Synthesis, fungicidal activity, structure-activity relationships (SARs) and density functional theory (DFT) studies of novel strobilurin analogues containing arylpyrazole rings

A series of novel strobilurin analogues (1a-1f, 2a-2e, 3a-3e) containing arylpyrazole rings were synthesized and characterized by NMR spectroscopy. The structures of 1f, 2b and 3b were also determined by single crystal X-ray diffraction analysis. These analogues were collected together with other twenty-eight similar compounds 4a-4f, 5a-5h, 6a-6h and 7a-7f from our previous studies, for in vitro bioassays and thorough structure-activity relationships (SARs) studies. Most compounds exhibited excellent-to-good fungicidal activity against Rhizoctonia solani, especially 5c, 7a, 6c, and 3b with 98.94%, 83.40%, 71.40% and 65.87% inhibition rates at 0.1 μg mL−1, respectively, better than commercial pyraclostrobin. Comparative molecular field analysis (CoMFA) was employed to study three-dimensional quantitative structure-activity relationships (3D-QSARs). Density functional theory (DFT) calculation was also carried out to provide more information regarding SARs. The present work provided some hints for developing novel strobilurin fungicides.

Intermediates N-arylpyrazoles I were synthesized from arylhydrazines via addition-cyclization and oxidation, which could then afford 4-bromo-N-arylpyrazoles IV by bromination 17 . Intermediate benzyl bromide (E)-methyl 2-(2-(bromomethyl)phenyl)-2-(methoxyimino)acetate II was prepared from 1-(o-tolyl)ethanone via four steps including oxidation, esterification, oximation and bromination 8 . A previous report by Kim et al. 18 described that intermediate (E)-methyl 3-methoxy-2-(o-tolyl)acrylate III-c could be synthesized from 1-bromo-2-methylbenzene and (E)-methyl 3-methoxyacrylate via Suzuki-Miyaura coupling reaction (Fig. 3). However, this approach required Grignard reagent and costly catalyst Pd(PPh 3 ) 4 , which faced harsh reaction conditions and complicated processes. So in our procedure, readily accessible 2-(o-tolyl)acetic acid was used as starting material, and intermediate III-c could be obtained through three steps including esterification, condensation and methylation. The condensation of III-a with methyl formate was carried out under NaH alkaline condition, which gave III-b in 85% yield. A better yield (78%) of III-c was obtained in a molar ratio of III-b to dimethyl sulfate 1:1.2 equiv. in DMF as solvent, and with NaH as base.
In terms of the o-substituted pharmacophores, the sequence of fungicidal activity against Rhizoctonia solani was methoxyiminoacetate moiety (5a-5h) > methoxyiminoacetamide moiety (6a-6h) > methoxyacrylate moiety (3a-3e) in general, irrespective of difference in substituent R on the terminal phenyl ring. For example, within the series of R = 4-Cl derivatives, methoxyiminoacetate-derivative 5c displayed a much higher fungicidal activity than the corresponding methoxyiminoacetamide-derivative 6c, while the methoxyacrylate-derivative 3b showed the lowest. Similar speculation could apply to the compounds 5e, 6e and 3d (R = 3-CF 3 ). All the above three chloro-containing compounds 5c, 6c and 3b showed better activity than pyraclostrobin, which indicated the methoxycarbamate pharmacophore of pyraclostrobin might have no effective impact on the inhibition of Rhizoctonia solani. In addition, when changing the best o-substituted methoxyiminoacetate pharmacophore into the p-substituted, the results were unsatisfactory. Compounds 4a-4e showed much lower fungicidal activity than 5a-5h, which indicated the significant impact of pharmacophore position on the inhibition rates, and the o-substitution might be better.
To examine the electronic effect of substituent R on the phenyl ring, the electron-donating CH 3 and electron-withdrawing F, Cl, Br, I, CF 3 were introduced. Compounds with electron-withdrawing substituents displayed higher fungicidal activity against Rhizoctonia solani than that with electron-donating substituents, as seen in the comparison of 1d . According to the different electronic effect of electron-withdrawing substituent R, the sequence of fungicidal activity against Rhizoctonia solani is chloro-substituted > bromo-substituted, as seen in the compar- , and 6c (R = 4-Cl) vs. 6 h (R = 4-Br), and within the series of R = 3-CF 3 derivatives, the introduction of the fluoro group could make the fungicidal activity obvious improvement, as seen in the comparison of 1f 3 ). However, compounds 4c (R = 3-CF 3 ) and 4f (R = 3-CF 3 -4-F), 5d (R = 3-CF 3 -4-F) and 5e (R = 3-CF 3 ), 7e (R = 3-CF 3 ) and 7f (R = 3-CF 3 -4-F) are three pairs of exceptions: 7f exhibited weaker fungicidal activity against Rhizoctonia solani as compared with 7e, whereas 4f and 5d showed better activity than 4c and 5e at 10 μg mL −1 , respectively, and the results were just the opposite when the concentration was reduced to 1 μg mL −1 and 0.1 μg mL −1 . These differences in fungicidal activity might be due to variations in combination of methoxyiminoacetate pharmacophore, According to the different positions of chloro group, the sequence of fungicidal activity is Cl-substituted phenyl ring > Cl-substituted pyrazole ring, as seen in the comparison of 5c vs. 7a-7f. However, compound 7a only containing a chloro on the pyrazole ring displayed nearly equal fungicidal activity to the best 5c, and with the increasing number of chloro group, the fungicidal activity was decreased, as seen in the comparison of 7d vs. 5c and 7a. These observations revealed that the mono-chlorination has an important influence on the fungicidal activity.
To further investigate the effect of other halogen substituents X on the pyrazole ring, Br and I were introduced to the series of methoxyiminoacetate-derivatives, as compared with the non-substituted H and Cl. When the substituent R on the phenyl ring was the same, in most cases, the fungicidal trend of these four series against Rhizoctonia solani was H > Cl > Br > I. For example, within the series of R = 4-CF 3 derivatives, compound 5e (X = H) had better fungicidal activity than 7e (X = Cl) and 1e (X = Br) at 10 μg mL −1 and 1 μg mL −1 , respectively, whereas 2d (X = I) showed the weakest. Similar relationships could apply to the R = 3-CF 3 -4-F derivatives 5d (X = H), 7f (X = Cl), 1f (X = Br) and 2e (X = I), R = 4-OCF 3 derivatives 5g (X = H), 7c (X = Cl), 1c (X = Br) and 2c (X = I), as well as R = 3-CH 3 derivatives 5f (X = H), 7b (X = Cl), 1b (X = Br) and 2a (X = I). These results might indicate that the larger molecular volume (Br and I) was unfavorable for the intracellular uptake and transport in the fungus, and the non-substituted pyrazole ring might be the best.
In summary, the SARs study revealed that the improvement of fungicidal activity required a reasonable combination of both methoxyiminoacetate pharmacophore and electron-withdrawing substituent R, and the type and size of substituent X on the pyrazole ring were critical. The chloro group had an effective impact on the fungicidal activity whether it on the terminal phenyl ring or pyrazole ring. The present work indicated that 5c (98.94% at 0.1 μg mL −1 , methoxyiminoacetate pharmacophore, R = 4-Cl, X = H), 7a (83.40% at 0.1 μg mL −1 , methoxyiminoacetate pharmacophore, R = H, X = Cl), 6c (71.40% at 0.1 μg mL −1 , methoxyiminoacetamide pharmacophore, R = 4-Cl, X = H) and 3b (65.87% at 0.1 μg mL −1 , methoxyacrylate pharmacophore, R = 4-Cl, X = H) could be used as potential lead compounds for further studies of novel fungicides.
Three-dimensional quantitative structure-activity relationships (3D-QSARs). In order to obtain further insight into the structural requirements of novel strobilurin fungicides, we have performed a 3D-QSAR study of the above forty-four strobilurin analogues against Rhizoctonia solani using CoMFA technique. In CoMFA, it is assumed that the interaction between an analogue and its molecular target is preliminarily non-covalent and shape dependent in nature. The 3D-QSAR can be derived correlating the differences in steric and electrostatic fields surrounding a set of molecules to the fungicidal activity.
The toxicity baselines and EC 50 values were obtained from DPS data processing system, and the negative logarithm of EC 50 (pEC 50 ) was used as the biological activity in the 3D-QSAR study ( Table 3). The lowest energy conformers were selected and minimized using Powell method to rms 0.001 kcal mol −1 Å −1 . Alignment of the molecules was carried out using N-phenyl pyrazole ring as the common skeleton (Fig. 8, blue color), and the most active molecule 5c was used as a template molecule for database alignment according to the pEC 50 values. Thirty-three molecules were randomly selected as the training set to establish the CoMFA model, and the remaining eleven molecules were used as the test set to test the predictive ability of the model (Table 3, "*"). The CoMFA model was generated using Partial Least-Squares (PLS) approach. The cross-validation with the Leave-One-Out (LOO) option and the SAMPLS program was carried out to obtain the optimal number of components (n) and cross-validated coefficient (q 2 ). After n was determined, a non-cross validated analysis was performed without column filtering to obtain regression coefficient (R 2 ) and its standard error (SEE), as well as F-test value (F) for the model evaluation.  The alignment gave a conventional R 2 (R 2 ncv ) of 0.977 with 2 components, a predictive R 2 (R 2 pred ) of 0.816 and an F value of 94.553. The model generated with a good internal predictive ability (q 2 = 0.508) and a small standard error of estimation (SEE = 0.202) was selected as the best model to explain SARs and carry out further analysis. Observed and predicted fungicidal activity of the training and test sets were plotted in Fig. 9 and listed in Table 3. The results indicated that the observed and predicted data were in good agreement with each other, with a correlation coefficient R 2 of 0.936. Figure 10a displayed the steric contour plot. The green contours describe regions where sterically favorable groups enhance activity (80% contribution), and yellow contours describe regions of unfavorable steric effects (20% contribution). The pharmacophore and its linked phenyl ring were surrounded by the sterically favorable green contours. The most active molecule 5c had a methoxyiminoacetate moiety substituted phenyl ring embedded in this green region. Other sterically-favorable green contours were observed near the terminal phenyl ring. This green contour was surrounded by the unfavorable yellow region. A substitution on the 2-or 4-position of the terminal phenyl ring was favored, whereas any substitution on the adjacent 3-or 5-position was unfavorable. This also suggested that the introduction of the chloro group to the 4-position was important for fungicidal activity. Figure 10b displayed the electrostatic contour plot. The blue contours describe regions where positively charged groups enhance activity (80% contribution), and red contours describe regions where negatively charged groups enhance the activity (20% contribution). In compound 5c, the red contours were found near the methoxyiminoacetate pharmacophore and the 4-position of the terminal phenyl ring, suggesting that a high electron density in this region increased the activity. A large negative-charge unfavorable blue contour was found to surround the side chain CH 2 and the terminal phenyl ring. This indicated that substitutions in these regions with high electron density reduced activity and emphasized the necessity of positively charged groups. Overall, steric interactions (56.3% contribution) played a major role in the influence of fungicidal activity than electrostatic interactions (43.7% contribution).

Density functional theory (DFT) calculation. The frontier-orbital energies of a compound play an
important role in bioactivities 20 . E HOMO is a rough measure of the electron-donating ability of a compound and, normally, increasing its value can improve the biological activity, whereas the E LUMO acts in reverse 21,22 . Energy gap between HOMO and LUMO characterizes the molecular chemical stability and it is a critical parameter in determining molecular electrical transport properties because it is a measure of electron conductivity. It also affects the bioactivity of a compound. Thus, the study of the frontier-orbital energies may be helpful to the investigation of fungicidal activity. Compounds 5c, 7a and pyraclostrobin which had wide difference in activity were selected for DFT comparison.
The LUMO and HOMO maps of 5c, 7a and pyraclostrobin were shown in Fig. 11. Comparing the HOMO-LUMO gaps of the three molecules, the order was: pyraclostrobin >7a > 5c. The narrow HOMO-LUMO gap implies a high chemical reactivity because it is energetically favorable to add electrons to a low-lying LUMO or extract electrons from a high-lying HOMO, and so to form an activated complex in any potential reaction 23 . This suggested that compound 5c might possess a relatively high activity, which correlated well with the fungicidal activity results. In addition, the calculations indicated some similarities between 5c and 7a. In the HOMO,

Structure
No.  Table 2. Fungicidal activity of forty-four strobilurin analogues. a 0, No activity, and 100, total kill.   Table 3. Experimental and theoretical fungicidal activity of forty-four strobilurin analogues against Rhizoctonia solani.  Observed (X-axis) and predicted (Y-axis) biological activities of the training (blue dot) and test (red dot) sets. (Thirty-three molecules were randomly selected as the training set and eleven molecules ( Table 3, "*") were used as the test set to test the predictive ability of the model. The observed and predicted pEC 50 could be obtained according to the location of each dot; the values were listed in Table 3). the electrons were mainly delocalized on the terminal benzene ring and pyrazole ring (including CH 2 , O and Cl atoms). When electron transitions took place, some electrons in the HOMO would enter into the LUMO 24 ; then, in the LUMO, the electrons were similarly delocalized on the bridge benzene ring and oxime ester moiety. The HOMO-LUMO gaps of 5c (0.160 a.u) and 7a (0.161 a.u) were very close to each other. The similar electron distributions and energy gaps between 5c and 7a might cause both with excellent fungicidal activity. However, pyraclostrobin exhibited quite different electron distributions as compared with 5c and 7a. Whether in the HOMO or LUMO, its electrons were mainly delocalized on the terminal benzene ring and pyrazole ring. In the LUMO maps, the general trend of electron delocalization was: pyraclostrobin >7a > 5c, which represented a negative correlation with their fungicidal activity. As reported, the frontier molecular orbitals are located on the main groups, the atoms of which can easily bind with the receptor 12,13 . Moreover, the different degrees of delocalization may affect the orbital interaction 25 . Therefore, it seemed that the high electron delocalization of pyraclostrobin in the LUMO might potentially make the orbital interactions limited, which might lead to a decrease in activity. Figure 12 is the molecular electrostatic potential (MEP) of 5c, 7a and pyraclostrobin. The MEP simultaneously displays molecular size, shape as well as positive, negative and neutral electrostatic potential regions in terms of color grading. From MEP, we can know the rich electron region and the lack electron region, where potential increases in the order of red < orange < yellow < green < blue. As can be seen from the MEP of 5c and 7a, the carbonyl oxygen atom on the methoxyiminoacetate pharmacophore had the greatest negative charges. Thus, it seemed probable that the oxygen atom interacted with the receptor.

Experimental
Chemical synthesis. All reagents were used in analytical grades. All reaction were monitored by thin layer chromatography (TLC), visualization was effected by UV (254 nm). Column chromatography was performed on flash silica gel (300-400 mesh) using mixtures of petroleum ether with ethyl acetate as eluent. Melting points were measured on an X-4 microscope electrothermal apparatus (Taike China) and were uncorrected. NMR spectra were recorded on a Bruker AV-400 spectrometer ( 1 H NMR at 400 Hz, 13 C NMR at 100 Hz) in deuterated solvents using TMS as an internal standard. Chemical shifts (δ) were given in parts per million (ppm), and coupling constants (J) were given in Hertz (Hz). The synthetic procedures and detailed characterization data of intermediates I, II, III, IV and 5 can be found in the ESI.  General procedure for the synthesis of products 1a-1f and 3a-3e. To a solution of IV or I (1.0 mmol) in acetone (30 mL) was added K 2 CO 3 (1.5 mmol). The mixture was refluxed for 15 min, then II or III (1.1 mmol) was added slowly. The mixture was refluxed for about 4 h (monitored by TLC), then K 2 CO 3 was filtered off. The solvent was evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (eluent for 1a-1f: ethyl acetate/petroleum ether, 1: 5v/v; for 3a-3e: ethyl acetate/ petroleum ether, 1: 8v/v) to afford products.       3D-QSARs details. The 3D-QSARs studies were performed using SYBYL X 2.0 software with a standard Tripos force field 28 . The compounds were constructed from the fragments in the SYBYL database with standard bond lengths and bond angles. Geometry optimization was carried out using the standard Tripos force field with distance dependent dielectric function and energy gradient of 0.001 kcal mol −1 Å −1 . The lowest energy conformers were selected and minimized using the Powell method till root-mean-square (rms) deviation 0.001 kcal mol −1 Å −1 was achieved.

Conclusions
In summary, sixteen novel strobilurin analogues (1a-1f, 2a-2e, 3a-3e) were designed and synthesized. The structures of 1f, 2b and 3b were determined by single crystal X-ray diffraction analysis. Other twenty-eight similar compounds 4a-4f, 5a-5h, 6a-6h and 7a-7f from our previous studies were also collected together with the above sixteen analogues for in vitro bioassays and structure-activity relationships (SARs) study in details. Most compounds exhibited excellent-to-good fungicidal activity against Rhizoctonia solani, especially 5c, 7a, 6c, and 3b with 98.94%, 83.40%, 71.40% and 65.87% inhibition rates at 0.1 μg mL −1 , respectively, better than commercial pyraclostrobin. The SARs revealed that the improvement of fungicidal activity required a reasonable combination of both methoxyiminoacetate pharmacophore and electron-withdrawing substituent R, and the type and size of substituent X on the pyrazole ring was critical. The 3D-QSAR model for the above forty-four strobilurin analogues was also derived using CoMFA method, with high correlative and predictive abilities. The contour maps indicated that the electron rich substituent R on the 4-position of the terminal phenyl ring might improve activity, which was in good agreement with the SARs discussion. In addition, through DFT calculation, it seemed that the high electron delocalization of pyraclostrobin in LUMO might possibly make the orbital interactions limited, which might bring out a decrease in activity, whereas the similar electron distributions and narrow energy gaps between 5c and 7a might give both with excellent fungicidal activity. The present work indicated that 5c, 7a, 6c and 3b could be used as potential lead compounds for further studies of novel fungicides.