Medicinal Chemistry Journal by hilaris1

Medicinal Chemistry

Research Article Volume 10:3, 2020 DOI: 10.37421/mccr.2020.10.544

Open Access

ISSN: 2161-0444

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents Haicheng Liu1, Yushi Futamura2, Honghai Wu1, Aki Ishiyama4, Taotao Zhang1,3, Tao Shi1, Qunxiong Zheng3, Masato Iwatsuki4, Satoshi Ōmura4, Hongbin Zou1* and Hiroyuki Osada2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, P. R. China Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 3 Department of Applied Chemistry, Zhejiang Gongshang University, Hangzhou 310035, P. R. China. 4 Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan 1 2

Abstract Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50=1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization. Graphical Abstract

Keywords: Cinnamamide framework • Antimalarial activity • Phenotypic screen • Plasmodium falciparum 3D7 • 3-cinnamamido-N-substituted benzamides

Introduction Malaria caused by Plasmodium parasites and transmitted to humans via infected female anopheles mosquitoes, is the third most deadly disease after *Address for Correspondence: Hongbin Zou, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, P.R. China, E-mail: zouhb@zju.edu.cn Copyright: © 2020 Zou H, et al. This is an open-access article distributed under the terms of the creative commons attribution license which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Received 02 February, 2020; Accepted 20 February, 2020; Published 28 February, 2020

tuberculosis (TB) and AIDS [1]. According to the World Health Organization (WHO), approximately 219 million malaria cases and 435,000 malariarelated deaths were reported worldwide in 2017 [2]. The most malariasusceptible groups are children under the age of five and pregnant women, especially in the region of Africa where 90% of the clinical cases occurred [3]. Unfortunately, reported data also highlight that no significant progress has been made to control the global malaria prevalence from 2015-2017. To further complicate the problem, drug-resistance has been increasingly detected both in vitro and in vivo [4-6]. A rising failure rate of the artemisininbased combination therapy (ACT) has been observed in recent years [7-10], involving the treatment of several traditional antimalarial drugs, such as Quinine [11], Proguanil [12] (Figure 1) and their derivatives. On the other hand, the inadequate understanding of the Plasmodium life cycle at the molecular level makes the discovery of novel antimalarial candidates more difficult [13]. Hence, there is an urgent need to develop new anti-plasmodial

Liu H, et al.

Med Chem (Los Angeles), Volume 10:3, 2020

H H3C

CH3

O O O HO

N HO

MeO

CH3

H N

N H

O N

H3CO

N H

H N

CH3

O B2

DDD107498

N H

Proguanil

Quinine

CH3

Artemisinin O

HN H

1 IC50=1.20 µM

Figure 1. Examples of compounds with antimalarial activity.

therapies with potent modes of action against multiple life-cycle stages of the parasite [14]. In 2015, a phenotypic screen of the Dundee protein kinase scaffold library (over 4,731 compounds) was performed against the blood stage of the multidrug-sensitive Plasmodium falciparum (P. falciparum) strain 3D7, resulting in the identification of a series of compounds based on a 2,6-disubstituted quinoline-4-carboxamide scaffold [15]. Those compounds possessed sub-micromolar potency against the parasites, but are limited by poor physicochemical properties. Further chemical optimization led to the discovery of DDD107498 (Figure 1) with an improved physicochemical property and excellent inhibitory activities against the Plasmodium parasites (EC50= 1.0 nM) (100-fold increase in potency) at multiple life-cycle stages [16]. The discovery of DDD107498 highlighted phenotypic screen as a promising methodology for the development of antimalarial agents, by finding potential bioactive molecules from rapidly growing compound library and its corresponding information dataset. Furthermore, identifying the parent structures of lead compounds through a phenotypic screen might widen the scope of antimalarial candidates and enrich their diversity [17]. Inspired by the discovery of DDD107498, we performed a phenotypic screen with our in-house compound library. Among the retrieved hits, compound 1 with cinnamamide skeleton exhibited, for the first time, moderate antimalarial activity (IC50=1.20 µM) against chloroquine-sensitive P. falciparum 3D7, in addition to its well-known set of biological effects, including anti-convulsion, anti-mutagenesis, anti-tumor, anti-inflammatory, vasodilating, insecticidal, and bacteriostatic activities [18-20]. In order to obtain more potently active compounds, lead optimization was carried out, involving the design and synthesis of a series of derivatives with various substituents replacing functional groups B1 and B2 (Figure 1). The activity of the analogues was then evaluated in the structure-activity relationship (SAR) study. Further in vitro activity test of these compounds against P. falciparum 3D7 indicate that half of the derivatives, with declined IC50 values of 0.093 µM at the lowest, showed improved antimalarial activity in comparison with the precursor 1. Their cytotoxic effects on both human cancer and non-cancer cell lines were also assessed and their selectivity index was determined.

Materials and Methods Chemistry Chemicals were commercially obtained from commercial sources and used without further purification. Thin-layer chromatography was used for reaction monitoring and compounds were visualized by ultraviolet light (UV−254 nm). Column chromatography purifications were performed using 200-300 mesh silica gel. NMR spectra were recorded for 1H NMR at 500 MHz and for 13C NMR at 125 MHz on a Bruker Advance spectrometer at 500 MHz and chemical shifts were expressed in ppm (δ ). High resolution mass spectroscopy (HRMS) was obtained using Agilent 1290 HPLC-6224 TOF Spectrometer. Page 108 of 114

General procedure for the synthesis of cinnamic acids (A): Aldehyde (35.6 mmol) was refluxed at 115 ºC with malonic acid (7.4 g, 71.2 mmol) in pyridine (40 mL) and piperidine (10 drops) as a catalyst for 12 h. The reaction mixture was evaporated to remove pyridine and the residue was mixed with water (50 mL) and extracted by ethyl acetate (50 mL × 3). The organic layer was washed with dilute hydrochloric acid and evaporated to get crude solid which was further stirred with potassium hydroxide (1-1.5 times) water solution. It was then extracted by ethyl acetate (30 mL × 2) and the resultant aqueous layer was acidified until no white solid precipitated. This precipitate was then filtered, washed with cold water and dried to get A for further usage. General procedure for the synthesis of methyl phenylacrylamido benzoates (B): Intermediate A (22.4 mmol), 1-ethyl-(3dimethylaminopropyl) carbonyl diimide hydrochloride (EDC•HCl, 4.7 g, 24.6 mmol) and 1-hydroxybenzotriazole (HOBT, 3.6 g, 26.7 mmol) were dissolved in anhydrous DMF (20 mL), and stirred at room temperature for 2 hours. Methyl 3-aminobenzoate (4.0 g, 26.5 mmol) was then added into the reaction mixture which was further stirred at 50 ºC overnight. 1M hydrochloric acid (50 mL) was added to quench the reaction and the precipitate was collected, filtered, washed with cold water and dried to get B for the next step. General procedure for the synthesis of phenyl-acrylamido benzoic acids (C): Intermediate B (9.65 mmol) was refluxed with 1M sodium hydroxide (11 mL) in ethanol (30 mL) for 3 hours. It was then evaporated to remove the ethanol and additional 20 mL of water was added to the mixture, followed by the extraction with 30 mL ethyl acetate. The resultant aqueous layer was added with 1M hydrochloric acid in the ice bath. The precipitate was then filtered, washed with cold water and dried to get quantitative C for the next step. General procedure for the synthesis of 3-cinnamamido-Nsubstituted benzamides (1-40): Intermediate C (0.34mmol), 1-ethyl(3-dimethylaminopropyl) carbonyl diimide hydrochloride (EDC•HCl, 71 mg, 0.37 mmol) and 1-hydroxybenzotriazole (HOBT, 54 mg, 0.40 mmol) were dissolved in anhydrous DMF (2 mL), and then stirred at room temperature for 2 hours. Appropriate amine (0.51 mmol) was added into the reaction mixture which was stirred at 50 ºC over-night. 1M hydrochloric acid (5 mL) was added to quench the reaction and the precipitate was collected, filtered, washed with cold water and dried to get the final product. Further column chromatography purifications (hexane and ethyl acetate as eluent) were performed using 200-300 mesh silica gel if needed. (E)-3-(3-(4-Methoxyphenyl)acrylamido)-N-(pyridin-3-yl)benzamide (1): White solid (Yield: 48%); 1H NMR (500 MHz, DMSO-d6): δ 10.71 (1H, s), 10.41 (1H, s), 9.07 (1H, s), 8.43 (1H, d, J=4.5 Hz), 8.37 (1H, d, J =8.5 Hz), 8.28 (1H, s), 7.95 (1H, d, J=7.5 Hz), 7.69 (1H, d, J=8.0 Hz), 7.60 (3H, dd, J=8.5, 6.0 Hz), 7.53 (2H, dd, J=16.0, 11.5 Hz), 7.02 (2H, d, J=8.5 Hz), 6.73 (1H, d, J=16.0 Hz), 3.8 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 166.6, 164.6, 161.2, 142.7, 140.8, 140.2, 139.8, 137.2, 135.3, 130.3, 129.9, 129.4, 127.7, 125.2, 123.1, 122.7, 119.9, 119.2, 115.0, 55.8; HRMS-ESI [M+H]+ calcd. for C22H19N3O3: 374.1504, found: 374.1500. (E)-3-(3-(4-Methoxyphenyl)acrylamido)-N-(5-methylisoxazol-3-yl) benzamide (2): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 10.70 (1H, s), 10.61 (1H, s), 8.26 (1H, s), 8.08 (1H, d, J=8.5 Hz), 7.91 (2H, d, J =7.5 Hz), 7.74 (1H, d, J=15.5 Hz), 7.60 (1H, s), 7.55 (2H, d, J=8.5 Hz), 7.49 (1H, t, J=7.5 Hz), 7.27 (1H, d, J=7.5 Hz), 6.95 (1H, d, J=15.5 Hz), 3.51 (3H, s), 2.52 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.7, 164.6, 162.8, 161.2, 140.7, 140.1, 131.9, 129.9, 129.5, 127.7, 124.4, 123.7, 120.4, 120.0, 115.0, 55.8; HRMS-ESI [M+H]+ calcd. for C21H19N3O4: 378.1454, found: 378.1453. (E)-3-(3-(4-Methoxyphenyl)acrylamide)-N-(thiazol-2-yl)benzamide (3): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 12.64 (1H, s), 10.35 (1H, s), 8.34 (1H, s), 7.93 (1H, dd, J =8.5, 1.0 Hz), 7.80 (1H, d, J=8.0 Hz), 7.59 (3H, m), 7.58 (1H, d, J=15.5 Hz), 7.49 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=3.5 Hz), 7.02 (2H, d, J=8.5 Hz), 6.71 (1H, d, J=15.5 Hz),

Liu H, et al.

3.81 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 165.6, 164.6, 161.2, 159.2, 140.8, 140.1, 138.1, 133.5, 129.9, 129.5, 127.7, 123.5, 123.0, 119.9, 119.6, 115.0, 114.3, 55.8; HRMS-ESI [M+H]+ calcd. for C20H17N3O3S: 380.1069, found: 380.1066. (E)-3-(3-(4-Methoxyphenyl)acrylamido)-N-(quinolin-7-yl) benzamide (4): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 10.73 (1H, s), 10.66 (1H, s), 8.94 (1H, s), 8.55 (1H, s), 8.36 (1H, d, J=8.0 Hz), 8.29 (1H, s), 8.10 (3H, m), 7.94(2H, d, J =8.0 Hz), 7.89(2H, d, J=8.0 Hz), 7.85 (1H, d, J=8.0 Hz), 7.77 (1H, d, J=16.0 Hz), 7.64 (1H, t, J=8.0 Hz), 7.56 (1H, dd, J=8.0,3.5 Hz), 6.97 (1H, d, J=16.0 Hz), 3.81 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.3, 164.7, 161.2, 145.9, 144.5, 140.7, 140.3, 135.3, 130.2, 129.9, 129.4, 127.7, 125.8, 124.0, 123.2, 123.0, 120.7, 120.0, 119.3, 115.0, 55.8; HRMS-ESI [M+H]+ calcd. for C26H21N3O3: 424.1661, found: 424.1658. (E)-3-(3-(4-Methoxyphenyl)acrylamido)-N-(3-(trifluoromethyl) phenyl)benzamide (5): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 10.54 (1H, s), 10.28 (1H, s), 8.82 (1H, s), 8.43 (1H, d, J=2.0 Hz), 8.15 (1H, d, J=8.0 Hz), 8.07 (1H, d, J=7.5 Hz), 7.98 (2H, d, J =8.0 Hz), 7.91 (2H, d, J=8.0 Hz), 7.82 (1H, d, J=16.0 Hz), 7.80 (1H, m), 7.70 (1H, t, J=8.0 Hz), 7.61 (1H, t, J=8.0 Hz), 7.50 (1H, d, J=7.5 Hz), 7.01 (1H, d, J=16.0 Hz), 3.85 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 164.6, 161.2, 140.8, 140.5, 140.1, 135.7, 130.4, 129.9, 129.4, 127.7, 124.2, 122.9, 122.7, 119.9, 119.2, 116.8, 116.8, 115.0, 55.8; HRMS-ESI [M+H]+ calcd. for C24H19F3N2O3: 441.1426, found: 441.1423.

Med Chem (Los Angeles), Volume 10:3, 2020

δ 12.64 (1H, s), 10.53 (1H, s), 8.35 (1H, s), 7.95 (1H, d, J=8.0 Hz), 7.87 (2H, d, J =8.5 Hz), 7.83 (3H, t, J =8.0 Hz), 7.70 (1H, d, J=16.0 Hz), 7.57 (1H, d, J=3.5 Hz), 7.51 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=3.5 Hz), 6.98 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 163.8, 139.8, 139.2, 133.6, 130.1, 129.9, 129.5, 128.9, 126.4, 126.3, 125.6, 125.4, 123.6, 123.5, 123.4, 119.8, 114.3; HRMS-ESI [M+H]+ calcd. for C20H14F3N3O2S: 418.0837, found: 418.0831. (E)-N-(Quinolin-7-yl)-3-(3-(4-(trifluoromethyl)phenyl)acrylamido) benzamide (11): White solid (Yield: 55%); 1H NMR (500 MHz, DMSO-d6): δ 10.65 (1H, s), 10.56 (1H, s), 8.86 (1H, s), 8.59 (1H, s), 8.31 (1H, d, J=8.0 Hz), 8.27 (1H, s), 7.98 (3H, m), 7.87 (2H, d, J =8.0 Hz), 7.82 (2H, d, J=8.0 Hz), 7.75 (1H, d, J=8.0 Hz), 7.71 (1H, d, J=16.0 Hz), 7.55 (1H, t, J=8.0 Hz), 7.45 (1H, dd, J=8.0, 4.0 Hz), 7.00 (1H, d, J =16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 163.8, 151.4, 148.8, 140.5, 139.8, 139.3, 139.2, 136.1, 136.0, 129.5, 128.9, 128.8, 126.4, 126.4, 125.6, 125.3, 125.1, 123.1, 122.9, 121.6, 120.7, 119.4, 117.8; HRMS-ESI [M+H]+ calcd. for C26H18F3N3O2: 462.1429, found: 462.1425. (E)-N-(3-(Trifluoromethyl)phenyl)-3-(3-(4-(trifluoromethyl)phenyl) acrylamido)benzamide (12): White solid (Yield: 60%); 1H NMR (500 MHz, DMSO-d6): δ 10.60 (1H, s), 10.55 (1H, s), 8.24 (2H, d, J=2.0 Hz), 8.06 (1H, d, J=8.0 Hz), 7.96 (1H, d, J=7.5 Hz), 7.87 (2H, d, J =8.0 Hz), 7.82 (2H, d, J=8.0 Hz), 7.70 (2H, dd, J=11.5, 4.0 Hz), 7.61 (1H, t, J=8.0 Hz), 7.54 (1H, t, J=8.0 Hz), 7.47 (1H, d, J=7.5 Hz), 6.99 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 163.8, 157.1, 140.4, 139.8, 139.3, 135.8, 130.4, 129.5, 128.9, 126.4, 125.3, 124.2, 123.0, 120.4, 119.3, 116.8; HRMS-ESI [M+H]+ calcd. for C24H16F6N2O2: 479.1194, found: 479.1190.

(E)-4-(3-(3-(4-Methoxyphenyl)acrylamido)benzamido)-Nmethylpicolinamide(6): White solid (Yield: 70%); 1H NMR (500 MHz, DMSO-d6): δ 10.84 (1H, s), 10.53 (1H, s), 9.11 (1H, s), 8.52 (1H, d, J=4.5 Hz), 8.43 (1H, d, J =8.5 Hz), 8.19 (1H, s), 7.95 (1H, d, J=8.0 Hz), 7.77 (1H, d, J=8.0 Hz), 7.68 (3H, dd, J=8.5, 5.5 Hz), 7.59 (1H, d, J=16.0 Hz), 7.57(1H, m), 7.09 (2H, d, J=8.5 Hz), 6.78 (1H, d, J=16.0 Hz), 3.80 (3H, s), 2.72 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.7, 164.7, 149.2, 148.3, 141.4, 140.2, 131. 8, 129.5, 128.3, 127.8, 126.6, 126.5, 125.0, 124.4, 123.6, 122.5, 120.3, 119.6, 119.1, 116.2, 111.4, 110.1, 56.4, 56.0; HRMSESI [M+H]+ calcd. for C24H22N4O4: 431.1719, found: 431.1717.

(E)-N-methyl-4-(3-(3-(4-(trifluoromethyl)phenyl)acrylamido) benzamido)picolinamide (13): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 12.54 (1H, s), 10.45 (1H, s), 8.24 (1H, s), 7.89(2H, m), 7.68 (2H, d, J=8.0 Hz), 7.61 (2H, d, J=8.5 Hz), 7.59 (1H, d, J=8.0 Hz), 7.55 (3H, m), 7.41 (1H, d, J=16.0 Hz), 7.24 (1H, m), 6.92 (1H, d, J =16.0 Hz), 2.89 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.6, 163.7, 139.8, 139.2, 131.9, 129.6, 128.9, 128.3, 126.4, 126.4, 125.4, 124.8, 123.8, 120.5, 110.0; HRMS-ESI [M+H]+ calcd. for C24H19F3N4O3: 469.1487, found: 469.1482.

(E)-N-(4-Chloropyridin-2-yl)-3-(3-(4-methoxyphenyl)acrylamido) benzamide (7): White solid (Yield: 25%); 1H NMR (500 MHz, DMSO-d6): δ 10.56 (1H, s), 10.44 (1H, s), 8.49 (1H, s), 8.37 (1H, d, J=7.5 Hz), 8.24 (1H, m), 8.10 (1H, s), 7.98 (1H, d, J=7.5 Hz), 7.73 (2H, d, J=8.0 Hz), 7.61 (1H, t, J=8.5 Hz), 7.53(1H, d, J=16.0 Hz), 7.21 (1H, m), 7.10 (2H, d, J=8.0 Hz), 6.91 (1H, d, J=16.0 Hz), 3.81 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 164.5, 161.2, 154.8, 150.9, 143.3, 140.0, 134.9, 130.3, 129.9, 129.3, 127.8, 127.7, 126.3, 123.0, 120.8, 119.9, 119.5, 115.0, 109.5, 105.5, 98.8, 55.8; HRMS-ESI [M+H]+ calcd. for C22H18ClN3O3: 408.1115, found: 408.1112.

(E)-N-(Pyridin-3-yl)-3-(3-(3-(trifluoromethyl)phenyl)acrylamido) benzamide (14): White solid (Yield: 35%); 1H NMR (500 MHz, DMSO-d6): δ 10.53 (1H, s), 10.50 (1H, s), 8.95 (1H, d, J=2.0 Hz), 8.34 (1H, d, J=4.0 Hz), 8.25 (1H, s), 8.22 (1H, d, J =8.0 Hz), 8.00 (1H, s), 7.96 (2H, m), 7.78 (1H, d, J=8.0 Hz), 7.72 (1H, d, J =16.0 Hz), 7.71 (2H, t, J=7.0 Hz), 7.53 (1H, t, J=8.0 Hz), 7.43 (1H, dd, J=8.5, 4.5 Hz), 6.99 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 163.8, 145.1, 142.5, 139.8, 139.2, 136.3, 136.3, 135.7, 132.0, 130.7, 129.5, 127.8, 126.6, 124.7, 124.0, 123.0, 122.9, 119.3; HRMS-ESI [M+H]+ calcd. for C22H16F3N3O2: 412.1273, found: 412.1271.

(E)-N-(Pyridin-3-yl)-3-(3-(4-(trifluoromethyl)phenyl)acrylamido) benzamide (8): White solid (Yield: 45%); 1H NMR (500 MHz, DMSO-d6): δ 10.82 (1H, s), 10.64 (1H, s), 9.14 (1H, d, J=2.5 Hz), 8.47 (1H, m), 8.31 (1H, t, J =2.0 Hz), 7.98 (1H, m), 7.87 (2H, d, J=8.5 Hz), 7.83 (2H, d, J=8.5 Hz), 7.74 (1H, d, J=8.5 Hz), 7.70 (1H, d, J=16.0 Hz), 7.69 (1H, t, J=8.0 Hz), 7.56 (1H, t, J=7.5 Hz), 7.02 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 163.8, 142.6, 139.9, 139.7, 139.2, 137.2, 135.3, 130.4, 129.5, 128.9, 126.4, 126.4, 125.4, 125.2, 123.2, 123.1, 119.4; HRMS-ESI [M+H]+ calcd. for C22H16F3N3O2: 412.1273, found: 412.1269.

(E)-N-(5-Methylisoxazol-3-yl)-3-(3-(3-(trifluoromethyl)phenyl) acrylamido)benzamide (15): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 10.84 (1H, s), 10.63 (1H, s), 8.67 (1H, s), 8.01 (1H, s), 7.96 (1H, d, J=8.0 Hz), 7.78 (2H, m), 7.69 (1H, d, J=16.0 Hz), 7.67 (1H, t, J=8.5 Hz), 7.60 (1H, d, J=8.0 Hz), 7.45 (1H, t, J=8.5 Hz), 7.23 (1H, s), 6.99 (1H, d, J=16.0 Hz), 2.59 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 169.9, 165.8, 164.6, 161.2, 159.1, 140.8, 140.1, 134.5, 129.9, 129.4, 127.7, 123.2, 122.9, 119.9, 119.6, 115.0, 97.5, 55.8; HRMS-ESI [M+H]+ calcd. for C21H16F3N3O3: 416.1222, found: 416.1220.

(E)-N-(5-Methylisoxazol-3-yl)-3-(3-(4-(trifluoromethyl)phenyl) acrylamido)benzamide(9): White solid (Yield: 30%); 1H NMR (500 MHz, DMSO-d6): δ 12,49 (1H, s), 10.68 (1H, s), 8.19 (1H, s), 7.94 (1H, d, J=8.5 Hz), 7.83 (2H, d, J =7.5 Hz), 7.74 (1H, d, J=16.0 Hz), 7.68 (1H, s), 7.66 (1H, d, J=7.5 Hz), 7.60 (2H, d, J=8.5 Hz), 7.49 (1H, t, J=7.5 Hz), 6.94 (1H, d, J=16.0 Hz), 2.58 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.6, 163.7, 142.5, 139.8, 139.2, 139.2, 131.9, 130.1, 129.9, 129.6, 129.3, 128.9, 128.3, 127.8, 126.4, 126.3, 125.3, 124.8, 123.8, 123.5, 120.5, 119.6, 110.0, 63.6; HRMS-ESI [M+H]+ calcd. for C21H16F3N3O3: 416.1222, found: 416.1218.

(E)-N-(Thiazol-2-yl)-3-(3-(3-(trifluoromethyl)phenyl)acrylamido) benzamide (16): White solid (Yield: 80%); 1H NMR (500 MHz, DMSO-d6): δ 12.65 (1H, s), 10.50 (1H, s), 8.34 (1H, s), 7.99 (1H, s), 7.95 (1H, d, J=8.0 Hz), 7.76 (1H, dd, J =8.0, 1.5 Hz), 7.81 (1H, d, J =8.0 Hz), 7.76 (1H, d, J =7.5 Hz), 7.70 (1H, m), 7.69 (1H, d, J=16.0 Hz), 7.55 (1H, d, J=3.5 Hz), 7.49 (1H, t, J=8.0 Hz), 7.28 (1H, d, J=3.5 Hz), 6.97 (1H, d, J=15.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 163.8, 139.9, 139.2, 136.3, 132.0, 130.7, 130.4, 130.2, 129.6, 127.8, 126.6, 125.6, 124.7, 124.6, 124.6, 123.5, 123.4, 119.7, 114.3; HRMS-ESI [M+H]+ calcd. for C20H14F3N3O2S: 418.0837, found: 418.0832.

(E)-N-(Thiazol-2-yl)-3-(3-(4-(trifluoromethyl)phenyl)acrylamido) benzamide (10): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): Page 109 of 114

(E)-N-(Quinolin-7-yl)-3-(3-(3-(trifluoromethyl)phenyl)acrylamido)

Liu H, et al.

benzamide (17): White solid (Yield: 58%); 1H NMR (500 MHz, DMSO-d6): δ 10.65 (1H, s), 10.56 (1H, s), 8.86 (1H, s), 8.59 (1H, s), 8.31 (1H, d, J=8.0 Hz), 8.27 (1H, s), 7.98 (3H, m), 7.87 (2H, d, J =8.0 Hz), 7.82 (2H, d, J=8.0 Hz), 7.75 (1H, d, J=8.0 Hz), 7.71 (1H, d, J=16.0 Hz), 7.55 (1H, t, J=8.0 Hz), 7.45 (1H, dd, J=8.0, 4.0 Hz), 7.00 (1H, d, J =16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 167.2, 164.0, 145.6, 145.4, 144.6, 140.2, 140.0, 139.1, 136.4, 135.3, 131.9, 130.6, 130.4, 130.2, 130.1, 129.5, 126.6, 125.8, 125.6, 124.9, 124.6, 124.1, 123.4, 123.3, 120.7, 119.4, 108.7; HRMS-ESI [M+H]+ calcd. for C26H18F3N3O2: 462.1428, found: 462.1426. (E)-N-(3-(Trifluoromethyl)phenyl)-3-(3-(3-(trifluoromethyl)phenyl) acrylamido)benzamide (18): White solid (Yield: 90%); 1H NMR (500 MHz, DMSO-d6): δ 10.59 (1H, s), 10.49 (1H, s), 8.23 (2H, d, J=1.5 Hz), 8.04 (1H, d, J=8.0 Hz), 7.99 (1H, s), 7.94 (2H, t, J=7.0 Hz), 7.76 (1H, d, J=8.0 Hz), 7.71 (1H, d, J=16.0 Hz), 7.69 (2H, m), 7.60 (1H, t, J=8.0 Hz), 7.52 (1H, t, J=8.0 Hz), 7.45 (1H, d, J=8.0 Hz), 6.97 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 163.8, 157.1, 140.4, 139.9, 139.2, 136.3, 135.8, 132.0, 130.7, 130.4, 130.2, 130.0, 129.7, 129.5, 126.6, 124.7, 124.6, 124.3, 123.0, 120.5, 119.2, 116.8; HRMS-ESI [M+H]+ calcd. for C24H16F6N2O2: 479.1194, found: 479.1190.

Med Chem (Los Angeles), Volume 10:3, 2020

(E)-3-(3-(4-Chlorophenyl)acrylamido)-N-(3-(trifluoromethyl) phenyl)benzamide (24): White solid (Yield: 71%); 1H NMR (500 MHz, DMSO-d6): δ 10.69 (1H, s), 10.60 (1H, s), 8.38 (2H, d, J=2.0 Hz), 8.20 (1H, d, J=8.0 Hz), 8.04 (1H, d, J=7.5 Hz), 7.98 (2H, d, J =8.0 Hz), 7.95 (2H, d, J=8.0 Hz), 7.73 (1H, d, J=16.0 Hz), 7.71 (1H, m) , 7.59 (1H, t, J=8.0 Hz), 7.52 (1H, t, J=8.0 Hz), 7.45 (1H, d, J=7.5 Hz), 6.91 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 164.1, 157.1, 140.4, 139.9, 139.7, 135.8, 134.8, 134.1, 130.4, 130.0, 129.6, 129.5, 124.3, 123.3, 123.0, 122.9, 120.5, 119.3, 116.8, 116.8; HRMS-ESI [M+H]+ calcd. for C23H16ClF3N2O2: 445.0930, found: 445.0921. (E)-4-(3-(3-(4-Chlorophenyl)acrylamido)benzamido)-Nmethylpicolinamide (25): White solid (Yield: 35%); 1H NMR (500 MHz, DMSO-d6): δ 12.01 (1H, s), 10.72 (1H, s), 8.21 (1H, s), 8.09 (2H, m), 7.87 (1H, d, J=8.0 Hz), 7.73 (2H, d, J=8.0 Hz), 7.68 (1H, m), 7.61 (2H, d, J=8.5 Hz), 7.49 (2H, m), 7.41 (1H, d, J=16.0 Hz), 7.07 (1H, m), 6.98 (1H, d, J=16.0 Hz), 2.62 (3H, s); 13C NMR (125 MHz, DMSO-d6) δ =166.0, 164.1, 140.0, 139.7, 134.8, 134.1, 131.0, 130.0, 129.8, 129.6, 124.5, 124.1, 123.2, 120.2, 61.3; HRMS-ESI [M+H]+ calcd. for C23H19ClN4O3: 435.1224, found: 435.1219.

(E)-N-Methyl-4-(3-(3-(3-(trifluoromethyl)phenyl)acrylamido) benzoamido)picolinamide (19): White solid (Yield: 60%); 1H NMR (500 MHz, DMSO-d6): δ 12.40 (1H, s), 10.24 (1H, s), 8.12 (1H, s), 7.72(3H, s), 7.59 (1H, d, J=8.0 Hz), 7.45 (2H, d, J=8.5 Hz), 7.39 (1H, d, J=16.0 Hz), 7.34 (1H, d, J=3.5 Hz), 7.29(2H, m), 7.28 (1H, d, J=16.0 Hz), 7.06 (1H, d, J=3.5 Hz), 6.63 (1H, d, J=16.0 Hz), 2.27 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 164.1, 162.8, 139.9, 139.7, 134.8, 134.1, 133.5, 130.0, 129.5, 123.6, 123.3, 119.7, 114.3; HRMS-ESI [M+H]+ calcd. for C24H19F3N4O3: 469.1487, found: 469.1484.

(E)-3-(3-(4-Chlorophenyl)acrylamido)-N-(4-chloropyridin-2-yl) benzamide (26): White solid (Yield: 15%); 1H NMR (500 MHz, DMSO-d6): δ 10.38 (1H, s), 10.11 (1H, s), 8.36 (1H, s), 8.29 (1H, d, J=7.5 Hz), 8.21 (1H, m), 8.02 (1H, s), 7.89 (1H, d, J=7.5 Hz), 7.76 (2H, d, J=8.0 Hz), 7.63 (2H, d, J=8.0 Hz), 7.58(1H, d, J=16.0 Hz), 7.48 (1H, t, J=8.5 Hz), 7.17 (1H, m), 6.69 (1H, d, J=16.0 Hz) ; 13C NMR (125 MHz, DMSO-d6): δ 166.8, 164.0, 153.8, 149.9, 144.4, 139.8, 139.6, 135.0, 134.8, 134.1, 130.0, 129.6, 129.4, 123.3, 123.3, 123.2, 120.3, 119.6, 114.6; HRMS-ESI [M+H]+ calcd. for C21H15Cl2N3O2: 412.0619, found: 412.0617.

(E)-3-(3-(4-Chlorophenyl)acrylamido)-N-(pyridin-3-yl)benzamide (20): White solid (Yield: 30%); 1H NMR (500 MHz, DMSO-d6): δ 10.77 (1H, s), 10.59 (1H, s), 9.08 (1H, d, J=2.5 Hz), 8.38 (1H, m), 8.25 (1H, t, J =2.0 Hz), 7.90 (1H, m), 7.82 (2H, d, J=8.5 Hz), 7.75 (2H, d, J=8.5 Hz), 7.68 (1H, d, J=8.5 Hz), 7.61 (1H, d, J=16.0 Hz), 7.52 (1H, t, J=8.0 Hz), 7.44 (1H, t, J=8.0 Hz), 7.05 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 164.1, 145.1, 142.5, 139.9, 139.7, 136.3, 135.7, 134.8, 134.1, 130.0, 129.6, 129.5, 127.8, 124.0, 123.3, 122.9, 122.9, 119.3; HRMS-ESI [M+H]+ calcd. for C21H16ClN3O2: 378.1009, found: 378.1005.

(E)-3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido)-N(pyridin-3-yl)benzamide (27): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 10.50 (2H, s), 8.93 (1H, d, J =2.0 Hz), 8.32 (1H, m), 8.24 (1H, s), 8.20 (1H, d, J=8.5 Hz), 8.11 (1H, s), 7.95 (2H, t, J=6.5 Hz), 7.81 (1H, d, J=8.5 Hz), 7.71 (2H, m), 7.53 (1H, t, J=8.0 Hz), 7.41 (1H, dd, J=8.5, 5.0 Hz), 6.97 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 163.7, 145.0, 142.4, 139.8, 138.2, 136.3, 135.7, 135.0, 133.3, 132.8, 131.9, 129.5, 127.8, 127.4, 127.3, 125.2, 124.0, 123.0, 122.9, 119.3; HRMS-ESI [M+H]+ calcd. for C22H15ClF3N3O2: 446.0883, found: 446.0879.

(E)-3-(3-(4-Chlorophenyl)acrylamido)-N-(5-methylisoxazol-3-yl) benzamide (21): White solid (Yield: 37%); 1H NMR (500 MHz, DMSO-d6): δ 10.39 (1H, s), 10.12 (1H, s), 8.32 (1H, s), 7.99 (1H, d, J=8.5 Hz), 7.68 (1H, s), 7.65 (2H, d, J =7.5 Hz), 7.60 (1H, d, J=15.5 Hz), 7.56 (1H, d, J=7.5 Hz), 7.52 (2H, d, J=8.5 Hz), 7.47 (1H, t, J=7.5 Hz), 6.83 (1H, d, J=15.5 Hz), 2.73 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.6, 164.0, 139.9, 139.6, 134.8, 134.1, 131.9, 129.9, 129.5, 124.7, 123.8, 123.4, 120.5, 55.8; HRMS-ESI [M+H]+ calcd. for C20H16ClN3O3: 382.0958, found: 382.0956.

(E)-3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido)-N-(5methylisoxazol-3-yl)benzamide (28): White solid (Yield: 31%); 1H NMR (500 MHz, DMSO-d6): δ 10.79 (1H, s), 10.34(1H, s), 8.59 (1H, d, J=2.0 Hz), 7.93 (1H, s), 7.86 (1H, d, J=8.0 Hz), 7.69 (2H, m), 7.58 (1H, d, J=16.0 Hz), 7.56 (1H, t, J=8.0 Hz), 7.30 (1H, d, J=8.0 Hz), 7.11 (1H, s), 6.71 (1H, d, J=16.0 Hz), 2.47 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.6, 163.6, 139.8, 138.1, 135.0, 133.2, 132.8, 132.0, 131.9, 129.6, 127.8, 127.6, 127.3, 125.3, 124.8, 124.2, 123.8, 122.1, 120.5; HRMS-ESI [M+H]+ calcd. for C21H15ClF3N3O3: 450.0832, found: 450.0829.

(E)-3-(3-(4-Chlorophenyl)acrylamido)-N-(thiazol-2-yl)benzamide (22): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 11.95 (1H, s), 10.48 (1H, s), 8.31 (1H, s), 8.05 (1H, d, J=8.0 Hz), 7.90 (2H, d, J =8.0 Hz), 7.85 (2H, d, J =8.0 Hz), 7.80 (1H, d, J=7.5 Hz ), 7.70 (1H, d, J=16.0 Hz), 7.57 (1H, d, J=3.5 Hz), 7.51 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=3.5 Hz), 6.98 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 164.1, 157.1, 139.9, 139.7, 134.8, 134.1, 130.0, 129.6, 129.5, 123.5, 123.3, 123.3, 119.7; HRMS-ESI [M+H]+ calcd. for C19H14ClN3O2S: 384.0573, found: 384.0567. (E)-3-(3-(4-Chlorophenyl)acrylamide)-N-(quinolin-7-yl)benzamide (23): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 10.67 (1H, s), 10.51 (1H, s), 8.87 (1H, dd, J =4.5, 1.5 Hz), 8.58 (1H, s), 8.30 (1H, d, J=8.0 Hz), 8.25 (1H, s), 7.98 (3H, d, J=15.0 Hz), 7.73 (1H, d, J=7.5 Hz), 7.68 (2H, d, J=8.5 Hz), 7.63 (1H, d, J=16.0 Hz), 7.54 (3H, m), 7.45 (1H, dd, J=8.5, 4.0 Hz), 6.87 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 164.1, 151.4, 148.8, 140.5, 139.9, 139.7, 136.1, 136.0, 136.8, 134.1, 130.0, 129.6, 129.4, 128.8, 125.1, 123.3, 123.0, 122.9, 121.6, 120.7, 119.3, 117.8; HRMS-ESI [M+H]+ calcd. for C25H18ClN3O2: 428.1166, found: 428.1159. Page 110 of 114

(E)-3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido)-N(thiazol-2-yl)benzamide (29): White solid (Yield: 52%); 1H NMR (500 MHz, DMSO-d6): δ 12.64 (1H, s), 10.51 (1H, s), 8.35 (1H, s), 8.12 (1H, s), 7.96 (1H, d, J=8.5 Hz), 7.92 (1H, d, J =8.0 Hz), 7.82 (2H, t, J =7.5 Hz), 7.71 (1H, d, J=16.0 Hz), 7.57 (1H, d, J=3.5 Hz), 7.51 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=3.5 Hz), 6.98 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 163.7, 162.8, 139.8, 138.2, 135.0, 133.3, 132.8, 131.9, 129.5, 127.9, 127.6, 127.4, 127.3, 125.3, 124.3, 123.6, 123.4, 122.1, 119.7, 114.3; HRMS-ESI [M+H]+ calcd. for C20H13ClF3N3O2S: 452.0447, found: 452.0441. (E)-3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido)-N(quinolin-7-yl)benzamide (30): Light-yellow solid (Yield: 53%); 1H NMR (500 MHz, DMSO-d6): δ 10.66 (1H, s), 10.54 (1H, s), 8.88 (1H, d, J=2.5 Hz), 8.59 (1H, s), 8.31 (1H, d, J=8.5 Hz), 8.27 (1H, s), 8.13 (1H, s), 7.98 (4H, d, J =4.5 Hz), 7.83 (1H, d, J=8.5 Hz), 7.74 (1H, d, J=8.0 Hz), 7.72 (1H, d, J=16.0 Hz), 7.56 (1H, t, J=8.0 Hz), 7.46 (1H, dd, J=8.0, 4.0 Hz), 7.00 (1H, d, J=15.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 163.7, 157.1, 151.4, 148.8, 140.5, 139.8, 138.2, 136.1, 136.0, 135.0, 133.3, 132.9, 129.5, 128.8,

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125.2, 125.1, 123.1, 122.8, 121.6, 120.7, 119.3, 117.7; HRMS-ESI [M+H]+ calcd. for C26H17ClF3N3O2: 496.1039, found: 496.1038. (E)-3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido)-N-(3trifluoromethyl)phenyl)benzamide (31): White solid (Yield: 38%); 1H NMR (500 MHz, DMSO-d6): δ 10.41 (1H, s), 10.32 (1H, s), 8.28 (2H, d, J=1.5 Hz), 7.99 (1H, d, J=8.0 Hz), 7.90 (1H, s), 7.81 (1H, d, J=7.5 Hz), 7.73 (1H, d, J=8.0 Hz), 7.66 (1H, d, J=16.0 Hz), 7.61 (2H, m), 7.54 (1H, t, J=8.0 Hz), 7.43 (1H, t, J=8.0 Hz), 7.38 (1H, d, J=8.0 Hz), 6.89 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 163.7, 162.8, 140.4, 139.8, 138.1, 135.8, 135.0, 133.3, 132.8, 131.9, 130.3, 130.0, 129.8, 129.5, 127.9, 127.6, 127.3, 127.3, 125.7, 125.3, 124.3, 123.0, 120.5, 119.3, 116.9, 116.8; HRMS-ESI [M+H]+ calcd. for C24H15ClF6N2O2: 513.0804, found: 513.0800. (E)-4-(3-(3-(4-Chloro-3-(trifluoromethyl)phenyl)acrylamido) benzamido)-N-methylpicolinamide (32): White solid (Yield: 49%); 1H NMR (500 MHz, DMSO-d6): δ 11.68 (1H, s), 10.12 (1H, s), 8.11 (1H, s), 7.85 (2H, m), 7.85 (1H, d, J=8.0 Hz), 7.76 (1H, d, J=8.0 Hz), 7.68 (1H, d, J=8.0 Hz), 7.65 (1H, t, J=8.5 Hz), 7.48 (2H, m), 7.43 (1H, d, J=16.0 Hz), 7.41 (1H, d, J=8.5 Hz), 7.07 (1H, m), 6.93(1H, d, J=16.0 Hz), 2.88 (3H, s); 13 C NMR (125 MHz, DMSO-d6): δ 167.6, 163.6, 162.8, 139.8, 138.1, 135.0, 133.3, 132.8, 131.9, 131.9, 129.6, 127.8, 127.6, 127.4, 127.3, 125.3, 124.8, 124.2, 123.8, 122.1, 120.4; HRMS-ESI [M+H]+ calcd. for C24H18ClF3N4O3: 503.1098, found: 503.1095. (E)-3-(3-(4-Fluoro-3-nitrophenyl)acrylamido)-N-(pyridin-3-yl) benzamide (33): Yellow solid (Yield: 60%); 1H NMR (500 MHz, DMSO-d6): δ 10.50 (1H, s), 10.44 (1H, s), 8.93 (1H, d, J=2.5 Hz), 8.32 (1H, dd, J=5.0, 1.0 Hz), 8.23 (1H, s), 8.20 (1H, m), 8.17 (1H, d, J =2.0 Hz), 7.94 (1H, m), 7.92 (1H, dd, J=8.5, 2.5 Hz), 7.69 (1H, d, J =7.5 Hz), 7.62 (1H, d, J=16.0 Hz), 7.52 (1H, t, J=8.0 Hz), 7.44 (1H, d, J=9.0 Hz), 7.41 (1H, m), 6.84 (1H, d, J=16.0 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 164.0, 162.8, 152.5, 145.1, 142.5, 140.2, 139.9, 138.6, 136.3, 135.7, 133.9, 129.4, 127.8, 127.7, 124.4, 124.0, 122.9, 122.9, 122.8, 119.2, 116.1; HRMS-ESI [M+H]+ calcd. for C21H15FN4O4: 407.1155, found: 407.1154. (E)-3-(3-(4-Fluoro-3-nitrophenyl)acrylamido)-N-(thiazol-2-yl) benzamide (34): Light-red solid (Yield: 70%); 1H NMR (500 MHz, DMSO-d6): δ 12.64 (1H, s), 10.44 (1H, s), 8.35 (1H, s), 8.18 (1H, d, J=2.0 Hz), 7.93 (2H, m), 7.82 (1H, d, J =8.0 Hz), 7.63 (1H, d, J=16.0 Hz), 7.57 (1H, d, J=3.5 Hz), 7.50 (1H, t, J=8.0 Hz), 7.44 (1H, d, J=8.5 Hz), 7.29 (1H, d, J=3.5 Hz), 6.84 (1H, d, J=16.0 Hz) ; 13C NMR (125 MHz, DMSO-d6): δ 164.0, 162.8, 152.5, 140.2, 140.0, 138.6, 133.9, 129.5, 127.7, 124.4, 123.5, 123.2, 122.8, 119.6, 116.1, 114.3; HRMS-ESI [M+H]+ calcd. for C19H13FN4O4S: 413.0720, found: 413.0715. (E)-3-(3-(4-Fluoro-3-nitrophenyl)acrylamido)-N-(quinolin-7-yl) benzamide (35): Light-red solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 10.51 (1H, s), 10.37 (1H, s), 8.94 (2H, m), 8.67 (1H, s), 8.53 (1H, dd, J=7.5, 2.5 Hz), 8.21 (1H, d, J =7.5 Hz), 8.10 (1H, s), 7.98 (3H, m), 7.84 (1H, d, J=8.0 Hz), 7.79 (1H, d, J=15.5 Hz), 7.78 (1H, m), 7.69 (1H, t, J=8.0 Hz), 7.58 (1H, t, J=8.0 Hz), 7.13 (1H, d, J=15.5 Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.7, 164.1, 163.5, 162.8, 157.1, 152.5, 150.3, 141.3, 140.2, 140.0, 138.5, 136.0, 133.8, 129.4, 129.1, 127.7, 125.3, 124.4, 122.8, 122.1, 120.7, 119.3, 116.1; HRMS-ESI [M+H]+ calcd. for C25H17FN4O4: 457.1312, found: 457.1310. (E)-3-(3-(4-Fluoro-3-nitrophenyl)acrylamido)-N-(3-(trifluoromethyl) phenyl)benzamide (36): Yellow solid (Yield: 37%); 1H NMR (500 MHz, DMSO-d6): δ 10.60 (1H, s), 10.44 (1H, s), 8.25 (1H, s), 8.23 (1H, s), 8.17(1H, d, J=2.0 Hz), 8.05 (1H, d, J =8.0 Hz), 7.93 (2H, m), 7.69 (1H, d, J=8.0 Hz), 7.63 (1H, d, J =15.5 Hz), 7.61 (1H, t, J=8.0 Hz), 7.52 (1H, t, J=7.5 Hz), 7.45 (2H, m), 6.84 (1H, d, J=15.5Hz); 13C NMR (125 MHz, DMSO-d6): δ 166.4, 164.0, 152.5, 140.4, 140.2, 139.9, 138.6, 135.8, 133.9, 130.4, 130.0, 129.7, 129.5, 127.7, 125.7, 124.4, 124.2, 122.9, 122.9, 122.8, 120.4, 119.2, 116.8, 116.8, 116.1; HRMS-ESI [M+H] + calcd. for C23H15F4N3O4: 474.1077, found: 474.1075. (E)-3-(3-(4-Hydroxy-3-methoxyphenyl)acrylamido)-N-(pyridin-3-yl) benzamide (37): White solid (Yield: 35%); 1H NMR (500 MHz, DMSO-d6): Page 111 of 114

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δ 10.47 (1H, s), 10.42 (1H, s), 9.63 (1H, s), 8.99 (1H, d, J=2.5 Hz), 8.44 (1H, d, J=4.0 Hz), 8.28 (1H, s), 8.20 (1H, m), 8.13 (1H, d, J =8.0 Hz), 7.95 (1H, m), 7.89 (1H, dd, J=8.5, 2.0 Hz), 7.72 (1H, d, J =7.5 Hz), 7.68 (1H, d, J=16.0 Hz), 7.55 (1H, t, J=8.0 Hz), 7.47 (1H, d, J=8.0 Hz), 7.43 (1H, m), 6.89 (1H, d, J=16.0 Hz), 3.86 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 164.7, 149.3, 148.4, 145.1, 142.5, 141.5, 140.2, 136.3, 135.7, 129.3, 127.8, 126.6, 124.0, 122.8, 122.6, 122.5, 119.1, 116.3, 111.5, 56.1; HRMSESI [M+H]+ calcd. for C22H19N3O4: 390.1454, found: 390.1452. ( E) - 3 - ( 3 - ( 4 - H y dr ox y - 3 - m e t hox y phe ny l) a c r y l amido)-N-(5methylisoxazol-3-yl)benzamide (38): White solid (Yield: 30%); 1H NMR (500 MHz, DMSO-d6): δ 10.51 (1H, s), 10.14 (1H, s), 9.69 (1H, s), 8.29 (1H, s), 7.99 (1H, s), 7.91 (1H, d, J=8.0 Hz), 7.61 (1H, d, J=7.5 Hz), 7.54 (1H, d, J=15.5 Hz), 7.44 (1H, t, J=8.0 Hz), 7.13 (1H, d, J=2.0 Hz), 7.04 (1H, dd, J=8.5, 1.5 Hz), 6.88 (1H, d, J=8.0 Hz), 6.62 (1H, d, J=15.5 Hz), 3.81 (3H, s), 2.58 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 167.7, 164.7, 149.2, 148.3, 141.5, 140.2, 131.9, 129.5, 126.5, 124.4, 123.6, 122.5, 120.3, 119.0, 116.2, 111.3, 56.0; HRMS-ESI [M+H]+ calcd. for C21H19N3O5: 394.1403, found: 394.1394. (E)-3-(3-(4-Hydroxy-3-methoxyphenyl)acrylamido)-N-(thiazol-2-yl) benzamide (39): White solid (Yield: 50%); 1H NMR (500 MHz, DMSO-d6): δ 12.31 (1H, s), 10.78 (1H, s), 9.43 (1H, s), 8.49 (1H, s), 8.08 (1H, s), 7.92(1H, d, J=8.0 Hz), 7.88 (1H, d, J =8.0 Hz), 7.80 (2H, t, J =7.5 Hz), 7.66 (1H, d, J=16.0 Hz), 7.52 (1H, d, J=3.5 Hz), 7.47(1H, t, J=8.5 Hz), 7.31 (1H, d, J=3.5 Hz), 7.06 (1H, d, J=16.0 Hz) , 3.81 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 165.7, 164.8, 159.3, 149.3, 148.4, 141.4, 140.3, 137.7, 133.4, 129.4, 126.6, 123.4, 122.9, 122.5, 119.5, 119.2, 116.2, 114.4, 111.5, 56.0; HRMS-ESI [M+H]+ calcd. for C20H17N3O4S: 396.1018, found: 396.1011. (E)-3-(3-(4-Hydroxy-3-methoxyphenyl)acrylamido)-N -(3-(trifluoromethyl)phenyl)benzamide (40): White solid (Yield: 40%); 1H NMR (500 MHz, DMSO-d6): δ 10.31 (1H, s), 10.09 (1H, s), 9.45 (1H, s), 8.36 (2H, d, J=1.5 Hz), 7.99 (1H, d, J=8.0 Hz), 7.87 (2H, t, J=7.0 Hz), 7.78 (1H, d, J=8.0 Hz), 7.70 (1H, d, J=16.0 Hz), 7.68 (2H, m), 7.59 (1H, t, J=8.0 Hz), 7.45 (1H, dd, J=7.5,2.0 Hz), 7.29 (1H, d, J=8.0 Hz), 6.96 (1H, d, J=16.0 Hz), 3.83 (3H, s); 13C NMR (125 MHz, DMSO-d6): δ 166.5, 164.7, 149.3, 148.4, 141.5, 140.5, 140.2, 135.8, 130.4, 129.4, 126.6, 124.3, 122.8, 122.6, 122.5, 120.4, 119.1, 119.1, 116.8, 116.8, 116.2, 111.5, 56.1; HRMS-ESI [M+H]+ calcd. for C24H19F3N2O4: 457.1375, found: 457.1366.

Biological evaluation In vitro antimalarial activity assay: The antimalarial assays using P. falciparum 3D7 were conducted as previously reported [21,22]. Plasmodium falciparum 3D7 were cultured at 37°C under 5.0% CO2 and 5% O2 in 3% hematocrit-type A human red blood cells (Japanese Red Cross Society) in RPMI1640 containing 25 mM HEPES, 24 mM NaHCO3 and 0.03% l-glutamine (Thermo Fisher Scientific), supplemented with 0.4% glucose, 20 µg/ml hypoxathine, 24 µg/ml gentamicin, and 0.25% AlbuMax II (Sigma-Aldrich). To perform the P. f. growth assay, 50 µL of 0.3%-parasitized red blood cells and 2% hematocrit were dispensed into 384-well plate. Following 72-h exposure to a test sample, plates were frozen at -70 °C overnight and then thawed at room temperature for at least 4 h. To evaluate LDH activity, 25 µL of freshly made reaction mix (300 mM sodium l-lactate, 300 µM 3-acetyl pyridine adenine dinucleotide, 374 µM Nitro Blue tetrazolium chloride, 270 µg/mL diaphorase (22.5 U/mL), 1.5% Tween 20, 209 mM Tris-HCl, pH 8.0) was added. Plates were shaken to ensure mixing and absorbance at 620 nm was monitored in a plate reader after 10 min of incubation at room temperature. Artemisinin was used as positive control for this assay with IC50 of 0.02 μM against Plasmodium falciparum 3D7. In vitro cytotoxicity assay: Maintenance of WI-38 cells, and cytotoxicity assay using these cells were conducted as previously described [23]. The human promyelocytic leukemia cell line WI-38 was obtained from RIKEN Cell Bank, and verified to be free of mycoplasma contamination by Hoechst staining. WI-38 was cultured at 37 °C in RPMI1640 (Thermo Fisher Scientific), supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich). Cells (3.75 x 103 cells/well) were seeded into a

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384-well plate. Following 48-h exposures to samples, cell proliferations were determined using a Cell Count Reagent SF (Nacalai Tesque) according to the manufacturer’s instructions. Briefly, a 1/10 volume of WST-8 solution was added to each well, and the plates were incubated for 2 h. Then, cell growth was measured as the absorbance at 450 nm on a microplate reader (Thermo Fisher Scientific). In vivo antimalarial activity assay: Evaluation of the in vivo antimalarial activity was performed based on the Peters 4-day suppressive test [24-26]. Male CD-1 (ICR) mice were purchased from Charles River Japan Inc., Japan. Mice at a weight of ca. 18~22 g were intravenously infected with 2 x 106 parasitized red blood cells of P. berghei N strain. Test compounds were prepared by 10%DMSO/0.5% Tween80 aqueous solution and sonicated for 30 min prior to intraperitoneal administration. Treatment started two hours after the infection (Day 0) and continued daily for 3 days (Day1-3). Each compound was given at the dosages of 10 and 30 mg/kg/day. Five mice were used per treated or control group. On Day 4, blood smears were collected from each mouse to determine parasitaemia. Percentage inhibition was calculated using the following formula: Percentage inhibition=Treated mice parasitaemia / Untreated (vehicle) mice parasitaemia x 100 The statistical analysis (Dunnett’s test) was performed using JMP statistical software (JMP® 8 SAS Institute Inc., Cary, NC, USA) and P values < 0.05 were considered statistically significant.

Results and Discussion Chemistry The derivatives of 3-cinnamamido-N-substituted benzamide were prepared following a very simple synthetic pathway outlined in Scheme 1 (see detailed R1 and R2 in Table 1). Substituted cinnamic acids (A) were synthesized by the well-known Knoevenagel condensation reaction of benzaldehydes and malonic acid in the presence of piperidine as a weak basic catalyst in pyridine at 115ºC for 12 h. Upon the concurrent decarboxylation of malonic acid, the newly formed α, β-unsaturated cinnamic acids were then coupled with methyl 3-aminobenzoate. The obtained product (B) was subsequently hydrolyzed in an ethanol-water solvent system with one equivalent of NaOH. Further addition of the hydrochloric acid to the water-dissolved reaction mixture resulted in the formation of the precipitates of the acid in the ice bath, which can be easily filtered to obtain the desired intermediate C. Triggered by EDC·HCl and HOBT, the second amidation, similar to the second step of the reaction,

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took place between the acid intermediates (C) and the substituted aromatic amines in DMF to provide the 3-cinnamamido-N-substituted benzamides (1-40, Table 1) in moderate to good yields. In the present work, 40 compounds (Table 1) were synthesized by replacing the methoxy group on the phenyl ring with hydroxy, fluorine, chlorine, trifluoromethyl or nitro groups in a mono- or di-substituted manner (B1, Figure 1), while the aromatic moiety B2 (Figure 1) was replaced by diversified heterocycles such as oxazole, thiazole, quinoline, pyridine, or substituted benzene. In vitro antimalarial activity: The obtained derivatives were then evaluated against P. falciparum 3D7 cultures to determine their antimalarial activity, which could be indicated by their IC50 values. The assay was performed in triplicate and the IC50 values were calculated from the results of at least two repetitions. Artemisinin was used as the positive control with IC50 of 0.020 μM against P. falciparum 3D7. As a result, the majority of the compounds with the 3-cinnamamido-N-substituted benzamide framework exhibited equal or slightly better antimalarial activity than compound 1 (Table 2). Among them, compounds 11, 23, 30 and 31 were found to be the most potent against P. falciparum 3D7. Further SAR study of these molecules was conducted to determine the effects of different substituents of R1 or R2 (Table 2). The results demonstrated that four substituted groups on phenyl ring of R1, namely, 4-trifluoromethyl (8), 3-trifluoromethyl (14), 4-Chloro (20) and 4-chloro3-trifluoromethyl (27) groups, slightly enhanced the antimalarial effect, while 4-hydroxy-3-methoxy substituent (37), by contrast, invoked a drastic fourfold decrease in potency. Meanwhile, the 4-fluoro-3-nitro substituted compound (33) exhibited comparable antimalarial activity as compound 1. On the other hand, the SAR results of R2 substitution represented derivatives with quinolone (compound 30) and 3-trifluoromethyl benzene (compound 31) as the two most potent compounds with IC50 values of 0.093 and 0.10 µM, respectively. A decrease in antimalarial potency was observed when N-methyl formamide moiety was introduced to the pyridine of R2. Meanwhile, the thiazole moiety of R2 led to a moderate decrease of antimalarial effect against P. falciparum 3D7, while the methyl-substituted 5-membered isoxazole exerted no appreciable antimalarial activity. In vitro selectivity test: The most potent compounds 11, 23, 30 and 31 further underwent selectivity study, wherein their anti-proliferative activities were evaluated against mammalian cell lines, bacteria, and fungi. Aa a result, all compounds showed weak cytotoxicity against human normal fibroblast WI-38 cells (IC50 values: 18 µM for 11, 3.7 µM for 23, 8.7 µM for 30, and 7.7 µM for 31), no cytotoxicity against mammalian cell lines HeLa,

Scheme 1. Synthesis of 3-cinnamamido-N-substituted benzamides. Reagents and conditions: (i) pyridine, piperidine, reflux, 12 h; (ii) EDC•HCl, HOBT, DMF, overnight; (iii) NaOH, ethanol, reflux, 3 h; then 1 M HCl; (iv) EDC•HCl, HOBT, DMF, overnight. Page 112 of 114

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Table 1. In vitro antimalarial activity against P. falciparum 3D7 (IC50: µM). O R1

N H

R1 R2

H3CO

F3C

H N

Cl CF3

F NO2

HO OCH3

CF3

N H

CH3 Cl

1.20 ± 0.1

>30

1.70 ± 0.1

0.91 ± 0.05

0.84 ± 0.04

11 ± 0.8

4.80 ± 0.2

—[a]

16 ± 1.1

1.90 ± 0.1

0.11 ± 0.04

0.29 ± 0.02

18 ± 1.0

—

0.99 ± 0.06

>30

0.35 ± 0.02

0.60 ± 0.03

7.30 ± 0.6

—

0.96 ± 0.07

>30

3.90 ± 0.2

0.19 ± 0.01

0.84 ± 0.05

9.50 ± 0.5

0.89 ± 0.06

—

0.53 ± 0.03

18 ± 0.9

1.70 ± 0.1

0.093 ± 0.006

0.10 ± 0.008

30 ± 1.6

—

1.10 ± 0.1

—

1.40 ± 0.09

0.95 ± 0.05

0.46 ± 0.02

—

4.50 ± 0.3

>30

1.10 ± 0.05

—

8.10 ± 0.4

—

0.59 ± 0.03

CF3

Table 2. Antimalarial activity of 30 and 31. (a) 10%DMSO/0.5% Tween 80 suspension, (b) 10%DMSO/0.5% Tween 80 solution. Compound

in vitro activity[µM] P.f. 3D7

WI-38

Compound

0.093 ± 0.006

8.7 ± 0.5

30 (a)

0.1 ± 0.008

7.7 ± 0.4

31 (b)

Artemisinin

0.02 ± 0.001

>0.3

Artesunate

MG-63, and srcts-NRK (IC50 up to 30 µM), and no antibacterial or antifungal activities toward any tested strain, suggesting encouraging selectivity profiles against malaria. In vivo antimalarial assay: Next, we evaluated the in vivo antimalarial activity of the most active compounds 30 and 31 using the method of Peters’ 4-day suppressive test [24]. Each test compound, as well as positive control artesunate, was intraperitoneally (i.p.) administered at the dosage of 10 or 30 mg/kg. The results showed that artesunate significantly suppressed malaria parasites infection, whereas compounds 30 and 31 only reduced approximately 10% of parasites growth at 10 mg/kg/day in comparison. The activity further diminished at a higher dose, presumably due to the poor solubility of the compounds in the formulation since compound residues were observed in the abdominal cavity during the necropsy on Day 4 [25-27]. Page 113 of 114

in vivo activity[M] Dosage[mg/Kg]

Route

Inhibition[%]

10 30 10 30 10 30

i.p. i.p. i.p. i.p. i.p. i.p.

13.3 1.2 11.7 0 85.4(P<0.0001) 98.1(P<0.0001)

Conclusion The elusive mechanism of P. falciparum hinders the discovery of antimalarial agents. However, cell-based screen for novel bioactive compounds has made new contribution to the development of antimalarial drugs in recent years. A phenotypic screen of our compound library resulted in the identification of 3-cinnamamido-N-substituted benzamide as a hit showing moderate inhibitory activity against P. falciparum 3D7 strain. To our best knowledge, it is the first report on the antimalarial effect of the cinnamamide framework. Further derivatization of this skeleton led to 40 cinnamamides, most of which showed similar or slightly better antimalarial activity in comparison with the hit molecule. Among these derivatives, compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values ranging from 0.093 to 0.19 µM. Notably, the poor solubility of these analogues could possibly affect the reliability and accuracy of the in vitro and in vivo results, the

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degree of which warrants further studies. Nevertheless, our work sets another compelling example of phenotypic screen-based antimalarial drug discovery by identifying potentially novel promising candidates for further development of antimalarial therapies.

Ethics Approval Participate

and

Consent

All animal experiments were approved by the Animal Care and Use Committee of Kitasato University, Tokyo, Japan.

Human and Animal Rights No humans were used in the study. The research was conducted in accordance with the ethical standards. All care and use of laboratory animals were followed.

Conflict of Interest The authors declare no conflict of interest, financial or otherwise.

Acknowledgements We would like to thank Dr. J. Otaka, Ms. H. Aono, Ms. M. Tanaka and Mr. K. Yamamoto in RIKEN for supporting the activity tests. This work was supported by the National Key R&D Program of China (No. 2017YFE0102200) and the Zhejiang Provincial Fund for Distinguished Young Scholars (LR15B020001).

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How to cite this article: Haicheng Liu, Yushi Futamura, Honghai Wu, and Aki Ishiyama, et al. “Discovery of 3-Cinnamamido-NSubstituted Benzamides as Potential Antimalarial Agents.” Med Chem (Los Angeles) 10 (2020). doi: 10.37421/mccr.2020.10.544 Page 114 of 114