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Design, synthesis and structure-activity relationship study of novel indole-2-carboxamide derivatives as anti-inflammatory agents for the treatment of sepsis Zhiguo Liu, Longguang Tang, Heping Zhu, Tingting Xu, Chenyu Qiu, Suqing Zheng, Yugui Gu, Jian-Peng Feng, Yali Zhang, and Guang Liang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b02006 • Publication Date (Web): 04 May 2016 Downloaded from http://pubs.acs.org on May 7, 2016

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Journal of Medicinal Chemistry

Design, synthesis and structure-activity relationship study of novel indole-2-carboxamide derivatives as anti-inflammatory agents for the treatment of sepsis

Zhiguo Liu 1, #, Longguang Tang

2, #

, Heping Zhu

1, #

, Tingting Xu 3, Chenyu Qiu 1,

Suqing Zheng 1, Yugui Gu 4, Jianpeng Feng 1, Yali Zhang1, *, Guang Liang1, *

1

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou

Medical University, 1210 University Town, Wenzhou, Zhejiang 325035, China 2

Center for Molecular Imaging and Translational Medicine, State Key Laboratory of

Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China. 3

The Second Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang

325035, China 4

Chemical Biology Section in WMU-WU Joint Research Centre, Wenzhou

University, Wenzhou, Zhejiang 325035, China

#

These authors contribute equally to this work.

* Corresponding author: Yali Zhang, Associate Professor School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang 325035, China Tel: (+86)-577-86699892; Fax: (+86)-577-86699892.

ACS Paragon Plus Environment


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E-mail: [email protected] * Corresponding author: Guang Liang, Ph.D, Professor Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang 325035, China Tel: (+86)-577-86699396; Fax: (+86)-577-86699396. E-mail: [email protected]


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ABSTRACT

Sepsis is characterized by a systemic inflammatory response syndrome. Derivatives of indole have been reported to exhibit diverse biological activities. This study reports on the design and synthesis of a new series of indole-2-carboxamide derivatives, which are screened for their anti-inflammatory activities in RAW 264.7 macrophages. A majority of these derivatives effectively inhibited lipopolysaccharides (LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Preliminary structure-activity relationship analysis was also conducted. The results indicate that the most promising compounds in the prepared series were 14f and 14g. They were found to effectively reduce LPS-induced pulmonary inflammation and over-expression of a series of inflammatory mediators. Furthermore, in vivo administration of 14f and 14g resulted in remarkable lung histopathological improvements in mice, without toxicity in organs. Taken together, these data indicate that the newly discovered indole-2-carboxamide derivatives could be particularly useful for further treatment in inflammatory diseases.

Keywords:

Indole-2-carboxamide;

Sepsis,

Lung

Anti-inflammation.


injury;

Drug

design;


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INTRODUCTION

Sepsis is the leading cause of acute lung injury (ALI) and death in critical care patients globally,1 and is characterized by a systemic inflammatory response syndrome (SIRS). At a minimum, SIRS results in clinical manifestations of abnormalities in body temperature (hypothermia or hyperthermia),2, 3 respiratory rate (tachypnea),4 heart rate (tachycardia),5 and the white blood cell count (leukocytopenia or leukocytosis).6, 7 It is estimated that sepsis affects about 750,000 people in the US alone, of which more than 210,000 die every year.8 Despite tremendous clinical and scientific effort in this field, curative therapy in sepsis still remains a considerable challenge in critical care medicine.9,

10

Hence, the development of new therapeutic approaches for sepsis is

invaluable for hospitalized patients. Pro-inflammatory cytokines play a crucial role in initiating an effective anti-infectious process;11 However, they are also associated with harmful effects that can lead to multiple types of inflammatory diseases, such as sepsis and ALI.12-14 Two key cytokines, Interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), have been shown to exert modulating effects on the pathogenesis of sepsis through a series of cytokine signaling pathways.15, 16 These observations suggest that the process of acute inflammation results in sepsis, and emphasize that the suppression or blockade of the proinflammatory activities of IL-6 and TNF-α may pave the way for new therapies to combat sepsis.17-19 Indole, a planer heterocyclic molecule, widespread and in abundance in natural products also is a component in the skeletal structure of several well-known drugs.20, 21 Therefore, indole derivatives have captured the attention of the scientific community due to their wide spectrum of biological activities, which include anti-inflammatory,22,


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23

anti-tuberculosis,24 anti-tumor,25,

26

anti-convulsion and anti-cardiovascular

effects.27-29 Over the past few decades, several research groups and pharmaceutical companies have reported on important drugs molecules containing the indole moiety. These have been applied for the treatment of acute or chronic inflammation as well as a variety of inflammatory diseases. Indomethacin (1, Figure 1) is a successful nonsteroidal anti-inflammatory drug (NSAID) in preventing sepsis and ALI,30 and has led to the exploration of Tenidap (2),31 ORG27569 (3),32, 33 LM-1685 (4),34 SCIO-469 (5),35 and a series of indole-based anti-inflammatory agents. Many of these candidates have been commonly used to reduce fever, pain, stiffness, and swelling by inhibiting the production of prostaglandins, as well as molecules known to cause these symptoms.36 However, to the best of our knowledge, these drugs have been accompanied by potential side effects, including headache,37 upset stomach, heartburn, diarrhea, skin rash, or a feeling of bowel fullness.38, 39

Please insert Figure 1

Considering that the inclusion of the indole moiety contributes to the potency of anti-inflammation, our group previously designed a series of imidazopyridine analogues through the application of isosteres in a drug design. Amongst these analogues, the compound 5-(4-Methylpiperazin-1-yl)-3-propyl-2-(1H-pyrrolo[2,3-b] pyridin-3-yl)-3H-imidazo[4,5-b]pyridine (X12, 6)40 showed beneficial effects for the prevention and treatment of sepsis in vitro and in vivo. In our ongoing studies into potent anti-inflammatory drugs targeting sepsis therapies, we detailed a drug design campaign that led to the discovery of a series of novel indole-2-carboxamide analogues. Previous investigations have demonstrated that indole-2-carboxamide has served as a



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drug core structure with a potentially safe and rational clinical profile.41, 42 Thus, we herein further evaluated the in vivo toxicity of synthetic analogues, investigated their inhibitory effect against LPS-induced expressions of TNF-α and IL-6, as well as the structure-activity relationships. In addition, we reported on the anti-inflammatory properties and in vivo sepsis treatment effects of 14f and 14g, two of the representative compounds.


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RESULTS AND DISCUSSION

Chemistry. The syntheses of indole-2-carboxamide derivatives (10a-10w, 13a-13h and 14a-14g) discussed in this paper are illustrated in Scheme 1. Hydrolysis of commercially available ethyl 5-nitro-1H-indole-2-carboxylate (7) under basic conditions provided by 5-nitro-1H-indole-2-carboxylic acid (8), which coupled with appropriate

aromatic

amines

9a-9w

to

afford

the

corresponding

5-nitro-indole-2-carboxamides 10a-10w. The subsequent N-alkylations placed substituents at N1 on the indole-skeleton of the amine 10j, which were easily performed under weak basic conditions due to its relatively acidic character, thus affording 13a-13h.43 Furthermore, preparation of 5-amino-indole-2-carboxamide 14a was achieved through the reduction of the nitro group of 13g using reduced iron and calcium chloride in EtOH. The successful conversion of 14a into 5-amino substituted indole-2-carboxamides (14b-14g) in the presence of acyl chlorides was catalyzed by N,N-Diisopropylethylamine (DIPEA).

Please insert Figure Scheme 1.

The key intermediates 9i, 9j and 9k-9w, with morpholinyl, 4-methyl piperidinyl, hetero- or bulky aromatic moieties at R1 and R2, were obtained by a two- or three-step synthetic route (Scheme S1). The nucleophilic substitutions of 1-fluoro-4-nitrobenzene with morpholine and 4-methyl piperidine followed by the hydrogen reduction furnished the corresponding N-arylated products 9i and 9j in excellent yields. Scheme S1 also shows the preparation of 2-substituted-4-aminophenol derivatives 9k-9w, treatment of



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the 2-substituted-4-nitrophenol 17 with various alkyl halides in the presence of K2CO3 gave aryl ethers 18, and these were reduced to anilines in 62-70% yields by using activated zinc power as a reducing agent. All new indole-2-carboxamides were fully characterized by proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), and electrospray ionisation mass spectrometry (ESI-MS).

Initial Evaluation against LPS-Induced TNF-α and IL-6 Release Lipopolysaccharide (LPS), a major biologically active agent of gram-negative bacteria, plays a key role in the development of a systemic inflammation response, especially in the expression of proinflammatory cytokines such as IL-6 and TNF-α.44 Hence, the enzyme-linked immunosorbent assay (ELISA) was used to screen the inhibition of all synthetic compounds (10a-10w) toward LPS-induced TNF-α and IL-6 release in RAW 264.7 mouse macrophages. Macrophages were pre-incubated for 60 minutes with the test compounds at a concentration of 10 µM, indomethacin or 6 (6) (10 µM), which were used as the positive controls, and DMSO which was used as the control medium. Subsequently, the cells were treated with LPS (0.5 mg/mL) for 22 h at 37 oC. The ability (% inhibition) of the tested compounds to reduce proinflammatory cytokines IL-6 and TNF-α is summarized in Table 1. Several indole-2-carboxamide derivatives showed promising potency against both LPS-induced expression of TNF-α and IL-6, such as 10e, 10g, 10j and 10k, and their inhibitory abilities were similar, but significantly higher than that of the positive controls, indomethacin or 6. Notably, 10j, with an installment of a 4-methyl piperidinyl group at R2, displayed the most potent effect on LPS-induced IL-6 and TNF-α expression with its inhibitory rates reaching 98.76% and 78.29%, respectively.


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Please insert Table 1

Structure-Activity Studies To the best of our knowledge, there are very few studies that report on the cytokine-inhibitory effects of indole-2-carboxamide derivatives and discuss their structure-activity relationship (SAR).45 It can be observed that compounds 10a and 10b, with a substituted or unsubstituted N-benzyl group at the amide substituent had very weak potency in either IL-6 or TNF-α (inhibitory rates < 12%). However, after replacement of the benzyl group with corresponding phenyl moieties, compounds 10c and 10d were found to dramatically improve activities, with inhibitory rates ranging from 18.2% (for TNF-α ) to 52.8% (for IL-6). After the modification from a methoxy group of 10d to an ethoxy group (10e), similar potency was observed with slightly improved inhibition of TNF-α. With the benzene ring in place, we next incorporated a methoxy group at the meta- or para-position yielding compounds 10f and 10g. Comparatively, greater activity occurred when the methoxy group was located at the para-position (10g, with 62.3% and 45.8% inhibitory rates for IL-6 and TNF-α, respectively), indicating that the position of the substituent was also a determinant of inhibitory potency. The introduction of morpholine, 4-methylpiperazine and various bulk alkoxy substituents at the para-position of the phenyl ring in compound 10c led to compounds 10i, 10j and 10k-10o. In general, bulk alkoxy-substituted analogues exhibited very weak or no activity. For example, respectively, about 5 and 20-fold lower IL-6 and TNF-α inhibitory profiles were observed for 101-10o compared to 10c. In addition, a similar trend was observed in compounds 10p-10w with similar alkoxy substituents



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and an additional chlorine at the ortho-position of the phenyl ring. However, analogue 10k with an O-isobutenyl moiety at the para-position of the phenyl ring resulted in a dramatic maintenance in inhibitory activity (56.5% and 38.6% inhibitory rates for IL-6 and TNF-α, respectively). Interestingly, replacement of the alkoxy groups of 10l-10o with morpholine (10i), 4-methylpiperazine (10j), or bearing the C3,4- pyrrole moiety (10h) improved potency, and in particular compound 10j showed the highest potent inhibitory capacity against IL-6 and TNF-α releases, indicating that a relatively large polar group at this position could be beneficial to anti-inflammatory activity.

Initial SAR at the N1-Position of 10j Following these positive indicators, we chose compound 10j for further follow-up studies. Optimization of the N-1 substituents on the indole core was performed to improve the inhibitory effect, and in particular the inhibition on TNF-α. These results are shown in Table 2. N-methylation of compound 10j led to compound 13a, which was equipotent in IL-6 inhibition with 10j, and resulted in an approximate 4-fold decrease in TNF-α activity. But N-allylation of compound 10j provided compound 13b, which significantly reduced the inhibition rates of both IL-6 (58.2%) and TNF-α (12.7%). Incorporation of substituted benzyl moieties at the same position provided N-benzylation compounds 13c-13h. Except for 13h, which has an electron-donating group (4-methoxy) on the benzyl group, these compounds exhibited significantly decreased inhibitory ability targeting IL-6, with an inhibitory rate of 64.3%. All other derivatives, including those with 2,6-dichlorobenzyl (13c), 3-fluorobenzyl (13d), 4-chlorobenzyl (13e), 4-bromobenzyl (13f), and 4-trifluoromethylbenzyl (13g) groups, displayed comparable IL-6 inhibitory activity to 10j, suggesting that the potency for suppressing IL-6 expression is directly related to the electrophilicity of the substituent


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on the benzyl group. Furthermore, in the brief analysis of halogen substitutions on the benzyl moiety, the profile of TNF-α inhibition with electron-withdrawing ability displayed the following pattern: 4-trifluoromethyl (13g) > 4-chloro (13e) > 2,6-dichloro (13c) > 4-bromo (13f). In the case of fluorine substitution, the 3-fluorobenzyl analogue, 13d, was 3-fold less potent than the corresponding 4-chlorinated compound, 13e, suggesting that the position of the substituent on the benzyl moiety may have a potential effect. Surprisingly, compound 13g, which has a 4-trifluoromethyl methyl substituent, showed the most potent effect on LPS-induced TNF-α and IL-6 production with its inhibitory rates reaching 98.7% and 75.1%, respectively. With good overall properties, tetrahydro-2H-pyranylmethyl was chosen for further exploration.


Optimization at the 5-Position of Indole to Obtain 14f and 14g. To further improve the anti-inflammatory activity of 13g, a variety of 5-substituted indole-2-carboxamide derivatives were evaluated, and their IC50 values were identified as summarized in Table 3. Reduction of the 5-nitro group of 13g gave the 5-amino indole-2-carboxamide 14a, which led to a dramatic loss in inhibitory activity, indicating that a hydrophobic group at this position maybe preferred for the activity. Branching at the α-position to the nitrogen of 14a, such as with a propenylcarbonyl group (14b) diminished IL-6 activity (IC50 = 6.28 µM) compared to lead compound 13g, while a linear chain such as the n-propylcarbonyl group (14c) or 3-cholo-propylcarbonyl was better tolerated (14d). However, the opposite trend was



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observed for the TNF-α inhibition capabilities of 14b, 14c and 14d, where the IC50 values were 1.05, 4.10 and 6.02 µM, respectively. Furthermore, compound 14e with a methylsulfonyl group at the 5-position provided similar inhibitory activity to 13g. Notably, incorporation of α-furancarbonyl at this position led to compound 14f, which displayed a significant increase in potency for IL-6. Taken together, the structure-activity relationships in this series are complex. In particular, compound 14g with a dimethylaminocarbonyl substitution, the most potent among the series, had an IC50 (IL-6) of 1.24 µM, about 4-fold greater than that of the lead compound, 13g. Moreover, all of the compounds, except for 14c and 14d, had IC50 values that were 1.5 to 4.6 times lower than that of 6 in blocking TNF-α production. All of these findings provide new evidence for the anti-inflammatory effects of these indole-2-carboxamide derivatives.


Inhibition of LPS-Induced Cytokine Release and MPO Activity by Active Compounds Before the in vivo experiments, we studied the acute toxicity of compounds 14f and 14g in vivo. According to the histopatholical results in Figure S1, neither 14f nor 14g had negative effects on liver, kidney and heart tissues. Moreover, when compared with the control group, the body weight of mice treated with compounds 14f and 14g showed no obvious differences (Figure S2). Myeloperoxidase (MPO) serves as a marker of infiltrating leukocytes (macrophages and neutrophils), and it plays a central role in the initiation and propagation of acute and chronic inflammatory disease.46 To confirm the effect of synthetic indole-2-carboxamide analogues on neutrophil accumulation, the


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most promising compounds 14f and 14g were selected to assess the inhibitory effects on MPO activity in lung tissues, as well as LPS-induced TNF-α release in serum. When compared to sham-operated mice, LPS-stimulation caused a significant increase in the MPO level in control mice. Upon treatment with 14f, 14g and 6, a remarkable reduction of MPO activity was observed when caused by LPS or without LPS injection (Figure 2A). Under the same experimental conditions, active compounds 14f and 14g displayed a potent inhibitory effect on suppressing LPS-induced TNF-α expression in serum, almost equal to that of 6 (Figure 2B). These results suggest that these compounds possess significant anti-inflammatory activity.


Effect of 14f and 14g on Histopathological Changes in the Lung Tissue of Mice Sepsis is the major cause of death in intensive care, and the lung is one of the earliest organs affected by sepsis that is characterized by ALI.47 To investigate the potential effect of 14f and 14g on lung injury of LPS-treated mice, the histopathology changes of C57BL/6 lung were observed using hematoxylin and eosin staining (H&E staining) 6 h after the injection of LPS. As can be observed in Figure 3, a normal pulmonary structure was observed in the sham group, while the lungs of mice exposed to LPS showed significant proinflammatory alterations characterized by lung edema and neutrophilic infiltration. Comparatively, administration of 14f, 14g and 6 (10 mg/kg) effectively reduced airspace inflammation and amended the tissue structure of pulmonary lobules. The results indicated that 14f and 14g have a remarkable protective effect against lung injury in mice treated with LPS.



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The Effects of 14f and 14g on Pulmonary Macrophage Infiltration in Septic Mice To confirm the aforementioned expression profile, we further enriched immunohistochemistry analysis with F4/80, a macrophage marker. As shown in Figure 4, lung tissues from the sham group showed normal histological features. However, in the LPS group, mice exhibited marked increases in macrophage infiltration. In contrast, macrophage infiltration was significantly ameliorated in the 6, 14f and 14g treated groups. These results were consistent with our previous H&E staining analysis.


Compounds 14f and 14g Suppressed LPS-induced mRNA Expressions in Mice To determine the efficacy of compounds 14f and 14g on cytokine production, we next performed a real-time quantitative PCR (RT-qPCR) analysis of the mRNA levels of six inflammatory cytokines in murine lung injury models: TNF-α, IL-6, IL-1β, cyclooxygenase-2 (COX-2), IL-12 and IL-33. The results, summarized in Figure 5, clearly demonstrate that LPS alone caused a progressive increase in mRNA accumulation for all of the cytokines. Except for the inhibitory effects against IL-1β and COX-2, 6 pretreatment was found to effectively down-regulate the expressions of TNF-α, IL-6, IL-12 and IL-33 with statistical significance. Meanwhile, injection with 14f and 14g resulted in more potently reduced mRNA expressions of those inflammatory mediators compared to those injected with 6. Particularly, compound 14g, which was the most active compound in inhibiting the expression of the aforementioned inflammatory mediators in mouse lung tissue, was most effective at the


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mRNA level. These data suggest that the anti-inflammatory activity of indole-2-carboxamides 14f and 14g partly affect the cytokine profile at the mRNA level.

Please insert Figure 5 CONCLUSION Sepsis represents the host's systemic inflammatory response to a severe infection. Despite an overall modest decline in the proportional mortality from sepsis, the total number of patients dying from sepsis is greater than in the past.48 Due to the resistance of such diseases to conventional treatments, as well as the side effects of presently available non-steroid anti-inflammatory drugs (NSAIDs), there is a pressing need to identify new anti-inflammatory compound and drug targets as part of a comprehensive medical-countermeasure strategy to prevent or mitigate sepsis. Collectively, this investigation provides evidence that indole-2-carboxamide is a viable template for the development of enhanced activity drug treatment with less side effects compared to traditional anti-inflammatory approaches.49 In the present study, we have designed and synthesized a novel series of indole-2-carboxamide analogues and evaluated their anti-inflammatory activity. The majority of the synthetic compounds exhibited significant inhibitory activities against LPS-induced TNF-α and IL-6 expression in RAW 264.7 macrophages. The preliminary structure-activity relationship (SAR) study indicated that a relatively large polar group at the amino carbonyl is beneficial to anti-inflammatory activity. Moreover, introducing an electron withdrawing group (EWG), especially on the para-position of the benzyl group, into the N-1 position on the indole skeleton is favored for efficacy. Furthermore, a hydrophobic group on the 5-position of the indole moiety generally



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improves activity. Clarification of the structural determinants for potency can be used as a guide in the design of novel potent molecules for future development. After initial anti-inflammatory screening, compounds 14f and 14g were identified as the most potent compounds, especially 14g, which displayed potent inhibition toward IL-6 and TNF-α with IC50 values of 1.24 and 2.67 µM, respectively. Additionally, pretreatment with active compounds 14f and 14g significantly decreased the TNF-α level in serum, lung MPO activity, pulmonary histopathological changes, and macrophage infiltration in a model of sepsis-induced lung injury. More importantly, the most active compound 14g was found to be extremely active in inhibiting the mRNA levels of multiple inflammatory cytokines in lung tissues. Thus, 14g is a promising potential compound as an anti-inflammatory agent that could improve common sepsis therapies.


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EXPERIMENTAL PROCEDURES Chemistry General In general, all commercial chemicals and solvents were reagent grade and were used without further purification. Thin-layer chromatography (TLC) was conducted on Kieselgel 60 F254 plates and flash column chromatography (medium pressure liquid chromatography) purifications were performed using Merck silica gel 60 (230-400 mesh ASTM) (Merck KGaA, Darmstadt, Germany). Melting points were determined on a Fisher-Johns melting apparatus and were uncorrected. 1H NMR and

13

C NMR

spectra were recorded in DMSO-d6 solution with a Bruker instruments at 500 or 600 MHz, and peak positions are given in parts per million (δ) down field from tetramethylsilane as internal standard, J values are given in hertz. Electron-spray ionization mass spectra in positive mode (ESI-MS) data were recorded on a Bruker Esquire 3000t spectrometer. Analytical HPLC analyses were performed at ambient temperature on a Agilent 1260 liquid chromatograph fitted with a Inertex C18 column (4.6 mm × 150 mm, 5 µm particle size) with CH3CN-H2O (A) and 0.1% TFA in CH3CN-H2O (B) solvent mixtures and equipped with a G1314A VWD detector. All general chemicals were the highest available grade, and the purity of all synthetic compounds was determined by HPLC analysis and was greater than 95%.



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Synthesis of 5-nitro-1H-indole-2-carboxylic acid (8). To a stirred solution of 5-nitro-1H-indole-2-carboxylate (7) (25.0 g, 0.11 mol) in 500 mL of THF-MeOH (1:1, v/v) was added a solution of NaOH (40 g, 1.0 mol) in 300 mL of water. The resulting deep-red-brown solution was stirred for 3 h and then quenched by water, the solution was removed THF in vicuo, and the solution was acidated to pH=1 with dilute HCl. The precipitated product was collected by vacuum filtration, and the remaining dissolved product was extracted with THF/ethyl acetate (1:2, v/v, 2 × 400 mL). The precipitate was dissolved in THF, and its solution was combined with organic layers from the extractions. Drying over MgSO4, filtration, concentration in vacuo, and crystallization with THF/EtOAc/Hexane afforded 19.6 g (90%) of 5-nitro-1H-indole-2-carboxylic acid (8) as a white solid. mp >300°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 11.50 (s, 1H), 7.20 (d, 1H, J = 8.4 Hz), 6.85 (s, 1H), 6.70-6.73 (m, 2H). ESI-MS m/z: 205.2 (M+H)-.

General Procedure A for the Preparation of the Indole-2-carboxamides 10a-10w. To a mixture of 5-nitroindole-2-carboxylic acid (8) (100 mg, 0.5 mmol) and aromatic amines 9a-9w (0.5 mmol) in DMA (6 mL) were added TBTU (0.4 g, 1.3 mmol) and DIPEA (0.1 mL, 0.6 mmol) slowly. The reaction mixture was stirred overnight, concentrated, and the mixture was diluted with 10 mL of EtOAc and 50 mL of NaHCO3 (satd), and the solid was suspended between the two layers. The solid compound was filtered, washed with water, and then resuspended with 1 M NaH2PO4, pH 3.0, filtered, washed again with water and 10% MeOH in water, dried under oil pump vacuum.

5-Nitro-N-phenethyl-1H-indole-2-carboxamide

(10a):


Following

general

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procedure A, the crude residue was purified by recrystallization, to furnish 10a as a white powder. Yield: 72.8%, mp: 141.3-143.5°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.293 (s, 1H), 8.833 (t, J = 6.0 Hz, 1H), 8.703 (s, 1H), 8.065 (dd, J = 2.4, 9.0 Hz, 1H), 7.566 (d, J = 9.0 Hz, 1H), 7.368 (s, 1H), 7.257-7.311 (m, 4H), 7.202 (t, J = 7.2 Hz, 1H), 3.519-3.554 (m, 2H), 2.881 (t, J = 7.2 Hz, 2H).

13

C NMR (125 MHz,

DMSO-d6) δ: 160.13, 141.16, 139.34, 139.22, 135.29, 128.64×2, 128.32×2, 126.34, 126.11, 119.08, 118.27, 112.74, 104.60, 40.49, 35.03 ppm. ESI-MS m/z: 310.10 (M+H)+.

N-(4-Methoxyphenethyl)-5-nitro-1H-indole-2-carboxamide

(10b):

Following

general procedure A, the crude residue was purified by recrystallization, to furnish 10b as a white powder. Yield: 70.6%, mp: 150.6-153.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.288 (s, 1H), 8.798 (t, J = 6.0 Hz, 1H), 8.703 (s, 1H), 8.065 (dd, J = 2.4, 9.0 Hz, 1H), 7.565 (d, J = 9.0 Hz, 1H), 7.368 (s, 1H), 7.171 (d, J = 8.4 Hz, 2H), 6.856 (d, J = 8.4 Hz, 2H), 3.711 (s, 3H), 3.474-3.508 (m, 2H), 2.808 (t, J = 7.8 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 160.12, 157.70, 141.20, 139.23, 135.34, 131.21×2, 129.60, 126.37, 119.06, 118.27, 113.76×2, 112.75, 104.59, 54.95, 34.17, 25.30 ppm. ESI-MS m/z: 339.1 (M)+.

5-Nitro-N-phenyl-1H-indole-2-carboxamide (10c): Following general procedure A, the crude residue was purified by flash chromatography to furnish 10c as a white powder. Yield: 77.6%, mp: 145.2-147.3°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm)



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10.498 (s, 1H), 8.755 (s, 1H), 8.086 (d, J = 9.0 Hz, 1H), 7.820 (d, J = 7.2 Hz, 2H), 7.678 (s, 1H), 7.607 (d, J = 9.0 Hz, 1H), 7.385 (t, J = 7.2 Hz, 2H), 7.128 (t, J = 7.2 Hz, 2H). 13

C NMR (150 MHz, DMSO-d6) δ: 159.11, 141.14, 138.66, 128.73×3, 126.46, 123.83,

120.27×3, 119.29, 118.48, 113.12, 106.14 ppm. ESI-MS m/z: 282.07 (M+H)+.

N-(4-Methoxyphenyl)-5-nitro-1H-indole-2-carboxamide (10d): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10d as a yellow powder. Yield: 75.6%, mp: 140.6-142.2°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.400 (s, 1H), 8.736 (s, 1H), 8.071 (d, J = 7.8 Hz, 1H), 7.715 (d, J = 9.0 Hz, 2H), 7.614 (s, 1H), 7.595 (d, J = 9.6 Hz, 1H), 6.959 (d, J = 8.4 Hz, 2H), 3.757 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 158.81, 155.66, 141.04, 131.70, 126.53, 121.84×2, 119.16×2, 118.27, 113.88×3, 113.10, 105.70, 55.19 ppm. ESI-MS m/z: 312.08 (M+H)+.

N-(4-Ethoxyphenyl)-5-nitro-1H-indole-2-carboxamide (10e): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10e as a pale yellow powder. Yield: 83.5%, mp: 155.6-157.8°C.

1

H NMR (600 MHz,

DMSO-d6): δ (ppm) 12.440 (s, 1H), 10.353 (s, 1H), 8.760 (d, J = 2.4 Hz, 1H), 8.101 (dd, J = 1.8, 9.0 Hz, 1H), 7.687 (d, J = 9.0 Hz, 2H), 7.648 (s, 1H), 7.608 (d, J = 9.0 Hz, 1H), 6.948 (d, J = 9.0 Hz, 2H), 4.004-4.039 (m, 2H), 1.320 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 158.52, 155.01, 141.33, 139.50, 135.23, 131.49, 126.34, 121.93×2, 119.25, 114.42×2, 114.05, 112.86, 105.69, 63.10, 14.69 ppm. ESI-MS m/z: 326.1 (M+H)+.


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5-Amino-N-(4-methoxyphenethyl)-1H-indole-2-carboxamide

(10f):

Following

general procedure A, the crude residue was purified by recrystallization, to furnish 10f as a pale yellow powder. Yield: 80.5%, mp: 338.1-339.8°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.487 (s, 1H), 10.384 (s, 1H), 8.780 (s, 1H), 8.114 (d, J = 9.0 Hz, 1H), 7.696 (s, 1H), 7.616 (d, J = 9.0 Hz, 1H), 7.105 (s, 2H), 6.308 (s, 1H), 3.765 (s, 6H).

13

C NMR (125 MHz, DMSO-d6) δ: 160.44×2, 158.90, 141.36, 140.23, 139.57,

134.93, 126.25, 119.38, 118.77, 112.88, 106.09, 98.59×2, 95.76, 55.12×2. ppm. ESI-MS m/z: 341.3 (M+H)+.

5-Nitro-N-(3,4,5-trimethoxyphenyl)-1H-indole-2-carboxamide (10g): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10g as a yellow powder. Yield: 83.5%, mp: 250.4-253.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.455 (s, 1H), 10.379 (s, 1H), 8.777 (s, 1H), 8.112 (dd, J = 1.8, 9.0 Hz, 1H), 7.668 (s, 1H), 7.616 (d, J = 9.0 Hz, 1H), 7.232 (s, 2H), 3.797 (s, 9H). 13C NMR (150 MHz, DMSO-d6) δ: 158.74, 152.70×2, 141.39, 139.55, 135.06, 134.68, 133.98, 126.33, 119.37, 118.76, 112.90, 105.88, 98.17×2, 60.14, 55.80×2 ppm. ESI-MS m/z: 371.3 (M)+.

N-(2-Methyl-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide (10h): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10h as a pale yellow solid. Yield: 89.2 %, mp: 257.8-259.7°C. 1H NMR (600 MHz,



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DMSO-d6): δ (ppm) 12.405 (s, 1H), 10.888 (s, 1H), 10.264 (s, 1H), 8.749 (d, J = 2.4 Hz, 1H), 8.100 (dd, J = 2.4, 9.0 Hz, 1H), 7.845 (s, 1H), 7.666 (s, 1H), 7.617 (d, J = 9.0 Hz, 1H), 7.334 (dd, J = 1.8, 9.0 Hz, 1H), 7.253 (d, J = 9.0 Hz, 1H), 6.125 (s, 1H), 2.380 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.45, 141.28, 159.45, 136.43, 135.71,

133.33, 130.09, 128.44, 126.43, 119.14, 118.45, 114.75, 112.82, 111.38, 110.21, 105.36, 99.28, 13.44 ppm. ESI-MS m/z: 375.6 (M+H)+.

N-(4-Morpholinophenyl)-5-nitro-1H-indole-2-carboxamide

(10i):

Following

general procedure A, the crude residue was purified by recrystallization, to furnish 10i as a pale yellow powder. Yield: 80.8%, mp: 328.1-329.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.428 (s, 1H), 10.309 (s, 1H), 8.753 (d, J = 1.8 Hz, 1H), 8.098 (dd, J = 2.4, 9.0 Hz, 1H), 7.655 (d, J = 9.0 Hz, 2H), 7.641 (s, 1H), 7.606 (d, J = 9.0 Hz, 1H), 6.972 (d, J = 9.0 Hz, 2H), 3.747 (t, J = 5.6 Hz, 4H), 3.088 (t, J = 5.6 Hz, 4H). 13C NMR (150 MHz, DMSO-d6) δ: 158.40, 147.72, 141.32, 139.49, 135.34, 130.62, 126.37, 121.46×2, 119.22, 118.56, 115.30×2, 112.84, 105.57, 66.10×2, 48.77×2 ppm. ESI-MS m/z: 367.5 (M+H)+.

N-[4-(4-Methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-carboxamide

(10j):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10j as a pale yellow powder. Yield: 82.5%, mp: 259.1-262.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.422 (s,1H), 10.291 (s, 1H), 8.749 (d, J = 2.4 Hz, 1H), 8.096 (dd, J = 2.4, 9.0 Hz, 1H), 7.639 (s, 1H), 7.630 (d, J = 7.2 Hz, 2H), 7.606 (d, J =


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9.0 Hz, 1H), 6.955 (d, J = 9.0 Hz, 2H), 3.112 (t, J = 4.8 Hz, 4H), 2.456 (t, J = 4.8 Hz, 4H), 2.222 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.59, 147.92, 141.53,

139.71,135.59, 130.50, 126.60, 121.68×2, 119.43, 118.77, 115.74×2, 113.06, 105.76, 54.83×2, 48.60×2, 45.98 ppm. ESI-MS m/z: 380.2 (M+H)+.

N-{4-[(3-Methylbut-2-en-1-yl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide (10k): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10k as a pale yellow powder. Yield: 85.5%, mp: 275.2-277.8°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.556 (s, 1H), 10.436 (s, 1H), 8.763 (s, 1H), 8.100 (d, J = 8.5 Hz, 1H), 7.697 (d, J = 8.0 Hz, 2H), 7.650 (s, 1H), 7.615 (d, J = 8.5 Hz, 1H), 6.956 (d, J = 8.0 Hz, 2H), 5.439 (s, 1H), 4.515 (d, J = 5.0 Hz, 2H), 1.748 (s, 3H), 1.712 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ: 158.47, 154.89, 141.27, 139.50, 136.90, 135.26, 131.52, 126.30, 121.85×2, 120.02, 119.21, 118.55, 114.59×2, 112.85, 105.78, 64.40, 25.39, 17.98. ppm. ESI-MS m/z: 365.3 (M)+.

N-{4-[(4-Methylbenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide

(10l):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10l as a pale yellow powder. Yield: 85.5%, mp: 269.1-272.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.446 (s, 1H), 10.366 (s, 1H), 8.761 (d, J = 1.8 Hz, 1H), 8.103 (d, J = 9.0 Hz, 1H), 7.690 (d, J = 9.0 Hz, 2H), 7.647 (s, 1H), 7.608 (d, J = 9.0 Hz, 1H), 7.340 (d, J = 7.8 Hz, 2H), 7.201 (d, J = 7.8 Hz, 2H), 7.026 (d, J = 9.0 Hz, 2H), 5.052 (s, 2H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.54, 154.82, 141.33, 139.51,



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137.03, 135.20, 134.09, 131.73, 128.96×2, 127.78×2, 126.34, 121.90×2, 119.27, 118.62, 114.90×2, 112.87, 105.72, 69.28, 20.77 ppm. ESI-MS m/z: 401.1 (M)+.

N-{4-[(4-Chlorobenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide

(10m):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10m as a pale yellow powder. Yield: 83.8%, mp: 217.8-220.5°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.448 (s, 1H), 10.381 (s, 1H), 8.763 (d, J = 2.4 Hz, 1H), 8.103 (d, J = 9.0 Hz, 1H), 7.718 (d, J = 9.0 Hz, 2H), 7.650 (s, 1H), 7.608 (d, J = 9.0 Hz, 1H), 7.525 (s, 1H), 7.433 (d, J = 6.0 Hz, 1H), 7.428 (s, 1H), 7.389-7.408 (m, 1H), 7.051 (d, J = 9.0 Hz, 2H), 5.132 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 158.56, 154.51, 141.33, 139.77, 139.51, 135.16, 133.09, 131.98, 130.36, 127.70, 127.27, 126.33, 126.14, 121.92×2, 119.27, 118.63, 114.93×2, 112.86, 105.74, 68.44 ppm. ESI-MS m/z: 423.0 (M+H)+.

N-{4-[(3-Fluorobenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide

(10n):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10n as a pale yellow powder. Yield: 81.2%, mp: 299.3-301.2°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.483 (s, 1H), 10.412 (s, 1H), 8.779 (s, 1H), 8.112 (dd, J = 2.0, 9.0 Hz, 1H), 7.724 (d, J = 9.0 Hz, 2H), 7.667 (s, 1H), 7.615 (d, J = 9.0 Hz, 1H), 7.455 (q, J = 8.0 Hz, 1H), 7.319-7.290 (m, 2H), 7.175 (d, J = 9.0 Hz, 1H), 7.061 (d, J = 9.0 Hz, 2H), 5.145 (s, 2H). 13C NMR (125 MHz, DMSO-d6) δ: 163.14, 158.53, 154.50, 141.29, 140.07, 139.49, 135.14, 131.95, 130.39, 130.39, 126.30, 123.48, 121.88×2,


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119.27, 118.60, 114.88×2, 114.08, 112.83, 105.72, 68.48 ppm. ESI-MS m/z: 406.4 (M+H)+.

N-{4-[(3,5-Difluorobenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide

(10o):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10o as a pale yellow powder. Yield:

89.5%, mp: 312.6-314.8°C. 1H NMR

(600 MHz, DMSO-d6): δ (ppm) 12.448 (s, 1H), 10.388 (s, 1H), 8.763 (s, 1H), 8.103 (dd, J = 2.4, 9.0 Hz, 1H), 7.713 (d, J = 9.0 Hz, 1H), 7.649 (s, 1H), 7.609 (d, J = 9.6 Hz, 2H), 7.213 (d, J = 2.4 Hz, 1H), 7.188 (d, J = 7.8 Hz, 2H), 7.053 (d, J = 8.4 Hz, 2H), 5.151 (s, 2H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.58×2, 154.30, 141.34, 139.52, 135.15,

132.13, 126.33, 121.93×3, 119.28, 118.64, 114.97×3, 112.87, 110.45, 110.29, 109.31, 105.71, 68.01 ppm. ESI-MS m/z: 423.0 (M)+.

N-[4-(Benzyloxy)-3-chlorophenyl]-5-nitro-1H-indole-2-carboxamide

(10p):

Following general procedure A, the crude residue was purified by recrystallization, to furnish 10p as a pale yellow powder. Yield: 89.5%, mp: 238.7-239.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 11.409 (s, 1H), 8.649 (s, 1H), 8.054 (d, J = 2.4 Hz, 1H), 7.927 (d, J = 8.4 Hz, 1H), 7.712 (dd, J = 2.4, 9.0 Hz, 1H), 7.476-7.516 (m, 4H), 7.402-7.438 (m, 3H), 7.343 (t, J = 7.2 Hz, 1H), 7.242 (d, J = 9.0 Hz, 1H), 5.199 (s, 2H). 13

C NMR (150 MHz, DMSO-d6) δ: 149.75, 145.78, 136.98×2, 133.36, 128.69×3,

128.13×2, 127.73×3, 121.60, 121.49, 119.81×2, 119.11, 114.88×2, 105.76, 70.50 ppm. ESI-MS m/z: 422.1 (M)+.



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N-{3-Chloro-4-[(2,5-dimethylbenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxami de (10q): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10q as a pale yellow powder. Yield: 85.3%, mp: 230.5-232.7°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.499 (s, 1H), 10.509 (s, 1H), 8.796 (s, 1H), 8.119 (dd, J = 2.0, 9.0 Hz, 1H), 7.973 (s, 1H), 7.720 (d, J = 9.0 Hz, 1H), 7.670 (s, 1H), 7.623 (d, J = 9.0 Hz, 1H), 7.345 (d, J = 9.0 Hz, 1H), 7.280 (s, 1H), 7.131 (d, J = 7.5 Hz, 1H), 7.080 (d, J = 7.5 Hz, 1H), 5.134 (s, 2H), 2.315 (s, 3H), 2.288 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.70, 150.07, 141.34, 139.56,

134.77, 134.63, 134.22, 133.55, 132.42, 130.09, 129.25, 128.71, 126.25, 121.87, 121.13, 120.10, 119.39, 118.75, 114.42, 112.89, 106.203, 69.16, 20.28, 18.52 ppm. ESI-MS m/z: 449.1 (M)+.

N-{3-Chloro-4-[(4-methoxybenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide (10r): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10r as a yellow solid. Yield: 86.5%, mp: 301.2-303.7°C. 1

H NMR (600 MHz, DMSO-d6): δ (ppm) 12.593 (s, 1H), 10.625 (s, 1H), 8.759 (d, J =

2.4 Hz, 1H), 8.099 (dd, J = 2.4, 9.0 Hz, 1H), 7.983 (d, J = 2.4 Hz, 1H), 7.717 (dd, J = 2.4, 9.0 Hz, 1H), 7.671 (s, 1H), 7.614 (d, J = 9.0 Hz, 1H), 7.405 (d, J = 9.0 Hz, 2H), 7.274 (d, J = 9.0 Hz, 1H), 6.964 (d, J = 8.4 Hz, 2H), 5.116 (s, 2H), 3.761 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 159.32, 159.00, 150.21, 141.51, 140.00, 132.78, 129.63×3, 128.69, 126.53, 122.04, 121.51, 120.24, 119.55, 118.88, 114.94, 114.10×3, 113.23, 106.56,


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70.35 ppm. ESI-MS m/z: 452.0 (M)+.

N-{3-Chloro-4-[(3-methoxybenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide (10s): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10r as a pale yellow solid. Yield: 87.5%, mp: 269.3-272.1°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 12.495 (s, 1H), 10.499 (s, 1H), 8.794 (s, 1H), 8.114 (dd, J = 2.4, 9.0 Hz, 1H), 7.966 (d, J = 2.0 Hz, 1H), 7.686 (dd, J = 2.0, 9.0 Hz, 1H), 7.659 (s, 1H), 7.614 (d, J = 9.0 Hz, 1H), 7.328 (t, J = 8.0 Hz, 1H), 7.267 (d, J = 9.0 Hz, 1H), 7.051 (s, 1H), 7.043 (d, J = 8.0 Hz, 1H), 6.911 (d, J = 8.0 Hz, 1H), 5.194 (s, 2H), 3.769 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ: 159.30, 158.68, 149.86, 141.34, 139.55, 138.18, 134.75, 132.50, 129.57, 126.24, 121.83, 121.23, 120.03, 119.45, 119.38, 118.74, 114.54, 113.20, 112.96, 112.88, 106.03, 69.98, 54.98 ppm. ESI-MS m/z: 451.9 (M)+.

N-{3-Chloro-4-[(3,5-dimethoxybenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxa mide (10t): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10t as a pale yellow solid. Yield: 86.4%, mp: 275.7-277.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.469 (s, 1H), 10.476 (s, 1H), 8.781 (d, J = 2.4 Hz, 1H), 8.108 (dd, J = 2.4, 9.0 Hz, 1H), 7.958 (d, J = 2.4 Hz, 1H), 7.678 (d, J = 9.0 Hz, 1H), 7.649 (s, 2H), 7.611 (d, J = 9.0 Hz, 1H), 7.244 (d, J = 9.0 Hz, 1H), 6.634 (d, J = 2.4 Hz, 1H), 6.458 (m, J = 2.4 Hz, 1H), 5.155 (s, 2H), 3.748 (s, 6H). 13C NMR (150 MHz, DMSO-d6) δ: 160.79×2, 158.96, 150.09, 141.62,



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139.82, 139.24, 135.01, 132.79, 126.50, 122.10, 121.54, 120.31, 119.62, 119.00, 114.86, 113.15, 106.30, 105.44×2, 99.62, 70.26, 55.40×2 ppm. ESI-MS m/z:483.0 (M+H)+.

N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamide (10u): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10u as a pale yellow solid. Yield: 82.3%, mp: 295.6-297.9°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.471 (s, 1H), 10.487 (s, 1H), 8.781 (d, J = 1.8 Hz, 1H), 8.109 (dd, J = 2.4, 9.0 Hz, 1H), 7.972 (d, J = 2.4 Hz, 1H), 7.694 (dd, J = 2.4, 9.0 Hz, 1H), 7.654 (s, 1H), 7.614 (d, J = 9.0 Hz, 1H), 7.447-7.484 (m, 1H), 7.321 (d, J = 7.2 Hz, 1H), 7.305 (d, J = 12.0 Hz, 1H), 7.269 (d, J = 9.0 Hz, 1H), 7.162-7.195 (m, 1H), 5.239 ( s, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 161.61, 158.97, 149.96, 141.62, 139.83, 135.00, 132.94, 130.80, 126.50, 123.55, 122.13, 121.54, 120.32, 119.63, 119.01, 114.85×2, 114.32, 114.18, 113.15, 106.32, 69.62 ppm. ESI-MS m/z: 462.5 (M+Na)+.

N-{3-Chloro-4-[(2,6-dichlorobenzyl)oxy]phenyl}-5-nitro-1H-indole-2-carboxamid e (10v): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10v as a pale yellow solid. Yield: 84.5%, mp: 309.2-311.6°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.486 (s, 1H), 10.523 (s, 1H), 8.790 (d, J = 2.4 Hz, 1H), 8.114 (dd, J = 2.4, 9.0 Hz, 1H), 7.961 (d, J = 2.4 Hz, 1H), 7.749 (dd, J = 2.4, 9.0 Hz, 1H), 7.670 (s, 1H), 7.620 (d, J = 9.0 Hz, 1H), 7.585 (d, J =


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8.4 Hz, 2H), 7.494 (t, J = 8.4 Hz, 1H), 7.419 (d, J = 9.0 Hz, 1H), 5.316 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 159.01, 150.35, 141.63, 139.83, 136.41×2, 134.99, 133.37, 131.99, 131.54, 129.05×2, 126.51, 122.14, 121.88, 120.40, 119.65, 119.03, 115.45, 113.16, 106.36, 66.72 ppm. ESI-MS m/z: 490.5 (M+H)+.

N-{3-Chloro-4-{[4-(trifluoromethyl)benzyl]oxy}phenyl}-5-nitro-1H-indole-2-carb oxamide (10w): Following general procedure A, the crude residue was purified by recrystallization, to furnish 10w as a pale yellow solid. Yield: 80.4%, mp: 277.5-279.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 12.469 (s, 1H), 10.491 (s, 1H), 8.779 (d, J = 1.8 Hz, 1H), 8.108 (dd, J = 2.4, 9.0 Hz, 1H), 7.980 (d, J = 2.4 Hz, 1H), 7.794 (d, J = 8.4 Hz, 2H), 7.684-7.707 (m, 3H), 7.651 (s, 1H), 7.614 (d, J = 9.0 Hz, 1H), 7.273 (d, J = 9.0 Hz, 1H), 5.333 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 158.98, 149.91, 141.76, 141.63, 139.82, 134.99, 133.00, 128.05×3, 126.50, 125.64, 125.34, 122.15, 121.53, 120.33, 119.62, 119.01, 114.84, 113.15, 106.32, 69.60 ppm. ESI-MS m/z: 489.6 (M+H)+.

General

Procedure

B

for

the

Preparation

of

N-Substituted-5-nitro-1H-indole-2-carboxamides 13a-13h. A solution of N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-carboxamide (10j) (0.758g, 2.0 mmol) in dry acetonitrile (5.0 mL) was added K2CO3 (0.83g, 6.0 mmol) portionwise, followed by alkyl or arylalkyl halide (6.0 mmol) at room temperature. The reaction mixture was stirred at 50 °C for 5 h. The reaction mixture was then filtered and the filtercake was washed with acetonitrile (10 mL). The filtrate



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was concentrated under reduced pressure and the residue was dissolved in EtOAc (50 mL), washed with water and brine. The organic layer was dried over MgSO4, filtered, and the filtrate was concentrated in vacuo to give the crude residue.

1-Methyl-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-carboxamid e (13a): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13a as a white powder. Yield: 72.8%, mp: 253.1-255.2°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.392 (s, 1H), 8.731 (d, J = 2.4 Hz, 1H), 8.151 (dd, J = 2.4, 9.0 Hz, 1H), 7.787 (d, J = 9.0 Hz, 1H), 7.616 (d, J = 9.0 Hz, 2H), 7.497 (s, 1H), 6.937 (d, J = 9.0 Hz, 2H), 4.065 (s, 3H), 3.106 (t, J = 4.8 Hz, 4H), 2.450 (t, J = 4.8 Hz, 4H), 2.219 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 159.09, 147.74, 141.48, 141.01, 136.01, 130.39, 124.79, 121.41×2, 118.90, 118.45, 115.44×2, 111.38, 107.27, 54.62×2, 48.39×2, 45.78, 32.13 ppm. ESI-MS m/z: 394.4 (M+H)+.

1-Allyl-N-(4-(4-methylpiperazin-1-yl)phenyl)-5-nitro-1H-indole-2-carboxamide (13b): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13b as a white powder. Yield: 70.8%, mp: 253.2-254.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.415 (s, 1H), 8.751 (d, J = 1.8 Hz, 1H), 8.142 (dd, J = 2.4, 9.0 Hz, 1H), 7.765 (d, J = 9 Hz, 1H), 7.605 (d, J = 9.0 Hz, 2H), 7.534 (s, 1H), 6.933 (d, J = 9 Hz, 2H), 6.045-5.982 (m, 1H), 5.295 (d, J = 5.4 Hz, 2H), 5.092 (dd, J = 1.2, 10.8 Hz, 1H), 4.884 (dd, J = 1.8, 17.4 Hz, 1H), 3.103 (t, J = 4.8 Hz, 4H), 2.450 (t, J = 4.8 Hz, 4H), 2.219 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 158.99, 147.74, 141.63, 140.51, 135.49, 134.21, 130.35, 124.97,


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121.38×2, 118.99, 118.60, 116.28, 115.43×2, 111.60, 107.71, 54.59×2, 48.39×2, 46.62, 45.76 ppm. ESI-MS m/z: 420.4 (M+H)+.

1-(2,6-Dichlorobenzyl)-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2 -carboxamide (13c): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13c as a white powder. Yield: 72.5%, mp: 252.1-253.8°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.415 (s, 1H), 8.751 (d, J = 1.8 Hz, 1H), 8.142 (dd, J = 2.4, 9.0 Hz, 1H), 7.765 (d, J = 9.0 Hz, 1H), 7.605 (d, J = 9.0 Hz, 2H), 7.534 (s, 1H), 6.933 (d, J = 9.0 Hz, 2H), 6.045-5.982 (m, 1H), 5.295 (d, J = 5.4 Hz, 2H), 5.092 (dd, J = 1.2, 10.8 Hz, 1H), 4.884 (dd, J = 1.8, 17.4 Hz, 1H), 3.103 (t, J = 4.8 Hz, 4H), 2.450 (t, J = 4.8 Hz, 4H), 2.219 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 159.44, 147.80, 141.60, 140.67, 137.58, 135.07×2, 131.44, 130.58, 130.27, 129.31×2, 125.44, 121.48×2, 119.00, 118.76, 115.45×2, 111.12, 107.40, 54.60×2, 48.38×2, 45.77, 44.76 ppm. ESI-MS m/z: 538.1 (M+H)+.

1-(3-Fluorobenzyl)-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-car boxamide (13d): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13d as a white powder. Yield: 69.2%, mp: 248.2-250.3°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 10.469 (s, 1H), 8.776 (d, J = 2.4 Hz, 1H), 8.131 (dd, J = 2.4, 9.6 Hz, 1H), 7.795 (d, J = 9.0 Hz, 1H), 7.599 (s, 1H), 7.573 (d, J = 9.0 Hz, 2H), 7.340-7.303 (m, 1H), 7.070-7.043 (m, 1H), 6.924 (d, J = 9.0 Hz, 4H), 5.939 (s, 2H), 3.097 (t, J = 4.8 Hz, 4H), 2.445 (t, J = 4.8 Hz,



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4H), 2.215 (s, 3H).

13

C NMR (125 MHz, DMSO-d6) δ: 161.18, 159.02, 147.76,

141.84, 140.66, 135.38, 130.64, 130.57, 130.19, 125.13, 122.55, 121.41×2, 119.09, 118.93, 115.41×2, 113.46, 113.29, 111.70, 108.23, 54.52×2, 48.28×2, 47.02, 45.69 ppm. ESI-MS m/z: 488.1 (M+H)+.

1-(4-Chlorobenzyl)-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-ca rboxamide (13e): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13e as a white powder. Yield: 68.5%, mp: 224.8-226.5°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.446 (s, 1H), 8.767 (d, J = 2.4 Hz, 1H), 8.125 (dd, J = 2.4, 9.0 Hz, 1H), 7.787 (d, J = 9.6 Hz, 1H), 7.599 (s, 1H), 7.573 (d, J = 9.0 Hz, 2H), 7.341 (d, J = 8.4 Hz, 2H), 7.127 (d, J = 9.0 Hz, 2H), 6.923 (d, J = 9.0 Hz, 2H), 5.911 (s, 2H), 3.100 (t, J = 4.8 Hz, 4H), 2.456 (t, J = 4.8 Hz, 4H), 2.223 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 159.03, 147.80,

141.85, 140.68, 136.93, 135.35, 131.93, 130.21, 128.54×2, 125.16, 121.48×2, 125.16, 121.48×2, 119.11, 115.45×2, 111.74, 108.27, 54.56×2, 48.33×2, 46.94, 45.72 ppm. ESI-MS m/z: 504.1 (M+H)+.

1-(4-Bromobenzyl)-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-ca rboxamide (13f): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13a as a white powder. Yield: 70.6%, mp: 242.3-244.1°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.447 (s, 1H), 8.770 (d, J = 2.4 Hz, 1H), 8.125 (dd, J = 2.4, 9.0 Hz, 1H), 7.785 (d, J = 9.0 Hz, 1H),


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7.603 (s, 1H), 7.574 (d, J = 8.4 Hz, 2H), 7.475 (d, J = 8.4 Hz, 2H), 7.062 (d, J = 7.8 Hz, 2H), 6.923 (d, J = 9.0 Hz, 2H), 5.895 (s, 2H), 3.096 (t, J = 4.8 Hz, 4H), 2.443 (t, J = 4.8 Hz, 4H), 2.214 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.99, 147.79,

141.83, 140.66, 137.35, 135.32, 131.44×2, 130.18, 128.80×2, 125.13, 121.45×2, 120.40, 119.09, 118.91, 115.41×2, 111.72, 108.25, 54.58×2, 48.35×2, 46.99, 45.76 ppm. ESI-MS m/z: 548.4 (M)+.

N-[4-(4-Methylpiperazin-1-yl)phenyl]-5-nitro-1-[4-(trifluoromethyl)benzyl]-1H-i ndole-2-carboxamide (13g): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13g as a white powder. Yield: 72.6%, mp: 231.8-233.3°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.447 (s, 1H), 8.793 (d, J = 2.4 Hz, 1H), 8.130 (dd, J = 2.4, 9.0 Hz, 1H), 7.773 (d, J = 9.0 Hz, 1H), 7.664 (s, 1H), 7.649 (s, 2H), 7.561 (d, J = 9.0 Hz, 2H), 7.276 (d, J = 8.4 Hz, 2H), 6.914 (d, J = 9.0 Hz, 2H), 6.027 (s, 2H), 3.091 (t, J = 4.8 Hz, 4H), 2.442 (t, J = 4.8 Hz, 4H), 2.213 (s, 3H).

13

C NMR (150 MHz, DMSO-d6) δ: 158.93, 145.42, 142.77,

141.92, 140.74, 136.97, 135.28, 130.15, 127.15×2, 125.49×2, 125.16, 121.48×2, 119.16, 119.03, 117.39, 115.40×2, 111.68, 108.32, 54.57×2, 48.34×2, 47.32, 45.75 ppm. ESI-MS m/z: 538.0 (M+H)+.

1-(4-Methoxybenzyl)-N-[4-(4-methylpiperazin-1-yl)phenyl]-5-nitro-1H-indole-2-c arboxamide (13h): Following general procedure B, the crude residue was purified by flash silica gel column chromatography, to furnish 13h as a white powder. Yield:



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

75.7%, mp: 235.6-237.3°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 10.456 (s, 1H), 8.746 (s, 1H), 8.122 (d, J = 9.0 Hz, 1H), 7.840 (d, J = 9.0 Hz, 1H), 7.605 (d, J = 8.0 Hz, 2H), 7.537 (s, 1H), 7.109 (d, J = 8.0 Hz, 2H), 6.936 (d, J = 8.0 Hz, 2H), 6.824 (d, J = 8.0 Hz, 2H), 5.846 (s, 2H), 3.666 (s, 3H), 3.103 (t, J = 4.8 Hz, 4H), 2.450 (t, J = 4.8 Hz, 4H), 2.219 (s, 3H).

13

C NMR (125 MHz, DMSO-d6) δ: 159.17, 158.52,

147.77, 141.66, 140.57, 135.49, 130.28, 129.74, 128.16×2, 125.11, 121.39×2, 118.98, 118.68, 115.42×2, 113.92×2, 111.83, 108.10, 54.99, 54.57×2, 48.36×2, 46.81, 45.74 ppm. ESI-MS m/z: 500.21 (M+H)+.

5-Amino-N-[4-(4-methylpiperazin-1-yl)phenyl]-1-[4-(trifluoromethyl)benzyl]-1Hindole-2-carboxamide (14a): Compound 13g (0.115g, 0.21 mmol) was dissolved in ethanol (5 mL), and to this solution was added the actived zinc powder (0.23 g, 6.9 mmol), followed by ammonium chloride (0.23 g, 4.2 mmol) at 25 °C. The temperature was raised to 50 °C, and the stirring was continued for 6 h. Progress of the reaction was followed by TLC. Upon completion of the reaction, 15 mL of DCM: MeOH (1:1) was added to the reaction mixture and inorganic residues were removed by filtration. The filtrate was evaporated, and the residue was purified by flash column chromatography to afford a white powder of 14a. Yield: 72.6%, mp: 218.8-220.3°C. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 10.063 (s, 1H), 7.627 (d, J = 8.0 Hz, 2H), 7.569 (d, J = 8.0 Hz, 2H), 7.237-7.187 (m, 4H), 7.143 (s, 1H), 6.893 (d, J = 9.0 Hz, 2H), 6.783 (s, 1H), 6.669 (d, J = 9.0 Hz, 1H), 5.852 (s, 2H), 3.076 (t, J = 4.8 Hz, 4H), 2.439 (t, J = 4.8 Hz, 4H), 2.211 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ: 160.00, 147.39, 144.13, 142.88, 141.34, 132.31, 130.99, 130.78, 127.03, 126.79, 125.24×2, 125.22×2, 121.23×2, 115.54,


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115.42×2, 111.02, 104.81, 103.20, 54.59×2, 48.46×2, 46.59, 45.74. ESI-MS m/z: 508.3 (M+H)+.

General Procedure C for the Preparation of Indole-2-carboxamides 14b-14g. To a solution of compound 14a (53.7 mg, 1.0 mmol) in N,N-dimethylacetamide (5 mL) was added DIEA (2.0 mmol) and alkyl chloride (1.0 mmol) slowly. The reaction mixture was then stirred for 2 h at room temperature. The reaction mixture was cooled to ambient temperature and extracted by EtOAc (10 mL×2), the organic layer was washed sequentially with water (20 mL) and brine (20 mL). The organic layer was dried, filtered, and concentrated under reduced pressure.

5-Acrylamido-N-[4-(4-methylpiperazin-1-yl)phenyl]-1-[4-(trifluoromethyl)benzyl ]-1H-indole-2-carboxamide (14b): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b as a white powder. Yield: 65.7%, mp: 231.2-233.2°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.190 (s, 1H), 10.152 (s, 1H), 8.202 (d, J = 1.8 Hz, 1H), 7.637 (d, J = 8.4 Hz, 2H), 7.569 (d, J = 9.0 Hz, 2H), 7.484 (d, J = 9.0 Hz, 1H), 7.412 (dd, J = 2.4, 9.0 Hz, 1H), 7.382 (s, 1H), 7.250 (d, J = 7.8 Hz, 2H), 6.905 (d, J = 9.0 Hz, 2H), 6.494-6.449 (m, 1H), 6.250 (dd, J = 1.8, 16.8 Hz, 1H), 5.935 (s, 2H), 5.730 (dd, J = 1.8, 10.2 Hz, 1H), 3.090 (t, J = 4.8 Hz, 4H), 2.459 (t, J = 4.8 Hz, 4H), 2.225 (s, 3H).

13

C NMR (150

MHz, DMSO-d6) δ: 162.89, 159.71, 147.53, 143.69, 135.14, 132.67, 132.25, 132.11, 130.57, 127.08×2, 126.20, 125.69, 125.36×2, 121.38×2, 118.11, 115.45×2, 111.76,



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110.99, 108.83, 106.85, 106.23, 54.55×2, 48.38×2, 46.79, 45.68 ppm. ESI-MS m/z: 562.2 (M+H)+.

N-[4-(4-Methylpiperazin-1-yl)phenyl]-5-propionamido-1-[4-(trifluoromethyl)ben zyl]-1H-indole-2-carboxamide (14c): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b as a white powder. Yield: 68.5%, mp: 222.2-224.1°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.167 (s, 1H), 9.801 (s, 1H), 8.092 (d, J = 1.8 Hz, 1H), 7.631 (d, J = 7.8 Hz, 2H), 7.565 (d, J = 9.0 Hz, 2H), 7.443 (d, J = 9.0 Hz, 1H), 7.350 (s, 1H), 7.333 (dd, J = 1.8, 9.0 Hz, 1H), 7.242 (d, J = 7.8 Hz, 1H), 6.900 (d, J = 9.0 Hz, 2H), 5.925 (s, 2H), 3.459-3.424 (m, 1H), 3.081 (t, J = 4.8 Hz, 4H), 2.438 (t, J = 4.8 Hz, 4H), 2.320 (q, J = 7.2 Hz, 2H), 2.209 (s, 3H), 1.098 (t, J = 7.8 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 171.59, 159.75, 147.55, 143.73, 134.90, 133.04, 132.09, 130.55, 127.70, 127.49, 127.06×2, 125.66, 125.32×2, 125.10, 121.37×2, 118.10, 115.42×2, 111.40, 110.80, 106.12, 56.00, 54.60×2, 48.44×2, 46.75, 45.75 ppm. ESI-MS m/z: 564.4 (M+H)+.

5-(2-Chloroacetamido)-N-[4-(4-methylpiperazin-1-yl)phenyl]-1-[4-(trifluorometh yl)benzyl]-1H-indole-2-carboxamide (14d): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b as a white powder. Yield: 65.3%, mp: 216.2-218.1°C.

1

H NMR (500 MHz,

DMSO-d6): δ (ppm) 10.147 (s, 1H), 10.045 (s, 1H), 8.156(s, 1H), 8.139 (d, J = 9.0 Hz, 1H), 7.783 (d, J = 9.0 Hz, 1H), 7.666 (d, J = 9.0 Hz, 2H), 7.659 (s, 1H), 7.574 (d, J =


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8.0 Hz, 1H), 7.279 (d, J = 8.0 Hz, 1H), 6.925 (d, J = 9.0 Hz, 1H), 5.933 (s, 2H), 4.232 (s, 2H), 3.105 (t, J = 4.8 Hz, 4H), 2.506 (t, J = 4.8 Hz, 4H), 2.235 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 164.37, 161.39, 148.13, 147.54, 143.67, 143.64, 132.55, 130.60, 127.75, 127.09×2, 125.37×2, 123.31, 121.51×2, 121.43, 115.92, 115.48, 112.46, 111.99, 54.49×2, 48.31×2, 46.84, 43.60, 42.30 ppm. ESI-MS m/z: 564.4 (M+H)+.

N-[4-(4-Methylpiperazin-1-yl)phenyl]-5-(methylsulfonamido)-1-[4-(trifluorometh yl)benzyl]-1H-indole-2-carboxamide (14e): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b as a white powder. Yield: 65.7%, mp: 231.2-233.2°C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.194 (s, 1H), 9.446 (s, 1H), 7.642 (d, J = 8.4 Hz, 2H), 7.567 (s, 1H), 7.552 (s, 1H), 7.517 (d, J = 9.0 Hz, 1H), 7.371 (s, 1H), 7.275 (d, J = 8.4 Hz, 2H), 7.154 (dd, J = 1.8, 9.0 Hz, 2H), 6.907 (d, J = 9.0 Hz, 2H), 5.928 (s, 2H), 3.084 (t, J = 4.8 Hz, 4H), 2.912 (s, 3H), 2.440 (t, J = 4.8 Hz, 4H), 2.211 (s, 3H).

13

C NMR (150 MHz,

DMSO-d6) δ: 159.65, 147.63, 143.54, 135.88, 132.63, 131.47, 130.45, 127.17×2, 126.01, 125.37, 126.01, 125.37×2, 121.42×2, 120.23, 115.43×2, 114.45, 111.50, 106.02, 54.60×2, 48.42×2, 46.84, 45.76, 38.67 ppm. ESI-MS m/z: 562.2 (M+H)+. . 5-(Furan-2-carboxamido)-N-[4-(4-methylpiperazin-1-yl)phenyl]-1-[4-(trifluorom ethyl)benzyl]-1H-indole-2-carboxamide (14f): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

as a white powder. Yield: 67.2%, mp: 203.5-205.2 °C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.195 (s, 1H), 10.159 (s, 1H), 8.165 (s, 1H), 7.927 (s, 1H), 7.641 (d, J = 7.8 Hz, 2H), 7.576 (d, J = 9.6 Hz, 2H), 7.549 (d, J = 1.8 Hz, 1H), 7.504 (d, J = 9.0 Hz, 1H), 7.406 (s, 1H), 7.322 (d, J = 3.6 Hz, 1H), 7.259 (d, J = 7.8 Hz, 2H), 6.907 (d, J = 9.0 Hz, 2H), 6.700 (q, J = 1.8 Hz, 1H), 5.952 (s, 2H), 3.091 (t, J = 4.8 Hz, 4H), 2.460 (t, J = 4.8 Hz, 4H), 2.224 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 159.72, 156.14, 147.80, 147.53, 145.44, 143.69, 135.53, 132.25, 131.96, 130.57, 127.08×2, 125.59, 125.34×2, 125.11, 119.29×2, 115.45×2, 114.28, 113.14, 112.07, 110.81, 106.25, 56.01, 54.54×2, 48.38×2, 46.81, 45.67, 18.54 ppm. ESI-MS m/z: 602.3 (M+H)+.

5-(3,3-Dimethylureido)-N-[4-(4-methylpiperazin-1-yl)phenyl]-1-[4-(trifluorometh yl)benzyl]-1H-indole-2-carboxamide (14g): Following general procedure C, the crude residue was purified by flash silica gel column chromatography, to furnish 14b as a white powder. Yield: 68.4%, mp: 132.8-134.9 °C. 1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.140 (s, 1H), 8.196 (s, 1H), 7.789 (d, J = 1.8 Hz, 1H), 7.631 (d, J = 8.4 Hz, 2H), 7.567 (d, J = 9.0 Hz, 2H), 7.385 (d, J = 9.0 Hz, 1H), 7.325 (s, 1H), 7.307 (dd, J = 1.8, 9.0 Hz, 1H), 7.239 (d, J = 8.4 Hz, 2H), 6.900 (d, J = 9.0 Hz, 2H), 5.922 (s, 2H), 3.085 (t, J = 4.8 Hz, 4H), 2.931 (s, 6H), 2.453 (t, J = 4.8 Hz, 4H), 2.220 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 159.82, 156.23, 147.48, 143.86, 134.67, 134.16, 131.73, 130.62, 127.04×2, 125.63, 125.31×2, 121.36×2, 119.88, 115.44×2,


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112.35, 110.28, 105.97, 56.00, 54.56×2, 48.41×2, 46.74, 45.69, 36.21×2, 18.53 ppm. ESI-MS m/z: 579.2 (M+H)+.

Animals. 18-22 g male C57BL/6 mice were obtained from the Animal Center of Wenzhou Medical University (Wenzhou, People’s Republic of China). Animals were housed at a constant room temperature with a 12/12-hour light-dark cycle and fed with a standard rodent diet and water. The animals were acclimatized to the laboratory for at least 7 days before use in the experiments. Animal protocols were approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Approval documents: wydw2014-0001). All animal care and experiments were performed in accordance with the approved protocols and the ‘The Detailed Rules and Regulations of Medical Animal Experiments Administration and Implementation’ (Document No. 1998-55, Ministry of Public Health, China).

Reagents. Lipopolysaccharide (LPS) were purchased from Sigma (Sigma, St. Louis, MO, USA). Saline was prepared as a 0.9% NaCl solution. eBioscience, Inc. (San Diego, CA, USA) was the source of the mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit and mouse TNF-α ELISA kit. Trizol-reagent, the two-step M-MLV and Platinum SYBR Green qPCR SuperMix-UDG kit were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Anti-F4/80 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).



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Cell Culture. Mouse RAW 264.7 macrophages were obtained from the American Type Culture Collection (ATCC, U.S.). RAW264.7 macrophages were incubated in DMEM medium (Gibco, Eggenstein, Germany) supplemented with 10% FBS (Hyclone, Logan, UT), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 oC with 5% CO2.

Determination of TNF-α and IL-6. The TNF-α and IL-6 levels in the medium and serum were determined by ELISA analysis as previously described.50 RAW 264.7 macrophages were seeded into 6-well plates at a density of 400,000 cells per well in DMEM medium. Cells were incubated at 37 °C in 5% CO2 for 24 hours. Macrophages were pretreated with compounds for 30 minutes, which were followed by the treatment of 0.5 µg/mL LPS. After treatment, the cells were incubated for 24 hours. The media were collected to measure the amount of TNF-α and IL-6. The total amount of the inflammatory factors in the culture medium was normalized to the total protein quantity of the viable cell pellets.

Real-Time Quantitative PCR. Cells were homogenized in TRIZOL kit (Invitrogen, Carlsbad, CA) for extraction of RNA according to each manufacturer’s protocol. Both reverse transcription and quantitative PCR were carried out using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Carlsbad, CA). Eppendorf Mastercycler ep realplex detection system (Eppendorf, Hamburg, Germany) was used for q-PCR analysis. The primers of genes including TNF-α, IL-6, IL-12, IL-1β, COX-2, IL-33 and


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β-actin were synthesized by Invitrogen. Details have been described previously.51 The primer sequences of mouse genes used are shown as follows: Mouse TNF-α sense primer, 5’-TGGAACTGGCAGAAGAGG-3’; Mouse TNF-α antisense primer, 5’-AGACAGAAGAGCGTGGTG-3’; Mouse IL-6 sense primer, 5’-GAGGATACCACTCCCAACAGACC-3’; Mouse IL-6 antisense primer, 5’-AAGTGCATCATCGTTGTTCATACA-3’; Mouse COX-2 sense primer, 5’-TGGTGCCTGGTCTGATGATG-3’; Mouse COX-2 antisense primer, 5’-GTGGTAACCGCTCAGGTGTTG-3’; Mouse IL-12 sense primer, 5’-GGAAGCACGGCAGCAGAATA-3’; Mouse IL-12 antisense primer, 5’-AACTTGAGGGAGAAGTAGGAATGG-3’; Mouse IL-33 sense primer, 5’-ACTATGAGTCTCCCTGTCCTG-3’; Mouse IL-33 antisense primer, 5’-ACGTCACCCCTTTGAAGC-3’; Mouse IL-1β sense primer, 5’-ACTCCTTAGTCCTCGGCCA-3’; Mouse IL-1β antisense primer, 5’-CCATCAGAGGCAAGGAGGAA-3’; Mouse β-actin sense primer, 5’-TGGAATCCTGTGGCATCCATGAAAC-3’; Mouse β-actin antisense primer, 5’-TAAAACGCAGCTCAGTAACAGTCCG-3’. The amount of each gene was determined and normalized by the amount of β-actin.

Lipopolysaccharide-induced Sepsis in Mice.52 18-22 g male C57BL/6 mice were randomly divided into eight groups: control group, LPS group, 6 group, 14f group, 14g group, 6 plus LPS group, 14f plus LPS group, and 14g plus LPS group. Mice were treated with 10 mg/kg 6, 14f or 14g solution by tail vein injection 15 minutes before a 20 mg/kg LPS injection. Control group animals were received only an equal volume of saline. 6, 14f and 14g groups were treated with compounds only. Mice were anesthetized and sacrificed 6 hours after LPS



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injection. Blood samples were collected from the right ventricle orbital veniplex using a heparinized syringe with a needle. Lung tissues were harvested.

Lung Histopathology Analysis Lung tissue were fixed in 4% paraformaldehyde solution, embedded in paraffin, and sectioned at 5 µm. After dehydration, sections were stained with hematoxylin and eosin (H&E) according to the previously reported method.53 A pathologist blindly scored each lung injury according to the following four categories: alveolar congestion, hemorrhage, neutrophil infiltration into the airspace or vessel wall, and thickness of alveolar wall/hyaline membrane formation. Each category was graded on a 0- to 4-point scale: 0 = no injury; 1 = injury up to 25% of the field; 2 = injury up to 50% of the field; 3 = injury up to 75% of the field; and 4 = diffuse injury.

Lung Immunohistochemistry Analysis.54 Tissue sections (5 µm thickness) were prepared, deparaffinized in xylene, and hydrated using an ethanol gradient. A Pressure-cooker was used for heat-induced antigen retrieval (10 mM sodium citrate buffer, pH 6.5). After treatment with 30% of hydrogen peroxide, all sections were blocked in 5% bovine serum albumin (BSA) and incubated with primary anti-F4/80 antibody overnight at 4 °C. The slides were then incubated with HRP-labeled secondary antibody for 10 min. After the sections were incubated

with

3,3-diaminobenzidine

tetrahydrochloride

(DAB)

for

color

development and counterstained with hematoxylin, the slides were evaluated under a microscope (200× amplification; Nikon). The percentage of F4/80-positive inflammatory cells was calculated in 10 randomly chosen fields (200×) per section.


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Measurement of Myeloperoxidase (MPO) Activity MPO activity in the lungs was assessed as an index of tissue neutrophil inﬁltration. 55

For MPO activity, the supernatants were incubated with 0.01% H2O2 in the presence

of O-dianisidine dihydrochloride (0.167 mg/mL) for 30 min. The change in absorbance at 460 nm for each sample was recorded with a plate reader (Bio-Tek Instruments Inc.). MPO activity was characterized as the quantity of enzyme degrading 1 µmol peroxide/min at 37 °C and is expressed in units per gram lung tissue.

Statistical Analysis Unless indicated otherwise, results are presented as the mean ± SEM. The differences between different treatments were analyzed using the two-sided Student’s t test. P values less than 0.05 were considered significant.



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ASSOCIATED CONTENT Supporting information Supporting Information Available: Synthetic routes for compounds in Scheme S1, acute toxicity study for active compounds in Figure S1and S2, 1H-NMR,

13

C-NMR

spectral and HPLC assessment of synthetic compounds.

AUTHOR INFORMATION Corresponding authors Y.Z.: phone, (+86)-577-86699892; E-mail: [email protected] G.L.: phone, (+86)-577-86699396; E-mail: [email protected]

Author contributions The manuscript was written with contributions from all of the authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This study was supported by the National Natural Science Funding of China (21472142, 21502144), The Scientific Research Foundation of Wenzhou Medical University (QTJ13003), Public Welfare Science and Technology Project of Wenzhou (Y20140736). ABBREVIATIONS USED

ALI, acute lung injury; IL-6, Interleukin-6; TNF-α, tumor necrosis factor alpha; NSAID, nonsteroidal anti-inflammatory drug; 1H NMR, proton nuclear magnetic resonance;

13

C NMR, Carbon nuclear magnetic resonance; MS, mass spectroscopy;

LPS, Lipopolysaccharide; ELISA, enzyme linked immunosorbent assay; SAR, structure-activity relationship; IC50, half maximal inhibitory concentration; MPO, Myeloperoxidase; DMF, N,N-dimethylformamide; THF, tetrahydrofuran; MeOH, methanol; EtOAc, ethyl acetate; HCl, hydrochloric acid; HPLC, high-performance liquid chromatography; K2CO3, potassium carbonate; MgSO4, magnesium sulfate; NaOH, sodium hydroxide; DMSO, dimethyl sulphoxide; Pd/C, palladium/carbon; DCM, dichloromethane; DMA, dimethyl adipate; TBTU, tributylthiourea; DIPEA, N,N-Diisopropylethylamine; NaHCO3, sodium bicarbonate; NaH2PO4, sodium dihydrogen phosphate.



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effects of quercetin on acute lung injury and biomarkers of inflammation and oxidative stress in the rat model of sepsis. Inflammation 2015. DOI: 10.1007/s10753-015-0296-9. 48. Martin, G. S.; Mannino, D. M.; Eaton, S.; Moss, M. The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 2003, 348, 1546-1554. 49. Sato, A.; McNulty, L.; Cox, K.; Kim, S.; Scott, A.; Daniell, K.; Summerville, K.; Price, C.; Hudson, S.; Kiakos, K.; Hartley, J. A.; Asao, T.; Lee, M. A novel class of in

vivo

active

anticancer

agents:

seco-hydroxycyclopropylbenz[e]indolone

achiral

(seco-CBI)

seco-aminoanalogues

of

and the

duocarmycins and CC-1065. J. Med. Chem. 2005, 48, 3903-3918. 50. Wu, J.; Li, J.; Cai, Y.; Pan, Y.; Ye, F.; Zhang, Y.; Zhao, Y.; Yang, S.; Li, X.; Liang, G. Evaluation and discovery of novel synthetic chalcone derivatives as anti-inflammatory agents. J. Med. Chem. 2011, 54, 8110-8123. 51. Chen, G.; Jiang, L.; Dong, L.; Wang, Z.; Xu, F.; Ding, T.; Fu, L.; Fang, Q.; Liu, Z.; Shan, X.; Liang, G. Synthesis and biological evaluation of novel indole-2-one and 7-aza-2-oxindole derivatives as anti-inflammatory agents. Drug Des. Dev. Ther. 2014, 8, 1869-1892. 52. Ando, H.; Takamura, T.; Ota, T.; Nagai, Y.; Kobayashi, K. Cerivastatin improves survival of mice with lipopolysaccharide-induced sepsis. J. Pharmacol. Exp. Ther. 2000, 294, 1043-1046. 53. Li, J.; Li, D.; Liu. X.; Tang, S.; Wei, F. Human umbilical cord mesenchymal stem cells reduce systemic inflammation and attenuate LPS-induced acute lung injury in rats. J. Inflammation (London, U. K.). 2012, 9, 1-11. 54.

Zhuang, P.; Shen, J.; Zhu, X.; Zhang, J.; Tang, Z.; Qin, L.; Sun, H. Direct transformation of lung microenvironment by interferon-α treatment counteracts



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growth of lung metastasis of hepatocellular carcinoma. PLoS One 2013, 8, e58913. 55. Noldin, V. F.; Vigil, S. V.; De, Liz. R.; Cechinel-Filho, V.; Fröde, T. S.; Creczynski-Pasa, T. B. N-phenylmaleimide derivatives as mimetic agents of the pro-inflammatory process: myeloperoxidase activation. Pharmacol. Rep. 2011, 63, 772-780.


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Figure Legends

Tabel 1. Anti-Inflammatory Screening of Compounds 10a-10w.

Tabel 2. Anti-Inflammatory Screening of Compounds 13a-13h.

Tabel 3. Anti-Inflammatory Screening of Compounds 14a-14g.

Figure 1. Structures of 1-6 and drug design conception.

Figure 2. Active compounds attenuate the LPS-induced lung inflammation in mice. Mice were injected intravenously with LPS. 6 hours later, mice were anaesthetized and killed. Serum and lung tissues were collected for further tests. (A) MPO activity in lung tissues; (B) Serum level of the cytokine TNF-α.

Figure 3. Effect of active compounds on histopathological changes in lung tissues in mice (×400). (A) H&E staining; (B) The histogram of lung injury scores.

Figure 4. 14f and 14g inhibited macrophages in filtration through F4/80-staining. (A) F4/80 immunohistochemical staining; (B) The histogram of F4/80-positive cells in A.

Figure 5. Active compounds inhibited the inflammatory genes expression induced



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by LPS in mouse lung tissue. (A) TNF-α; (B) IL-6; (C) IL-1β; (D) COX-2; (E) IL-12; (F) IL-33.

Scheme 1. Synthetic Routes of Indole-2-carboxamide Derivatives 10a-10w, 13a-13h and 14a-14g. α

Reagents and conditions: (a) NaOH, THF-MeOH (1:1, v/v), rt, 3h, 90-95%; (b)

Various amines (9a-9w), TBTU, DIPEA, DMA, rt, 80-85%; (c) alkyl/arylalkyl halide, K2CO3, MeCN, 50 oC, 8h, 65-75%; (d) Fe, NH4Cl, EtOH, H2O, 85oC, 2h, 72.6%; (e) Various acyl chlorides, DIPEA, DMA, 0 oC-rt, 2h, 68-75%.


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Table 1.



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Table 2.

O2N

HN N R

N

N

O 13a-13h (%)Inhibition (10 M)a

Comp.

R

IL-6

TNF-

10j

H

91.05 ± 3.25

13a

Me

87.23 ± 4.58 16.00 ± 1.69

13b

Allyl

58.19 ± 2.36

12.70 ± 0.89

13c

(2,6-diCl)Benzyl

93.31 ± 7.92

52.73 ± 2.21

13d

(3-F)Benzyl

86.40 ± 1.80

22.83 ± 1.52

13e

(4-Cl)Benzyl

97.24 ± 2.49

67.00 ± 2.67

13f

(4-Br)Benzyl

93.04 ± 3.68

31.10 ± 1.76

13g

(4-CF3)Benzyl

98.67 ± 2.64

75.09 ± 2.55

13h

(4-OMe)Benzyl

64.33 ± 3.53

41.37 ± 3.31

aValues

62.49 ± 2.24

are mean of at least n = 3 independent experiments ± SEM


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Table 3. H RN

HN N

N

O

F3C

14a-14g IC50 ( M)b

Comp.

R

13g

b

IL-6

TNF-

5.09 ± 1.52

2.74 ± 0.83

NAa

NA

14a

H

14b

Acryloyl

6.28 ± 1.72

1.05 ± 0.63

14c

Propionyl

3.33 ± 1.21

4.10 ± 1.68

14d

2-Chloracetyl

2.99 ± 0.85

6.03 ± 1.59

14e

Methanesulfonyl

4.91 ± 1.12

1.97 ± 0.58

14f

Furan-2-carbonyl

2.90 ± 0.73

2.66 ± 0.85

14g

N,N-dimethylamino carbonyl

1.24 ± 0.43

2.67 ± 0.76

1.62 ± 2.38

4.79 ± 1.15

6 a

N

NA means not activity . Values are mean of at least n = 3 independent experiments ± SEM.



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Figure 1.


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Figure 2.

MPO activity

A) ** ** **

B)

TNF-

Cytokine amount(pg/ml) in serum

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100

**

** **

50

ns

0

ShamLPS6Vehicle Vehicle Vehicle

ns

ns 6LPS

14fVehicle

14fLPS

14gVehicle


14gLPS


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Figure 3.

Figure 4.


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Figure 5.

*

*

**

**

*

Relative amount of mRNA

D)


C) * ***

E)

**

F)

*** ***

***

*

IL-33 Relative amount of mRNA


*


B)

A)


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100

ns

**

50

ns

**

0

ShamLPS6Vehicle Vehicle Vehicle


6LPS

14fVehicle

14fLPS

ns

14gVehicle

**

14gLPS


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1.


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Table of Contents graphic 247x89mm (96 x 96 DPI)


Design, Synthesis, and StructureâActivity ... - ACS Publications

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