted by centrifugation, and resuspended in PI/RNase A staining solution (50 \u0026micro;g/ml PI, 75 KU/mL RNase A). After 30 min dark incubation at 25\u0026deg;C, DNA content was measured via flow cytometry (Beckman Coulter Epics Elite XL). Cell cycle phases were quantified from 15,000 events using EXPO32 ADC software, with triplicate experiments ensuring analytical validity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eHit Discovery for Potential Anti-Cancer Agents\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eIn our quest for novel anti-cancer drugs, we screened our in-house library of natural products and synthetic compounds using the CCK-8 assay. The results revealed that several chemotypes exhibited notable cytotoxic activities, with a common structural feature: the presence of pyrroloindoline scaffolds. Given our extensive prior work on the synthesis of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines, we decided to systematically evaluate their cytotoxic activities and conduct an in-depth investigation into their pharmacological properties and potential applications as therapeutic agents.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChemistry.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePreviously, we developed an efficient method for synthesizing \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines via a cascade oxidative trifluorination and halogenative cyclization reaction of tryptamine-derived isocyanides (Wu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, all tested compounds were selected from our established \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindoline library, with their structures depicted in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 38 \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines with diverse functional group substitutions were investigated, including 6-Cl, Br, F, Me (\u003cb\u003e2\u003c/b\u003e\u0026ndash;\u003cb\u003e5\u003c/b\u003e); 5-Cl, OMe, CN, Me (\u003cb\u003e6\u003c/b\u003e\u0026ndash;\u003cb\u003e9\u003c/b\u003e); 4-Cl, Br (\u003cb\u003e10\u003c/b\u003e and \u003cb\u003e11\u003c/b\u003e); and 7-Me (\u003cb\u003e12\u003c/b\u003e). Additionally, compounds with 2-aryl (\u003cb\u003e13\u003c/b\u003e\u0026ndash;\u003cb\u003e15\u003c/b\u003e) and 2-alkyl (\u003cb\u003e16\u003c/b\u003e\u0026ndash;\u003cb\u003e18\u003c/b\u003e) substitutions were included. Two phenylene-bridged bipyrroloindolines (\u003cb\u003e19\u003c/b\u003e and \u003cb\u003e20\u003c/b\u003e) were also examined.\u003c/p\u003e \u003cp\u003eFurthermore, the influence of different 1-\u003cem\u003eN\u003c/em\u003e-protecting groups was investigated, including sulfonyl groups (\u003cb\u003e21\u003c/b\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cb\u003e14\u003c/b\u003e), tert-butyloxycarbonyl (Boc, \u003cb\u003e25\u003c/b\u003e), carbobenzyloxy (Cbz, \u003cb\u003e26\u003c/b\u003e), 2,2,2-trichloroethoxycarbonyl (Troc, \u003cb\u003e27\u003c/b\u003e), and allyloxycarbonyl (Alloc, \u003cb\u003e28\u003c/b\u003e). Substituents such as 3-chloro (\u003cb\u003e29\u003c/b\u003e) and 3-iodo (\u003cb\u003e30\u003c/b\u003e) on the pyrroloindoline core, as well as an \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyridoindole (\u003cb\u003e31\u003c/b\u003e), were also included in the study. Lastly, a series of C3-substituted \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindoline derivatives (\u003cb\u003e32\u003c/b\u003e\u0026ndash;\u003cb\u003e38\u003c/b\u003e) were also tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cytotoxicity of the synthesized compounds was evaluated using the CCK-8 assay against three human cancer cell lines: renal carcinoma 786-O, A498 and hepatoma HepG2, with staurosporine (STS) serving as the positive control. Initially, we carried out an assessment to evaluate the growth inhibition rate of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines derivatives against cancer cells at a concentration of 10 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results of the present study demonstrated that these compounds effectively reduced the viability of renal carcinoma and hepatoma cells. Based on these findings, the GI\u003csub\u003e50\u003c/sub\u003e values of several preferred compounds (\u003cb\u003e6\u003c/b\u003e, \u003cb\u003e7\u003c/b\u003e, \u003cb\u003e8\u003c/b\u003e, \u003cb\u003e19\u003c/b\u003e, \u003cb\u003e20\u003c/b\u003e, and \u003cb\u003e31\u003c/b\u003e) against tumor cells were further investigated. GI\u003csub\u003e50\u003c/sub\u003e values, defined as the concentrations required to inhibit 50% of cell growth, were calculated and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results indicated that \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines analogues exhibit moderate cytotoxicity against 786-O, A498, and HepG2 cell lines at micromolar concentrations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe cell viability of the \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCpd.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eCell Viability (100%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCpd.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eCell Viability (100%)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e786-O\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA498\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e786-O\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA498\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e24\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e25\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e26\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e29\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e32\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e35\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e38\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003ea\u003c/sup\u003e\u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines derivatives (10 \u0026micro;M) against 786-O, A498 and HepG2 cells cells. Staurosporine (1 \u0026micro;M) was used as a positive control. Every experiment was repeated independently at least three times.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytotoxicity of the synthesized compounds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCpd.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eGI\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e786-O\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA498\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.933\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.744\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e31\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003eGI\u003csub\u003e50\u003c/sub\u003e values were defined as the compound concentrations that resulted in 50% cell growth inhibition. Every experiment was repeated independently at least three times.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eColony Formation and Cell Scrape Assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that cancer metastasis significantly contributes to the high mortality rate and poor prognosis of cancer patients, the potential inhibitory effect of compound \u003cb\u003e31\u003c/b\u003e on metastasis provides promising avenues for the development of more effective cytotoxic therapies. To validate the cytotoxic efficacy of \u003cb\u003e31\u003c/b\u003e, a colony formation assay was carried out using 786-O cells. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, after a 14-day incubation period, prominent disparities in the growth of colony formation were observed. At a concentration of 5.0 \u0026micro;M, \u003cb\u003e31\u003c/b\u003e manifested a pronounced inhibitory effect on colony growth, with the inhibition displaying a clear dose-dependent trend. These results were consistent with those obtained from the CCK-8 assay. Furthermore, scrape assays were performed to evaluate the potential of \u003cb\u003e31\u003c/b\u003e in suppressing the metastasis of cancer cells. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, the migration of 786-O cells was significantly impeded by \u003cb\u003e31\u003c/b\u003e in a dose-dependent manner. These findings suggest that \u003cb\u003e31\u003c/b\u003e possesses the capability to effectively suppress cancer cell metastasis, underscoring its potential significance in advancing cancer treatment strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCell Apoptosis and Cell Cycle Arrest.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the induction of apoptosis by \u003cb\u003e31\u003c/b\u003e, flow cytometry assays were performed on 786-O cells. After being incubated with \u003cb\u003e31\u003c/b\u003e for 24 h, the apoptotic cells were measured by using Annexin\u0026ndash;FITC/PI double-staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, 31 could significantly induce cell apoptosis in 786-O cell lines in a dose-dependent manner. The apoptotic cells were obviously observed even after being incubated at low concentration (0.625 \u0026micro;M). Upon incubation at a concentration of 1.25 \u0026micro;M, the total apoptotic cells (including both early and late apoptotic cells) accounted for 27.91% in 786-O cells. Subsequently, when the concentration was elevated to 5 \u0026micro;M, the proportion of total apoptotic cells in 786-O cells surged to 62.60%. This evidently indicates that \u003cb\u003e31\u003c/b\u003e can markedly induce apoptosis in 786-O cells. To examine the effect of \u003cb\u003e31\u003c/b\u003e on cellular events, we applied a flow cytometry assay on 786-O cells. Firstly, the percentage of cells was determined in each phase of the cell cycle. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, after 48 h treatment with increasing concentrations of \u003cb\u003e31\u003c/b\u003e, 786-O cells in S phase showed an observable increase from 3.42\u0026ndash;17.29%, and an obvious decrease of cells in G0/G1 phase from 57.72\u0026ndash;47.50%. These results suggested that \u003cb\u003e31\u003c/b\u003e induced a cell cycle arrest in S phase, and its effect on cell cycle was found to be concentration dependent. This is basically consistent with our previous research. \u003cb\u003e31\u003c/b\u003e causes cell cycle arrest in the S phase, which may further lead to the obstruction of intracellular DNA synthesis, changes in metabolic activities and then induce apoptosis, thus further exerting cytotoxic effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo deeply investigate the pharmacological activities and potential applications of \u003cem\u003eN\u003c/em\u003e-CF₃-containing pyrroloindolines, a diverse range of their derivatives was synthesized, and their cytotoxic activities were rigorously evaluated. CCK-8 assays demonstrated that \u003cem\u003eN\u003c/em\u003e-CF₃-containing pyrroloindolines exhibited potent antiproliferative effects against three human cancer cell lines (786-O, A498, and HepG2). Among them, compound \u003cb\u003e31\u003c/b\u003e demonstrated significantly enhanced efficacy against the renal clear cell adenocarcinoma cell line (786-O). The cytotoxic effects of these compounds were further validated through colony formation assay, cell scrape assay, flow cytometric analysis, and studies on apoptosis induction and cell cycle arrest in 786-O cells. These findings highlight the potential of \u003cem\u003eN\u003c/em\u003e-CF₃-containing pyrroloindoline derivatives as promising candidates for anti-cancer drug development, warranting further exploration and advancement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Data curation, Yu Zhang; Formal analysis, Qigang Sun, Jun-Yunzi Wu and Yu Zhang; Funding acquisition, Yu Zhang and Qingjiang Li; Investigation, Qigang Sun and Jun-Yunzi Wu; Project administration, Qingjiang Li; Supervision, Qingjiang Li; Writing – original draft, Yu Zhang; Writing – review \u0026amp; editing, Qingjiang Li. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAUTHOR INFORMATION\u003c/p\u003e\n\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003e*E-mail: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQigang Sun: 0009-0001-4708-0613\u003c/p\u003e\n\u003cp\u003eJun-Yunzi Wu: 0009-0007-4436-8615\u003c/p\u003e\n\u003cp\u003eYu Zhang: 0000-0003-3179-0538\u003c/p\u003e\n\u003cp\u003eQingjiang Li: 0000-0001-5535-6993\u003c/p\u003e\n\u003cp\u003eDeclaration\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (22171293), the Guangdong Basic and Applied Basic Research Foundation (2024A1515012178), the Hunan Provincial Natural Science Foundation of China (2024JJ6397), and the Hunan Provincial Department of Education Project (23B0414).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlley, M. 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Chem Rev 116(2), 422-518.\u003c/li\u003e\n\u003c/ol\u003e"}],"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":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"N-CF3 moiety, pyrroloindoline, natural product, cytotoxic activity","lastPublishedDoi":"10.21203/rs.3.rs-6126144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6126144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePyrroloindoline scaffolds are widely found in numerous biologically active and medicinally significant natural products. Meanwhile, the potential applications of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e compounds in medicinal chemistry have garnered increasing attention due to their unique properties. However, the pharmacological activities and therapeutic potential of \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines remain underexplored. Building on our previously developed method for constructing \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindoline scaffolds, this study systematically evaluated the antitumor activities of a diverse series of \u003cem\u003eN\u003c/em\u003e-CF₃-containing pyrroloindoline derivatives. Cell-based assays demonstrated that these derivatives exhibited significant antiproliferative activities against human cancer cell lines, including 786-O, A498, and HepG2. Further investigations revealed that \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines not only induce apoptosis in cancer cells but also inhibit colony formation. Moreover, they showed potential to suppress cancer cell metastasis and proliferation, primarily by inducing apoptosis and cell cycle arrest. These findings suggest that \u003cem\u003eN\u003c/em\u003e-CF\u003csub\u003e3\u003c/sub\u003e-containing pyrroloindolines hold great promise as potential anticancer agents and merit further development.\u003c/p\u003e","manuscriptTitle":"Discovery and cytotoxic activity of N-CF3-containing pyrroloindolines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 11:00:25","doi":"10.21203/rs.3.rs-6126144/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-06T13:44:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T12:34:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-03-27T02:04:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T02:12:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-03-23T22:12:55+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2025-03-14T08:43:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8ed762ff-6059-4590-932b-96f3683642c2","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T16:04:08+00:00","versionOfRecord":{"articleIdentity":"rs-6126144","link":"https://doi.org/10.1007/s43450-025-00653-9","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2025-05-24 15:57:20","publishedOnDateReadable":"May 24th, 2025"},"versionCreatedAt":"2025-04-22 11:00:25","video":"","vorDoi":"10.1007/s43450-025-00653-9","vorDoiUrl":"https://doi.org/10.1007/s43450-025-00653-9","workflowStages":[]},"version":"v1","identity":"rs-6126144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6126144","identity":"rs-6126144","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}