Design, synthesis and biological evaluation of novel 1,2,3-triazoles chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors

Design, synthesis and biological evaluation of novel 1,2,3-triazoles chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors | 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 Design, synthesis and biological evaluation of novel 1,2,3-triazoles chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors Zi-Han Fan, Ri-Zhen Huang, Jia-Jia Liu, Mei-Shan Li, Xiao-Teng Jing, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5283388/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract A series of chromone-oxime derivatives containing 1,2,3-triazole moieties were designed, synthesized and evaluated for their IDO1 inhibitory activities. These compounds displayed moderate to good inhibitory activity against IDO1 with IC 50 values in low micromolar range. Among them, compound D20 displayed the most potent IDO1 inhibitory activities (hIDO1 IC 50 = 0.084 µM, HeLa IDO1 IC 50 = 0.059 µM) and was selected for further investigation. Surface plasmon resonance analysis confirmed that compound D20 directly interacted with IDO1 protein with a K D value of 0.57 µM. Molecular docking study revealed the oxygen atom in chromone-oxime moiety of compound D20 coordinated to the heme iron, and the 1,2,3-triazole group formed a hydrogen bond with the key residue ARG231. The UV spectra showed that D20 induced a Soret peak shift from 404 to 415 nm. Furthermore, compound D20 exhibited no cytotoxicity at its effective concentration in MTT assay. In summary, our study suggested that chromone-oxime derivatives containing 1,2,3-triazole moieties might serve as a potential agent for the further development of IDO1 inhibitors. Indoleamine 2 3-dioxygenase 1 Chromone-oxime 1 2 3-triazole Inhibitors Cancer immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Over the past decade, cancer immunotherapy has revolutionized cancer treatment with enduring efficacy in a variety of cancers. In particular, immune checkpoint inhibitors, including anti-PD1 (programmed cell death protein 1), anti-PDL1 (programmed death ligand 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have gained remarkable clinical success [ 1 , 2 ]. However, the overall response rate of these novel antitumor therapies remains low, only a subset of patients acquire the substantial benefit, limiting their potential to benefit broad patient population [ 3 , 4 ]. This is partially due to the fact that tumor cells could escape immune destruction through evolving various tactics to avoid, subvert, or manipulate innate and adaptive immunity, in addition to the immune checkpoint system in the immunosuppressive tumor microenvironment [ 5 , 6 ]. Therefore, the search for other cancer immunotherapy options able to restore or activate the immune system’s response to cancer is urgently need. A large body of evidences indicate that the immune regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a critical role in tumor immune escape through the control of the kynerenine pathway in the tumor microenvironment [ 7 – 9 ]. IDO1 is a heme-containing enzyme catalyzing the initial and rate limiting step of tryptophan (Trp) catabolism resulting in the generation of kynurenine (Kyn) and depletion of Trp, which can further activate aryl hydrocarbon receptor (AhR) [ 10 , 11 ]. This tumor microenvironment triggers several pathways that involve in suppression of effector T cell (Teff) proliferation and promotion of regulatory T cells (Treg) differentiation [ 12 ]. Actually, constitutive overexpression of IDO1 is related to tumor cells evading the immune response and survive and has been observed in a variety of cancer types, which is well correlated with poor prognosis and low survival of patients [ 9 , 13 ]. Moreover, plenty of evidences revealed that IDO1 could contribute to the arising of resistance in immune checkpoint blockade therapies [ 14 , 15 ], and correspondingly, the combination of checkpoint inhibitors with IDO1 inhibitors offers an advanced strategy in cancer immunotherapy and showed synergetic efficiency in clinical trials. Therefore, IDO1 has been regarded as an attractive target for cancer immunotherapy. A number of small molecule IDO1 inhibitors, such as indoximod, epacadostat, navoximod, BMS-986205 and PF-06840003 (Fig. 1 ), have been developed and subjected to clinic trials as monotherapy or in combination with other therapeutic treatments [ 16 – 18 ]. However, none of them have been approved for the treatment of malignancies by any regulatory agencies. Epacadostat, the most advanced compound in clinical development, showed promising anticancer activity in its early phase I/II trials, but disappointingly failed to reach the primary end point in subsequent pivotal phase III trial in combination with pembrolizumab [ 19 ]. Although the clinical results of a single trial are not a decisive factor in this field, further exploration of the role of IDO1 in tumor immune escape should be done, and there is an urgent need to develop novel and structurally diverse IDO1 inhibitors. Our group has focused on the discovery of potent IDO1 inhibitors for several years. Recently, we reported the identification of a series of sulfonamide chromone-oxime derivatives as potential IDO1 inhibitors [ 20 ]. The docking studies showed that the oxygen atom of oxime moiety can coordinate to the heme iron. Besides, substituted 1,2,3-triazoles have been found to be useful drug scaffolds with wide applications in drug discovery [ 21 – 23 ]. Moreover, 1,2,3-triazole based compounds have been reported as nanomolar IDO1 inhibitors [ 24 – 26 ]. In our continuous efforts to identify more potent IDO1 inhibitors, we designed and synthesized a series of novel chromone-oxime derivatives containing 1,2,3-triazole moieties and evaluated their IDO1 inhibitory activities. Results and discussion Chemistry The detailed synthetic route to all the precursors for the target 1,2,3-triazoles chromone oxime derivatives was outlined in Schemes 1. Intermediates 4 were synthesized by published method [27]. Intermediates 3 were prepared by the reaction of commercially available 2-hydroxy-4 -methoxy benzophenone ( 1 ) with diethyl oxalate ( 2 ) in the presence of sodium methoxide in dioxane, at 120 °C, which were subsequently cyclized under acidic conditions to provide intermediates 4 . The aromatic azides ( 6 ) are prepared by addition of substituted phenyl amine ( 5 ) in the presence of MeCN, this intermediate was then reacted with tertbutyl nitrite and TMSN3. Compound 7 was reacted with different substituted aromatic azides ( 6 ), ascorbate acid and CuSO4·5H2O in MeOH to yield compounds 8 . Compounds 8 were subjected to deprotection with HCl/dioxane to afford intermediates 9 . Subsequently, the important intermediates 10 were obtained by the reaction of the corresponding intermediates 4 with 9 in the presence of HOBT, EDCI and triethylamine in trichloromethane at 25 °C. Finally, intermediates 10 were converted to the target compounds D1 - D26 in the presence of hydroxylamine hydrochloride in methanol. Inhibition of IDO1 activity The inhibitory potency of all synthesized compounds toward IDO1 were measured as hIDO1 IC 50 from enzymatic assays with purified recombinant human IDO1 proteins. All synthesized compounds were also evaluated in a cellular IDO1 inhibition assay. For cellular-based assay, the HeLa cells stimulated with recombinant human IFN-γ were untreated/treated with different concentrations of each compound for 48 h and the amount of generated kynurenine was measured by the absorbance at 480 nm using a microplate reader [28]. Epacadostat was used as a positive control. As shown in Table 1, most compounds displayed potent IDO1 inhibitory activities with the IC 50 values at low micromolar range. Compound D20 with methoxy group at R 1 and 3-fluorine group at R 2 showed the highest inhibitory activity (hIDO1 IC 50 = 0.084 μM, HeLa IDO1 IC 50 = 0.059 μM), which was 2-fold weaker than epacadostat (hIDO1 IC 50 = 0.048 μM, HeLa IDO1 IC 50 = 0.019 μM). The preliminary SAR at the side chain bearing different substituted benzene was first investigated, of which the 2-OCH 3 substitution ( D1 ) yielded a moderate inhibitory activity against IDO1 (hIDO1 IC 50 = 7.62 μM, HeLa IDO1 IC 50 = 5.70 μM). Compound D2 (hIDO1 IC 50 = 4.91 μM, HeLa IDO1 IC 50 = 3.69 μM) with the 3-OCH 3 substituted group retained moderate activity as that of compound D1 . Moving the methoxy group from ortho -position to para -position ( D3 ) resulted in a significant drop of inhibitory activity. Compound D4 obtained by the replacement of 2-OCH 3 group at R 2 of D1 with an electron withdrawing functional group (2-F), which led to a significantly improvement in inhibitory activity (hIDO1 IC 50 = 1.64 μM, HeLa IDO1 IC 50 = 1.07 μM). Substitution with 2-Cl at R 2 position ( D7 , hIDO1 IC 50 = 4.20 μM, HeLa IDO1 IC 50 = 3.26 μM) also displayed potent inhibitory activities despite slight loss of potency than D4 . In addition, a 2 to 6-fold decrease in inhibitory potency was observed accompanied after the replacement chlorine or fluorine with a bromine ( D10 ), respectively. Generally, compounds with electron-withdrawing groups displayed more potent inhibitory activity compared with compounds with electron-donating groups. Compound D5 with 3-fluorine group (hIDO1 IC 50 = 0.78 μM, HeLa IDO1 IC 50 = 0.63 μM) demonstrated improvement in inhibitory activity against IDO1 over methoxy substituted derivative D2 (hIDO1 IC 50 = 4.91 μM, HeLa IDO1 IC 50 = 3.69 μM). In addition, compared to D12 and D19 , moving the halogen group from ortho -position to meta -position resulted in an improved inhibitory activity ( D13 vs D12 , D20 vs D19 , respectively), while changing substituents from meta -position to para -position significantly decreased the enzyme inhibitory activity ( D5 vs D6 , D8 vs D9 , respectively). However, complete loss of activity was obtained by introducing a 4-methoxy or 4-chlorine at R 2 position to respectively give compounds D3 and D9 (IC 50 > 10 μM). In addition, the inhibitory activities of different small substituents at R 1 position were also studied. As shown in Table 1, R 1 substituents at the C-7 position of the chromone group have a significant effect on inhibitory activity. Compounds in which the hydrogen at R 1 was replaced with a methyl group ( D11 vs D1 , D14 vs D6 , D16 vs D10 , respectively) resulted in a significant increase in potency. Additionally, compared with methyl derivative D13 , the substitution with a methoxyl group ( D20 ) at R 1 caused a 4-fold improvement of activity, while a 3-fold improvement in inhibitory potency was observed accompanied after the replacement of hydrogen atom in compound D10 with fluorine ( D26 ). Furthermore, the same compound ( D26 ) displayed similar inhibitory activity with compound D16 . These results suggested the small substituents at R 1 position may occupy the hydrophobic A pocket to obtain more potent inhibitory activity, which is consistent with other published results [29]. Table 1 . Structures and IDO1 inhibitory activities of title compounds D1 - D26 . Compd. R1 R2 Enzymatic assay hIDO1 IC 50 (μM) a Cellular assay Hela (IDO1) IC 50 (μM) a D1 H 2-OCH 3 7.62 ± 1.15 5.70 ± 1.36 D2 H 3-OCH 3 4.91 ± 0.92 3.69 ± 0.80 D3 H 4-OCH 3 >10 >10 D4 H 2-F 1.64 ± 0.83 1.07 ± 0.37 D5 H 3-F 0.78 ± 0.46 0.63 ± 0.14 D6 H 4-F 5.23 ± 2.33 4.02 ± 0.85 D7 H 2-Cl 4.20 ± 0.89 3.26 ± 0.74 D8 H 3-Cl 3.16 ± 0.96 1.89 ± 0.29 D9 H 4-Cl >10 >10 D10 H 2-Br 9.84 ± 1.21 8.86 ± 2.13 D11 CH 3 2-OCH 3 1.45 ± 0.78 0.98 ± 0.43 D12 CH 3 2-F 0.82 ± 0.37 0.74 ± 0.22 D13 CH 3 3-F 0.36 ± 0.08 0.27 ± 0.11 D14 CH 3 4-F 3.11 ± 0.91 2.69 ± 0.62 D15 CH 3 3-Cl 1.23 ± 0.55 0.91 ± 0.23 D16 CH 3 2-Br 2.79 ± 0.42 2.11 ± 0.51 D17 OCH 3 3-OCH 3 0.43 ± 0.21 0.32 ± 0.12 D18 OCH 3 2-Br 1.47 ± 0.35 0.87 ± 0.15 D19 OCH 3 2-F 0.52 ± 0.15 0.48 ± 0.09 D20 OCH 3 3-F 0.084 ± 0.04 0.059 ± 0.02 D21 F 2-OCH 3 0.76 ± 0.14 0.54 ± 0.17 D22 F 4-OCH 3 3.53 ± 0.75 2.26 ± 0.63 D23 F 2-F 0.68 ± 0.26 0.57 ± 0.27 D24 F 2-Cl 2.84 ± 0.63 2.05 ± 0.76 D25 F 4-Cl 5.27 ± 1.72 5.04 ± 1.08 D26 F 2-Br 2.71 ± 0.89 1.92 ± 0.51 Epacadostat - - 0.048 ± 0.01 0.019 ± 0.01 a Data are mean ± SD values from three independent experiments. Surface plasmon resonance analysis To validate the direct interaction of compound D20 with IDO1 protein, we performed a commonly used surface plasmon resonance (SPR) based binding assay using Biacore T200 instrument. This analytic technique facilitates measurement of the kinetic and thermodynamic parameters of ligand-protein complex formation and is widely utilized to investigate enzyme/inhibitor interactions [30]. Association and dissociation measurements were taken and the binding affinity of compound D20 for IDO1 was determined with the aid of Biacore evaluation software. As shown in Figure 2, the discernible exponential curves revealed the concentration-dependent responses for the association and dissociation after compound D20interacting with the immobilized protein, indicative of the binding of compound D20 to and dissociation from the IDO1 protein. The equilibrium dissociation constant (K D , representing binding affinity) between compound D20 and IDO1 was 0.57 μM, which indicated its strong binding affinity to the target IDO1 protein. These data supplied definitive evidence of compound D20 directly binding to IDO1 protein. Molecular docking To further study the structure-activity relationship (SAR) and elucidate the binding mode between chromone oxime derivatives and IDO1, we performed a molecular docking study for the most potent compound D20 with IDO1 using SYBYL-X 2.1 software and the crystal structure of IDO1/inhibitor (PDB: 6KOF) was selected as the template. As illustrated in Figure 3, the oxime group of compound D20 coordinated to the heme iron via an oxygen atom and formed a hydrogen bond with the porphyrin ring of heme, which contributed to its inhibitory activity against IDO1. The benzene ring of chromone-oxime skeleton inserted deep into the vertical region of the heme called “Pocket A” formed by the hydrophobic residues TYR126, VAL130, PHE163, PHE164, SER263, and LEU234 (Pocket A). The methoxy group located deep into Pocket A. This may be the reason that replacing the H atom with methoxy group led to the improvement of activities. In addition, benzene ring formed π−π interaction with amino acid residues PHE164, TYR126, which may be essential for potent IDO1 inhibitory activity. Notably, the triazole group formed a hydrogen bond with the key residue ARG231. More importantly, terminal benzene moiety extended into the active site entrance region (Pocket B) formed by PHE227, ARG231, SER235 and ASN240, where the substitutional F atom formed halogen hydrogen bonds with the side chains of ARG231 and SER235, respectively. The preliminary docking results may support the fact that the compound D20 was potent IDO1 inhibitor. UV−Visible IDO1 Binding Study In order to further validate the binding mode of compound D20 within the IDO1 active site, we conducted a heme binding study using UV−visible absorption spectroscopy (Fig. 4). IDO1 is a heme-containing protein, which comprises a porphyrin ring with an iron atom at the center, and has characteristic light absorption at around λ = 400 nm (Soret band). The optical properties of the heme group are highly sensitive to the local surroundings upon the ligand/substrate binding, which changes the spectral properties of the heme [31]. The use of this unique UV absorption spectral changes in the Soret peak of the heme is an effective and rapid means of assessing the direct interaction between compounds and IDO1. As shown in Fig. 4, in the absence of compound D20 , the absorption spectrum of rhIDO1 exhibited a Soret peak at 404 nm, consistent with the previous literature. An obviously red shift in the Soret band was observed from 404 nm to 415 nm for IDO1 incubated with compound D20 , indicating coordination of the heme iron in the active site by compound D20 . Cytotoxicity Since the inhibition of IDO1 could simply be an effect of the cytotoxicity of the tested compounds, we evaluated their cytotoxicity on A2780, Hct-116 and HeLa cancer cells via the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As summarized in Table 2, most of the compounds exhibited an IC 50 value of micromolar level (most IC 50 values were over 50 μM), which was significantly higher than their IC 50 values against IDO1. We also observed that few compounds showed moderate cytotoxicity against Hct-116. These results indicated that most of the compounds displayed no/negligible influences on cells at their effective concentration against IDO1. Table 2 . Cytotoxicity of compounds D1 - D26 against cancer cell lines. IC 50 ( μM ) a Compd. R 1 R 2 A2780 Hct-116 HeLa D1 H 2-OCH 3 >50 48.70 ± 2.23 >50 D2 H 3-OCH 3 >50 >50 >50 D3 H 4-OCH 3 >50 29.22 ± 1.41 >50 D4 H 2-F >50 >50 >50 D5 H 3-F >50 >50 >50 D6 H 4-F >50 44.43 ± 1.56 >50 D7 H 2-Cl >50 >50 >50 D8 H 3-Cl >50 >50 >50 D9 H 4-Cl >50 >50 >50 D10 H 2-Br >50 >50 >50 D11 CH 3 2-OCH 3 >50 >50 >50 D12 CH 3 2-F >50 >50 >50 D13 CH 3 3-F >50 >50 >50 D14 CH 3 4-F >50 >50 >50 D15 CH 3 3-Cl >50 >50 >50 D16 CH 3 2-Br >50 >50 >50 D17 OCH 3 3-OCH 3 >50 >50 >50 D18 OCH 3 2-Br >50 >50 >50 D19 OCH 3 2-F >50 >50 >50 D20 OCH 3 3-F 46.42 ± 1.43 44.35 ± 2.69 >50 D21 F 2-OCH 3 >50 >50 >50 D22 F 4-OCH 3 >50 23.76 ± 1.37 >50 D23 F 2-F >50 25.82 ± 1.68 >50 D24 F 2-Cl >50 >50 >50 D25 F 4-Cl >50 >50 >50 D26 F 2-Br >50 >50 >50 Epacadostat - - >50 >50 >50 Conclusion In conclusion, a series of novel chromone-oxime derivatives containing triazole moieties were designed and synthesized as IDO1 inhibitors. Most of the derivatives showed potent IDO1 inhibitory activity with IC 50 values at the level of submicromolar concentrations. Encouragingly, compound D20 displayed the most potent IDO1 inhibitor among these derivatives, with an IC 50 value of 0.084 µM and 0.059 µM in the enzymatic and cellular assay against IDO1, respectively. Moreover, the SPR analysis confirmed the interaction between compound D20 and IDO1 protein with an equilibrium dissociation constant (K D ) value of 0.57 µM. Molecular docking study illustrated key interactions between the most active compound D20 and IDO1 in which the chromone-oxime moiety coordinated to the heme iron and formed a hydrogen bond with the porphyrin ring of heme, while the triazole moiety formed a hydrogen bond with the key residue ARG231. Further results of the UV spectra of IDO1 showed a Soret peak shift from 403 to 415 nm in presence of compound D20 , which supported the notion that compound D20 bound directly to the heme iron. Additionally, compound D20 did not exhibited cytotoxicity at its effective concentration in MTT assay. Consequently, our findings suggested that the rational design of chromone-oxime triazole derivatives offers significant potential for the discovery of a new class of IDO1 inhibitors for cancer therapy. Experimental procedure Chemistry Compounds 4 was synthesized according to the literature [27]. All the chemical reagents and solvents used were of analytical grade. Silica gel (200-300 mesh) used in column chromatography was provided by Tsingtao Marine Chemistry Co. Ltd. 1 H NMR spectra were recorded on a 400 MHz ( 1 H, 400 MHz; 13 C, 101 MHz) Bruker spectrometer with TMS as an internal standard in DMSO- d 6 . High-resolution ESI-MS spectra were recorded on an Agilent 1290-6545 UHPLC-QTOF mass spectrometer. G eneral synthetic procedure for compounds D1-D26 . In brief, substituted ortho-hydroxyacetophenone ( 1 ,0.19 g, 1.16 mmol) ) was dissolved in dry 1,4-dioxane (2 ml) followed by adding diethyl oxalate ( 2 , 474 µL, 3.49 mmol) and sodium methoxide solution (531 µL, 2.32 mmol). The mixture was stirred at 120 °C under reflux for 30 min. Then, hydrochloric acid solution (3 mL, 18 mmol) was added to the reaction, which was stirred at 120 °C under reflux for another 30 min. After the reaction, the mixture was poured into water (50 ml), and the precipitate was filtered off. The crude product was washed with dichloromethane and further vacuum dried to obtain compound 4 . The aromatic azides ( 6 ) was prepared by drop tertbutyl nitrite (1.5 mmol) and azide trimethylsilane (1.5 mmol) to the stirring dry acetonitrile solution of substituted aniline ( 5 , 1 mmol) at 0 °C. The content was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the aryl azides which were used in the next step without further purification. To a solution of aromatic azides ( 6 ) and compound 4 in MeOH (5 mL) were added aqueous solution (0.5 ml) of ascorbic acid (35 mg, 0.2 mmol) and CuSO 4 · 5H 2 O (25 mg, 0.1 mmol), respectively. The mixture was stirred at 60 °C for 6 h and evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel eluted with petroleum ether/ethylacetate (V:V = 1:1) to afford compound 8 . To a solution of compound 8 in ethyl acetate (5 mL) were added hydrochloric acid-dioxane solution (5 mL), and stirred at room temperature for 3 h till completion of the reaction (monitored by TLC analysis). The above mixture was evaporated under reduced pressure to afford compound 9 which was used in the next step without further purification. To a solution of compound 4 (1 mmol) and triethylamine (2 mmol) in chloroform (10 mL) was added 1-hydroxybenzotriazole (1.3 mmol, 0.176 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.3 mmol, 0.249 g). The mixture was stirred under ice bath conditions for 6 h followed by adding compound 9 (1 mmol), allowed to stir for another 48 h. After the reaction, the mixture was washed with 1 M HCl (2 × 10 mL), saturated NaHCO 3 aqueous solution (2 × 10 mL) and brine (2 × 10 mL), dried over anhydrous Na 2 SO 4 , and concentrated, followed by purification through column chromatography to yield compound 10 . To a solution of compound 3 (1 mmol) in absolute methanol (10 mL) was added p -toluenesulfonic acid (1.5 mmol). The mixture was refluxed at 70 °C for 0.5 h, an excess amount of hydroxylamine hydrochloride (10 mmol) was added. The reaction was allowed to reflux at 70 °C for 12 h, and remove solvent under vacuum. 2 M NaOH solution (10 mL) was added, then filtered and washed with water (10 mL), ether (10 mL) and absolute methanol (5 mL). The solid was collected and dried to afford compounds D1-D26 . The structures were confirmed by 1 H NMR, 13 C NMR and HR-MS. (E)-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D1 ). Yield: 56.1%, white solid. m.p. 284.3~285.7 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.30 (s, 1H), 9.40 (t, J = 5.9 Hz, 1H), 8.34 (s, 1H), 7.90 – 7.83 (m, 1H), 7.62 – 7.58 (m, 1H), 7.53 (s, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.25 (s, 1H), 7.14 (t, J = 7.7 Hz, 1H), 4.62 (d, J = 5.9 Hz, 2H), 3.85 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.2, 152.1, 150.9, 147.6, 144.6, 142.0, 131.3, 131.1, 126.2, 126.2, 125.9, 125.5, 125.4, 122.7, 121.3, 119.0, 118.3, 113.5, 99.1, 56.6, 35.0. HR-MS (m/z) (ESI): calcd for C 20 H 18 N 5 O 4 [M + H] + :392.1281; found:392.1187. (E)-4-(hydroxyimino)-N-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D2 ). Yield:43.1%. white solid. m.p.183.6~184.3℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.31 (s, 1H), 9.42 (s, 1H), 8.76 (s, 1H), 7.87 (dd, J = 8.0, 1.7 Hz, 1H), 7.60 – 7.44 (m, 4H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 – 7.25 (m, 2H), 7.09 – 7.00 (m, 1H), 4.62 (s, 2H), 3.85 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.6, 160.3, 150.9, 147.6, 146.0, 142.0, 138.2, 131.4, 131.3, 125.9, 122.7, 121.9, 119.0, 118.3, 114.8, 112.4, 105.9, 99.1, 56.1, 35.1. HR-MS (m/z) (ESI): calcd for C 20 H 17 KN 5 O 4 [M + K] + :430.0918; found:430.0829. (E)-4-(hydroxyimino)-N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D3 ). Yield:63.9%. white solid. m.p.258.6~261.7 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.30 (s, 1H), 9.40 (t, J = 5.9 Hz, 1H), 8.62 (s, 1H), 7.87 (dd, J = 8.0, 1.6 Hz, 1H), 7.82 (d, J = 8.9 Hz, 2H), 7.57 – 7.50 (m, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 – 7.23 (m, 2H), 7.12 (d, J = 9.0 Hz, 2H), 4.61 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.2, 159.6, 150.9, 147.6, 145.8, 142.0, 131.3, 130.5, 125.9, 122.7, 122.1, 121.7, 119.0, 118.3, 115.3, 99.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C 20 H 17 K 5 O 4 [M + K] + : 430.0918; found: 430.0171. (E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D4 ). Yield: 66.9%. white solid. m.p. 254.9~257.1℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.31 (s, 1H), 9.43 (t, J = 5.8 Hz, 1H), 8.51 (d, J = 2.1 Hz, 1H), 7.91 – 7.79 (m, 2H), 7.62 – 7.50 (m, 4H), 7.44 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 – 7.24 (m, 2H), 4.64 (d, J = 5.8 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 155.5, 153.0, 150.9, 147.6, 145.5, 142.0, 131.7, 131.6, 131.4, 126.4, 126.0, 126.0, 125.9, 125.3, 125.2, 125.2, 125.1, 122.7, 119.0, 118.3, 117.7, 117.5, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 15 FN 4 O 3 [M + H] + : 380.1159; found: 380.0195. (E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D5 ). Yield:43.1%. white solid. m.p.228.2~231.5℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.31 (s, 1H), 9.43 (t, J = 5.9 Hz, 1H), 8.80 (s, 1H), 7.85 (ddd, J = 17.6, 7.9, 1.9 Hz, 3H), 7.64 (td, J = 8.2, 6.2 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.41 – 7.23 (m, 4H), 4.62 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.1, 161.7, 160.3, 150.9, 147.5, 146.3, 142.0, 138.4, 138.3, 132.3, 132.2, 131.4, 125.9, 122.7, 122.0, 119.0, 118.3, 116.3, 116.3, 115.8, 115.6, 108.0, 107.7, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C 19 H 14 FKN 5 O 3 [M + K] + : 418.0718; found: 418.0648. (E)-N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D6 ). Yield: 55.3%. white solid. m.p.236.5~236.9 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.30 (s, 1H), 9.42 (t, J = 5.9 Hz, 1H), 8.71 (s, 1H), 8.02 – 7.92 (m, 2H), 7.87 (dd, J = 8.0, 1.7 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.44 (t, J = 8.8 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 – 7.23 (m, 2H), 4.61 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 163.2, 160.8, 160.3, 150.9, 147.6, 146.1, 142.0, 133.7, 131.4, 125.9, 122.8, 122.7, 122.0, 119.0, 118.3, 117.3, 117.0, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C 19 H 14 FN 5 NaO 3 [M + Na] + : 402.0978; found: 402.0994. (E)-N-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D7 ). Yield:49.3%. grey solid. m.p. 242.6~244.3 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.30 (s, 1H), 9.44 (t, J = 5.9 Hz, 1H), 8.46 (s, 1H), 7.87 (dd, J = 8.0, 1.6 Hz, 1H), 7.77 (dt, J = 7.9, 1.1 Hz, 1H), 7.69 – 7.50 (m, 3H), 7.37 (dd, J = 8.4, 1.1 Hz, 1H), 7.32 – 7.25 (m, 2H), 4.64 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.6, 145.0, 142.0, 135.0, 132.0, 131.4, 131.0, 128.9, 128.9, 128.8, 125.9, 125.7, 122.7, 119.0, 118.3, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 14 ClN 5 NaO 3 [M + Na] + : 418.0683; found: 418.0646 (E)-N-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D8 ). Yield: 60.7%. white solid. m.p. 256.1~257.3℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.31 (s, 1H), 9.43 (t, J = 5.9 Hz, 1H), 8.82 (s, 1H), 8.07 (s, 1H), 7.91 (dd, J = 29.6, 8.1 Hz, 2H), 7.69 – 7.46 (m, 3H), 7.43 – 7.22 (m, 3H), 4.62 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.5, 146.3, 142.0, 138.1, 134.7, 132.1, 131.2, 128.8, 125.9, 122.7, 121.9, 120.1, 118.9, 118.3, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C 19 H 15 ClN 5 O 3 [M + H] + : 396.0863; found: 396.1263. (E)-N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D9 ). Yield: 40.1%. white solid. m.p. 266.5~267.8 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.31 (s, 1H), 9.43 (t, J = 5.9 Hz, 1H), 8.77 (s, 1H), 8.00 – 7.94 (m, 2H), 7.87 (dd, J = 7.9, 1.7 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.53 (ddd, J = 8.6, 7.2, 1.6 Hz, 1H), 7.37 (dd, J = 8.4, 1.2 Hz, 1H), 7.33 – 7.25 (m, 2H), 4.62 (d, J = 5.8 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.6, 146.3, 142.0, 135.9, 133.3, 131.4, 130.3, 125.9, 122.7, 122.1, 121.9, 119.0, 118.3, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C 19 H 15 ClN 5 O 3 [M + H] + : 396.0863; found: 396.0854. (E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D10 ). Yield:55.4%.yellow solid.m.p.203.5~206.3℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.29 (s, 1H), 9.43 (t, J = 5.9 Hz, 1H), 8.43 (s, 1H), 7.94 – 7.83 (m, 2H), 7.64 – 7.59 (m, 2H), 7.54 (ddt, J = 8.9, 7.2, 2.8 Hz, 2H), 7.37 (d, J = 8.3 Hz, 1H), 7.33 – 7.23 (m, 2H), 4.64 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 151.0, 147.6, 144.9, 142.0, 136.7, 134.1, 132.3, 131.4, 129.4, 129.1, 125.9, 125.7, 122.7, 119.2, 119.0, 118.3, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 14 BrN 5 NaO 3 [M+Na] + : 462.0178; found: 462.0746 (E)-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methyl-4H-chromene-2-carboxamide ( D11 ). Yield: 53.2%. white solid. m.p. 258.8~259.6 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.17 (s, 1H), 9.35 (t, J = 5.9 Hz, 1H), 8.34 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.64 – 7.57 (m, 1H), 7.57 – 7.48 (m, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (s, 1H), 7.19 (s, 1H), 7.17 – 7.08 (m, 2H), 4.61 (d, J = 5.9 Hz, 2H), 3.85 (s, 3H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 152.1, 150.9, 147.6, 144.5, 142.0, 141.4, 131.1, 127.0, 126.2, 126.2, 125.5, 122.5, 121.3, 118.2, 116.3, 113.5, 99.0, 56.6, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C 21 H 19 N 5 NaO 4 [M + Na] + : 428.1335; found: 428.0955. (E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide ( D12 ). Yield: 64.7%. white solid. m.p. 183.1~185.2 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.17 (s, 1H), 9.38 (t, J = 5.9 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H), 7.83 (td, J = 7.8, 1.6 Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.66 – 7.52 (m, 2H), 7.48 – 7.41 (m, 1H), 7.24 (s, 1H), 7.18 (s, 1H), 7.12 (dd, J = 8.1, 1.7 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 155.5, 153.0, 150.9, 147.5, 145.4, 142.0, 141.4, 131.7, 131.6, 127.0, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 125.1, 122.5, 118.2, 117.7, 117.5, 116.3, 99.0, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C 20 H 17 FN 5 O 3 [M + H] + : 394.1315; found: 394.1265. (E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide ( D13 ). Yield: 46.9%. white solid. m.p. 213.7~214.3 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.17 (s, 1H), 9.38 (t, J = 5.9 Hz, 1H), 8.79 (s, 1H), 7.91 – 7.79 (m, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.70 – 7.59 (m, 1H), 7.34 (td, J = 8.5, 2.5 Hz, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.12 (d, J = 8.2 Hz, 1H), 4.61 (d, J = 5.9 Hz, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.1, 161.7, 160.3, 150.9, 147.5, 146.3, 142.0, 141.4, 138.4, 138.3, 132.3, 132.2, 127.0, 122.5, 122.0, 118.2, 116.3, 115.8, 115.6, 108.0, 107.7, 99.0, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C 20 H 17 FN 5 O 3 [M + H] + : 394.1315; found: 394.0732. (E)-N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide ( D14 ). Yield: 32.1%. white solid. m.p. 221.7~222.6 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.17 (s, 1H), 9.39 (t, J = 6.0 Hz, 1H), 8.44 (d, J = 12.8 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.75 (dd, J = 11.9, 7.9 Hz, 1H), 7.69 – 7.50 (m, 3H), 7.24 (s, 1H), 7.19 (s, 1H), 7.12 (d, J = 8.1 Hz, 1H), 4.63 (d, J = 5.9 Hz, 2H), 2.37 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.6, 144.8, 142.0, 141.4, 136.7, 134.1, 132.3, 132.0, 131.0, 129.4, 129.1, 128.9, 128.8, 127.0, 125.7, 122.5, 119.2, 118.2, 116.3, 99.1, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C 20 H 17 FN 5 O 3 [M + H] + : 394.1315; found: 394.0934. (E)-N-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide ( D15 ). Yield: 31.2%. white solid. m.p. 155.7~157.3 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.18 (s, 1H), 9.39 (t, J = 5.9 Hz, 1H), 8.81 (s, 1H), 8.07 (t, J = 2.0 Hz, 1H), 7.98 – 7.92 (m, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 8.1 Hz, 1H), 7.58 – 7.53 (m, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.12 (dd, J = 8.2, 1.7 Hz, 1H), 4.61 (d, J = 5.8 Hz, 2H), 2.37 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.5, 146.3, 142.0, 141.4, 138.1, 134.7, 132.1, 128.8, 127.0, 122.5, 121.9, 120.1, 119.0, 118.2, 116.3, 99.1, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C 20 H 17 ClN 5 O 3 [M + H] + : 410.1020; found: 410.2033. (E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide ( D16 ). Yield: 49.3%. yellow solid. m.p. 239.0~240.6 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.17 (s, 1H), 9.38 (d, J = 6.0 Hz, 1H), 8.71 (s, 1H), 7.96 (dd, J = 8.7, 4.7 Hz, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 8.6 Hz, 2H), 7.29 – 7.07 (m, 3H), 4.61 (d, J = 5.5 Hz, 2H), 2.36 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 160.3, 150.9, 147.5, 146.1, 142.0, 141.4, 133.7, 127.0, 122.8, 122.7, 122.5, 122.1, 118.2, 117.3, 117.0, 116.3, 99.0, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C 20 H 16 BrN 5 NaO 3 [M + Na] + : 476.0334; found: 476.3274. (E)-4-(hydroxyimino)-7-methoxy-N-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D17 ). Yield: 36.2%. white solid. m.p.216.4~222.7 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.06 (s, 1H), 9.38 (s, 1H), 8.76 (s, 1H), 7.76 (d, J = 9.5 Hz, 1H), 7.56 – 7.41 (m, 3H), 7.25 (s, 1H), 7.05 (dt, J = 5.2, 2.6 Hz, 1H), 6.91 (dt, J = 4.9, 2.5 Hz, 2H), 4.61 (d, J = 5.7 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 161.6, 160.6, 160.2, 152.2, 147.5, 145.9, 141.9, 138.2, 131.3, 123.9, 121.9, 114.8, 114.0, 112.4, 111.7, 106.0, 101.8, 99.1, 56.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C 21 H 19 N 5 NaO 5 [M + Na] + :444.1284; found:444.0661. (E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide ( D18 ). Yield: 65.1%. yellow solid. m.p. 116.4~120.7 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.06 (s, 1H), 9.41 (t, J = 5.9 Hz, 1H), 8.43 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.80 – 7.70 (m, 1H), 7.61 (d, J = 4.5 Hz, 2H), 7.59 – 7.51 (m, 1H), 7.24 (s, 1H), 6.90 (d, J = 7.5 Hz, 2H), 4.63 (d, J = 5.8 Hz, 2H), 3.82 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 161.6, 160.2, 152.1, 147.6, 144.9, 141.8, 136.7, 134.1, 132.3, 129.4, 129.1, 125.7, 123.9, 119.2, 114.0, 111.7, 101.8, 99.1, 56.0, 34.9. HR-MS (m/z) (ESI): calcd for C 20 H 16 BrKN 5 O 4 [M + K] + : 508.0023; found: 508.0137. (E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide ( D19 ). Yield: 41.2%. white solid. m.p. 240.8~241.5 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.06 (s, 1H), 9.40 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.76 (d, J = 9.5 Hz, 1H), 7.65 – 7.53 (m, 2H), 7.48 – 7.40 (m, 1H), 7.24 (s, 1H), 6.90 (dq, J = 5.5, 2.6 Hz, 2H), 4.63 (d, J = 5.9 Hz, 2H), 3.82 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 161.6, 160.3, 155.5, 153.0, 152.1, 147.5, 145.4, 141.8, 131.7, 131.6, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 125.1, 123.8, 117.7, 117.5, 114.0, 111.7, 101.8, 99.1, 56.0, 34.9. HR-MS (m/z) (ESI): calcd for C 21 H 17 FN 5 O 4 [M + H] + : 410.1265; found: 410.1256. (E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide ( D20 ). Yield: 63.5%, white solid, m.p: 194.9~195.5 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.07 (s, 1H), 9.40 (s, 1H), 8.80 (s, 1H), 7.93 – 7.71 (m, 3H), 7.64 (q, J = 7.7 Hz, 1H), 7.41 – 7.19 (m, 2H), 6.91 (d, J = 6.1 Hz, 2H), 4.69 – 4.55 (m, 2H), 3.82 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.1, 161.7, 161.6, 160.3, 152.1, 147.5, 146.2, 141.8, 138.3, 132.3, 132.2, 123.9, 122.0, 116.3, 116.3, 115.9, 115.6, 114.0, 111.7, 108.0, 107.7, 101.8, 99.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C 21 H 17 FN 5 O 4 [M + H] + : 410.1265; found: 410.1266. (E)-7-fluoro-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D21 ). Yield: 45.5%. white solid. m.p. 267.3~268.1 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 9.37 (s, 1H), 8.35 (s, 1H), 7.90 (s, 1H), 7.70 – 7.45 (m, 2H), 7.39 – 7.07 (m, 5H), 4.62 (s, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.6, 162.1, 159.9, 152.1, 151.9, 151.7, 147.6, 144.4, 141.4, 131.2, 126.2, 126.2, 125.6, 124.9, 124.8, 121.3, 115.8, 115.8, 113.9, 113.7, 113.5, 105.4, 105.1, 99.3, 56.6, 35.0. HR-MS (m/z) (ESI): calcd for C 20 H 17 FN 5 O 4 [M + H] + : 410.1265; found: 410.1258. (E)-7-fluoro-4-(hydroxyimino)-N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide ( D22 ). Yield: 30.9%. white solid. m.p. 250.3~252.4 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.34 (s, 1H), 9.35 (s, 1H), 8.61 (s, 1H), 7.90 (dd, J = 9.6, 6.3 Hz, 1H), 7.85 – 7.77 (m, 2H), 7.26 (s, 1H), 7.18 (ddd, J = 8.5, 4.3, 1.8 Hz, 2H), 7.15 – 7.09 (m, 2H), 4.60 (d, J = 5.8 Hz, 2H), 3.83 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.6, 162.1, 156.0, 159.6, 151.9, 151.7, 147.5, 145.6, 141.4, 130.5, 124.9, 124.8, 122.1, 121.8, 115.8, 115.3, 113.9, 113.7, 105.3, 105.1, 99.3, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C 20 H 17 FN 5 O 4 [M + H] + : 410.1265; found: 410.1249. (E)-7-fluoro-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D23 ). Yield: 57.9%. white solid. m.p. 264.5~266.8 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 9.40 (t, J = 5.9 Hz, 1H), 8.52 (d, J = 2.1 Hz, 1H), 7.90 (dd, J = 9.8, 6.2 Hz, 1H), 7.83 (td, J = 7.8, 1.6 Hz, 1H), 7.59 (dqt, J = 11.7, 7.4, 2.2 Hz, 2H), 7.48 – 7.40 (m, 1H), 7.26 (d, J = 1.5 Hz, 1H), 7.18 (dt, J = 8.6, 2.2 Hz, 2H), 4.64 (d, J = 5.8 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.6, 162.1, 160.0, 155.5, 153.0, 151.8, 151.7, 147.5, 145.4, 141.3, 131.7, 131.6, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 124.9, 124.8, 117.7, 117.5, 115.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 14 F 2 N 5 O 3 [M + H] + : 398.1065; found: 398.2318. (E)-N-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D24 ). Yield: 69.5%. white solid. m.p. 239.4~242.3 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 9.42 (t, J = 6.0 Hz, 1H), 8.47 (s, 1H), 7.89 (td, J = 11.2, 10.5, 6.6 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.71 – 7.53 (m, 3H), 7.26 (s, 1H), 7.18 (t, J = 9.7 Hz, 2H), 4.64 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 162.1, 160.0, 151.9, 151.7, 147.5, 145.0, 144.9, 141.3, 135.0, 132.0, 131.0, 128.9, 128.8, 125.8, 124.9, 124.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 14 ClFN 5 O 3 [M + H] + : 414.0769; found: 414.0789. (E)-N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D25 ). Yield: 40.3%, white solid, m.p: 250.7~251.4 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 9.40 (t, J = 5.8 Hz, 1H), 8.77 (s, 1H), 8.00 – 7.94 (m, 2H), 7.90 (dd, J = 9.7, 6.3 Hz, 1H), 7.72 – 7.61 (m, 2H), 7.27 (s, 1H), 7.22 – 7.15 (m, 2H), 4.62 (d, J = 5.8 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 164.6, 162.1, 160.0, 151.8, 151.7, 147.5, 146.1, 141.4, 135.9, 133.3, 130.3, 124.9, 124.8, 122.1, 121.9, 115.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.1. HR-MS (m/z) (ESI): calcd for C 19 H 14 ClFN 5 O 3 [M + H] + : 414.0769; found: 414.0805. (E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide ( D26 ). Yield: 37.1%, yellow solid, m.p: 246.7~251.7 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.35 (s, 1H), 9.41 (t, J = 5.9 Hz, 1H), 8.44 (s, 1H), 7.90 (t, J = 8.2 Hz, 2H), 7.61 (d, J = 4.5 Hz, 2H), 7.55 (dt, J = 8.8, 4.6 Hz, 1H), 7.26 (s, 1H), 7.18 (t, J = 7.8 Hz, 2H), 4.64 (d, J = 5.9 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 162.1, 160.0, 151.9, 151.7, 147.5, 144.8, 141.3, 136.7, 134.1, 132.3, 129.4, 129.1, 125.7, 124.9, 124.8, 119.2, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C 19 H 14 BrFN 5 O 3 [M + H] + : 458.0264; found: 458.1380. B iological assays The biological experimental procedures including enzymatic assays, cellular IDO1 activity assays, SPR experiments, UV−visible spectra and cell viability assays were carried out according to our previous work[20, 32, 33] and described in the Supplementary Information (SI). Molecular docking Molecular docking was carried out in Sybyl-X 2.1 on a Windows workstation and described in the SI. The crystal structure of IDO1 protein with inhibitor was retrieved from the RCSB Protein Data Bank (IDO1: 6KOF) [34]. Declarations Conflicts of interest There are no conflicts to declare. Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 82104008 and 21977021), the Guangxi Science and Technology Base and Talents Program (AD20297059), the China Postdoctoral Science Foundation (2021MD703847), State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (No. CMEMR2023-B17), Middle-aged and Young Teachers' Basic Ability Promotion Project of Guangxi (2021KY0942). References Yang Y (2015) J Clin Invest 125: 3335−3337. Zou W (2005) Nat Rev Cancer 5: 263−274. Majzner RG, Mackall CL (2019) Nat Med 25: 1341-55. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, Iyer AK (2017) Front Pharmacol 8: 561−575. Munn DH, Mellor AL (2016) Trends Immunol 37: 193-207. Binnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, Coussens LM, Gabrilovich DI, Ostrand-Rosenberg S, Hedrick CC, Vonderheide RH, Pittet MJ, Jain RK, Zou W, Howcroft TK, Woodhouse EC, Weinberg RA, Krummel MF (2018) Nat Med 24: 541-550. Prendergast GC, Mondal A, Dey S, Laury-Kleintop LD, Muller AJ (2018) Trends Cancer 4: 38−58. Uyttenhove C, Pilotte L, Théta I, Stroobant V, Colau D, Parmentier N, Boon T, Eynde BJV (2003) Nat Med 9: 1269-1274. Godin-Ethier J, Hanafi LA, Piccirillo CA, Lapointe R (2011) Clin Cancer Res 17: 6985-6991. Opitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, Trump S, Schumacher T, Jestaedt L, Schrenk D, Weller M, Jugold M, Guillemin GJ, Miller CL, Lutz C, Radlwimmer B, Lehmann I, von Deimling A, Wick W, Platten M (2011) Nature 478: 197-203. Sugimoto H, Oda S, Otsuki T, Hino T, Yoshida T, Shiro Y (2006) Proc Natl Acad Sci U.S.A. 103: 2611-2616. Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, Mellor AL (2005) Immunity 22: 633−642. Hornyák L, Dobos N, Koncz G, Karányi Z, Páll D, Szabó Z, Halmos G, Székvölgyi L (2018) Front Immunol 9: 151. Ricciuti B, Leonardi GC, Puccetti P, Fallarino F, Bianconi V, Sahebkar A, Baglivo S, Chiari R, Pirro M (2019) Pharmacol Ther 196: 105−116. Li A, Barsoumian HB, Schoenhals JE, Cushman TR, Caetano MS, Wang X, Valdecanas DR, Niknam S, Younes AI, Li G, Woodward WA, Cortez MA, Welsh JW (2018) Cancer Lett 431: 54-463. Weng T, Qiu X, Wang J, Li Z, Bian J (2018) Eur J Med Chem 143: 656-669. Platten M, Nollen EAA, Röhrig UF, Fallarino F, Opitz CA (2019) Nat Rev Drug Discovery 18: 379−401. Feng X, Liao D, Liu D, Ping A, Li Z, Bian J (2020) J Med Chem 63: 15115-15139. Long GV, Dummer R, Hamid O, Gajewski TF, Caglevic C, Dalle S, Arance A, Carlino MS, Grob J-J, Kim TM, Demidov L, Robert C, Larkin J, Anderson JR, Maleski J, Jones M, Diede SJ, Mitchell TC (2019) Lancet Oncol. 20: 1083−1097. Wang K, Song L-H, Liang Q-L, Zhang Y, Ma X-L, Wang Q, Zhang H-Y, Jiang C-N, Wei J-H, Huang R-Z. (2023) Eur J Med Chem 254: 115349. Zhao S, Liu J, Lv Z, Zhang G, Xu Z (2023) Eur J Med Chem 251: 115254. Bonandi E, Christodoulou MS, Fumagalli G, Perdicchia D, Rastelli G, Passarella D (2017) Drug Discov Today 22: 1572−1581. Huang R-Z, Liang G-B, Li M-S, Fang Y-L, Zhao S-F, Zhou M-M, Liao Z-X, Sun J, Wang H-S. (2019) MedChemCommun 10: 584-597. Röhrig UF, Awad L, Grosdidier A, Larrieu P, Stroobant V, Colau D, Cerundolo V, Simpson AJ, Vogel P, van den Eynde BJ, Zoete V, Michielin O (2010) J Med Chem 53: 1172−1189. Röhrig UF, Majjigapu SR, Grosdidier A, Bron S, Stroobant V, Pilotte L, Colau D, Vogel P, van den Eynde B J, Zoete V, Michielin O (2012) J Med Chem 55: 5270−5290. He X, He G, Chu Z, Wu H, Wang J, Ge Y, Shen H, Zhang S, Shan J, Peng K, Wei Z, Zou Y, Xu Y, Zhu Q (2021) J Med Chem 64: 17950−17968. Gaspar A, Matos MJ, Garrido J, Uriarte E, Borges F (2014) Chem. Rev. 114: 4960−4992. Ye K, Wang K, Wang T, Tang H, Wang L, Zhang W, Jiang S, Zhang X, Zhang K (2023) Eur J Med Chem 250: 115217. Röhrig UF, Majjigapu SR, Vogel P, Zoete V, Michielin O (2015) J Med Chem 58: 9421−9437. Zhang H, Liu C, Chen Q, Shen L-A, Xiao W, Li J, Wang Y, Zhu D, Zhang Q, Li J (2023) J Med Chem 66: 1349−1379. Zou Y, Hu Y, Ge S, Zheng Y, Li Y, Liu W, Guo W, Zhang Y, Xu Q, Lai Y (2019) Eur J Med Chem 184: 111750. Huang R, Jing X, Huang X, Pan Y, Fang Y, Liang G, Liao Z, Wang H, Chen Z, Zhang Y (2020) J Med Chem 63: 1544−1563. Liu S-Q, Mao Z-C, Xu Y-L, Chen X-M, Wang H-L, Wang Q, Wei J-H, Huang R-Z, Zhang Y (2023) Bioorg Chem 131: 106323. Peng YH, Liao FY, Tseng CT, Kuppusamy R., Li AS, Chen CH, Fan YS, Wang SY, Wu MH, Hsueh CC, Chang JY, Lee LC, Shih C, Shia KS, Yeh TK, Hung MS, Kuo CC, Song JS, Wu SY, Ueng SH (2020) J Med Chem 63: 1642-1659. Supplementary Files scheme.png Schemes 1. Reagents and reaction conditions:（a）dioxane, diethyl oxalate and sodium methoxide, 120 ℃, 30 min; (b) hydrochloric acid, 120 ℃, 30 min; (c) t-BuONO, TMSN 3 , MeCN, 3 h, r.t; (d) CuSO 4 , ascorbate acid, MeOH, H 2 O, 6 h, 60 ℃; (e) ethyl acetate, HCl/dioxane, 4 h, r.t; (f) trichloromethane, EDCI, HOBT, triethylamine, r.t, 48 h; (g) methanol, hydroxylamine hydrochloride, 12 h, 70 ℃. GraphicalAbstract.png Graphical abstract Suporttinginformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 12 Dec, 2024 Reviewers invited by journal 06 Dec, 2024 Editor assigned by journal 19 Oct, 2024 First submitted to journal 17 Oct, 2024 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5283388","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":387277717,"identity":"4e3e29b0-0675-4cd3-9fc2-712b7b7404db","order_by":0,"name":"Zi-Han Fan","email":"","orcid":"","institution":"Guilin Normal College","correspondingAuthor":false,"prefix":"","firstName":"Zi-Han","middleName":"","lastName":"Fan","suffix":""},{"id":387277718,"identity":"acc17002-3405-4b0f-b1e3-5075edbd59ab","order_by":1,"name":"Ri-Zhen Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIie3PsQrCMBCA4UihLiddU6w+Q0WIg0JfJUXoJjh2qKIg6WDFV3F0bBHicjrXTR/AwU0HQUVxbDMK5h+OG+4bjhCd7ieD9/S2cXbkYaROHILYd48o1UmP5JzZp5lRDjrLnaS39QgqSRqE/sQkVjznhcRJ94G9wC0Y1anM/bVDKO5WhYRWEkZrQoIJaZD7aBKXDkqIAcy+PwlQzoa+MBSICaxeExHQJyFqBKDdbYgUXMA+5Sih/BcLW4ezGHtuNc4u1zBqWvGimHzafDdQOX81Vj3U6XS6f+wBTFdFjhxKrUYAAAAASUVORK5CYII=","orcid":"","institution":"Guilin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ri-Zhen","middleName":"","lastName":"Huang","suffix":""},{"id":387277719,"identity":"c25f507f-ec73-48b1-b601-86e94932b24b","order_by":2,"name":"Jia-Jia Liu","email":"","orcid":"","institution":"Guangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Jia","middleName":"","lastName":"Liu","suffix":""},{"id":387277720,"identity":"ea65f6b9-a977-43d8-b9a0-9f1615c83aa9","order_by":3,"name":"Mei-Shan Li","email":"","orcid":"","institution":"Guangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mei-Shan","middleName":"","lastName":"Li","suffix":""},{"id":387277721,"identity":"3ee22888-266c-41c6-9cfd-501da9df22a2","order_by":4,"name":"Xiao-Teng Jing","email":"","orcid":"","institution":"Guilin Normal College","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Teng","middleName":"","lastName":"Jing","suffix":""},{"id":387277722,"identity":"293d5d99-6052-4e5a-948e-c638d2c56501","order_by":5,"name":"Heng-Shan Wang","email":"","orcid":"","institution":"Guangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Heng-Shan","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-17 13:54:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5283388/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5283388/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70928193,"identity":"54967add-a780-4828-944b-fdf34c7c3ba4","added_by":"auto","created_at":"2024-12-09 09:26:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68910,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of representative IDO1 inhibitors in clinical trial\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/1c0da1b23d1a1819a6c1a2a4.png"},{"id":70928341,"identity":"ce6d79bd-b904-45f2-b5c0-11f1ef038128","added_by":"auto","created_at":"2024-12-09 09:34:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114629,"visible":true,"origin":"","legend":"\u003cp\u003eBiophysical binding data supports the interaction of compound \u003cstrong\u003eD20\u003c/strong\u003e with human IDO1 protein. Dose response curve determined by SPR for the binding of IDO1 with \u003cstrong\u003eD20\u003c/strong\u003eis shown.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/810bb8cb36268cc26f1e8906.png"},{"id":70928187,"identity":"9f7ef29c-2f64-4d71-b2c7-b72bbcb47f36","added_by":"auto","created_at":"2024-12-09 09:26:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":383523,"visible":true,"origin":"","legend":"\u003cp\u003eBinding modes of compound \u003cstrong\u003eD20\u003c/strong\u003e in the active site of IDO1 (PDB: 6KOF). Ligands and the important residues for binding interactions are represented by stick and line models. The hydrogen bonds are shown as yellow dotted lines (color figure online).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/3437f8e5627031a7d381b4bb.png"},{"id":70928343,"identity":"ecbc3b83-cda5-4a5d-837a-0f778c31fa0f","added_by":"auto","created_at":"2024-12-09 09:34:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70403,"visible":true,"origin":"","legend":"\u003cp\u003eUV spectra of ferric IDO1 without (black) and with 2 mM concentration of compound \u003cstrong\u003e20\u003c/strong\u003e (red). In the presence of \u003cstrong\u003e20\u003c/strong\u003e, the Soret peak shifts from 404 to 415 nm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/6c16c021bea82b9cecb535b2.png"},{"id":70930479,"identity":"cc486caa-b836-40eb-8188-e395f628d851","added_by":"auto","created_at":"2024-12-09 09:58:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1569352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/fe56433f-5001-4c92-a5d4-72f9883883d0.pdf"},{"id":70928185,"identity":"73389628-9deb-4cf7-94e2-90f7fe27bb71","added_by":"auto","created_at":"2024-12-09 09:26:24","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":73522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchemes 1\u003c/strong\u003e. Reagents and reaction conditions:（a）dioxane, diethyl oxalate and sodium methoxide, 120 ℃, 30 min; (b) hydrochloric acid, 120 ℃, 30 min; (c) t-BuONO, TMSN\u003csub\u003e3\u003c/sub\u003e, MeCN, 3 h, r.t; (d) CuSO\u003csub\u003e4\u003c/sub\u003e, ascorbate acid, MeOH, H\u003csub\u003e2\u003c/sub\u003eO, 6 h, 60 ℃; (e) ethyl acetate, HCl/dioxane, 4 h, r.t; (f) trichloromethane, EDCI, HOBT, triethylamine, r.t, 48 h; (g) methanol, hydroxylamine hydrochloride, 12 h, 70 ℃.\u003c/p\u003e","description":"","filename":"scheme.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/25cbfec4702af32a75cedd5e.png"},{"id":70928188,"identity":"0314df74-dadd-4f8e-b540-e131dbacd657","added_by":"auto","created_at":"2024-12-09 09:26:24","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":209482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/4b4b9d19ccb04dae90972893.png"},{"id":70928218,"identity":"181d5400-71c8-46c9-91ab-0e105cb4c6ef","added_by":"auto","created_at":"2024-12-09 09:26:26","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6396366,"visible":true,"origin":"","legend":"","description":"","filename":"Suporttinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5283388/v1/0655948c52efd98a6a11e5e2.docx"}],"financialInterests":"","formattedTitle":"Design, synthesis and biological evaluation of novel 1,2,3-triazoles chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the past decade, cancer immunotherapy has revolutionized cancer treatment with enduring efficacy in a variety of cancers. In particular, immune checkpoint inhibitors, including anti-PD1 (programmed cell death protein 1), anti-PDL1 (programmed death ligand 1) and anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have gained remarkable clinical success [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, the overall response rate of these novel antitumor therapies remains low, only a subset of patients acquire the substantial benefit, limiting their potential to benefit broad patient population [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This is partially due to the fact that tumor cells could escape immune destruction through evolving various tactics to avoid, subvert, or manipulate innate and adaptive immunity, in addition to the immune checkpoint system in the immunosuppressive tumor microenvironment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the search for other cancer immunotherapy options able to restore or activate the immune system\u0026rsquo;s response to cancer is urgently need.\u003c/p\u003e \u003cp\u003eA large body of evidences indicate that the immune regulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays a critical role in tumor immune escape through the control of the kynerenine pathway in the tumor microenvironment [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. IDO1 is a heme-containing enzyme catalyzing the initial and rate limiting step of tryptophan (Trp) catabolism resulting in the generation of kynurenine (Kyn) and depletion of Trp, which can further activate aryl hydrocarbon receptor (AhR) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This tumor microenvironment triggers several pathways that involve in suppression of effector T cell (Teff) proliferation and promotion of regulatory T cells (Treg) differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Actually, constitutive overexpression of IDO1 is related to tumor cells evading the immune response and survive and has been observed in a variety of cancer types, which is well correlated with poor prognosis and low survival of patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, plenty of evidences revealed that IDO1 could contribute to the arising of resistance in immune checkpoint blockade therapies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and correspondingly, the combination of checkpoint inhibitors with IDO1 inhibitors offers an advanced strategy in cancer immunotherapy and showed synergetic efficiency in clinical trials. Therefore, IDO1 has been regarded as an attractive target for cancer immunotherapy.\u003c/p\u003e \u003cp\u003eA number of small molecule IDO1 inhibitors, such as indoximod, epacadostat, navoximod, BMS-986205 and PF-06840003 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), have been developed and subjected to clinic trials as monotherapy or in combination with other therapeutic treatments [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, none of them have been approved for the treatment of malignancies by any regulatory agencies. Epacadostat, the most advanced compound in clinical development, showed promising anticancer activity in its early phase I/II trials, but disappointingly failed to reach the primary end point in subsequent pivotal phase III trial in combination with pembrolizumab [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although the clinical results of a single trial are not a decisive factor in this field, further exploration of the role of IDO1 in tumor immune escape should be done, and there is an urgent need to develop novel and structurally diverse IDO1 inhibitors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur group has focused on the discovery of potent IDO1 inhibitors for several years. Recently, we reported the identification of a series of sulfonamide chromone-oxime derivatives as potential IDO1 inhibitors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The docking studies showed that the oxygen atom of oxime moiety can coordinate to the heme iron. Besides, substituted 1,2,3-triazoles have been found to be useful drug scaffolds with wide applications in drug discovery [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, 1,2,3-triazole based compounds have been reported as nanomolar IDO1 inhibitors [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our continuous efforts to identify more potent IDO1 inhibitors, we designed and synthesized a series of novel chromone-oxime derivatives containing 1,2,3-triazole moieties and evaluated their IDO1 inhibitory activities.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eChemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe detailed synthetic route to all the precursors for the target 1,2,3-triazoles chromone oxime derivatives was outlined in Schemes 1. Intermediates 4 were synthesized by published method [27]. Intermediates 3 were prepared by the reaction of commercially available 2-hydroxy-4 -methoxy benzophenone (\u003cstrong\u003e1\u003c/strong\u003e) with diethyl oxalate (\u003cstrong\u003e2\u003c/strong\u003e) in the presence of sodium methoxide in dioxane, at 120 \u0026deg;C, which were subsequently cyclized under acidic conditions to provide intermediates \u003cstrong\u003e4\u003c/strong\u003e. The aromatic azides (\u003cstrong\u003e6\u003c/strong\u003e) are prepared by addition of substituted phenyl amine (\u003cstrong\u003e5\u003c/strong\u003e) in the presence of MeCN, this intermediate was then reacted with tertbutyl nitrite and TMSN3. Compound \u003cstrong\u003e7\u003c/strong\u003e was reacted with different substituted aromatic azides (\u003cstrong\u003e6\u003c/strong\u003e), ascorbate acid and CuSO4\u0026middot;5H2O in MeOH to yield compounds \u003cstrong\u003e8\u003c/strong\u003e. Compounds \u003cstrong\u003e8\u003c/strong\u003e were subjected to deprotection with HCl/dioxane to afford intermediates \u003cstrong\u003e9\u003c/strong\u003e. Subsequently, the important intermediates \u003cstrong\u003e10\u003c/strong\u003e were obtained by the reaction of the corresponding intermediates \u003cstrong\u003e4\u003c/strong\u003e with \u003cstrong\u003e9\u003c/strong\u003e in the presence of HOBT, EDCI and triethylamine in trichloromethane at 25 \u0026deg;C. Finally, intermediates \u003cstrong\u003e10\u003c/strong\u003e were converted to the target compounds \u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003eD26\u003c/strong\u003e in the presence of hydroxylamine hydrochloride in methanol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of IDO1 activity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inhibitory potency of all synthesized compounds toward IDO1 were measured as hIDO1 IC\u003csub\u003e50\u003c/sub\u003e from enzymatic assays with purified recombinant human IDO1 proteins. All synthesized compounds were also evaluated in a cellular IDO1 inhibition assay. For cellular-based assay, the HeLa cells stimulated with recombinant human IFN-\u0026gamma; were untreated/treated with different concentrations of each compound for 48 h and the amount of generated kynurenine was measured by the absorbance at 480 nm using a microplate reader [28]. Epacadostat was used as a positive control. As shown in Table 1, most compounds displayed potent IDO1 inhibitory activities with the IC\u003csub\u003e50\u003c/sub\u003e values at low micromolar range. Compound \u003cstrong\u003eD20\u003c/strong\u003e with methoxy group at R\u003csub\u003e1\u003c/sub\u003e and 3-fluorine group at R\u003csub\u003e2\u003c/sub\u003e showed the highest inhibitory activity (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.084 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.059 \u0026mu;M), which was 2-fold weaker than epacadostat (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.048 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.019 \u0026mu;M). The preliminary SAR at the side chain bearing different substituted benzene was first investigated, of which the 2-OCH\u003csub\u003e3\u003c/sub\u003e substitution (\u003cstrong\u003eD1\u003c/strong\u003e) yielded a moderate inhibitory activity against IDO1 (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 7.62 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 5.70 \u0026mu;M). Compound \u003cstrong\u003eD2\u003c/strong\u003e (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 4.91 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 3.69 \u0026mu;M) with the 3-OCH\u003csub\u003e3\u003c/sub\u003e substituted group retained moderate activity as that of compound \u003cstrong\u003eD1\u003c/strong\u003e. Moving the methoxy group from \u003cem\u003eortho\u003c/em\u003e-position to \u003cem\u003epara\u003c/em\u003e-position (\u003cstrong\u003eD3\u003c/strong\u003e) resulted in a significant drop of inhibitory activity. Compound \u003cstrong\u003eD4\u003c/strong\u003e obtained by the replacement of 2-OCH\u003csub\u003e3\u003c/sub\u003e group at R\u003csub\u003e2\u003c/sub\u003e of \u003cstrong\u003eD1\u003c/strong\u003e with an electron withdrawing functional group (2-F), which led to a significantly improvement in inhibitory activity (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 1.64 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 1.07 \u0026mu;M). Substitution with 2-Cl at R\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eposition (\u003cstrong\u003eD7\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ehIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 4.20 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 3.26 \u0026mu;M) also displayed potent inhibitory activities despite slight loss of potency than \u003cstrong\u003eD4\u003c/strong\u003e. In addition, a 2 to 6-fold decrease in inhibitory potency was observed accompanied after the replacement chlorine or fluorine with a bromine (\u003cstrong\u003eD10\u003c/strong\u003e), respectively. Generally, compounds with electron-withdrawing groups displayed more potent inhibitory activity compared with compounds with electron-donating groups. Compound \u003cstrong\u003eD5\u003c/strong\u003e with 3-fluorine group (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.78 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 0.63 \u0026mu;M) demonstrated improvement in inhibitory activity against IDO1 over methoxy substituted derivative \u003cstrong\u003eD2\u0026nbsp;\u003c/strong\u003e(hIDO1 IC\u003csub\u003e50\u003c/sub\u003e = 4.91 \u0026mu;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e = 3.69 \u0026mu;M). In addition, compared to \u003cstrong\u003eD12\u003c/strong\u003e and \u003cstrong\u003eD19\u003c/strong\u003e, moving the halogen group from \u003cem\u003eortho\u003c/em\u003e-position to \u003cem\u003emeta\u003c/em\u003e-position resulted in an improved inhibitory activity (\u003cstrong\u003eD13\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD12\u003c/strong\u003e, \u003cstrong\u003eD20\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD19\u003c/strong\u003e, respectively), while changing substituents from \u003cem\u003emeta\u003c/em\u003e-position\u003cem\u003e\u0026nbsp;\u003c/em\u003eto \u003cem\u003epara\u003c/em\u003e-position significantly decreased the enzyme inhibitory activity (\u003cstrong\u003eD5\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD6\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;D8\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD9\u003c/strong\u003e, respectively). However, complete loss of activity was obtained by introducing a 4-methoxy or 4-chlorine at R\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eposition to respectively give compounds \u003cstrong\u003eD3\u003c/strong\u003e and \u003cstrong\u003eD9\u003c/strong\u003e (IC\u003csub\u003e50\u003c/sub\u003e \u0026gt; 10 \u0026mu;M).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, the inhibitory activities of different small substituents at R\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eposition were also studied. As shown in Table 1, R\u003csub\u003e1\u003c/sub\u003e substituents at the C-7 position of the chromone group have a significant effect on inhibitory activity. Compounds in which the hydrogen at R\u003csub\u003e1\u003c/sub\u003e was replaced with a methyl group (\u003cstrong\u003eD11\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD1\u003c/strong\u003e, \u003cstrong\u003eD14\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD6\u003c/strong\u003e, \u003cstrong\u003eD16\u003c/strong\u003e \u003cem\u003evs\u003c/em\u003e \u003cstrong\u003eD10\u003c/strong\u003e, respectively) resulted in a significant increase in potency. Additionally, compared with methyl derivative \u003cstrong\u003eD13\u003c/strong\u003e, the substitution with a methoxyl group (\u003cstrong\u003eD20\u003c/strong\u003e) at R\u003csub\u003e1\u003c/sub\u003e caused a 4-fold improvement of activity, while a 3-fold improvement in inhibitory potency was observed accompanied after the replacement of hydrogen atom in compound \u003cstrong\u003eD10\u003c/strong\u003e with fluorine (\u003cstrong\u003eD26\u003c/strong\u003e). Furthermore, the same compound (\u003cstrong\u003eD26\u003c/strong\u003e) displayed similar inhibitory activity with compound \u003cstrong\u003eD16\u003c/strong\u003e. These results suggested the small substituents at R\u003csub\u003e1\u0026nbsp;\u003c/sub\u003eposition may occupy the hydrophobic A pocket to obtain more potent inhibitory activity, which is consistent with other published results [29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Structures and IDO1 inhibitory activities of title compounds \u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003eD26\u003c/strong\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003eCompd.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eEnzymatic assay hIDO1\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;IC\u003csub\u003e50\u003c/sub\u003e (\u0026mu;M)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003eCellular assay\u003c/p\u003e\n \u003cp\u003eHela (IDO1)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; IC\u003csub\u003e50\u003c/sub\u003e (\u0026mu;M)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e7.62 \u0026plusmn; 1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e5.70 \u0026plusmn; 1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e4.91 \u0026plusmn; 0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e3.69 \u0026plusmn; 0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.64 \u0026plusmn; 0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e1.07 \u0026plusmn; 0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.78 \u0026plusmn; 0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.63 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e5.23 \u0026plusmn; 2.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e4.02 \u0026plusmn; 0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e4.20 \u0026plusmn; 0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e3.26 \u0026plusmn; 0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3.16 \u0026plusmn; 0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e1.89 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e9.84 \u0026plusmn; 1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e8.86 \u0026plusmn; 2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.45 \u0026plusmn; 0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.98 \u0026plusmn; 0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.82 \u0026plusmn; 0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.74 \u0026plusmn; 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.27 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3.11 \u0026plusmn; 0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e2.69 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.23 \u0026plusmn; 0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.91 \u0026plusmn; 0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2.79 \u0026plusmn; 0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e2.11 \u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.43 \u0026plusmn; 0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.32 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.47 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.87 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.52 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.48 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.084 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.059 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.76 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.54 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e3.53 \u0026plusmn; 0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e2.26 \u0026plusmn; 0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.68 \u0026plusmn; 0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.57 \u0026plusmn; 0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2.84 \u0026plusmn; 0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e2.05 \u0026plusmn; 0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e5.27 \u0026plusmn; 1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e5.04 \u0026plusmn; 1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2.71 \u0026plusmn; 0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e1.92 \u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEpacadostat\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.048\u0026nbsp;\u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e0.019\u0026nbsp;\u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Data are mean \u0026plusmn; SD values from three independent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurface plasmon resonance analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the direct interaction of compound D20 with IDO1 protein, we performed a commonly used surface plasmon resonance (SPR) based binding assay using Biacore T200 instrument. This analytic technique facilitates measurement of the kinetic and thermodynamic parameters of ligand-protein complex formation and is widely utilized to investigate enzyme/inhibitor interactions [30]. Association and dissociation measurements were taken and the binding affinity of compound \u003cstrong\u003eD20\u003c/strong\u003e for IDO1 was determined with the aid of Biacore evaluation software. As shown in Figure 2, the discernible exponential curves revealed the concentration-dependent responses for the association and dissociation after compound D20interacting with the immobilized protein, indicative of the binding of compound \u003cstrong\u003eD20\u003c/strong\u003e to and dissociation from the IDO1 protein. The equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e, representing binding affinity) between compound \u003cstrong\u003eD20\u003c/strong\u003e and IDO1 was 0.57 \u0026mu;M, which indicated its strong binding affinity to the target IDO1 protein. These data supplied definitive evidence of compound \u003cstrong\u003eD20\u003c/strong\u003e directly binding to IDO1 protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further study the structure-activity relationship (SAR) and elucidate the binding mode between chromone oxime derivatives and IDO1, we performed a molecular docking study for the most potent compound \u003cstrong\u003eD20\u003c/strong\u003e with IDO1 using SYBYL-X 2.1 software and the crystal structure of IDO1/inhibitor (PDB: 6KOF) was selected as the template. As illustrated in Figure 3, the oxime group of compound \u003cstrong\u003eD20\u003c/strong\u003e coordinated to the heme iron via an oxygen atom and formed a hydrogen bond with the porphyrin ring of heme, which contributed to its inhibitory activity against IDO1. The benzene ring of chromone-oxime skeleton inserted deep into the vertical region of the heme called \u0026ldquo;Pocket A\u0026rdquo; formed by the hydrophobic residues TYR126, VAL130, PHE163, PHE164, SER263, and LEU234 (Pocket A). The methoxy group located deep into Pocket A. This may be the reason that replacing the H atom with methoxy group led to the improvement of activities. In addition, benzene ring formed \u0026pi;\u0026minus;\u0026pi; interaction with amino acid residues PHE164, TYR126, which may be essential for potent IDO1 inhibitory activity. Notably, the triazole group formed a hydrogen bond with the key residue ARG231. More importantly, terminal benzene moiety extended into the active site entrance region (Pocket B) formed by PHE227, ARG231, SER235 and ASN240, where the substitutional F atom formed halogen hydrogen bonds with the side chains of ARG231 and SER235, respectively. The preliminary docking results may support the fact that the compound \u003cstrong\u003eD20\u003c/strong\u003e was potent IDO1 inhibitor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUV\u0026minus;Visible IDO1 Binding Study\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further validate the binding mode of compound \u003cstrong\u003eD20\u003c/strong\u003e within the IDO1 active site, we conducted a heme binding study using UV\u0026minus;visible absorption spectroscopy (Fig. 4). IDO1 is a heme-containing protein, which comprises a porphyrin ring with an iron atom at the center, and has characteristic light absorption at around \u0026lambda; = 400 nm (Soret band). The optical properties of the heme group are highly sensitive to the local surroundings upon the ligand/substrate binding, which changes the spectral properties of the heme [31]. The use of this unique UV absorption spectral changes in the Soret peak of the heme is an effective and rapid means of assessing the direct interaction between compounds and IDO1. As shown in Fig. 4, in the absence of compound \u003cstrong\u003eD20\u003c/strong\u003e, the absorption spectrum of rhIDO1 exhibited a Soret peak at 404 nm, consistent with the previous literature. An obviously red shift in the Soret band was observed from 404 nm to 415 nm for IDO1 incubated with compound \u003cstrong\u003eD20\u003c/strong\u003e, indicating coordination of the heme iron in the active site by compound \u003cstrong\u003eD20\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the inhibition of IDO1 could simply be an effect of the cytotoxicity of the tested compounds, we evaluated their cytotoxicity on A2780, Hct-116 and HeLa cancer cells via the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As summarized in Table 2, most of the compounds exhibited an IC\u003csub\u003e50\u003c/sub\u003e value of micromolar level (most IC\u003csub\u003e50\u003c/sub\u003e values were over 50 \u0026mu;M), which was significantly higher than their IC\u003csub\u003e50\u003c/sub\u003e values against IDO1. We also observed that few compounds showed moderate cytotoxicity against Hct-116. These results indicated that most of the compounds displayed no/negligible influences on cells at their effective concentration against IDO1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Cytotoxicity of compounds \u003cstrong\u003eD1\u003c/strong\u003e-\u003cstrong\u003eD26\u0026nbsp;\u003c/strong\u003eagainst cancer cell lines.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 473px;\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u003cstrong\u003e\u0026mu;M\u003c/strong\u003e)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCompd.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eR\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eR\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eA2780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eHct-116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eHeLa\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e48.70 \u0026plusmn; 2.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e29.22 \u0026plusmn; 1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e44.43 \u0026plusmn; 1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD17\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD19\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e46.42 \u0026plusmn; 1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e44.35 \u0026plusmn; 2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD21\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e23.76 \u0026plusmn; 1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD23\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e25.82 \u0026plusmn; 1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD24\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eD26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e2-Br\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEpacadostat\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026gt;50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a series of novel chromone-oxime derivatives containing triazole moieties were designed and synthesized as IDO1 inhibitors. Most of the derivatives showed potent IDO1 inhibitory activity with IC\u003csub\u003e50\u003c/sub\u003e values at the level of submicromolar concentrations. Encouragingly, compound D20 displayed the most potent IDO1 inhibitor among these derivatives, with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.084 \u0026micro;M and 0.059 \u0026micro;M in the enzymatic and cellular assay against IDO1, respectively. Moreover, the SPR analysis confirmed the interaction between compound \u003cb\u003eD20\u003c/b\u003e and IDO1 protein with an equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) value of 0.57 \u0026micro;M. Molecular docking study illustrated key interactions between the most active compound \u003cb\u003eD20\u003c/b\u003e and IDO1 in which the chromone-oxime moiety coordinated to the heme iron and formed a hydrogen bond with the porphyrin ring of heme, while the triazole moiety formed a hydrogen bond with the key residue ARG231. Further results of the UV spectra of IDO1 showed a Soret peak shift from 403 to 415 nm in presence of compound \u003cb\u003eD20\u003c/b\u003e, which supported the notion that compound \u003cb\u003eD20\u003c/b\u003e bound directly to the heme iron. Additionally, compound \u003cb\u003eD20\u003c/b\u003e did not exhibited cytotoxicity at its effective concentration in MTT assay. Consequently, our findings suggested that the rational design of chromone-oxime triazole derivatives offers significant potential for the discovery of a new class of IDO1 inhibitors for cancer therapy.\u003c/p\u003e"},{"header":"Experimental procedure","content":"\u003cp\u003e\u003cstrong\u003eChemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompounds \u003cstrong\u003e4\u003c/strong\u003e was synthesized according to the literature [27]. All the chemical reagents and solvents used were of analytical grade. Silica gel (200-300 mesh) used in column chromatography was provided by Tsingtao Marine Chemistry Co. Ltd. \u003csup\u003e1\u003c/sup\u003eH NMR spectra were recorded on a 400 MHz (\u003csup\u003e1\u003c/sup\u003eH, 400 MHz; \u003csup\u003e13\u003c/sup\u003eC, 101 MHz) Bruker spectrometer with TMS as an internal standard in DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e. High-resolution ESI-MS spectra were recorded on an Agilent 1290-6545 UHPLC-QTOF mass spectrometer. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e\u003cstrong\u003eeneral synthetic procedure for\u003c/strong\u003e \u003cstrong\u003ecompounds D1-D26\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn brief, substituted ortho-hydroxyacetophenone (\u003cstrong\u003e1\u003c/strong\u003e,0.19 g, 1.16 mmol) ) was dissolved in dry 1,4-dioxane (2 ml) followed by adding diethyl oxalate (\u003cstrong\u003e2\u003c/strong\u003e, 474 \u0026micro;L, 3.49 mmol) and sodium methoxide solution (531 \u0026micro;L, 2.32 mmol). The mixture was stirred at 120 \u0026deg;C under reflux for 30 min. Then, hydrochloric acid solution (3 mL, 18 mmol) was added to the reaction, which was stirred at 120 \u0026deg;C under reflux for another 30 min. After the reaction, the mixture was poured into water (50 ml), and the precipitate was filtered off. The crude product was washed with dichloromethane and further vacuum dried to obtain compound \u003cstrong\u003e4\u003c/strong\u003e. The aromatic azides (\u003cstrong\u003e6\u003c/strong\u003e) was prepared by drop tertbutyl nitrite (1.5 mmol) and azide trimethylsilane (1.5 mmol) to the stirring dry acetonitrile solution of substituted aniline (\u003cstrong\u003e5\u003c/strong\u003e, 1 mmol) at 0 \u0026deg;C. The content was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the aryl azides which were used in the next step without further purification. To a solution of aromatic azides (\u003cstrong\u003e6\u003c/strong\u003e) and compound \u003cstrong\u003e4 \u003c/strong\u003ein MeOH (5 mL) were added aqueous solution (0.5 ml) of ascorbic acid (35 mg, 0.2 mmol) and CuSO\u003csub\u003e4\u003c/sub\u003e \u0026middot; 5H\u003csub\u003e2\u003c/sub\u003eO (25 mg, 0.1 mmol), respectively. The mixture was stirred at 60 \u0026deg;C for 6 h and evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel eluted with petroleum ether/ethylacetate (V:V = 1:1) to afford compound \u003cstrong\u003e8\u003c/strong\u003e. To a solution of compound \u003cstrong\u003e8 \u003c/strong\u003ein ethyl acetate (5 mL) were added hydrochloric acid-dioxane solution (5 mL), and stirred at room temperature for 3 h till completion of the reaction (monitored by TLC analysis). The above mixture was evaporated under reduced pressure to afford compound \u003cstrong\u003e9 \u003c/strong\u003ewhich was used in the next step without further purification. To a solution of compound \u003cstrong\u003e4\u003c/strong\u003e (1 mmol) and triethylamine (2 mmol) in chloroform (10 mL) was added 1-hydroxybenzotriazole (1.3 mmol, 0.176 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.3 mmol, 0.249 g). The mixture was stirred under ice bath conditions for 6 h followed by adding compound \u003cstrong\u003e9\u003c/strong\u003e (1 mmol), allowed to stir for another 48 h. After the reaction, the mixture was washed with 1 M HCl (2 \u0026times; 10 mL), saturated NaHCO\u003csub\u003e3\u003c/sub\u003e aqueous solution (2 \u0026times; 10 mL) and brine (2 \u0026times; 10 mL), dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, and concentrated, followed by purification through column chromatography to yield compound \u003cstrong\u003e10\u003c/strong\u003e. To a solution of compound 3 (1 mmol) in absolute methanol (10 mL) was added \u003cem\u003ep\u003c/em\u003e-toluenesulfonic acid (1.5 mmol). The mixture was refluxed at 70 \u0026deg;C for 0.5 h, an excess amount of hydroxylamine hydrochloride (10 mmol) was added. The reaction was allowed to reflux at 70 \u0026deg;C for 12 h, and remove solvent under vacuum. 2 M NaOH solution (10 mL) was added, then filtered and washed with water (10 mL), ether (10 mL) and absolute methanol (5 mL). The solid was collected and dried to afford compounds \u003cstrong\u003eD1-D26\u003c/strong\u003e. The structures were confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR and HR-MS. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(E)-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD1\u003c/strong\u003e). \u003c/em\u003eYield: 56.1%, white solid. m.p. 284.3~285.7 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003e\u0026delta;\u003c/em\u003e 11.30 (s, 1H), 9.40 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.34 (s, 1H), 7.90 \u0026ndash; 7.83 (m, 1H), 7.62 \u0026ndash; 7.58 (m, 1H), 7.53 (s, 1H), 7.37 (d, \u003cem\u003eJ \u003c/em\u003e= 8.3 Hz, 1H), 7.25 (s, 1H), 7.14 (t, \u003cem\u003eJ \u003c/em\u003e= 7.7 Hz, 1H), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 3.85 (s, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.2, 152.1, 150.9, 147.6, 144.6, 142.0, 131.3, 131.1, 126.2, 126.2, 125.9, 125.5, 125.4, 122.7, 121.3, 119.0, 118.3, 113.5, 99.1, 56.6, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e [M + H]\u003csup\u003e+\u003c/sup\u003e:392.1281; found:392.1187.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-4-(hydroxyimino)-N-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD2\u003c/strong\u003e). \u003c/em\u003eYield:43.1%. white solid. m.p.183.6~184.3℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.31 (s, 1H), 9.42 (s, 1H), 8.76 (s, 1H), 7.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.0, 1.7 Hz, 1H), 7.60 \u0026ndash; 7.44 (m, 4H), 7.37 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.33 \u0026ndash; 7.25 (m, 2H), 7.09 \u0026ndash; 7.00 (m, 1H), 4.62 (s, 2H), 3.85 (s, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.6, 160.3, 150.9, 147.6, 146.0, 142.0, 138.2, 131.4, 131.3, 125.9, 122.7, 121.9, 119.0, 118.3, 114.8, 112.4, 105.9, 99.1, 56.1, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eKN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e [M + K]\u003csup\u003e+\u003c/sup\u003e:430.0918; found:430.0829.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-4-(hydroxyimino)-N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD3\u003c/strong\u003e). \u003c/em\u003eYield:63.9%. white solid. m.p.258.6~261.7 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.30 (s, 1H), 9.40 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.62 (s, 1H), 7.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.0, 1.6 Hz, 1H), 7.82 (d, \u003cem\u003eJ\u003c/em\u003e = 8.9 Hz, 2H), 7.57 \u0026ndash; 7.50 (m, 1H), 7.37 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.33 \u0026ndash; 7.23 (m, 2H), 7.12 (d, \u003cem\u003eJ\u003c/em\u003e = 9.0 Hz, 2H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H), 3.83 (s, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.2, 159.6, 150.9, 147.6, 145.8, 142.0, 131.3, 130.5, 125.9, 122.7, 122.1, 121.7, 119.0, 118.3, 115.3, 99.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eK\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e [M + K]\u003csup\u003e+\u003c/sup\u003e: 430.0918; found: 430.0171.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD4\u003c/strong\u003e). \u003c/em\u003eYield: 66.9%. white solid. m.p. 254.9~257.1℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.31 (s, 1H), 9.43 (t, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 1H), 8.51 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 1H), 7.91 \u0026ndash; 7.79 (m, 2H), 7.62 \u0026ndash; 7.50 (m, 4H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e = 7.6 Hz, 1H), 7.37 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.33 \u0026ndash; 7.24 (m, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 155.5, 153.0, 150.9, 147.6, 145.5, 142.0, 131.7, 131.6, 131.4, 126.4, 126.0, 126.0, 125.9, 125.3, 125.2, 125.2, 125.1, 122.7, 119.0, 118.3, 117.7, 117.5, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eFN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M + H]\u003csup\u003e+\u003c/sup\u003e: 380.1159; found: 380.0195.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD5\u003c/strong\u003e). \u003c/em\u003eYield:43.1%. white solid. m.p.228.2~231.5℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.31 (s, 1H), 9.43 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.80 (s, 1H), 7.85 (ddd, \u003cem\u003eJ\u003c/em\u003e = 17.6, 7.9, 1.9 Hz, 3H), 7.64 (td, J = 8.2, 6.2 Hz, 1H), 7.57 \u0026ndash; 7.50 (m, 1H), 7.41 \u0026ndash; 7.23 (m, 4H), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.1, 161.7, 160.3, 150.9, 147.5, 146.3, 142.0, 138.4, 138.3, 132.3, 132.2, 131.4, 125.9, 122.7, 122.0, 119.0, 118.3, 116.3, 116.3, 115.8, 115.6, 108.0, 107.7, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFKN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M + K]\u003csup\u003e+\u003c/sup\u003e: 418.0718; found: 418.0648.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD6\u003c/strong\u003e). \u003c/em\u003eYield: 55.3%. white solid. m.p.236.5~236.9 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.30 (s, 1H), 9.42 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.71 (s, 1H), 8.02 \u0026ndash; 7.92 (m, 2H), 7.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.0, 1.7 Hz, 1H), 7.57 \u0026ndash; 7.50 (m, 1H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e = 8.8 Hz, 2H), 7.37 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.33 \u0026ndash; 7.23 (m, 2H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 163.2, 160.8, 160.3, 150.9, 147.6, 146.1, 142.0, 133.7, 131.4, 125.9, 122.8, 122.7, 122.0, 119.0, 118.3, 117.3, 117.0, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e3\u003c/sub\u003e [M + Na]\u003csup\u003e+\u003c/sup\u003e: 402.0978; found: 402.0994. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD7\u003c/strong\u003e). \u003c/em\u003eYield:49.3%. grey solid. m.p. 242.6~244.3 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.30 (s, 1H), 9.44 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.46 (s, 1H), 7.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.0, 1.6 Hz, 1H), 7.77 (dt, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.1 Hz, 1H), 7.69 \u0026ndash; 7.50 (m, 3H), 7.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.4, 1.1 Hz, 1H), 7.32 \u0026ndash; 7.25 (m, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.6, 145.0, 142.0, 135.0, 132.0, 131.4, 131.0, 128.9, 128.9, 128.8, 125.9, 125.7, 122.7, 119.0, 118.3, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e3\u003c/sub\u003e [M + Na]\u003csup\u003e+\u003c/sup\u003e: 418.0683; found: 418.0646\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD8\u003c/strong\u003e). \u003c/em\u003eYield: 60.7%. white solid. m.p. 256.1~257.3℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.31 (s, 1H), 9.43 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.82 (s, 1H), 8.07 (s, 1H), 7.91 (dd, \u003cem\u003eJ\u003c/em\u003e = 29.6, 8.1 Hz, 2H), 7.69 \u0026ndash; 7.46 (m, 3H), 7.43 \u0026ndash; 7.22 (m, 3H), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.5, 146.3, 142.0, 138.1, 134.7, 132.1, 131.2, 128.8, 125.9, 122.7, 121.9, 120.1, 118.9, 118.3, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M + H]\u003csup\u003e+\u003c/sup\u003e: 396.0863; found: 396.1263.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD9\u003c/strong\u003e). \u003c/em\u003eYield: 40.1%. white solid. m.p. 266.5~267.8 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.31 (s, 1H), 9.43 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.77 (s, 1H), 8.00 \u0026ndash; 7.94 (m, 2H), 7.87 (dd, \u003cem\u003eJ\u003c/em\u003e = 7.9, 1.7 Hz, 1H), 7.71 \u0026ndash; 7.63 (m, 2H), 7.53 (ddd, \u003cem\u003eJ\u003c/em\u003e = 8.6, 7.2, 1.6 Hz, 1H), 7.37 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.4, 1.2 Hz, 1H), 7.33 \u0026ndash; 7.25 (m, 2H), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.6, 146.3, 142.0, 135.9, 133.3, 131.4, 130.3, 125.9, 122.7, 122.1, 121.9, 119.0, 118.3, 99.1, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e [M + H]\u003csup\u003e+\u003c/sup\u003e: 396.0863; found: 396.0854.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD10\u003c/strong\u003e). \u003c/em\u003eYield:55.4%.yellow solid.m.p.203.5~206.3℃.\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.29 (s, 1H), 9.43 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.43 (s, 1H), 7.94 \u0026ndash; 7.83 (m, 2H), 7.64 \u0026ndash; 7.59 (m, 2H), 7.54 (ddt, \u003cem\u003eJ\u003c/em\u003e = 8.9, 7.2, 2.8 Hz, 2H), 7.37 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.33 \u0026ndash; 7.23 (m, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 151.0, 147.6, 144.9, 142.0, 136.7, 134.1, 132.3, 131.4, 129.4, 129.1, 125.9, 125.7, 122.7, 119.2, 119.0, 118.3, 99.1, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eBrN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e3\u003c/sub\u003e [M+Na]\u003csup\u003e+\u003c/sup\u003e: 462.0178; found: 462.0746\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD11\u003c/strong\u003e). \u003c/em\u003eYield: 53.2%. white solid. m.p. 258.8~259.6 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.17 (s, 1H), 9.35 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.34 (s, 1H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.64 \u0026ndash; 7.57 (m, 1H), 7.57 \u0026ndash; 7.48 (m, 1H), 7.32 (d, \u003cem\u003eJ\u003c/em\u003e = 8.3 Hz, 1H), 7.23 (s, 1H), 7.19 (s, 1H), 7.17 \u0026ndash; 7.08 (m, 2H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 3.85 (s, 3H), 2.36 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 152.1, 150.9, 147.6, 144.5, 142.0, 141.4, 131.1, 127.0, 126.2, 126.2, 125.5, 122.5, 121.3, 118.2, 116.3, 113.5, 99.0, 56.6, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e4\u003c/sub\u003e[M + Na]\u003csup\u003e+\u003c/sup\u003e: 428.1335; found: 428.0955.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD12\u003c/strong\u003e). \u003c/em\u003eYield: 64.7%. white solid. m.p. 183.1~185.2 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.17 (s, 1H), 9.38 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.50 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 1H), 7.83 (td, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.6 Hz, 1H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.66 \u0026ndash; 7.52 (m, 2H), 7.48 \u0026ndash; 7.41 (m, 1H), 7.24 (s, 1H), 7.18 (s, 1H), 7.12 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.1, 1.7 Hz, 1H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 2.36 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 155.5, 153.0, 150.9, 147.5, 145.4, 142.0, 141.4, 131.7, 131.6, 127.0, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 125.1, 122.5, 118.2, 117.7, 117.5, 116.3, 99.0, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 394.1315; found: 394.1265.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD13\u003c/strong\u003e). \u003c/em\u003eYield: 46.9%. white solid. m.p. 213.7~214.3 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.17 (s, 1H), 9.38 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.79 (s, 1H), 7.91 \u0026ndash; 7.79 (m, 2H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 2H), 7.70 \u0026ndash; 7.59 (m, 1H), 7.34 (td, \u003cem\u003eJ\u003c/em\u003e = 8.5, 2.5 Hz, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.12 (d, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 1H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 2.36 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.1, 161.7, 160.3, 150.9, 147.5, 146.3, 142.0, 141.4, 138.4, 138.3, 132.3, 132.2, 127.0, 122.5, 122.0, 118.2, 116.3, 115.8, 115.6, 108.0, 107.7, 99.0, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 394.1315; found: 394.0732.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(4-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD14\u003c/strong\u003e). \u003c/em\u003eYield: 32.1%. white solid. m.p. 221.7~222.6 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.17 (s, 1H), 9.39 (t, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 1H), 8.44 (d, \u003cem\u003eJ\u003c/em\u003e = 12.8 Hz, 1H), 7.91 (d, \u003cem\u003eJ\u003c/em\u003e = 7.9 Hz, 1H), 7.75 (dd, \u003cem\u003eJ\u003c/em\u003e = 11.9, 7.9 Hz, 1H), 7.69 \u0026ndash; 7.50 (m, 3H), 7.24 (s, 1H), 7.19 (s, 1H), 7.12 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 2.37 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.6, 144.8, 142.0, 141.4, 136.7, 134.1, 132.3, 132.0, 131.0, 129.4, 129.1, 128.9, 128.8, 127.0, 125.7, 122.5, 119.2, 118.2, 116.3, 99.1, 35.0, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 394.1315; found: 394.0934.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD15\u003c/strong\u003e). \u003c/em\u003eYield: 31.2%. white solid. m.p. 155.7~157.3 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.18 (s, 1H), 9.39 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.81 (s, 1H), 8.07 (t, \u003cem\u003eJ\u003c/em\u003e = 2.0 Hz, 1H), 7.98 \u0026ndash; 7.92 (m, 1H), 7.75 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.62 (t, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.58 \u0026ndash; 7.53 (m, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.12 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.2, 1.7 Hz, 1H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H), 2.37 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.5, 146.3, 142.0, 141.4, 138.1, 134.7, 132.1, 128.8, 127.0, 122.5, 121.9, 120.1, 119.0, 118.2, 116.3, 99.1, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 410.1020; found: 410.2033.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methyl-4H-chromene-2-carboxamide (\u003cstrong\u003eD16\u003c/strong\u003e). \u003c/em\u003eYield: 49.3%. yellow solid. m.p. 239.0~240.6 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.17 (s, 1H), 9.38 (d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 1H), 8.71 (s, 1H), 7.96 (dd, \u003cem\u003eJ\u003c/em\u003e = 8.7, 4.7 Hz, 2H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e = 8.1 Hz, 1H), 7.44 (t, \u003cem\u003eJ\u003c/em\u003e = 8.6 Hz, 2H), 7.29 \u0026ndash; 7.07 (m, 3H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.5 Hz, 2H), 2.36 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 160.3, 150.9, 147.5, 146.1, 142.0, 141.4, 133.7, 127.0, 122.8, 122.7, 122.5, 122.1, 118.2, 117.3, 117.0, 116.3, 99.0, 35.1, 21.4. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eBrN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e3 \u003c/sub\u003e[M + Na]\u003csup\u003e+\u003c/sup\u003e: 476.0334; found: 476.3274.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-4-(hydroxyimino)-7-methoxy-N-((1-(3-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD17\u003c/strong\u003e). \u003c/em\u003eYield: 36.2%. white solid. m.p.216.4~222.7 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.06 (s, 1H), 9.38 (s, 1H), 8.76 (s, 1H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e = 9.5 Hz, 1H), 7.56 \u0026ndash; 7.41 (m, 3H), 7.25 (s, 1H), 7.05 (dt, \u003cem\u003eJ\u003c/em\u003e = 5.2, 2.6 Hz, 1H), 6.91 (dt, \u003cem\u003eJ\u003c/em\u003e = 4.9, 2.5 Hz, 2H), 4.61 (d, \u003cem\u003eJ\u003c/em\u003e = 5.7 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 161.6, 160.6, 160.2, 152.2, 147.5, 145.9, 141.9, 138.2, 131.3, 123.9, 121.9, 114.8, 114.0, 112.4, 111.7, 106.0, 101.8, 99.1, 56.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eNaO\u003csub\u003e5 \u003c/sub\u003e[M + Na]\u003csup\u003e+\u003c/sup\u003e:444.1284; found:444.0661. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide (\u003cstrong\u003eD18\u003c/strong\u003e). \u003c/em\u003eYield: 65.1%. yellow solid. m.p. 116.4~120.7 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.06 (s, 1H), 9.41 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.43 (s, 1H), 7.91 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.80 \u0026ndash; 7.70 (m, 1H), 7.61 (d, \u003cem\u003eJ\u003c/em\u003e = 4.5 Hz, 2H), 7.59 \u0026ndash; 7.51 (m, 1H), 7.24 (s, 1H), 6.90 (d, \u003cem\u003eJ\u003c/em\u003e = 7.5 Hz, 2H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H), 3.82 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 161.6, 160.2, 152.1, 147.6, 144.9, 141.8, 136.7, 134.1, 132.3, 129.4, 129.1, 125.7, 123.9, 119.2, 114.0, 111.7, 101.8, 99.1, 56.0, 34.9. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eBrKN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003e[M + K]\u003csup\u003e+\u003c/sup\u003e: 508.0023; found: 508.0137.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide (\u003cstrong\u003eD19\u003c/strong\u003e). \u003c/em\u003eYield: 41.2%. white solid. m.p. 240.8~241.5 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.06 (s, 1H), 9.40 (s, 1H), 8.51 (d, \u003cem\u003eJ\u003c/em\u003e = 2.0 Hz, 1H), 7.83 (d, \u003cem\u003eJ\u003c/em\u003e = 1.6 Hz, 1H), 7.76 (d, \u003cem\u003eJ\u003c/em\u003e = 9.5 Hz, 1H), 7.65 \u0026ndash; 7.53 (m, 2H), 7.48 \u0026ndash; 7.40 (m, 1H), 7.24 (s, 1H), 6.90 (dq, \u003cem\u003eJ\u003c/em\u003e = 5.5, 2.6 Hz, 2H), 4.63 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H), 3.82 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 161.6, 160.3, 155.5, 153.0, 152.1, 147.5, 145.4, 141.8, 131.7, 131.6, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 125.1, 123.8, 117.7, 117.5, 114.0, 111.7, 101.8, 99.1, 56.0, 34.9. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 410.1265; found: 410.1256. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(3-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-7-methoxy-4H-chromene-2-carboxamide (\u003cstrong\u003eD20\u003c/strong\u003e). \u003c/em\u003eYield: 63.5%, white solid, m.p: 194.9~195.5 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.07 (s, 1H), 9.40 (s, 1H), 8.80 (s, 1H), 7.93 \u0026ndash; 7.71 (m, 3H), 7.64 (q, \u003cem\u003eJ\u003c/em\u003e = 7.7 Hz, 1H), 7.41 \u0026ndash; 7.19 (m, 2H), 6.91 (d, \u003cem\u003eJ\u003c/em\u003e = 6.1 Hz, 2H), 4.69 \u0026ndash; 4.55 (m, 2H), 3.82 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.1, 161.7, 161.6, 160.3, 152.1, 147.5, 146.2, 141.8, 138.3, 132.3, 132.2, 123.9, 122.0, 116.3, 116.3, 115.9, 115.6, 114.0, 111.7, 108.0, 107.7, 101.8, 99.1, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 410.1265; found: 410.1266.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-7-fluoro-4-(hydroxyimino)-N-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD21\u003c/strong\u003e). \u003c/em\u003eYield: 45.5%. white solid. m.p. 267.3~268.1 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.36 (s, 1H), 9.37 (s, 1H), 8.35 (s, 1H), 7.90 (s, 1H), 7.70 \u0026ndash; 7.45 (m, 2H), 7.39 \u0026ndash; 7.07 (m, 5H), 4.62 (s, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.6, 162.1, 159.9, 152.1, 151.9, 151.7, 147.6, 144.4, 141.4, 131.2, 126.2, 126.2, 125.6, 124.9, 124.8, 121.3, 115.8, 115.8, 113.9, 113.7, 113.5, 105.4, 105.1, 99.3, 56.6, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 410.1265; found: 410.1258.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-7-fluoro-4-(hydroxyimino)-N-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-4H-chromene-2-carboxamide (\u003cstrong\u003eD22\u003c/strong\u003e). \u003c/em\u003eYield: 30.9%. white solid. m.p. 250.3~252.4 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.34 (s, 1H), 9.35 (s, 1H), 8.61 (s, 1H), 7.90 (dd, J = 9.6, 6.3 Hz, 1H), 7.85 \u0026ndash; 7.77 (m, 2H), 7.26 (s, 1H), 7.18 (ddd, \u003cem\u003eJ\u003c/em\u003e = 8.5, 4.3, 1.8 Hz, 2H), 7.15 \u0026ndash; 7.09 (m, 2H), 4.60 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H), 3.83 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.6, 162.1, 156.0, 159.6, 151.9, 151.7, 147.5, 145.6, 141.4, 130.5, 124.9, 124.8, 122.1, 121.8, 115.8, 115.3, 113.9, 113.7, 105.3, 105.1, 99.3, 56.0, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 410.1265; found: 410.1249.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-7-fluoro-N-((1-(2-fluorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD23\u003c/strong\u003e). \u003c/em\u003eYield: 57.9%. white solid. m.p. 264.5~266.8 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.36 (s, 1H), 9.40 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.52 (d, \u003cem\u003eJ\u003c/em\u003e = 2.1 Hz, 1H), 7.90 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.8, 6.2 Hz, 1H), 7.83 (td, \u003cem\u003eJ\u003c/em\u003e = 7.8, 1.6 Hz, 1H), 7.59 (dqt, \u003cem\u003eJ\u003c/em\u003e = 11.7, 7.4, 2.2 Hz, 2H), 7.48 \u0026ndash; 7.40 (m, 1H), 7.26 (d, \u003cem\u003eJ\u003c/em\u003e = 1.5 Hz, 1H), 7.18 (dt, \u003cem\u003eJ\u003c/em\u003e = 8.6, 2.2 Hz, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.6, 162.1, 160.0, 155.5, 153.0, 151.8, 151.7, 147.5, 145.4, 141.3, 131.7, 131.6, 126.4, 126.0, 126.0, 125.3, 125.2, 125.2, 124.9, 124.8, 117.7, 117.5, 115.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 398.1065; found: 398.2318.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD24\u003c/strong\u003e). \u003c/em\u003eYield: 69.5%. white solid. m.p. 239.4~242.3 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.36 (s, 1H), 9.42 (t, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, 1H), 8.47 (s, 1H), 7.89 (td, \u003cem\u003eJ\u003c/em\u003e = 11.2, 10.5, 6.6 Hz, 1H), 7.77 (d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 1H), 7.71 \u0026ndash; 7.53 (m, 3H), 7.26 (s, 1H), 7.18 (t, \u003cem\u003eJ\u003c/em\u003e = 9.7 Hz, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 162.1, 160.0, 151.9, 151.7, 147.5, 145.0, 144.9, 141.3, 135.0, 132.0, 131.0, 128.9, 128.8, 125.8, 124.9, 124.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eClFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 414.0769; found: 414.0789.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD25\u003c/strong\u003e). \u003c/em\u003eYield: 40.3%, white solid, m.p: 250.7~251.4 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.36 (s, 1H), 9.40 (t, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 1H), 8.77 (s, 1H), 8.00 \u0026ndash; 7.94 (m, 2H), 7.90 (dd, \u003cem\u003eJ\u003c/em\u003e = 9.7, 6.3 Hz, 1H), 7.72 \u0026ndash; 7.61 (m, 2H), 7.27 (s, 1H), 7.22 \u0026ndash; 7.15 (m, 2H), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e = 5.8 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 164.6, 162.1, 160.0, 151.8, 151.7, 147.5, 146.1, 141.4, 135.9, 133.3, 130.3, 124.9, 124.8, 122.1, 121.9, 115.8, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.1. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eClFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 414.0769; found: 414.0805.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003e(E)-N-((1-(2-bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)-7-fluoro-4-(hydroxyimino)-4H-chromene-2-carboxamide (\u003cstrong\u003eD26\u003c/strong\u003e). \u003c/em\u003eYield: 37.1%, yellow solid, m.p: 246.7~251.7 ℃. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 11.35 (s, 1H), 9.41 (t, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 1H), 8.44 (s, 1H), 7.90 (t, \u003cem\u003eJ\u003c/em\u003e = 8.2 Hz, 2H), 7.61 (d,\u003cem\u003e J\u003c/em\u003e = 4.5 Hz, 2H), 7.55 (dt, \u003cem\u003eJ\u003c/em\u003e = 8.8, 4.6 Hz, 1H), 7.26 (s, 1H), 7.18 (t, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, 2H), 4.64 (d, \u003cem\u003eJ\u003c/em\u003e = 5.9 Hz, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u0026delta; 162.1, 160.0, 151.9, 151.7, 147.5, 144.8, 141.3, 136.7, 134.1, 132.3, 129.4, 129.1, 125.7, 124.9, 124.8, 119.2, 115.8, 113.9, 113.7, 105.3, 105.1, 99.4, 35.0. HR-MS (m/z) (ESI): calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eBrFN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e3 \u003c/sub\u003e[M + H]\u003csup\u003e+\u003c/sup\u003e: 458.0264; found: 458.1380.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003cstrong\u003eiological assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biological experimental procedures including enzymatic assays, cellular IDO1 activity assays, SPR experiments, UV\u0026minus;visible spectra and cell viability assays were carried out according to our previous work[20, 32, 33] and described in the Supplementary Information (SI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking was carried out in Sybyl-X 2.1 on a Windows workstation and described in the SI. The crystal structure of IDO1 protein with inhibitor was retrieved from the RCSB Protein Data Bank (IDO1: 6KOF) [34].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Nos. 82104008 and 21977021), the Guangxi Science and Technology Base and Talents Program (AD20297059), the China Postdoctoral Science Foundation (2021MD703847), State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (No. CMEMR2023-B17), Middle-aged and Young Teachers\u0026amp;apos; Basic Ability Promotion Project of Guangxi (2021KY0942).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYang Y (2015) J Clin Invest 125: 3335\u0026minus;3337.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZou W (2005) Nat Rev Cancer 5: 263\u0026minus;274.\u003c/li\u003e\n \u003cli\u003eMajzner RG, Mackall CL (2019) Nat Med 25: 1341-55.\u003c/li\u003e\n \u003cli\u003eAlsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, Iyer AK (2017) Front Pharmacol 8: \u0026nbsp;561\u0026minus;575.\u003c/li\u003e\n \u003cli\u003eMunn DH, Mellor AL (2016) Trends Immunol 37: 193-207.\u003c/li\u003e\n \u003cli\u003eBinnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, Coussens LM, Gabrilovich DI, Ostrand-Rosenberg S, Hedrick CC, Vonderheide RH, Pittet MJ, Jain RK, Zou W, Howcroft TK, Woodhouse EC, Weinberg RA, Krummel MF (2018) Nat Med 24: 541-550.\u003c/li\u003e\n \u003cli\u003ePrendergast GC, Mondal A, Dey S, Laury-Kleintop LD, Muller AJ (2018) Trends Cancer 4: 38\u0026minus;58.\u003c/li\u003e\n \u003cli\u003eUyttenhove C, Pilotte L, Th\u0026eacute;ta I, Stroobant V, Colau D, Parmentier N, Boon T, Eynde BJV (2003) Nat Med 9: 1269-1274.\u003c/li\u003e\n \u003cli\u003eGodin-Ethier J, Hanafi LA, Piccirillo CA, Lapointe R (2011) Clin Cancer Res 17: 6985-6991.\u003c/li\u003e\n \u003cli\u003eOpitz CA, Litzenburger UM, Sahm F, Ott M, Tritschler I, Trump S, Schumacher T, Jestaedt L, Schrenk D, Weller M, Jugold M, Guillemin GJ, Miller CL, Lutz C, Radlwimmer B, Lehmann I, von Deimling A, Wick W, Platten M (2011) Nature 478: 197-203.\u003c/li\u003e\n \u003cli\u003eSugimoto H, Oda S, Otsuki T, Hino T, Yoshida T, Shiro Y (2006) Proc Natl Acad Sci U.S.A. 103: \u0026nbsp;2611-2616.\u003c/li\u003e\n \u003cli\u003eMunn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, Mellor AL (2005) Immunity 22: 633\u0026minus;642.\u003c/li\u003e\n \u003cli\u003eHorny\u0026aacute;k L, Dobos N, Koncz G, Kar\u0026aacute;nyi Z, P\u0026aacute;ll D, Szab\u0026oacute; Z, Halmos G, Sz\u0026eacute;kv\u0026ouml;lgyi L (2018) Front Immunol 9: 151.\u003c/li\u003e\n \u003cli\u003eRicciuti B, Leonardi GC, Puccetti P, Fallarino F, Bianconi V, Sahebkar A, Baglivo S, Chiari R, Pirro M (2019) Pharmacol Ther 196: 105\u0026minus;116.\u003c/li\u003e\n \u003cli\u003eLi A, Barsoumian HB, Schoenhals JE, Cushman TR, Caetano MS, Wang X, Valdecanas DR, Niknam S, Younes AI, Li G, Woodward WA, Cortez MA, Welsh JW (2018) Cancer Lett 431: 54-463.\u003c/li\u003e\n \u003cli\u003eWeng T, Qiu X, Wang J, Li Z, Bian J (2018) Eur J Med Chem 143: 656-669.\u003c/li\u003e\n \u003cli\u003ePlatten M, Nollen EAA, Röhrig UF, Fallarino F, Opitz CA (2019) Nat Rev Drug Discovery 18: 379\u0026minus;401.\u003c/li\u003e\n \u003cli\u003eFeng X, Liao D, Liu D, Ping A, Li Z, Bian J (2020) J Med Chem 63: 15115-15139.\u003c/li\u003e\n \u003cli\u003eLong GV, Dummer R, Hamid O, Gajewski TF, Caglevic C, Dalle S, Arance A, Carlino MS, Grob J-J, Kim TM, Demidov L, Robert C, Larkin J, Anderson JR, Maleski J, Jones M, Diede SJ, Mitchell TC (2019) Lancet Oncol. 20: 1083\u0026minus;1097.\u003c/li\u003e\n \u003cli\u003eWang K, Song L-H, Liang Q-L, Zhang Y, Ma X-L, Wang Q, Zhang H-Y, Jiang C-N, Wei J-H, Huang R-Z. (2023) Eur J Med Chem 254: 115349.\u003c/li\u003e\n \u003cli\u003eZhao S, Liu J, Lv Z, Zhang G, Xu Z (2023) Eur J Med Chem 251: 115254.\u003c/li\u003e\n \u003cli\u003eBonandi E, Christodoulou MS, Fumagalli G, Perdicchia D, Rastelli G, Passarella D (2017) Drug Discov Today 22: 1572\u0026minus;1581.\u003c/li\u003e\n \u003cli\u003eHuang R-Z, Liang G-B, Li M-S, Fang Y-L, Zhao S-F, Zhou M-M, Liao Z-X, Sun J, Wang H-S. (2019) MedChemCommun 10: 584-597.\u003c/li\u003e\n \u003cli\u003eRöhrig UF, Awad L, Grosdidier A, Larrieu P, Stroobant V, Colau D, Cerundolo V, Simpson AJ, Vogel P, van den Eynde BJ, Zoete V, Michielin O (2010) J Med Chem 53: 1172\u0026minus;1189.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRöhrig UF, Majjigapu SR, Grosdidier A, Bron S, Stroobant V, Pilotte L, Colau D, Vogel P, van den Eynde B J, Zoete V, Michielin O (2012) J Med Chem 55: 5270\u0026minus;5290.\u003c/li\u003e\n \u003cli\u003eHe X, He G, Chu Z, Wu H, Wang J, Ge Y, Shen H, Zhang S, Shan J, Peng K, Wei Z, Zou Y, Xu Y, Zhu Q (2021) J Med Chem 64: 17950\u0026minus;17968.\u003c/li\u003e\n \u003cli\u003eGaspar A, Matos MJ, Garrido J, Uriarte E, Borges F (2014) Chem. Rev. 114: 4960\u0026minus;4992.\u003c/li\u003e\n \u003cli\u003eYe K, Wang K, Wang T, Tang H, Wang L, Zhang W, Jiang S, Zhang X, Zhang K (2023) Eur J Med Chem 250: \u0026nbsp;115217.\u003c/li\u003e\n \u003cli\u003eRöhrig UF, Majjigapu SR, Vogel P, Zoete V, Michielin O (2015) J Med Chem 58: 9421\u0026minus;9437.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang H, Liu C, Chen Q, Shen L-A, Xiao W, Li J, Wang Y, Zhu D, Zhang Q, Li J (2023) J Med Chem 66: 1349\u0026minus;1379.\u003c/li\u003e\n \u003cli\u003eZou Y, Hu Y, Ge S, Zheng Y, Li Y, Liu W, Guo W, Zhang Y, Xu Q, Lai Y (2019) Eur J Med Chem 184: 111750.\u003c/li\u003e\n \u003cli\u003eHuang R, Jing X, Huang X, Pan Y, Fang Y, Liang G, Liao Z, Wang H, Chen Z, Zhang Y (2020) J Med Chem 63: 1544\u0026minus;1563.\u003c/li\u003e\n \u003cli\u003eLiu S-Q, Mao Z-C, Xu Y-L, Chen X-M, Wang H-L, Wang Q, Wei J-H, Huang R-Z, Zhang Y (2023) Bioorg Chem 131: 106323.\u003c/li\u003e\n \u003cli\u003ePeng YH, Liao FY, Tseng CT, Kuppusamy R., Li AS, Chen CH, Fan YS, Wang SY, Wu MH, Hsueh CC, Chang JY, Lee LC, Shih C, Shia KS, Yeh TK, Hung MS, Kuo CC, Song JS, Wu SY, Ueng SH (2020) J Med Chem 63: 1642-1659.\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":"monatshefte-fur-chemie-chemical-monthly","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mccm","sideBox":"Learn more about [Monatshefte für Chemie - Chemical Monthly](https://www.springer.com/journal/706)","snPcode":"706","submissionUrl":"https://www.editorialmanager.com/mccm/","title":"Monatshefte für Chemie - Chemical Monthly","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Indoleamine 2,3-dioxygenase 1, Chromone-oxime, 1,2,3-triazole, Inhibitors, Cancer immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-5283388/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5283388/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA series of chromone-oxime derivatives containing 1,2,3-triazole moieties were designed, synthesized and evaluated for their IDO1 inhibitory activities. These compounds displayed moderate to good inhibitory activity against IDO1 with IC\u003csub\u003e50\u003c/sub\u003e values in low micromolar range. Among them, compound \u003cb\u003eD20\u003c/b\u003e displayed the most potent IDO1 inhibitory activities (hIDO1 IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.084 \u0026micro;M, HeLa IDO1 IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.059 \u0026micro;M) and was selected for further investigation. Surface plasmon resonance analysis confirmed that compound \u003cb\u003eD20\u003c/b\u003e directly interacted with IDO1 protein with a K\u003csub\u003eD\u003c/sub\u003e value of 0.57 \u0026micro;M. Molecular docking study revealed the oxygen atom in chromone-oxime moiety of compound \u003cb\u003eD20\u003c/b\u003e coordinated to the heme iron, and the 1,2,3-triazole group formed a hydrogen bond with the key residue ARG231. The UV spectra showed that \u003cb\u003eD20\u003c/b\u003e induced a Soret peak shift from 404 to 415 nm. Furthermore, compound \u003cb\u003eD20\u003c/b\u003e exhibited no cytotoxicity at its effective concentration in MTT assay. In summary, our study suggested that chromone-oxime derivatives containing 1,2,3-triazole moieties might serve as a potential agent for the further development of IDO1 inhibitors.\u003c/p\u003e","manuscriptTitle":"Design, synthesis and biological evaluation of novel 1,2,3-triazoles chromone-oxime derivatives as potent indoleamine 2,3-dioxygenase 1 inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 09:26:20","doi":"10.21203/rs.3.rs-5283388/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-12-13T03:47:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-06T21:10:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-19T14:29:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Monatshefte für Chemie - Chemical Monthly","date":"2024-10-17T09:50:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"monatshefte-fur-chemie-chemical-monthly","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mccm","sideBox":"Learn more about [Monatshefte für Chemie - Chemical Monthly](https://www.springer.com/journal/706)","snPcode":"706","submissionUrl":"https://www.editorialmanager.com/mccm/","title":"Monatshefte für Chemie - Chemical Monthly","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1c3755b3-3992-4edd-adc5-dd76bf8d3094","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-04T13:04:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-09 09:26:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5283388","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5283388","identity":"rs-5283388","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}