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Novel α,β-unsaturated amide derivatives bearing α-amino phosphonate moiety as potential antiviral agents
Bioorganic & Medicinal Chemistry Letters 27 (2017) 4270–4273
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel a,b-unsaturated amide derivatives bearing a-amino phosphonate moiety as potential antiviral agents Xianmin Lan, Dandan Xie, Limin Yin, Zhenzhen Wang, Jin Chen, Awei Zhang, Baoan Song, Deyu Hu ⇑ State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China
a r t i c l e
i n f o
Article history: Received 11 June 2017 Revised 16 August 2017 Accepted 21 August 2017 Available online 24 August 2017 Keywords: Ferulic acid a,b-Unsaturated amide a-Aminophosphonate SARs Antiviral activity
a b s t r a c t Based on flexible construction and broad bioactivity of ferulic acid, a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety were designed, synthesized and systematically evaluated for their antiviral activity. Bioassay results indicated that some compounds exhibited good antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) in vivo. Especially, compound g18 showed excellent curative and protective activities against CMV, with halfmaximal effective concentration (EC50) values of 284.67 lg/mL and 216.30 lg/mL, which were obviously superior to that of Ningnanmycin (352.08 lg/mL and 262.53 lg/mL). Preliminary structure-activity relationships (SARs) analysis revealed that the introduction of electron-withdrawing group at the 2-position or 4-position of the aromatic ring is favorable for antiviral activity. Present work provides a promising template for development of potential inhibitor of plant virus. Ó 2017 Elsevier Ltd. All rights reserved.
Plant virus caused dramatic loss in agriculture and horticulture all over the world.1 As the most common plant viruses, cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) which were widely distributed in the plant kingdom could infect many plants, including cucumbers, tomatoes, tobacco, pepper, and ornamental plants.2 Ningnanmycin is the most effective commercially available anti-plant virus agent and is widely used to prevent plant virus disease. However, the use of this agent for field trial is largely limited by its poor efficacy and high control cost.3–5 Accordingly, many efforts have been made to develop novel and more potent antiviral agents, some natural and synthesized products6–11 have been investigated about their antiviral activity. However, few of them have been successfully developed and applied in agriculture. Thus, the development of novel and more practical antiviral agents is increasingly required. Natural product-based antiviral agents possess many advantages, such as low mammalian toxicity, easy decomposition, friendly to environment, unique mode of action, and so on.12,13 Therefore, using natural products as lead compounds to develop new pesticides is a potential alternative. As a natural compound, ferulic acid (Fig. 1) widely distributed in plant14 and exhibited diverse physiological activities such as reduction of serum cholesterol levels15 antioxidant properties16 antibacterial17 and
⇑ Corresponding author. E-mail address: [email protected] (D. Hu). http://dx.doi.org/10.1016/j.bmcl.2017.08.048 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
anticancer18,19 activities. Intriguingly, ferulic acid possess a unique skeleton with A ring and a,b-unsaturated carboxylic acid (Fig. 1). Moreover, previous structure-activity relationship study implied that the a,b-unsaturated carboxylic acid fragment of ferulic acid is important for the antiviral activity.20 Additionally, as naturally occurring amino acid analogues, a-aminophosphonate and their derivatives possess broad spectrum of biological activities.21–28 In our previous work, some substituted amino aryl phosphonate derivatives exhibited remarkable inhibitory effect against plant virus.29–34 It is noteworthy that phosphonates possess more lipophilic nature and cell permeability along with physiological stability as the phosphorus-carbon bond which is not susceptible to enzymatic degradation by phosphatases.35,36 Considering the above, we proposed to replace the a,b-unsaturated carboxylic acid fragment of ferulic acid with a,b-unsaturated amide bearing a-aminophosphonate based on the bioisosterism, which is an effective approach for developing and optimizing bioactive lead compounds. Moreover, many studies have revealed that the introduction of substituted benzyl groups into the phenolic hydroxyl group (ring B) might favour bioactivity of the whole molecule (Fig. 1).37–40 Inspired by it, we continued to place various substituted benzyl groups at the hydroxy group on ring A (Fig. 1), and synthesized a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety. Their antiviral activity against CMV and TMV in vivo were evaluated and their structure-activity relationships were preliminarily analyzed. To the best
X. Lan et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4270–4273
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Fig. 1. Design of the target compounds.
of our knowledge, it’s the first report on the antiviral activity of a,bunsaturated amide derivatives bearing a-aminophosphonate moiety. The synthetic route for the target compounds g1–g26 was summarized in Scheme 1. Intermediates c1–c2 were synthesized according to literature method as described.41 Intermediate d was prepared by literature method.42 Intermediates f1–f13 were prepared by literature method.40 Target compounds g1–g26 were synthesized via dehydration condensation reaction between intermediates c1–c2 and f1–f13. All the target compounds were characterized by 1H NMR, 13C NMR, 31P NMR, IR and HRMS, the experimental details and data were provided in the Supplementary data and the representative data for g1 were shown as below. Data for diethyl (E)-((3-(4-(benzyloxy)-3-methoxyphenyl)acrylamido)(phenyl) methyl) phosphonate (g1). White solid; m.p. 118– 120 °C; yield, 56.2%; IR (KBr, cm 1) m: 3275.5, 2987.3, 1672.3, 1626.7, 1514.6, 1264.3, 1139.2, 1026.3, 978.5; 1H NMR (500 MHz, DMSO-d6, ppm) d: 9.01 (d, J = 8.9 Hz, 1H, CO-NH), 7.48–7.30
(m, 11H, Ar-H, Ar-CH@CH), 7.16 (s, 1H, Ar-H), 7.06 (dd, J = 15.7, 7.7 Hz, 2H, Ar-H), 6.77 (d, J = 15.8 Hz, 1H, CH@CH-CO), 5.54 (dd, J = 21.3, 9.8 Hz, 1H, Ar-CH), 5.09 (s, 2H, Ar-OCH2), 4.01–3.79 (m, 7H, OCH2CH3, Ar-OCH3), 1.15 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.06 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C NMR (125 MHz, CDCl3, ppm) d: 165.55, 150.04, 149.78, 142.19, 136.70, 134.20, 134.04, 129.58, 128.92, 128.72, 128.09, 128.02, 127.29, 122.02, 117.78, 113.56, 110.47, 70.95, 63.53 (d, JC-P = 20.0 Hz), 56.10, 50.35, 49.12, 16.57, 16.27; 31P NMR (202 MHz, DMSO-d6, ppm) d: 21.84; HRMS (ESI): Calculated for C28H33O6NP [M+H]+: 510.2040, found: 510.2037. The anti-CMV activities of compounds g1–g26 were tested using the half-leaf method.4 The inhibitory effects against CMV of the target compounds were summarized in Table 1. As shown in Table 1, most of the target compounds exhibited good to excellent curative activities against CMV at 500 lg/mL. Compounds g3, g5, g8, g16, g18, g21, and g23 showed higher curative activities (54.2%, 54.7%, 54.9%, 55.6%, 58.2%, 57.1%, and 55.0%, respectively) against CMV than Ningnanmycin (53.8%). Meanwhile, compounds
Scheme 1. Synthetic route of the target compounds.
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X. Lan et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4270–4273
Table 1 Antiviral activity of the target compounds against CMV in vivo.
a b
Compd.
Curative activitya (%)
Protection activitya(%)
Inactivation activitya (%)
Curative activity EC50 (lg/mL)
Protection activity EC50 (lg/mL)
g1 g2 g3 g4 g5 g6 g7 g8 g9 g10 g11 g12 g13 g14 g15 g16 g17 g18 g19 g20 g21 g22 g23 g24 g25 g26 Controlb
39.4 ± 2.5 50.2 ± 1.7 54.2 ± 2.4 34.8 ± 3.2 54.7 ± 2.5 38.6 ± 3.6 51.1 ± 2.2 54.9 ± 3.1 28.5 ± 2.6 50.1 ± 2.9 48.2 ± 3.3 43.1 ± 3.4 33.4 ± 2.4 43.4 ± 2.6 51.2 ± 3.4 55.6 ± 2.8 33.8 ± 5.3 58.2 ± 3.3 36.2 ± 3.9 52.2 ± 3.2 57.1 ± 2.3 30.2 ± 4.7 55.0 ± 2.6 51.1 ± 3.6 46.4 ± 3.4 40.3 ± 3.4 53.8 ± 2.1
41.2 ± 2.7 52.5 ± 3.2 56.3 ± 2.9 35.6 ± 4.2 57.5 ± 2.7 31.2 ± 4.2 49.2 ± 3.3 54.2 ± 3.4 28.2 ± 3.8 48.7 ± 2.6 40.4 ± 3.9 46.2 ± 2.8 35.2 ± 2.6 46.4 ± 2.0 55.2 ± 5.3 57.4 ± 4.3 36.6 ± 5.6 59.7 ± 2.2 30.6 ± 3.4 53.6 ± 3.1 58.6 ± 3.2 27.3 ± 3.2 56.2 ± 2.8 55.4 ± 3.2 54.2 ± 2.8 36.5 ± 2.6 57.2 ± 1.9
55.8 ± 2.4 69.2 ± 3.1 68.2 ± 4.8 56.2 ± 2.7 75.5 ± 5.4 49.2 ± 2.2 65.5 ± 4.5 85.4 ± 2.4 46.2 ± 3.2 65.5 ± 3.1 73.6 ± 2.8 66.5 ± 2.6 43.2 ± 2.4 66.8 ± 2.2 65.6 ± 4.1 78.6 ± 3.6 55.4 ± 2.6 80.5 ± 3.6 65.2 ± 2.6 77.3 ± 4.2 83.2 ± 2.7 45.3 ± 2.5 78.6 ± 3.2 79.6 ± 4.4 76.5 ± 2.6 50.2 ± 2.5 92.7 ± 2.8
996.14 ± 7.05 456.73 ± 7.83 332.06 ± 5.46 1505.81 ± 11.23 342.36 ± 4.55 851.67 ± 10.6 428.12 ± 6.33 347.77 ± 4.15 1504.95 ± 17.61 436.73 ± 6.92 534.02 ± 7.32 398.27 ± 8.37 1411.34 ± 12.64 777.43 ± 8.69 408.37 ± 6.47 323.27 ± 6.18 1544.82 ± 7.37 284.67 ± 4.35 900.72 ± 9.92 400.78 ± 7.22 296.99 ± 4.35 1511.53 ± 12.7 347.89 ± 5.57 396.61 ± 5.66 505.25 ± 4.45 874.09 ± 4.63 352.08 ± 5.11
826.71 ± 6.19 306.32 ± 4.44 292.82 ± 5.16 / 254.53 ± 5.11 1613.66 ± 12.39 558.39 ± 8.78 287.64 ± 4.82 / / / / / / 273.43 ± 5.59 236.90 ± 4.15 / 216.30 ± 4.09 / 314.77 ± 5.62 227.04 ± 3.43 1128.75 ± 13.34 278.15 ± 5.33 308.70 ± 6.11 302.46 ± 7.03 / 262.53 ± 4.19
Average of three replicate at 500 lg/mL. The commercial anti-plant virus agent Ningnanmycin was used as control.
g5, g16, g18, and g21 also displayed comparative or even higher protective effects (57.5%, 57.4%, 59.7%, and 58.6%, respectively) against CMV compared to Ningnanmycin (57.2%). To gain insight into the potential inhibitory capacity of these compounds against CMV, the EC50 values of curative and protective activities of some target compounds were evaluated and listed in Table 1. Compounds g3, g5, g8, g16, and g23 exerted comparative curative activities (with EC50 values of 332.06, 342.36, 347.77, 323.27, and 347.89 lg/mL, respectively) against CMV compared to that of Ningnanmycin (352.08 lg/mL). Especially, compounds g18 and g21 possessed obviously higher curative activities (with EC50 values of 284.67 and 296.99 lg/mL, respectively) against CMV than that of Ningnanmycin (352.08 lg/mL). Moreover, compounds g5, g16, g18, and g21 exerted more potent protective effects (with EC50 values of 254.53, 236.90, 216.30, and 227.04 lg/mL, respectively) against CMV than that of Ningnanmycin (262.53 lg/mL). The anti-TMV activities of compounds g1–g26 were also tested using the half-leaf method and the results of the preliminary bioassays were listed in Table 2. As shown in Table 2, compounds g5, g16, g18, and g23 showed similar curative activities against TMV to that of Ningnanmycin (56.2%) at 500 lg/mL, with the values of 51.7%, 52.6%, 53.7%, and 52.2%, respectively. Compounds g5, g9, g16, g18, g21, and g24 exhibited 68.3%, 67.6%, 70.4%, 72.5%, 68.7%, and 71.4% protective effects at 500 lg/mL, respectively, which were comparable to that of Ningnanmycin (73.3%). In addition, compounds g16 and g24 displayed comparable inactivating efficacies (89.6% and 90.5%, respectively) to that of Ningnanmycin (92.5%). Based on the preliminary biological evaluation, The EC50 values of curative and protective activities against TMV of some target compounds were evaluated. As shown in Table 2, compound g18 possessed excellent curative activity against TMV, with EC50 value of 285.42 lg/mL, which was similar to that of Ningnanmycin (254.91 lg/mL). Compounds g5, g16, g18, and g24 exhibited remarkable protective activities against TMV, with EC50 values of
190.74, 191.04, 180.37, and 183.16 lg/mL, respectively, which were comparable to that of Ningnanmycin (165.95 lg/mL). Bioassays results showed that the a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety exhibited excellent curative activities against CMV. Accordingly, the structureactivity relationships (SARs) analysis was deduced on the basis of the EC50 values of the anti-CMV activities. The results indicated that the compounds bearing electron-withdrawing group at the 4-position of the substituted aromatic rings (R2) are favorable for antiviral activity, for examples g2 (4-F-Ph), g3 (4-Cl-Ph), g5 (4CF3-Ph) > g1 (Ph) > g6 (4-CH3-Ph), and g15 (4-F-Ph), g16 (4-ClPh), g18 (4-CF3-Ph) > g14 (Ph) > g19 (4-CH3-Ph). Meanwhile, we found the similar trend of 2-position on R2 relative to 4-position, such as, g7 (2-Cl-Ph), g8 (2-CF3-Ph) > g9 (2-CH3-Ph) and g20 (2Cl-Ph), g21 (2-CF3-Ph) > g22 (2-CH3-Ph). Unfortunately, the compounds containing two electron-withdrawing groups on R2 and R1 bearing H are unfavorable for antiviral activity compared to one electron-withdrawing group on R2, this trend was confirmed with the orders g11 (2,4-diF-Ph), g12 (2-Cl-4-F-Ph) < g2 (4-F-Ph) and g10 (2,4-diCl-Ph), g12 (2-Cl-4-F-Ph) < g3 (4-Cl-Ph). The introduction of pyridine heterocyclic instead of benzene led to significantly decreased inhibitory activities against CMV. Additionally, most of compounds whose R1 are Cl atoms showed higher antiCMV activities than corresponding compounds whose R1 are H atoms except for compounds g17, g19, g22, and g25. These interesting results generally demonstrated that the introduction of Cl atom at the 4-position of the benzene ring belong to the aaminophosphonate moiety did somewhat contribute to antiviral activity. In summary, on the basis of the structure of naturally occurring ferulic acid, a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety were designed, synthesized, and systematically evaluated for their antiviral activity against CMV and TMV in vivo. The bioassay results indicated that some compounds exhibited good antiviral activities, of which compound g18 possessed excellent curative and protective activities against
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X. Lan et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4270–4273 Table 2 Antiviral activity of the target compounds against TMV in vivo.
a b
Compd.
Curative activitya (%)
Protection activitya(%)
Inactivation activitya (%)
Curative activity EC50 (lg/mL)
Protection activity EC50 (lg/mL)
g1 g2 g3 g4 g5 g6 g7 g8 g9 g10 g11 g12 g13 g14 g15 g16 g17 g18 g19 g20 g21 g22 g23 g24 g25 g26 Control
40.2 ± 4.1 45.2 ± 3.2 49.7 ± 2.7 38.8 ± 3.5 51.7 ± 2.5 32.6 ± 3.6 43.8 ± 4.0 45.8 ± 2.5 34.5 ± 5.0 45.6 ± 4.2 48.2 ± 3.7 44.4 ± 4.1 23.4 ± 4.1 45.4 ± 2.6 49.2 ± 3.4 52.6 ± 2.8 40.5 ± 4.4 53.7 ± 3.6 34.6 ± 3.7 49.5 ± 3.2 47.9 ± 2.5 30.5 ± 3.7 52.2 ± 3.9 50.2 ± 3.4 48.4 ± 3.5 38.4 ± 3.5 56.6 ± 2.6
55.2 ± 2.5 62.6 ± 2.1 65.8 ± 3.7 45.6 ± 4.2 68.3 ± 2.9 34.4 ± 3.3 57.7 ± 5.2 60.7 ± 2.8 67.6 ± 3.7 56.1 ± 3.2 48.8 ± 2.3 54.6 ± 2.8 33.6 ± 2.8 46.4 ± 2.4 65.2 ± 3.7 70.4 ± 4.3 48.6 ± 5.6 72.5 ± 3.2 46.2 ± 3.5 63.6 ± 3.8 68.7 ± 3.2 52.3 ± 3.6 58.8 ± 1.7 71.4 ± 2.2 57.6 ± 3.9 46.5 ± 3.9 73.3 ± 1.7
60.2 ± 3.7 79.3 ± 4.5 65.2 ± 4.0 75.6 ± 2.5 78.4 ± 3.4 69.2 ± 2.7 85.5 ± 3.5 74.4 ± 2.4 76.2 ± 3.0 67.5 ± 3.4 75.3 ± 2.7 66.4 ± 2.7 43.4 ± 2.7 67.4 ± 2.7 81.7 ± 2.3 89.6 ± 2.2 86.4 ± 4.1 86.6 ± 3.1 75.9 ± 2.3 87.2 ± 2.2 76.2 ± 2.2 75.3 ± 3.5 88.7 ± 3.1 90.5 ± 4.2 68.2 ± 2.4 40.2 ± 2.3 92.5 ± 2.3
981.21 ± 10.66 369.94 ± 6.98 310.32 ± 5.27 / 302.09 ± 6.29 1511.97 ± 11.64 / 319.39 ± 6.22 771.25 ± 8.99 / 410.31 ± 7.01 / / / / 296.84 ± 6.14 / 285.42 ± 5.56 / 350.23 ± 4.87 / / 300.44 ± 6.11 310.66 ± 6.51 / / 254.91 ± 3.07
448.67 ± 6.15 242.49 ± 6.51 215.21 ± 4.52 / 190.74 ± 3.67 / / 267.59 ± 4.19 210.34 ± 6.14 / 528.81 ± 8.10 / / / 227.98 ± 5.52 191.04 ± 4.25 / 180.37 ± 5.57 / 247.92 ± 6.21 202.38 ± 4.55 735.74 ± 9.18 320.72 ± 6.77 183.16 ± 5.10 / / 165.95 ± 3.12
b
Average of three replicate at 500 lg/mL. The commercial anti-plant virus agent Ningnanmycin was used as control.
CMV with EC50 values of 284.67 and 216.30 lg/mL, which were obviously superior to that of Ningnanmycin (352.08 and 262.53 lg/mL). SARs analysis implied that the introduction of electron-withdrawing groups at the 2-position or 4-position of the R2 are favorable for antiviral activity. In addition, the introduction of Cl atom at the 4-position of the benzene ring belong to the aaminophosphonate moiety did somewhat contribute to antiviral activity. It is the first report on the antiviral activity of a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety and present work provides a promising template for development of potential inhibitor of plant virus. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21672044 and 21562013) and Subsidy Project for Outstanding Key Laboratory of Guizhou Province in China (20154004) and the Provincial University Cooperation Plan of Guizhou Province in China (No. 20147001) and Collaborative Innovation Center for Natural Products and Biological Drugs of Yunnan for supporting the project. A. Supplementary data Supplementary data associated (containing information on the synthesis, characterization, and bioactivity test methods of the title compounds) with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.048. References 1. Bos L. Crop Prot. 1982;1:263. 2. Hu Q, Niu YP, Zhang K, Liu Y, Zhou XP. Virol J. 2011;8:1. 3. Wang ZW, Wang L, Ma S, Liu YX, Wang LZ, Wang QM. J Agric Food Chem. 2012;60:5825. 4. Long CW, Li P, Chen MH, Dong LR, Hu DY, Song BA. Eur J Med Chem. 2015;102:639.
5. Ji XF, Wang ZW, Dong J, Liu YX, Lu AD, Wang QM. J Agric Food Chem. 2016;64:9143. 6. Reichman M, Devash Y, Suhadolnik RJ, Sela I. Virology. 1983;128:240. 7. Ouyang GP, Cai XJ, Chen Z, et al. J Agric Food Chem. 2008;56:10160. 8. Song HJ, Liu YX, Liu YX, Huang YQ, Li YQ, Wang QM. Bioorg Med Chem Lett. 2014;24:5228. 9. Chen MH, Li P, Hu DY, et al. Bioorg Med Chem Lett. 2016;26:168. 10. Chen MH, Hu DY, Li XY, et al. Bioorg Med Chem Lett. 2015;25:3840. 11. Su B, Cai CL, Deng M, Liang DM, Wang LZ, Wang QM. Bioorg Med Chem Lett. 2014;24:2881. 12. Qian XH, Lee PW, Cao S. J Agric Food Chem. 2010;58:2613. 13. Seiber JN. J Agric Food Chem. 2011;59:1. 14. Tanaka A. Tokyo Government Chem Indus Res Inst Rep. 1972;20:792. 15. Guardiola F, Codony R, Addis PB, Rafecas M, Oatella J. Food Chem Toxicol. 1996;34:193. 16. Nystrom L, Makinen M, Lampi A, Piiromen V. J Agric Food Chem. 2005;53:2503. 17. Watt EVD, Pretorius JC. J Ethnopharmacol. 2001;76:87. 18. Hudson EA, Dinh PA, Kokubun T, Simmonds MSJ, Gescher A. Cancer Epidem Biomar. 2000;9:1163. 19. Yasukawa K, Akihisa T, Kimura Y, Tamura T, Takido M. Biol Pharm Bull. 1998;21:1072. 20. Wu M, Wang ZW, Meng CS, et al. PLoS ONE. 2013;8:22. 21. Mcleod DA, Brinkworth RI, Ashley JA, Janda KD, Wirsching P. Bioorg Med Chem Lett. 1991;1:653. 22. Lintunen T, Yli-Kauhaluoma JT. Bioorg Med Chem Lett. 2000;10:1749. 23. Jin JH, Song BA, Zhang GP, et al. Bioorg Med Chem Lett. 2006;16:1537. 24. Jin LH, Chen J, Song BA, et al. Bioorg Med Chem Lett. 2006;16:5036. 25. Roberts LR, Bradley PA, Bunnage ME, et al. Bioorg Med Chem Lett. 2011;21:6515. 26. Jia C, Yang KW, Liu CC, et al. Bioorg Med Chem Lett. 2012;22:482. 27. Luo GL, Chen L, Civiello R, et al. Bioorg Med Chem Lett. 2012;22:2917. 28. Yasuno Y, Hamada M, Yoshida Y, et al. Bioorg Med Chem Lett. 2016;26:3543. 29. Song BA, Zhang GP, Hu DY, et al. 2005; Patent: CN 1687088 A. 30. Long N, Cai XJ, Song BA, et al. J Agric Food Chem. 2008;56:5242. 31. Liu JZ, Song BA, Fan HT, et al. Eur J Med Chem. 2010;45:5108. 32. Zhou J, Fan HT, Song BA, et al. Phosphorus, Sulfur Silicon Relat Elem. 2011;186:81. 33. Chen Z, Li GJ, Fan HT, et al. Agr Sci Bull. 2011;27:250. 34. Xiao H, Li P, Hu DY, Song BA. Bioorg Med Chem Lett. 2014;24:3452. 35. Chen JL, Tang W, Che JY, et al. J Agric Food Chem. 2015;63:7219. 36. Che JY, Xu XY, Tang ZL, Gu YC, Shi DQ. Bioorg Med Chem Lett. 2016;26:1310. 37. Rashad AA, El-Sabbagh OI, Baraka MM, et al. Med Chem Res. 2010;19:1025. 38. Feng JY, Liu ZQ. Eur J Med Chem. 2011;46:1198. 39. Chiaradia LD, Martins PGA, Cordeiro MNS, et al. J Med Chem. 2012;55:390. 40. Banoglu E, Celikoglu E, Volker S, et al. Eur J Med Chem. 2016;113:1. 41. Kaboudin B, Moradi K. Tetrahedron Lett. 2005;46:2989. 42. Khatkar A, Nand A, Kumar P, Narasimhan B. Res Chem Intermed. 2015;41:299.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Novel a,b-unsaturated amide derivatives bearing a-amino phosphonate moiety as potential antiviral agents Xianmin Lan, Dandan Xie, Limin Yin, Zhenzhen Wang, Jin Chen, Awei Zhang, Baoan Song, Deyu Hu ⇑ State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, China
a r t i c l e
i n f o
Article history: Received 11 June 2017 Revised 16 August 2017 Accepted 21 August 2017 Available online 24 August 2017 Keywords: Ferulic acid a,b-Unsaturated amide a-Aminophosphonate SARs Antiviral activity
a b s t r a c t Based on flexible construction and broad bioactivity of ferulic acid, a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety were designed, synthesized and systematically evaluated for their antiviral activity. Bioassay results indicated that some compounds exhibited good antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) in vivo. Especially, compound g18 showed excellent curative and protective activities against CMV, with halfmaximal effective concentration (EC50) values of 284.67 lg/mL and 216.30 lg/mL, which were obviously superior to that of Ningnanmycin (352.08 lg/mL and 262.53 lg/mL). Preliminary structure-activity relationships (SARs) analysis revealed that the introduction of electron-withdrawing group at the 2-position or 4-position of the aromatic ring is favorable for antiviral activity. Present work provides a promising template for development of potential inhibitor of plant virus. Ó 2017 Elsevier Ltd. All rights reserved.
Plant virus caused dramatic loss in agriculture and horticulture all over the world.1 As the most common plant viruses, cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) which were widely distributed in the plant kingdom could infect many plants, including cucumbers, tomatoes, tobacco, pepper, and ornamental plants.2 Ningnanmycin is the most effective commercially available anti-plant virus agent and is widely used to prevent plant virus disease. However, the use of this agent for field trial is largely limited by its poor efficacy and high control cost.3–5 Accordingly, many efforts have been made to develop novel and more potent antiviral agents, some natural and synthesized products6–11 have been investigated about their antiviral activity. However, few of them have been successfully developed and applied in agriculture. Thus, the development of novel and more practical antiviral agents is increasingly required. Natural product-based antiviral agents possess many advantages, such as low mammalian toxicity, easy decomposition, friendly to environment, unique mode of action, and so on.12,13 Therefore, using natural products as lead compounds to develop new pesticides is a potential alternative. As a natural compound, ferulic acid (Fig. 1) widely distributed in plant14 and exhibited diverse physiological activities such as reduction of serum cholesterol levels15 antioxidant properties16 antibacterial17 and
⇑ Corresponding author. E-mail address: [email protected] (D. Hu). http://dx.doi.org/10.1016/j.bmcl.2017.08.048 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
anticancer18,19 activities. Intriguingly, ferulic acid possess a unique skeleton with A ring and a,b-unsaturated carboxylic acid (Fig. 1). Moreover, previous structure-activity relationship study implied that the a,b-unsaturated carboxylic acid fragment of ferulic acid is important for the antiviral activity.20 Additionally, as naturally occurring amino acid analogues, a-aminophosphonate and their derivatives possess broad spectrum of biological activities.21–28 In our previous work, some substituted amino aryl phosphonate derivatives exhibited remarkable inhibitory effect against plant virus.29–34 It is noteworthy that phosphonates possess more lipophilic nature and cell permeability along with physiological stability as the phosphorus-carbon bond which is not susceptible to enzymatic degradation by phosphatases.35,36 Considering the above, we proposed to replace the a,b-unsaturated carboxylic acid fragment of ferulic acid with a,b-unsaturated amide bearing a-aminophosphonate based on the bioisosterism, which is an effective approach for developing and optimizing bioactive lead compounds. Moreover, many studies have revealed that the introduction of substituted benzyl groups into the phenolic hydroxyl group (ring B) might favour bioactivity of the whole molecule (Fig. 1).37–40 Inspired by it, we continued to place various substituted benzyl groups at the hydroxy group on ring A (Fig. 1), and synthesized a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety. Their antiviral activity against CMV and TMV in vivo were evaluated and their structure-activity relationships were preliminarily analyzed. To the best
X. Lan et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 4270–4273
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Fig. 1. Design of the target compounds.
of our knowledge, it’s the first report on the antiviral activity of a,bunsaturated amide derivatives bearing a-aminophosphonate moiety. The synthetic route for the target compounds g1–g26 was summarized in Scheme 1. Intermediates c1–c2 were synthesized according to literature method as described.41 Intermediate d was prepared by literature method.42 Intermediates f1–f13 were prepared by literature method.40 Target compounds g1–g26 were synthesized via dehydration condensation reaction between intermediates c1–c2 and f1–f13. All the target compounds were characterized by 1H NMR, 13C NMR, 31P NMR, IR and HRMS, the experimental details and data were provided in the Supplementary data and the representative data for g1 were shown as below. Data for diethyl (E)-((3-(4-(benzyloxy)-3-methoxyphenyl)acrylamido)(phenyl) methyl) phosphonate (g1). White solid; m.p. 118– 120 °C; yield, 56.2%; IR (KBr, cm 1) m: 3275.5, 2987.3, 1672.3, 1626.7, 1514.6, 1264.3, 1139.2, 1026.3, 978.5; 1H NMR (500 MHz, DMSO-d6, ppm) d: 9.01 (d, J = 8.9 Hz, 1H, CO-NH), 7.48–7.30
(m, 11H, Ar-H, Ar-CH@CH), 7.16 (s, 1H, Ar-H), 7.06 (dd, J = 15.7, 7.7 Hz, 2H, Ar-H), 6.77 (d, J = 15.8 Hz, 1H, CH@CH-CO), 5.54 (dd, J = 21.3, 9.8 Hz, 1H, Ar-CH), 5.09 (s, 2H, Ar-OCH2), 4.01–3.79 (m, 7H, OCH2CH3, Ar-OCH3), 1.15 (t, J = 7.0 Hz, 3H, OCH2CH3), 1.06 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C NMR (125 MHz, CDCl3, ppm) d: 165.55, 150.04, 149.78, 142.19, 136.70, 134.20, 134.04, 129.58, 128.92, 128.72, 128.09, 128.02, 127.29, 122.02, 117.78, 113.56, 110.47, 70.95, 63.53 (d, JC-P = 20.0 Hz), 56.10, 50.35, 49.12, 16.57, 16.27; 31P NMR (202 MHz, DMSO-d6, ppm) d: 21.84; HRMS (ESI): Calculated for C28H33O6NP [M+H]+: 510.2040, found: 510.2037. The anti-CMV activities of compounds g1–g26 were tested using the half-leaf method.4 The inhibitory effects against CMV of the target compounds were summarized in Table 1. As shown in Table 1, most of the target compounds exhibited good to excellent curative activities against CMV at 500 lg/mL. Compounds g3, g5, g8, g16, g18, g21, and g23 showed higher curative activities (54.2%, 54.7%, 54.9%, 55.6%, 58.2%, 57.1%, and 55.0%, respectively) against CMV than Ningnanmycin (53.8%). Meanwhile, compounds
Scheme 1. Synthetic route of the target compounds.
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Table 1 Antiviral activity of the target compounds against CMV in vivo.
a b
Compd.
Curative activitya (%)
Protection activitya(%)
Inactivation activitya (%)
Curative activity EC50 (lg/mL)
Protection activity EC50 (lg/mL)
g1 g2 g3 g4 g5 g6 g7 g8 g9 g10 g11 g12 g13 g14 g15 g16 g17 g18 g19 g20 g21 g22 g23 g24 g25 g26 Controlb
39.4 ± 2.5 50.2 ± 1.7 54.2 ± 2.4 34.8 ± 3.2 54.7 ± 2.5 38.6 ± 3.6 51.1 ± 2.2 54.9 ± 3.1 28.5 ± 2.6 50.1 ± 2.9 48.2 ± 3.3 43.1 ± 3.4 33.4 ± 2.4 43.4 ± 2.6 51.2 ± 3.4 55.6 ± 2.8 33.8 ± 5.3 58.2 ± 3.3 36.2 ± 3.9 52.2 ± 3.2 57.1 ± 2.3 30.2 ± 4.7 55.0 ± 2.6 51.1 ± 3.6 46.4 ± 3.4 40.3 ± 3.4 53.8 ± 2.1
41.2 ± 2.7 52.5 ± 3.2 56.3 ± 2.9 35.6 ± 4.2 57.5 ± 2.7 31.2 ± 4.2 49.2 ± 3.3 54.2 ± 3.4 28.2 ± 3.8 48.7 ± 2.6 40.4 ± 3.9 46.2 ± 2.8 35.2 ± 2.6 46.4 ± 2.0 55.2 ± 5.3 57.4 ± 4.3 36.6 ± 5.6 59.7 ± 2.2 30.6 ± 3.4 53.6 ± 3.1 58.6 ± 3.2 27.3 ± 3.2 56.2 ± 2.8 55.4 ± 3.2 54.2 ± 2.8 36.5 ± 2.6 57.2 ± 1.9
55.8 ± 2.4 69.2 ± 3.1 68.2 ± 4.8 56.2 ± 2.7 75.5 ± 5.4 49.2 ± 2.2 65.5 ± 4.5 85.4 ± 2.4 46.2 ± 3.2 65.5 ± 3.1 73.6 ± 2.8 66.5 ± 2.6 43.2 ± 2.4 66.8 ± 2.2 65.6 ± 4.1 78.6 ± 3.6 55.4 ± 2.6 80.5 ± 3.6 65.2 ± 2.6 77.3 ± 4.2 83.2 ± 2.7 45.3 ± 2.5 78.6 ± 3.2 79.6 ± 4.4 76.5 ± 2.6 50.2 ± 2.5 92.7 ± 2.8
996.14 ± 7.05 456.73 ± 7.83 332.06 ± 5.46 1505.81 ± 11.23 342.36 ± 4.55 851.67 ± 10.6 428.12 ± 6.33 347.77 ± 4.15 1504.95 ± 17.61 436.73 ± 6.92 534.02 ± 7.32 398.27 ± 8.37 1411.34 ± 12.64 777.43 ± 8.69 408.37 ± 6.47 323.27 ± 6.18 1544.82 ± 7.37 284.67 ± 4.35 900.72 ± 9.92 400.78 ± 7.22 296.99 ± 4.35 1511.53 ± 12.7 347.89 ± 5.57 396.61 ± 5.66 505.25 ± 4.45 874.09 ± 4.63 352.08 ± 5.11
826.71 ± 6.19 306.32 ± 4.44 292.82 ± 5.16 / 254.53 ± 5.11 1613.66 ± 12.39 558.39 ± 8.78 287.64 ± 4.82 / / / / / / 273.43 ± 5.59 236.90 ± 4.15 / 216.30 ± 4.09 / 314.77 ± 5.62 227.04 ± 3.43 1128.75 ± 13.34 278.15 ± 5.33 308.70 ± 6.11 302.46 ± 7.03 / 262.53 ± 4.19
Average of three replicate at 500 lg/mL. The commercial anti-plant virus agent Ningnanmycin was used as control.
g5, g16, g18, and g21 also displayed comparative or even higher protective effects (57.5%, 57.4%, 59.7%, and 58.6%, respectively) against CMV compared to Ningnanmycin (57.2%). To gain insight into the potential inhibitory capacity of these compounds against CMV, the EC50 values of curative and protective activities of some target compounds were evaluated and listed in Table 1. Compounds g3, g5, g8, g16, and g23 exerted comparative curative activities (with EC50 values of 332.06, 342.36, 347.77, 323.27, and 347.89 lg/mL, respectively) against CMV compared to that of Ningnanmycin (352.08 lg/mL). Especially, compounds g18 and g21 possessed obviously higher curative activities (with EC50 values of 284.67 and 296.99 lg/mL, respectively) against CMV than that of Ningnanmycin (352.08 lg/mL). Moreover, compounds g5, g16, g18, and g21 exerted more potent protective effects (with EC50 values of 254.53, 236.90, 216.30, and 227.04 lg/mL, respectively) against CMV than that of Ningnanmycin (262.53 lg/mL). The anti-TMV activities of compounds g1–g26 were also tested using the half-leaf method and the results of the preliminary bioassays were listed in Table 2. As shown in Table 2, compounds g5, g16, g18, and g23 showed similar curative activities against TMV to that of Ningnanmycin (56.2%) at 500 lg/mL, with the values of 51.7%, 52.6%, 53.7%, and 52.2%, respectively. Compounds g5, g9, g16, g18, g21, and g24 exhibited 68.3%, 67.6%, 70.4%, 72.5%, 68.7%, and 71.4% protective effects at 500 lg/mL, respectively, which were comparable to that of Ningnanmycin (73.3%). In addition, compounds g16 and g24 displayed comparable inactivating efficacies (89.6% and 90.5%, respectively) to that of Ningnanmycin (92.5%). Based on the preliminary biological evaluation, The EC50 values of curative and protective activities against TMV of some target compounds were evaluated. As shown in Table 2, compound g18 possessed excellent curative activity against TMV, with EC50 value of 285.42 lg/mL, which was similar to that of Ningnanmycin (254.91 lg/mL). Compounds g5, g16, g18, and g24 exhibited remarkable protective activities against TMV, with EC50 values of
190.74, 191.04, 180.37, and 183.16 lg/mL, respectively, which were comparable to that of Ningnanmycin (165.95 lg/mL). Bioassays results showed that the a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety exhibited excellent curative activities against CMV. Accordingly, the structureactivity relationships (SARs) analysis was deduced on the basis of the EC50 values of the anti-CMV activities. The results indicated that the compounds bearing electron-withdrawing group at the 4-position of the substituted aromatic rings (R2) are favorable for antiviral activity, for examples g2 (4-F-Ph), g3 (4-Cl-Ph), g5 (4CF3-Ph) > g1 (Ph) > g6 (4-CH3-Ph), and g15 (4-F-Ph), g16 (4-ClPh), g18 (4-CF3-Ph) > g14 (Ph) > g19 (4-CH3-Ph). Meanwhile, we found the similar trend of 2-position on R2 relative to 4-position, such as, g7 (2-Cl-Ph), g8 (2-CF3-Ph) > g9 (2-CH3-Ph) and g20 (2Cl-Ph), g21 (2-CF3-Ph) > g22 (2-CH3-Ph). Unfortunately, the compounds containing two electron-withdrawing groups on R2 and R1 bearing H are unfavorable for antiviral activity compared to one electron-withdrawing group on R2, this trend was confirmed with the orders g11 (2,4-diF-Ph), g12 (2-Cl-4-F-Ph) < g2 (4-F-Ph) and g10 (2,4-diCl-Ph), g12 (2-Cl-4-F-Ph) < g3 (4-Cl-Ph). The introduction of pyridine heterocyclic instead of benzene led to significantly decreased inhibitory activities against CMV. Additionally, most of compounds whose R1 are Cl atoms showed higher antiCMV activities than corresponding compounds whose R1 are H atoms except for compounds g17, g19, g22, and g25. These interesting results generally demonstrated that the introduction of Cl atom at the 4-position of the benzene ring belong to the aaminophosphonate moiety did somewhat contribute to antiviral activity. In summary, on the basis of the structure of naturally occurring ferulic acid, a series of novel a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety were designed, synthesized, and systematically evaluated for their antiviral activity against CMV and TMV in vivo. The bioassay results indicated that some compounds exhibited good antiviral activities, of which compound g18 possessed excellent curative and protective activities against
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a b
Compd.
Curative activitya (%)
Protection activitya(%)
Inactivation activitya (%)
Curative activity EC50 (lg/mL)
Protection activity EC50 (lg/mL)
g1 g2 g3 g4 g5 g6 g7 g8 g9 g10 g11 g12 g13 g14 g15 g16 g17 g18 g19 g20 g21 g22 g23 g24 g25 g26 Control
40.2 ± 4.1 45.2 ± 3.2 49.7 ± 2.7 38.8 ± 3.5 51.7 ± 2.5 32.6 ± 3.6 43.8 ± 4.0 45.8 ± 2.5 34.5 ± 5.0 45.6 ± 4.2 48.2 ± 3.7 44.4 ± 4.1 23.4 ± 4.1 45.4 ± 2.6 49.2 ± 3.4 52.6 ± 2.8 40.5 ± 4.4 53.7 ± 3.6 34.6 ± 3.7 49.5 ± 3.2 47.9 ± 2.5 30.5 ± 3.7 52.2 ± 3.9 50.2 ± 3.4 48.4 ± 3.5 38.4 ± 3.5 56.6 ± 2.6
55.2 ± 2.5 62.6 ± 2.1 65.8 ± 3.7 45.6 ± 4.2 68.3 ± 2.9 34.4 ± 3.3 57.7 ± 5.2 60.7 ± 2.8 67.6 ± 3.7 56.1 ± 3.2 48.8 ± 2.3 54.6 ± 2.8 33.6 ± 2.8 46.4 ± 2.4 65.2 ± 3.7 70.4 ± 4.3 48.6 ± 5.6 72.5 ± 3.2 46.2 ± 3.5 63.6 ± 3.8 68.7 ± 3.2 52.3 ± 3.6 58.8 ± 1.7 71.4 ± 2.2 57.6 ± 3.9 46.5 ± 3.9 73.3 ± 1.7
60.2 ± 3.7 79.3 ± 4.5 65.2 ± 4.0 75.6 ± 2.5 78.4 ± 3.4 69.2 ± 2.7 85.5 ± 3.5 74.4 ± 2.4 76.2 ± 3.0 67.5 ± 3.4 75.3 ± 2.7 66.4 ± 2.7 43.4 ± 2.7 67.4 ± 2.7 81.7 ± 2.3 89.6 ± 2.2 86.4 ± 4.1 86.6 ± 3.1 75.9 ± 2.3 87.2 ± 2.2 76.2 ± 2.2 75.3 ± 3.5 88.7 ± 3.1 90.5 ± 4.2 68.2 ± 2.4 40.2 ± 2.3 92.5 ± 2.3
981.21 ± 10.66 369.94 ± 6.98 310.32 ± 5.27 / 302.09 ± 6.29 1511.97 ± 11.64 / 319.39 ± 6.22 771.25 ± 8.99 / 410.31 ± 7.01 / / / / 296.84 ± 6.14 / 285.42 ± 5.56 / 350.23 ± 4.87 / / 300.44 ± 6.11 310.66 ± 6.51 / / 254.91 ± 3.07
448.67 ± 6.15 242.49 ± 6.51 215.21 ± 4.52 / 190.74 ± 3.67 / / 267.59 ± 4.19 210.34 ± 6.14 / 528.81 ± 8.10 / / / 227.98 ± 5.52 191.04 ± 4.25 / 180.37 ± 5.57 / 247.92 ± 6.21 202.38 ± 4.55 735.74 ± 9.18 320.72 ± 6.77 183.16 ± 5.10 / / 165.95 ± 3.12
b
Average of three replicate at 500 lg/mL. The commercial anti-plant virus agent Ningnanmycin was used as control.
CMV with EC50 values of 284.67 and 216.30 lg/mL, which were obviously superior to that of Ningnanmycin (352.08 and 262.53 lg/mL). SARs analysis implied that the introduction of electron-withdrawing groups at the 2-position or 4-position of the R2 are favorable for antiviral activity. In addition, the introduction of Cl atom at the 4-position of the benzene ring belong to the aaminophosphonate moiety did somewhat contribute to antiviral activity. It is the first report on the antiviral activity of a,b-unsaturated amide derivatives bearing a-aminophosphonate moiety and present work provides a promising template for development of potential inhibitor of plant virus. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21672044 and 21562013) and Subsidy Project for Outstanding Key Laboratory of Guizhou Province in China (20154004) and the Provincial University Cooperation Plan of Guizhou Province in China (No. 20147001) and Collaborative Innovation Center for Natural Products and Biological Drugs of Yunnan for supporting the project. A. Supplementary data Supplementary data associated (containing information on the synthesis, characterization, and bioactivity test methods of the title compounds) with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.048. References 1. Bos L. Crop Prot. 1982;1:263. 2. Hu Q, Niu YP, Zhang K, Liu Y, Zhou XP. Virol J. 2011;8:1. 3. Wang ZW, Wang L, Ma S, Liu YX, Wang LZ, Wang QM. J Agric Food Chem. 2012;60:5825. 4. Long CW, Li P, Chen MH, Dong LR, Hu DY, Song BA. Eur J Med Chem. 2015;102:639.
5. Ji XF, Wang ZW, Dong J, Liu YX, Lu AD, Wang QM. J Agric Food Chem. 2016;64:9143. 6. Reichman M, Devash Y, Suhadolnik RJ, Sela I. Virology. 1983;128:240. 7. Ouyang GP, Cai XJ, Chen Z, et al. J Agric Food Chem. 2008;56:10160. 8. Song HJ, Liu YX, Liu YX, Huang YQ, Li YQ, Wang QM. Bioorg Med Chem Lett. 2014;24:5228. 9. Chen MH, Li P, Hu DY, et al. Bioorg Med Chem Lett. 2016;26:168. 10. Chen MH, Hu DY, Li XY, et al. Bioorg Med Chem Lett. 2015;25:3840. 11. Su B, Cai CL, Deng M, Liang DM, Wang LZ, Wang QM. Bioorg Med Chem Lett. 2014;24:2881. 12. Qian XH, Lee PW, Cao S. J Agric Food Chem. 2010;58:2613. 13. Seiber JN. J Agric Food Chem. 2011;59:1. 14. Tanaka A. Tokyo Government Chem Indus Res Inst Rep. 1972;20:792. 15. Guardiola F, Codony R, Addis PB, Rafecas M, Oatella J. Food Chem Toxicol. 1996;34:193. 16. Nystrom L, Makinen M, Lampi A, Piiromen V. J Agric Food Chem. 2005;53:2503. 17. Watt EVD, Pretorius JC. J Ethnopharmacol. 2001;76:87. 18. Hudson EA, Dinh PA, Kokubun T, Simmonds MSJ, Gescher A. Cancer Epidem Biomar. 2000;9:1163. 19. Yasukawa K, Akihisa T, Kimura Y, Tamura T, Takido M. Biol Pharm Bull. 1998;21:1072. 20. Wu M, Wang ZW, Meng CS, et al. PLoS ONE. 2013;8:22. 21. Mcleod DA, Brinkworth RI, Ashley JA, Janda KD, Wirsching P. Bioorg Med Chem Lett. 1991;1:653. 22. Lintunen T, Yli-Kauhaluoma JT. Bioorg Med Chem Lett. 2000;10:1749. 23. Jin JH, Song BA, Zhang GP, et al. Bioorg Med Chem Lett. 2006;16:1537. 24. Jin LH, Chen J, Song BA, et al. Bioorg Med Chem Lett. 2006;16:5036. 25. Roberts LR, Bradley PA, Bunnage ME, et al. Bioorg Med Chem Lett. 2011;21:6515. 26. Jia C, Yang KW, Liu CC, et al. Bioorg Med Chem Lett. 2012;22:482. 27. Luo GL, Chen L, Civiello R, et al. Bioorg Med Chem Lett. 2012;22:2917. 28. Yasuno Y, Hamada M, Yoshida Y, et al. Bioorg Med Chem Lett. 2016;26:3543. 29. Song BA, Zhang GP, Hu DY, et al. 2005; Patent: CN 1687088 A. 30. Long N, Cai XJ, Song BA, et al. J Agric Food Chem. 2008;56:5242. 31. Liu JZ, Song BA, Fan HT, et al. Eur J Med Chem. 2010;45:5108. 32. Zhou J, Fan HT, Song BA, et al. Phosphorus, Sulfur Silicon Relat Elem. 2011;186:81. 33. Chen Z, Li GJ, Fan HT, et al. Agr Sci Bull. 2011;27:250. 34. Xiao H, Li P, Hu DY, Song BA. Bioorg Med Chem Lett. 2014;24:3452. 35. Chen JL, Tang W, Che JY, et al. J Agric Food Chem. 2015;63:7219. 36. Che JY, Xu XY, Tang ZL, Gu YC, Shi DQ. Bioorg Med Chem Lett. 2016;26:1310. 37. Rashad AA, El-Sabbagh OI, Baraka MM, et al. Med Chem Res. 2010;19:1025. 38. Feng JY, Liu ZQ. Eur J Med Chem. 2011;46:1198. 39. Chiaradia LD, Martins PGA, Cordeiro MNS, et al. J Med Chem. 2012;55:390. 40. Banoglu E, Celikoglu E, Volker S, et al. Eur J Med Chem. 2016;113:1. 41. Kaboudin B, Moradi K. Tetrahedron Lett. 2005;46:2989. 42. Khatkar A, Nand A, Kumar P, Narasimhan B. Res Chem Intermed. 2015;41:299.