Synthesis and biological activity of amide derivatives derived from natural product Waltherione F

Structural optimization based on natural products has become an effective way to develop new fungicides, which provides important guiding significance for practicing the new development concept and promoting the green development of pesticides. In this project, the target compounds containing 4-quinolone and piperazine substructures based on waltherione F were synthesized through the combination of the fungicidal amide lead compound X-I-4 discovered in our previous work and various of fungicidal piperazine derivatives. Screening of their biological activities suggested that products I-3, I-5, II-3, II-7, II-10, II-11 and II-13 displayed higher inhibition rates against Rhizoctonia solani than other tested compounds. The in vitro cellular cytotoxicity assay revealed that compounds II-6 and II-11 exhibited higher cytotoxicity against HepG2 than other tested compounds. The fluorescence characteristics investigation showed that the absolute fluorescence QY value of the methanol solution of the compound I-6 was higher than those of I-2, I-3, I-7 and I-8, which was further elucidated by TD-DFT. Graphical abstract Graphical abstract


Introduction
Fungicides are important agrochemicals that occupied nearly 30% of the agrochemical market, providing significant material support for modern agricultural development. However, the long-term and large-scale use of traditional chemical synthetic fungicides has produced some negative problems, such as environmental pollution, pathogen resistance, and poisoning to beneficial insects and microorganisms [1,2]. Therefore, the development of environmentally friendly fungicides with high-efficiency and low-toxicity has become an inevitable trend for pesticide innovation [3,4].
Natural products have attracted extensive attention due to their novel structures, unique mechanism of action, abundance, no cross-resistance, fast biodegradation, and ecofriendliness. For example, osthol, matrine, berberine, and eugenol have been successfully used in the control of agricultural pathogens [5,6]. While natural products also present defects such as low extraction rate, difficult chemical synthesis, poor environmental stability, and insufficient biological activity [7]. The structural optimization of natural products has become an effective way to develop new pesticides, which can effectively solve the abovementioned problems [7][8][9][10][11]. For example, natural strobilurin A-derived methoxyacrylate fungicides are the top-tier in the fungicide market. The commercial fungicides dimethomorph, pyrimorph and flumorph were developed successively with natural cinnamic acid as a scaffold; in additon, the neonicotinoid and pyrethroid insecticides were successfully formulated from the structural optimization of natural nicotine and pyrethrins (Fig. 1).
Waltherione F isolated from Waltheria indica L. of the Sterculiaceae family is a 4-quinolone alkaloid that exhibits good antifungal activity [12][13][14]. In our previous work, 4-quinolone derivatives QD and quinoline derivatives X-II derived from natural waltherione F were synthesized, in which compounds QD-1 and X-II-5 were discovered to present superior fungicidal activity (Fig. 2) [15]. In this project, combined with the fungicidal amide lead compound X-I-4 discovered in our previous work and fungicidal piperazine compounds P-1 and P-2 reported in literature [16][17][18][19][20][21], the amide derivatives containing 4-quinolone and piperazine substructures were prepared, following with the screening of their biological activities (Fig. 3).

Organic synthesis
The corresponding intermediates and amide derivatives I-1 -I-9, II-1 -II-13 containing the 4-quinolone group were synthesized in accordance with the procedures displayed in Scheme 1. First, the material 2-methoxy-5-methylaniline was reacted with dimethyl acetylene to prepare molecule 2, which was cyclized to give ester 3 via the Conrad-Limpach reaction using polyphosphoric acid (PPA) as the condensing reagent. The carboxylic acid 4 was obtained from the hydrolysis of the ester group with lithium hydroxide, and reacted with substituted amines to afford the target molecules I-1 -I-9 via the HATU/DIEA catalyzed condensation reaction. In the preparation of the target molecules II-1 -II-13, the intermediate 5 was produced by the condensation reaction of the carboxylic acid 4 and tert-butyl piperazine-1-carboxylate under analogous conditions, which was further deprotected with trifluoroacetic acid to provide the amide derivative 6. Finally, various types of carboxylic acids reacted with the resultant intermediate 6 to gain the target molecules II-1 -II-13 using EDCI/HOBt as the condensing reagent. Subsequently, the obtained molecules were characterized with 1 H and 13 C NMR, and HRMS. Moreover, crystal structure analysis helps to accurately and intuitively determine the structures of the target molecules, as well as to investigate their physical and chemical properties. The crystal of compound I-6 was cultivated from methanol and DCM, and determined on a Bruker D8 Venture diffractometer to provide several structural characteristics, in which π-π interactions occurred between the benzene rings and 4-quinolone rings of the adjacent molecules to strengthen the integration of the crystal molecules ( Fig. 4, CCDC Number 2112787). In addition, given the melting points and TLC monitoring results, the target molecules exhibited strong polarity characteristics.

Fungicidal activity and in vitro cellular cytotoxicity
Waltherione alkaloids were reported to display good antifungal activity and cytotoxicity [12]. In this work, the measurement of fungicidal activity and cytotoxicity can improve our understanding of the structure-activity relationship of amide derivatives derived from waltherine F in order to guide the design and synthesis of more active target molecules. Therefore, the in vitro fungicidal activity of the target compounds against common agricultural pathogens and the in vitro cellular cytotoxicity assay against HepG2 were investigated, respectively ( Table 1, 2). The compounds I-1 -I-9 and II-1 -II-13 showed weak fungicidal activity against the tested agricultural pathogens, which may be related to the strong polarity of the compounds. However, in a given category, several compounds displayed higher inhibition rates against the specifically tested pathogens than other compounds. For example, the compounds I-3, I-5, II-3, II-7, II-11 and II-13 exhibited better fungicidal activity against Rhizoctonia solani than other compounds. Moreover, the compounds I-8 and II-6 showed better fungicidal activity against Colletotrichum capsici than other compounds. From the perspective of structural characteristics, there was no significant difference in fungicidal activity between I-1 -I-9 and II-1 -II-13. On the other hand, compounds I-1 -I-9 and II-1 -II-13 showed weak cytotoxicity against HepG2 (Table 2). In contrast, the compounds II-6 and II-11 exhibited higher cytotoxicity than other tested compounds. Overall, the structural optimization of natural waltherine F resulted in the strong polarity of the obtained amide derivatives, which presumably affected the weak fungicidal activity and cytotoxicity.

Fluorescence characteristic investigation
In our previous work, 4-quinolone derivatives QD and quinoline derivatives X-II were unexpectedly found to exhibit excellent fluorescence characteristics [15]. Similarly, the target molecules obtained in this work also displayed fluorescence properties, whose investigation may provide important guiding significance for the development of fluorescent probes. Therefore, the UV − vis absorption and fluorescence emission spectra of the compounds I-1 -I-9 and II-1 -II-13 were recorded (Fig. 5). From the data,    (Table  3). It could be found that compound I-6 exhibited a higher QY than I-2, I-3, I-7, and I-8, with a value of 27.4%. Subsequently, the TD-DFT calculations of molecules I-6  and I-7 were performed to elucidate the obtained fluorescence properties (Fig. 6). The HOMO orbital energy and LUMO orbital energy of molecule I-6 were both higher than that of I-7, which was conducive to the energy level transitions of electrons in the HOMO orbital of molecule I-6. In the meantime, the molecule I-6 exhibited a larger energy gap than I-7, which was beneficial to absorb more energy to produce stronger fluorescence intensity.

Conclusion
In summary, twenty-two novel amide derivatives derived from the structural modification of waltherione F were synthesized. The obtained structures were characterized by 1 H NMR, 13 C NMR and HRMS. Several crystal structural characteristics were also revealed via X-ray crystal diffraction of compounds I-6. The bioassay results indicated  that the compounds I-1 -I-9 and II-1 -II-13 showed weak inhibitory activities against the tested agricultural pathogens. However, in a given category, several compounds displayed higher inhibition rates against Rhizoctonia solani than other compounds. The in vitro cellular cytotoxicity assay revealed that compounds II-6 and II-11 exhibited higher cytotoxicity against HepG2 than other tested compounds. The fluorescence characteristics investigation showed that the QY value of the methanol solution of the compound I-6 was higher than that of I-2, I-3, I-7 and I-8, which was further explained by TD-DFT.

Material and instruments
Analytical grade materials and reagents used in the organic synthesis reactions were purchased from Energy Chemical and Bide Pharmatech Ltd. Melting points were measured on an X-5 binocular microscope. 1 H-and 13 C-NMR were provided on a Bruker-500 MHz spectrometer. HRMS (Waters Xevo G2-XS QTof, USA) was used to record the relative molecular mass. X-ray crystal structure was determined on a Bruker D8 Venture diffractometer. The purification of target compounds was performed by the column chromatography on silica gel (200-300 mesh).
Compound II-2 -II-13 was provided in a similar manner.

Fungicidal activity measurement
The mycelial growth inhibition method was used to determine the in vitro inhibitory activities of the target molecules against common agricultural pathogens according to the previously reported procedures [22] using commercial fluopyram and carbendazim as positive controls. First, the target compound was dissolved in a small amount of DMSO and then diluted with an aqueous solution containing Tween 80 to obtain the test stock solution. Subsequently, different pathogens were inoculated, respectively, in a mixture comprising the PDA medium and test solution. The colony diameter was measured to calculate the inhibition rate after incubating at 25°C for 3 days. Each treatment was repeated three times. The test pathogens include Rhizoctonia solani

In vitro cellular cytotoxicity assays
The in vitro cell viability of hepatocellular carcinoma cell lines (HepG-2) was assessed by MTT colorimetric assay according to the reported methods [23,24]. First, the cells were seeded in 96-well plates, incubated in a CO 2 incubator at 37°C for 24 h, and then treated with freshly prepared culture mediums containing the tested compounds (100 μM) for 24 h. Second, a fresh solution of MTT (5 mg/ml) was added to every single well of the 96-well plate, which was further incubated in a CO 2 incubator for another 4 h. After removal of the medium, the cells were dissolved with 100 μL of DMSO and analyzed in a multiwall-plate reader (Bio-Rad iMark) at 490 nm.

Fluorescence characteristics measurement
The UV-Vis absorption spectra were recorded from 800 nm to 200 nm at 50 μM to determine the appropriate excitation wavelength. The fluorescence emission spectra were provided with EM slit of 5 nm, PMT voltage of 480 V at the same concentration (50 μM). Subsequently, the absolute fluorescence quantum yields (QY) were recorded on a FLS1000 spectrometer with the parameters referred to the obtained fluorescence emission and excitation spectra.

TD-DFT calculation
The singlet ground-states geometrical optimizations were performed, and the calculations were carried out using the spin-restricted DFT method with B3LYP [25,26], in conjunction with 6-31 + G(d,p) basis set. Based on the optimized geometries of the molecules, the molecular orbitals (MOs) were calculated at the same level. The HOMO energy (E HOMO ) of each compound was taken from the eigenvalues of the Kohn-Sham calculated from the DFT. TD-DFT calculation of the single excitation energies was performed at the ground states using B3LYP with a basis set of 6-31 + G(d,p). Then the energy gaps (E g ) were estimated based on the single-singlet electronic transition energies. The LUMO energy level (E LUMO ) can be obtained according to the equation of E LUMO = E HOMO (DFT) + E g (TDDFT), and the E LUMO value was in excellent agreement with the experiments for the compounds [27]. All the calculations of both ground and excited states were performed within the Gaussian 09 quantum chemical package [28].