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Heterocyclic lactam derivatives containing piperonyl moiety as potential antifungal agents
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Heterocyclic lactam derivatives containing piperonyl moiety as potential antifungal agents Shuangshuang Wang, Longzhu Bao, Di Song, Jingjing Wang, Xiufang Cao
T
⁎
College of Science, Huazhong Agricultural University, Wuhan 430070, China
ARTICLE INFO
ABSTRACT
Keywords: Lactam Piperonyl Synthesis Antifungal activities Plant pathogenic fungi
To study the novel functionalized heterocyclic molecules with highly potential biological activity, two series of heterocyclic lactam derivatives containing the piperonyl moiety were designed and synthesized. The newly obtained compounds have been identified on the basis of analytical spectral data, including 1H NMR, 13C NMR, and ESI-MS. The target compounds were evaluated for their potential antifungal activities in vitro against twelve species of the plant pathogen fungi (Sclerotinia sclerotiorum, Rhizoctonia solani, Rap Sclerotinia stemrot, Fusarium graminearum, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Alternaria tenuis Nees, Monilinia fructicola, Colletotrichum gloeosporioides, Phytophthora capsici, Magnaporthe oryzae). Preliminary bioassays suggested that all prepared compounds I1–14 displayed broad-spectrum and moderate antifungal activities compared with the positive control hymexazol, especially for Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, and Monilinia fructicola. In particular, the inhibition rate of compound I9 exhibited good inhibition activity reached 95.16% against Sclerotinia sclerotiorum, and compounds I5, I12 against Phytophthora capsici were 93.44%, 91.25%. Further studies revealed that compounds I5 (IC50 = 19.13 µM) and I12 (IC50 = 9.12 µM) exhibited obviously antifungal activities against Phytophthora capsici, which were better than that of commercial agricultural fungicide hymexazol (IC50 = 325.45 µM). Therefore, these target compounds could be further studied and explored as a lead skeleton for discovery of novel antifungal agents.
In order to tackle the problems of environmental safety, food security, resistance and toxicity to non-target organism brought by the misuse of pesticides, it is extremely urgent to develop green and efficient new pesticides. Agricultural production steadily and economic development healthily benefits from the invention of novel green pesticides, and many scientists have worked hard to develop pesticides molecular libraries with high efficiency, low toxicity, environment friendly and independent intellectual property rights.1,2 What’s more, studies on novel functionalized agrochemicals are the essential way to achieve green agriculture, and leading optimization based on natural products has been an effective method to develop new pesticides. In addition, most of the compounds have heterocyclic structures in the world pesticide patents, and the study of heterocyclic pesticides has become a hot trend in new pesticides research.3,4 Among them, nitrogen-containing heterocyclic compounds have been widely used in pharmaceuticals, dyes, fine chemicals, etc.5 Therefore, nitrogen containing heterocyclic compounds with diverse biological activities and complicated structures play a vital role in green pesticides exploration. Tetramic acid is a type of natural molecular skeleton with a core of lactam, and the natural occurring tetramic acid derivatives are widely
⁎
found in marine organisms and terrestrial organisms such as bacteria, fungi, cyanobacteria, sponges.6,7 Due to the active cyclic keto-enol skeleton in their molecule structures, the tetramic acid derivatives have broad and unique biological activities, including insecticidal, acaricidal, herbicidal, antibacterial, antiviral, antitumor activities,8–13 what’s more, many compounds containing lactam fragment with natural source have the features of complex and diverse structures, multiple reactive sites, and wonderful modifications. In addition, as a series of important active intermediates, the piperonyl derivatives are widely used in spices, medicine, and pesticide fields (Fig. 1).14 The piperonyl ring-oriented derivatives have a wide range of pharmacological and physiological activities, like anti-tumor, anti-inflammatory, antibacterial, hypolipidemic, anti-parasite and central regulation.15–19 According to numerous reports, variety of clinical drugs containing piperonyl group exhibited good effects on anti-oxidation, and inhibited acetylcholinesterase (AChE). Thus, it is a more challenging and applied-promising project to find new analogues with significant antifungal activities by utilizing natural lactam derivatives with piperonyl moiety as a precursor. In summary, a series of novel lactam derivatives were designed in
Corresponding author. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.bmcl.2019.126661 Received 5 May 2019; Received in revised form 8 August 2019; Accepted 1 September 2019 Available online 03 September 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
S. Wang, et al.
Fig. 1. Representative structures of drugs and pesticides containing piperonyl moiety.
Fig. 2. Design strategies for lactam derivatives containing piperonyl moiety.
Scheme 1. General synthetic route for target compounds I1-14. 2
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
52.99 21.66 30.73 58.35 67.22 52.17 33.41 48.87 46.81 37.74 55.06 53.41 55.06 58.15 63.30 50.93
37.26 16.49 37.01 42.04 74.86 50.14 22.10 22.72 21.47 27.08 40.38 69.25 62.40 46.19 78.81 57.20
45.09 26.19 41.11 52.25 66.78 59.22 45.89 29.97 23.01 32.95 47.88 65.58 63.00 67.57 89.65 42.50
29.86 27.65 29.20 34.05 40.44 29.86 23.02 24.78 42.21 22.58 45.74 54.56 21.48 39.78 70.88 79.71
84.57 43.00 79.84 81.28 78.19 84.36 48.97 67.49 86.21 78.60 88.68 86.21 84.16 84.57 86.42 74.69
12.34 11.70 15.32 20.85 33.62 26.17 14.26 18.72 22.13 20.64 36.17 27.02 25.53 27.23 70.21 64.26
46.61 11.61 31.52 22.98 93.44 10.73 0.88 38.96 68.49 22.55 28.45 91.25 −17.71 9.42 63.24 15.11
60.83 22.98 60.40 54.49 58.87 49.02 40.05 6.79 3.73 2.85 29.77 28.67 6.35 24.51 100.00 67.84
this research, which were modified by the active cyclic keto-enol framework of natural antibiotic thiolactomycin and piperonyl skeleton. Specifically, novel heterocyclic tetramic acid derivatives were constructed efficiently by using lead optimization and pharmacophore hybridization strategies, whose structures containing the ether and ester bond (Fig. 2). Hence, we have explored the highly efficient preparation methods of tetramic acid derivatives. The in vitro antifungal activities of the target compounds against 12 pathogenic fungi were systematically evaluated by the plate method. The relationships between the structure and biological activities were preliminarily analyzed, which were expected to screening the novel tetramic acid derivatives bearing piperonyl group with potential significant activities. We hope that it can provide a certain theoretical basis and technical support for the development of green pesticides and new antifungal agents. In this paper, a series of novel heterocyclic lactam derivatives I1-14 containing the piperonyl skeleton were synthesized, and the detailed structures and synthetic pathways of target molecules were shown in Scheme 1. The important intermediates 5 were synthesized with some mild and simple modifications according to the previously reported method,20,21 in brief, which could be obtained by treating the readily available starting materials (proline and pipecolinic acid, substituted phenylacetic acid) with four steps including esterification, acylation, Nacylation, and intramolecular heterocyclization. Subsequently, the key intermediate 5 were etherified with piperonyl chloride in a solution of potassium carbonate in dry acetonitrile to give the desired products I1–7, and the target molecules I8–14 were provided from intermediates 5 with piperonyloyl chloride in the presence of Et3N in CH2Cl2. All newly tetramic acid derivatives were firstly reported with good yields and fully characterized by satisfactory spectroscopic analysis, including 1 H NMR, 13C NMR and ESI-MS. The chemical structures and basic physicochemical properties of the compounds I1–14 and the representative spectroscopy studies of the compound I14 (Figs. S1 and S2) were summarized in Materials and methods (Supporting Information). All newly prepared tetramic acid derivatives with the piperonyl skeleton were evaluated for their antifungal effects in vitro against twelve common types of the plant pathogen fungi (Sclerotinia sclerotiorum, Rhizoctonia solani, Rap Sclerotinia stemrot, Fusarium graminearum, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Alternaria tenuis Nees, Monilinia fructicola, Colletotrichum gloeosporioides, Phytophthora capsici, Magnaporthe oryzae) at a concentration of 100 µg/ mL by the standard petri plate test for determining fungicide inhibition of mycelial growth method using commercial agricultural fungicide hymexazol as positive control.22 The preliminary screening results were outlined in Table 1 and Fig. S3 (Supporting Information). As shown in Table 1 and Fig. S3, the preliminary bio-assay indicated that some of the presented tetramic acid derivatives I1–14 showed moderate to good inhibitory effects against twelve plant pathogenic fungi, especially for Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, Monilinia fructicola. Notably, the compounds I5 and I12 exhibited outstanding antifungal effects against all tested fungus at 100 µg/mL compared with other compounds. Furthermore, the compounds I8, I9, I11, and I12 showed selective antifungal activity to Sclerotinia sclerotiorum, whose excellent inhibitory activities were 87.08%, 95.16%, 87.48%, and 84.25% respectively. Many title compounds exhibited higher inhibitory effects against Rap Sclerotinia stemrot, significantly, the inhibition rates of compounds I3, I6, I10 and I14 reached 85.77%, 88.50%, 87.53%, and 82.26%. Compound I5 also showed selective antifungal activity to Pestallozzia theae with 74.86% inhibition compared with the control hymexazol (57.20%). It was interesting to note that many compounds showed more significant inhibitory effects against Monilinia fructicola, particularly compounds I11 (88.68%) and I12 (86.21%) compared with hymexazol (74.69%). Moreover, compound I5 and I12 indicated excellent selective inhibitory effects to Phytophthora capsici, the inhibition rates of I5 (93.44%) and I12 (91.25%) were also better than the positive control hymexazol
The high activities data are represented in bold.
38.74 8.50 38.34 36.36 57.71 40.71 25.10 48.22 39.13 18.58 45.06 53.16 31.03 37.55 71.94 95.45 76.22 69.59 85.77 73.88 73.69 88.50 54.39 71.93 79.14 87.53 77.78 79.34 74.27 82.26 92.20 97.08 38.34 30.83 47.83 36.36 44.27 39.92 29.25 40.51 46.25 42.49 50.20 48.22 28.26 37.15 72.33 56.52 36.61 20.06 66.89 32.57 76.18 46.30 25.71 87.08 95.16 43.07 87.48 84.25 51.55 53.57 83.24 98.39 I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 I13 I14 Piperine Hymexazol
Phomopsis adianticola Fusarium graminearum Rap Sclerotinia stemrot Rhizoctonia solani Sclerotinia sclerotiorum
In vitro Fungicidal activity (%) Compd. No.
Table 1 In vitro fungicidal activity of novel tetramic acid derivatives I1-14 at 100 µg/mL.
Pestallozzia theae
Pestalotiopsis guepinii
Alternaria tenuis Nees
Monilinia fructicola
Colletotrichum gloeosporioides
Phytophthora capsici
Magnaporthe oryzae
S. Wang, et al.
3
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
S. Wang, et al.
Table 2 IC50 values of selective compounds I1-14 against the plant pathogen fungi. Compd. No.
I1 I3 I4 I5 I6 I8 I9 I11 I12 I13 I14 Piperine Hymexazol
Substituents
IC50a (µM)
R1
n
Sclerotinia sclerotiorum
Rap Sclerotinia stemrot
Phomopsis adianticola
Pestallozzia theae
Pestalotiopsis guepinii
Monilinia fructicola
Phytophthora capsici
Magnaporthe oryzae
H 2,4-Cl2 H 2-Me 2,4-Cl2 H 2-Me 4-F 2-Me 2,4-Cl2 4-MeO – –
1 1 2 2 2 1 1 1 2 2 2 – –
/b / / 109.63 / 280.84 37.33 307.87 10.27 / / 112.74 33.03
/ / / / 49.16 / / / 70.18 / / 114.04 260.00
/ / / 205.09 / / / / / / / 324.81 941.21
/ / / 151.15 / / / / 439.45 / / 53.90 /
/ / / / / / / / 380.15 / 251.78 27.02 /
19.46 / 33.60 / 106.64 / 151.80 101.51 70.59 678.97 186.37 10.02 106.17
/ / / 19.13 / / / / 9.12 / / 183.89 325.45
/ 36.82 / / / / / / / / / 77.56 35.15
The high activities data are represented in bold. a IC50 – compound concentration required to inhibit colony growth by 50%. b – No determination.
Fig. 3. Inhibitory effect of compounds I5, I12, piperine, and hymexazol on Phytophthora capsici.
(15.11%). Above all, compounds I5 and I12 could be used as potential heterocyclic skeleton for further studies on pharmaceutical molecular discovery. With the aim to evaluate the potential activities of compounds I1–14, the IC50 values were further investigated according to the above preliminary screening results. Some of the compounds with good activities were selected for the further exploration against eight fungus in agriculture (Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Monilinia fructicola, Phytophthora capsici, Magnaporthe oryzae) at different concentration (3.125, 6.25, 12.5, 25, 50, 100 µg/mL), and the commercial fungicide hymexazol was used as the positive control. The IC50 of the compounds were calculated by SPSS 22.0 software, and the values of IC50 for all tetramic acid derivatives bearing the piperonyl moiety were summarized in Table 2. As shown in Table 2, Compounds I5 and I12 showed broad spectrum effects against all tested plant pathogen fungi. Compound I12 (IC50 = 10.27 μM) showed the better inhibitory effect against Sclerotinia sclerotiorum than the piperine (IC50 = 112.74 µM), which had the same inhibition activities trend as hymexazol (IC50 = 33.03 μM). In addition, it could be found that compounds I-6 and I-12 also had the same trend as hymexazol against Rap Sclerotinia stemrot. Compound I1
exhibited the good inhibitory effect against Monilinia fructicola with an IC50 values of 19.46 μM. Especially, the IC50 values of the significant compounds I5, and I12 against Phytophthora capsici were up to 19.13 and 9.12 μM obviously better than piperine (IC50 = 183.39 μM) and hymexazol (IC50 = 325.45 μM). The above results suggested that the tetramic acid derivatives I5, and I12 could be regarded as the new structural templates for highly effective fungicides research. Furthermore, the comparison chart of Fig. 3 showed the inhibitory effects of title compounds I5, I12, piperine and hymexazol on Phytophthora capsici at the concentration of 3.125, 6.25, 12.5, 25, 50, 100 µg/mL. It could be seen from the Fig. 3 that the compounds I5 and I12 displayed the superior inhibitory activities on the Phytophthora capsici at different concentrations compared to the piperine and hymexazol. Based on the contents of the above-mentioned antifungal activity (Tables 1 and 2), the structure-activity relationships (SARs) of the target compound I1–14 with different substituents were discussed subsequently. Firstly, for the compounds bearing with different piperidinyl and piperonyl moieties, the results showed that the ether substituted compounds exhibited better activity than the ester substituted compounds. When different saturated cycloalkyl groups (n = 1, 2) were attached to the tetramic acid ring, it could be seen that the compounds 4
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
S. Wang, et al.
with a six-membered ring (n = 2) had better antibacterial activities than the five-membered ring compounds (n = 1). In addition, when the compounds with different substituents R1 (poly-substituted phenyl), it could be roughly concluded that in the compounds I1–7 containing ether bond, the structural activity relationships of the derivatives substituent with five-membered ring was 2,4-Cl2 > H > 2-Me. Correspondingly, the 2-methyl substituted compounds with six-membered ring had the best activities, followed by 2,4-dichloro substituted compounds, and 4-Methoxy substituted compounds had the worst. In the compounds I8–14 containing ester bond, the 2-methyl substituted compounds with six-membered ring displayed still the best activities, and the structural activity relationships of the other compounds with five-membered ring was 4-F > 2-Me > H, 2,4-Cl2. In the present study, a series of novel heterocyclic tetramic acid derivatives containing piperonyl moiety were designed, synthesized and evaluated for their potential fungicidal activities against twelve common plant pathogenic fungi. The structures of all obtained molecules were confirmed by 1H NMR. 13C NMR, and ESI-MS. Preliminary bioassays showed that all target compounds revealed broad-spectrum and moderate antifungal activities against the tested fungus, especially against Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, and Monilinia fructicola. It was interesting to note that compounds I5 (IC50 = 19.13 µM) and I12 (IC50 = 9.12 µM) presented better selective inhibitory activity against Phytophthora capsici than the positive control hymexazol (IC50 = 325.45 µM). Moreover, we had also summarized the potential structure-activity relationships of compounds preliminarily, it could be found that the compounds I1–7 with ether bonds exhibited better effects than compounds I8–14 with ester bond, and the inhibitory activities of six-membered substituted compounds were superior than that of compounds with five-membered ring. Therefore, compounds I5 and I12 could lay a foundation for the subsequent design synthesis and structural optimization of novel tetramic acid derivatives.
References [1]. Yang G. A brief introduction of pesticide and pest resistance. Agrochemicals. 2017;56:213–215. [2]. Liu J, He H, Feng X. Direction of chemical pesticide development green chemical pesticides. Pesticides. 2005;44:1–4. [3]. Newman DJ, Cragg GM, Snader KM. Natural products as sources of new drugs over the period 1981–2002. J Nat Prod. 2003;66:1022–1037. [4]. Cragg GM, Newman DJ, Snader KM. Natural product in drug discovery and development. J Nat Prod. 1997;60:52–60. [5]. Wang D. Important role of heterocyclic compounds in developing pesticides. Pesticides. 1995;34:6–9. [6]. Henning HG, Gelbin A. Advances in heterocyclic chemistry. Adv Heterocycl Chem. 1993;57:139–185. [7]. Liang X, Huang Z, Ma X, Qi S. Unstable tetramic acid derivatives from the deep-seaderived fungus Cladosporium sphaerospermum EIODSF 008. Mar Drugs. 2018;16:448. [8]. Mo X, Li Q, Ju J. Naturally occurring tetramic acid products: isolation, structure elucidation and biological activity. RSC Adv. 2014;4:50566–50593. [9]. Ghisalberti EL. Bioactive tetramic acid metabolites. Stud Nat Prod Chem. 2003;28:109–163. [10]. Liang Y, Ke S, Wang K, Yang Z. Progress in the studies on natural bioactive pyrrolidine-2,4-diones. Chin J Org Chem. 2010;30:1801–1810. [11]. Royles BJL. Naturally occurring tetramic acids: structure, isolation, and synthesis. Chem Rev. 1995;95:1981–2001. [12]. Schobert R, Schlenk A. Tetramic and tetronic acids: an update on new derivatives and biological aspects. Bioorg Med Chem. 2008;16:4203–4221. [13]. Popova MV, Dobrydnev AV, Dyachenko MS, Duhayon C, Listunov D, Volovenko YM. Synthesis of a series of tetraminic acid sulfone analogs. Monatsh Chem. 2017;148:939–946. [14]. Yang R, Lv M, Xu H. Synthesis of piperine analogs containing isoxazoline/pyrazoline scaffold and their pesticidal bioactivities. J Agric Food Chem. 2018;66:11254–11264. [15]. Fernandes IA, Almeida LD, Ferreira PE, et al. Synthesis and biological evaluation of novel piperidine-benzodioxole derivatives designed as potential leishmanicidal drug candidates. Bioorg Med Chem Lett. 2015;25:3346–3349. [16]. Franklim TN, Lima LF, Sá JDN, et al. Design, synthesis and trypanocidal evaluation of novel 1,2,4-triazoles-3-thiones derived from natural piperine. Molecules. 2013;18:6366–6382. [17]. Mu L, Wang B, Ren H, et al. Synthesis and inhibitory effect of piperine derivates on monoamine oxidase. Bioorg Med Chem Lett. 2012;22:3343–3348. [18]. Ali Y, Alam MS, Hamid H, et al. Design, synthesis and biological evaluation of piperic acid triazolyl derivatives as potent anti-inflammatory agents. Eur J Med Chem. 2015;92:490–500. [19]. Pathak N, Khandlewal S. Cytoprotective and immunomodulating properties of piperine on murine splinocytes: an in vitro study. Eur J Pharmacol. 2007;576:160–170. [20]. Wang S, Bao L, Wang W, Song D, Wang J, Cao X. Heterocyclic pyrrolizinone and indolizinones derived from natural lactam as potential antifungal agents. Fitoterapia. 2018;129:257–266. [21]. Ke S, Zhang Y, Shu W, et al. Structural diversity-guided convenient construction of functionalized polysubstituted butenolides and lactam derivatives. C R Chim. 2011;14:1071–1079. [22]. Lv P, Chen Y, Zhao Z, et al. Design, synthesis, and antifungal activities of 3–acyl thiotetronic acid derivatives: New fatty acid synthase inhibitors. J Agric Food Chem. 2018;66:1023–1032.
Acknowledgement We gratefully acknowledge the support of this work by the Fundamental Research Funds for the Central Universities (2662016PY112). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126661.
5
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Heterocyclic lactam derivatives containing piperonyl moiety as potential antifungal agents Shuangshuang Wang, Longzhu Bao, Di Song, Jingjing Wang, Xiufang Cao
T
⁎
College of Science, Huazhong Agricultural University, Wuhan 430070, China
ARTICLE INFO
ABSTRACT
Keywords: Lactam Piperonyl Synthesis Antifungal activities Plant pathogenic fungi
To study the novel functionalized heterocyclic molecules with highly potential biological activity, two series of heterocyclic lactam derivatives containing the piperonyl moiety were designed and synthesized. The newly obtained compounds have been identified on the basis of analytical spectral data, including 1H NMR, 13C NMR, and ESI-MS. The target compounds were evaluated for their potential antifungal activities in vitro against twelve species of the plant pathogen fungi (Sclerotinia sclerotiorum, Rhizoctonia solani, Rap Sclerotinia stemrot, Fusarium graminearum, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Alternaria tenuis Nees, Monilinia fructicola, Colletotrichum gloeosporioides, Phytophthora capsici, Magnaporthe oryzae). Preliminary bioassays suggested that all prepared compounds I1–14 displayed broad-spectrum and moderate antifungal activities compared with the positive control hymexazol, especially for Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, and Monilinia fructicola. In particular, the inhibition rate of compound I9 exhibited good inhibition activity reached 95.16% against Sclerotinia sclerotiorum, and compounds I5, I12 against Phytophthora capsici were 93.44%, 91.25%. Further studies revealed that compounds I5 (IC50 = 19.13 µM) and I12 (IC50 = 9.12 µM) exhibited obviously antifungal activities against Phytophthora capsici, which were better than that of commercial agricultural fungicide hymexazol (IC50 = 325.45 µM). Therefore, these target compounds could be further studied and explored as a lead skeleton for discovery of novel antifungal agents.
In order to tackle the problems of environmental safety, food security, resistance and toxicity to non-target organism brought by the misuse of pesticides, it is extremely urgent to develop green and efficient new pesticides. Agricultural production steadily and economic development healthily benefits from the invention of novel green pesticides, and many scientists have worked hard to develop pesticides molecular libraries with high efficiency, low toxicity, environment friendly and independent intellectual property rights.1,2 What’s more, studies on novel functionalized agrochemicals are the essential way to achieve green agriculture, and leading optimization based on natural products has been an effective method to develop new pesticides. In addition, most of the compounds have heterocyclic structures in the world pesticide patents, and the study of heterocyclic pesticides has become a hot trend in new pesticides research.3,4 Among them, nitrogen-containing heterocyclic compounds have been widely used in pharmaceuticals, dyes, fine chemicals, etc.5 Therefore, nitrogen containing heterocyclic compounds with diverse biological activities and complicated structures play a vital role in green pesticides exploration. Tetramic acid is a type of natural molecular skeleton with a core of lactam, and the natural occurring tetramic acid derivatives are widely
⁎
found in marine organisms and terrestrial organisms such as bacteria, fungi, cyanobacteria, sponges.6,7 Due to the active cyclic keto-enol skeleton in their molecule structures, the tetramic acid derivatives have broad and unique biological activities, including insecticidal, acaricidal, herbicidal, antibacterial, antiviral, antitumor activities,8–13 what’s more, many compounds containing lactam fragment with natural source have the features of complex and diverse structures, multiple reactive sites, and wonderful modifications. In addition, as a series of important active intermediates, the piperonyl derivatives are widely used in spices, medicine, and pesticide fields (Fig. 1).14 The piperonyl ring-oriented derivatives have a wide range of pharmacological and physiological activities, like anti-tumor, anti-inflammatory, antibacterial, hypolipidemic, anti-parasite and central regulation.15–19 According to numerous reports, variety of clinical drugs containing piperonyl group exhibited good effects on anti-oxidation, and inhibited acetylcholinesterase (AChE). Thus, it is a more challenging and applied-promising project to find new analogues with significant antifungal activities by utilizing natural lactam derivatives with piperonyl moiety as a precursor. In summary, a series of novel lactam derivatives were designed in
Corresponding author. E-mail address: [email protected] (X. Cao).
https://doi.org/10.1016/j.bmcl.2019.126661 Received 5 May 2019; Received in revised form 8 August 2019; Accepted 1 September 2019 Available online 03 September 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
S. Wang, et al.
Fig. 1. Representative structures of drugs and pesticides containing piperonyl moiety.
Fig. 2. Design strategies for lactam derivatives containing piperonyl moiety.
Scheme 1. General synthetic route for target compounds I1-14. 2
Bioorganic & Medicinal Chemistry Letters 29 (2019) 126661
52.99 21.66 30.73 58.35 67.22 52.17 33.41 48.87 46.81 37.74 55.06 53.41 55.06 58.15 63.30 50.93
37.26 16.49 37.01 42.04 74.86 50.14 22.10 22.72 21.47 27.08 40.38 69.25 62.40 46.19 78.81 57.20
45.09 26.19 41.11 52.25 66.78 59.22 45.89 29.97 23.01 32.95 47.88 65.58 63.00 67.57 89.65 42.50
29.86 27.65 29.20 34.05 40.44 29.86 23.02 24.78 42.21 22.58 45.74 54.56 21.48 39.78 70.88 79.71
84.57 43.00 79.84 81.28 78.19 84.36 48.97 67.49 86.21 78.60 88.68 86.21 84.16 84.57 86.42 74.69
12.34 11.70 15.32 20.85 33.62 26.17 14.26 18.72 22.13 20.64 36.17 27.02 25.53 27.23 70.21 64.26
46.61 11.61 31.52 22.98 93.44 10.73 0.88 38.96 68.49 22.55 28.45 91.25 −17.71 9.42 63.24 15.11
60.83 22.98 60.40 54.49 58.87 49.02 40.05 6.79 3.73 2.85 29.77 28.67 6.35 24.51 100.00 67.84
this research, which were modified by the active cyclic keto-enol framework of natural antibiotic thiolactomycin and piperonyl skeleton. Specifically, novel heterocyclic tetramic acid derivatives were constructed efficiently by using lead optimization and pharmacophore hybridization strategies, whose structures containing the ether and ester bond (Fig. 2). Hence, we have explored the highly efficient preparation methods of tetramic acid derivatives. The in vitro antifungal activities of the target compounds against 12 pathogenic fungi were systematically evaluated by the plate method. The relationships between the structure and biological activities were preliminarily analyzed, which were expected to screening the novel tetramic acid derivatives bearing piperonyl group with potential significant activities. We hope that it can provide a certain theoretical basis and technical support for the development of green pesticides and new antifungal agents. In this paper, a series of novel heterocyclic lactam derivatives I1-14 containing the piperonyl skeleton were synthesized, and the detailed structures and synthetic pathways of target molecules were shown in Scheme 1. The important intermediates 5 were synthesized with some mild and simple modifications according to the previously reported method,20,21 in brief, which could be obtained by treating the readily available starting materials (proline and pipecolinic acid, substituted phenylacetic acid) with four steps including esterification, acylation, Nacylation, and intramolecular heterocyclization. Subsequently, the key intermediate 5 were etherified with piperonyl chloride in a solution of potassium carbonate in dry acetonitrile to give the desired products I1–7, and the target molecules I8–14 were provided from intermediates 5 with piperonyloyl chloride in the presence of Et3N in CH2Cl2. All newly tetramic acid derivatives were firstly reported with good yields and fully characterized by satisfactory spectroscopic analysis, including 1 H NMR, 13C NMR and ESI-MS. The chemical structures and basic physicochemical properties of the compounds I1–14 and the representative spectroscopy studies of the compound I14 (Figs. S1 and S2) were summarized in Materials and methods (Supporting Information). All newly prepared tetramic acid derivatives with the piperonyl skeleton were evaluated for their antifungal effects in vitro against twelve common types of the plant pathogen fungi (Sclerotinia sclerotiorum, Rhizoctonia solani, Rap Sclerotinia stemrot, Fusarium graminearum, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Alternaria tenuis Nees, Monilinia fructicola, Colletotrichum gloeosporioides, Phytophthora capsici, Magnaporthe oryzae) at a concentration of 100 µg/ mL by the standard petri plate test for determining fungicide inhibition of mycelial growth method using commercial agricultural fungicide hymexazol as positive control.22 The preliminary screening results were outlined in Table 1 and Fig. S3 (Supporting Information). As shown in Table 1 and Fig. S3, the preliminary bio-assay indicated that some of the presented tetramic acid derivatives I1–14 showed moderate to good inhibitory effects against twelve plant pathogenic fungi, especially for Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, Monilinia fructicola. Notably, the compounds I5 and I12 exhibited outstanding antifungal effects against all tested fungus at 100 µg/mL compared with other compounds. Furthermore, the compounds I8, I9, I11, and I12 showed selective antifungal activity to Sclerotinia sclerotiorum, whose excellent inhibitory activities were 87.08%, 95.16%, 87.48%, and 84.25% respectively. Many title compounds exhibited higher inhibitory effects against Rap Sclerotinia stemrot, significantly, the inhibition rates of compounds I3, I6, I10 and I14 reached 85.77%, 88.50%, 87.53%, and 82.26%. Compound I5 also showed selective antifungal activity to Pestallozzia theae with 74.86% inhibition compared with the control hymexazol (57.20%). It was interesting to note that many compounds showed more significant inhibitory effects against Monilinia fructicola, particularly compounds I11 (88.68%) and I12 (86.21%) compared with hymexazol (74.69%). Moreover, compound I5 and I12 indicated excellent selective inhibitory effects to Phytophthora capsici, the inhibition rates of I5 (93.44%) and I12 (91.25%) were also better than the positive control hymexazol
The high activities data are represented in bold.
38.74 8.50 38.34 36.36 57.71 40.71 25.10 48.22 39.13 18.58 45.06 53.16 31.03 37.55 71.94 95.45 76.22 69.59 85.77 73.88 73.69 88.50 54.39 71.93 79.14 87.53 77.78 79.34 74.27 82.26 92.20 97.08 38.34 30.83 47.83 36.36 44.27 39.92 29.25 40.51 46.25 42.49 50.20 48.22 28.26 37.15 72.33 56.52 36.61 20.06 66.89 32.57 76.18 46.30 25.71 87.08 95.16 43.07 87.48 84.25 51.55 53.57 83.24 98.39 I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 I13 I14 Piperine Hymexazol
Phomopsis adianticola Fusarium graminearum Rap Sclerotinia stemrot Rhizoctonia solani Sclerotinia sclerotiorum
In vitro Fungicidal activity (%) Compd. No.
Table 1 In vitro fungicidal activity of novel tetramic acid derivatives I1-14 at 100 µg/mL.
Pestallozzia theae
Pestalotiopsis guepinii
Alternaria tenuis Nees
Monilinia fructicola
Colletotrichum gloeosporioides
Phytophthora capsici
Magnaporthe oryzae
S. Wang, et al.
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Table 2 IC50 values of selective compounds I1-14 against the plant pathogen fungi. Compd. No.
I1 I3 I4 I5 I6 I8 I9 I11 I12 I13 I14 Piperine Hymexazol
Substituents
IC50a (µM)
R1
n
Sclerotinia sclerotiorum
Rap Sclerotinia stemrot
Phomopsis adianticola
Pestallozzia theae
Pestalotiopsis guepinii
Monilinia fructicola
Phytophthora capsici
Magnaporthe oryzae
H 2,4-Cl2 H 2-Me 2,4-Cl2 H 2-Me 4-F 2-Me 2,4-Cl2 4-MeO – –
1 1 2 2 2 1 1 1 2 2 2 – –
/b / / 109.63 / 280.84 37.33 307.87 10.27 / / 112.74 33.03
/ / / / 49.16 / / / 70.18 / / 114.04 260.00
/ / / 205.09 / / / / / / / 324.81 941.21
/ / / 151.15 / / / / 439.45 / / 53.90 /
/ / / / / / / / 380.15 / 251.78 27.02 /
19.46 / 33.60 / 106.64 / 151.80 101.51 70.59 678.97 186.37 10.02 106.17
/ / / 19.13 / / / / 9.12 / / 183.89 325.45
/ 36.82 / / / / / / / / / 77.56 35.15
The high activities data are represented in bold. a IC50 – compound concentration required to inhibit colony growth by 50%. b – No determination.
Fig. 3. Inhibitory effect of compounds I5, I12, piperine, and hymexazol on Phytophthora capsici.
(15.11%). Above all, compounds I5 and I12 could be used as potential heterocyclic skeleton for further studies on pharmaceutical molecular discovery. With the aim to evaluate the potential activities of compounds I1–14, the IC50 values were further investigated according to the above preliminary screening results. Some of the compounds with good activities were selected for the further exploration against eight fungus in agriculture (Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, Phomopsis adianticola, Pestallozzia theae, Pestalotiopsis guepinii, Monilinia fructicola, Phytophthora capsici, Magnaporthe oryzae) at different concentration (3.125, 6.25, 12.5, 25, 50, 100 µg/mL), and the commercial fungicide hymexazol was used as the positive control. The IC50 of the compounds were calculated by SPSS 22.0 software, and the values of IC50 for all tetramic acid derivatives bearing the piperonyl moiety were summarized in Table 2. As shown in Table 2, Compounds I5 and I12 showed broad spectrum effects against all tested plant pathogen fungi. Compound I12 (IC50 = 10.27 μM) showed the better inhibitory effect against Sclerotinia sclerotiorum than the piperine (IC50 = 112.74 µM), which had the same inhibition activities trend as hymexazol (IC50 = 33.03 μM). In addition, it could be found that compounds I-6 and I-12 also had the same trend as hymexazol against Rap Sclerotinia stemrot. Compound I1
exhibited the good inhibitory effect against Monilinia fructicola with an IC50 values of 19.46 μM. Especially, the IC50 values of the significant compounds I5, and I12 against Phytophthora capsici were up to 19.13 and 9.12 μM obviously better than piperine (IC50 = 183.39 μM) and hymexazol (IC50 = 325.45 μM). The above results suggested that the tetramic acid derivatives I5, and I12 could be regarded as the new structural templates for highly effective fungicides research. Furthermore, the comparison chart of Fig. 3 showed the inhibitory effects of title compounds I5, I12, piperine and hymexazol on Phytophthora capsici at the concentration of 3.125, 6.25, 12.5, 25, 50, 100 µg/mL. It could be seen from the Fig. 3 that the compounds I5 and I12 displayed the superior inhibitory activities on the Phytophthora capsici at different concentrations compared to the piperine and hymexazol. Based on the contents of the above-mentioned antifungal activity (Tables 1 and 2), the structure-activity relationships (SARs) of the target compound I1–14 with different substituents were discussed subsequently. Firstly, for the compounds bearing with different piperidinyl and piperonyl moieties, the results showed that the ether substituted compounds exhibited better activity than the ester substituted compounds. When different saturated cycloalkyl groups (n = 1, 2) were attached to the tetramic acid ring, it could be seen that the compounds 4
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with a six-membered ring (n = 2) had better antibacterial activities than the five-membered ring compounds (n = 1). In addition, when the compounds with different substituents R1 (poly-substituted phenyl), it could be roughly concluded that in the compounds I1–7 containing ether bond, the structural activity relationships of the derivatives substituent with five-membered ring was 2,4-Cl2 > H > 2-Me. Correspondingly, the 2-methyl substituted compounds with six-membered ring had the best activities, followed by 2,4-dichloro substituted compounds, and 4-Methoxy substituted compounds had the worst. In the compounds I8–14 containing ester bond, the 2-methyl substituted compounds with six-membered ring displayed still the best activities, and the structural activity relationships of the other compounds with five-membered ring was 4-F > 2-Me > H, 2,4-Cl2. In the present study, a series of novel heterocyclic tetramic acid derivatives containing piperonyl moiety were designed, synthesized and evaluated for their potential fungicidal activities against twelve common plant pathogenic fungi. The structures of all obtained molecules were confirmed by 1H NMR. 13C NMR, and ESI-MS. Preliminary bioassays showed that all target compounds revealed broad-spectrum and moderate antifungal activities against the tested fungus, especially against Sclerotinia sclerotiorum, Rap Sclerotinia stemrot, and Monilinia fructicola. It was interesting to note that compounds I5 (IC50 = 19.13 µM) and I12 (IC50 = 9.12 µM) presented better selective inhibitory activity against Phytophthora capsici than the positive control hymexazol (IC50 = 325.45 µM). Moreover, we had also summarized the potential structure-activity relationships of compounds preliminarily, it could be found that the compounds I1–7 with ether bonds exhibited better effects than compounds I8–14 with ester bond, and the inhibitory activities of six-membered substituted compounds were superior than that of compounds with five-membered ring. Therefore, compounds I5 and I12 could lay a foundation for the subsequent design synthesis and structural optimization of novel tetramic acid derivatives.
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Acknowledgement We gratefully acknowledge the support of this work by the Fundamental Research Funds for the Central Universities (2662016PY112). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126661.
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