Chinese Chemical Letters  2014, Vol.25 Issue (07):1069-1072   PDF    
N-Fluorinated phenyl-N'-pyrimidyl urea derivatives:Synthesis, biological evaluation and 3D-QSAR study
Xia-Li Yue, Hu Li, Shuang-Shuang Liu, Qing-Ye Zhang , Jing-Jing Yao, Fei-Yan Wang    
College of Science, Huazhong Agricultural University, Wuhan 430070, China
Abstract: With the increase of herbicide-resistant weeds, novel, more selective and even more potent herbicides to control weeds are needed. In this paper, a series of N-Fluorinated phenyl-N'-pyrimidyl urea derivatives were synthesized and screened for their herbicidal activities against Amaranthus retroflexus (AR) and Setaria viridis (SV). Compound 25 (N-(3-trifluoromethylphenyl)-N'-(2-amino-4-chloro-6-methylpyrimidyl) urea) exhibited marked herbicidal activity against SV (IC50=11.67 mg/L) and is more potent than bensulfuron (IC50=27.45 mg/L), a commercially available herbicide. A statistically significant CoMFA model with high prediction abilities (q2=0.869, r2=0.989) was obtained.
Key words: Fluorinated phenyl     Pyrimidine     QSAR     Herbicidal activities    
1. Introduction

Pesticides have greatly benefited grain harvest by reducing harmful pests. The widespread use of herbicides in farming has led to annual increases in the dosage of herbicides used. Simultaneously,the problem of herbicide-resistance emerged and became more and more serious. The increasing of the amount of spraying has also caused a series of problems. For example,they have led to harm to human health,the degradation of water resource quality, and the reduction in the biodiversity of field communities [1, 2, 3]. Therefore,it is essential and urgent to develop novel,more selective,and even more potent herbicides to control weeds.

The urea group is an attractive structural unit due to its broad range of biological activities,and it can be widely found in natural products [4]. As such,urea derivatives have attracted a lot of attention for applications in herbicidal [5],antifungal [6], antibacterial [7],and plant growth regulator [8] chemicals. Pyrimidines are also considered important heterocyclic compounds for their wide range of biological activities.N-phenyl-N'-pyrimidyl urea derivatives are considered to be prospective compounds with high activity due to their containing both a carbamide bridge (NH-CO-NH) and a pyrimidyl group [9]. On the other hand,fluorine is a very important element for its high electronegativity and small volume. The high electronegativity of fluorine will affect the distribution of the electrons in the molecule. Biologically active molecules will have significantly improved lipophilicity if they contain fluorine atoms [10, 11, 12, 13, 14, 15]. It was speculated that the introduction of a fluorine atom into the compound could improve the compound’s physicochemical effects.

Inspired by these findings,we focused on designing and synthesizing a series of novel compound derivatives based on the essential fluorinatedN-phenyl-N'-pyrimidyl urea scaffold. All the structures of the synthesized target compounds were thoroughly characterized by IR and 1H NMR spectra. Then all the compounds were screened for their herbicidal activities againstAmaranthus retroflexus(AR) andSetaria viridis(SV). The biological testing results showed that compound 25(N-(3-trifluoromethylphenyl)-N'-(2-amino-4-chloro-6-methylpyrimidyl) urea) displayed the most potent biological activity againstSV(IC50= 11.67 mg/L),and was more potent than bensulfuron (IC50= 27.45 mg/L),a commercially available herbicide. With this data,in this present study,we also performed quantitative structure-activity relationship (QSAR) studies,and a statistically significant CoMFA model with high predictive abilities (q2= 0.869,r2= 0.989) was obtained. 2. Experimental

2.1. Chemistry

All chemical reagents were commercially available and treated with standard methods before use. Solvents were dried and redistilled before use. Melting points were determined with a digital melting point apparatus and were uncorrected. IR spectra were recorded on a Thermo Nicolet FT-IR Avatar 330 instrument. 1H NMR spectra were recorded in DMSO-d6on a Varian Mercury 600 spectrometer,and chemical shifts (δ) were given in ppm relative to tetramethylsilane. The progress of the reactions was monitored by thin layer chromatography (TLC) on silica gel plates visualized with UV light. 2.1.1. General procedure for the synthesis of target compounds 3-47

The synthetic route forN-fluorinated phenyl-N'-pyrimidyl urea derivatives is depicted in Scheme 1. At first,40 mL toluene solution which contained 5 mmol fluorine-containing aniline was slowly dropped into another toluene solution which contained 2 mmol BTC (triphosgene) and a few drops Et3N while mixing at 0°C for about 1 h. The mixture was stirred at room temperature for 1 h and then heated to 80°C until the white solid completely dissolved. The intermediate 1 was isolated and obtained by concentration under reduced pressure and flushing by nitrogen [16, 17]. To obtain the final target compounds,the unpurified intermediate 1 was added into a series of substituted pyrimidinamine (5 mmol,as shown in Table 1) solutions which contained a little tetrabutylammonium bromide that was used as phase transfer catalyst. Then,the mixture was agitated at 80-100°C for 6-9 h. The reaction was detected according to TLC. When the reaction was complete,the product was cooled to room temperature,filtered,and washed with 10% Na2CO3,water,and acetone. The final target compounds were purified by recrystallization (DMF/acetone). 2.1.2. Characterization data of some selected target compounds

N-(2,4-Difluorophenyl)-N'-(2-amino-4-chloro-6-methoxypyrimidyl) urea (10): White crystal,yield 52.6%,mp 230-232°C,IR (KBr,cm-1):n3414 (N-H),3032 (Ar-H),1701 (C55O),1236 (OCH3). 1H NMR (600 MHz,DMSO-d6): δ10.42 (s,1H,NH),8.17 (d,1H, J= 6.6 Hz,ArH),7.36 (t,1H,J= 9.0 Hz,ArH),7.07 (t,1H,J= 7.8 Hz, ArH),6.78 (s,1H,pyrH),3.96 (s,3H,OMe).

N-(3-Trifluoromethylphenyl)-N'-(2-amino-4-chloro-6-methylpyrimidyl) urea (25): White crystal,yield 58.5%,mp 191-192°C,IR (KBr,cm-1): n3430 (N-H),3141 (Ar-H),1691 (C55O). 1H NMR (600 MHz,DMSO-d6):δ11.39 (s,1H,NH),10.43 (s,1H,NH),7.89 (d, 1H,J= 7.2 Hz,ArH),7.74 (t,1H,J= 8.4 Hz,ArH),7.26 (m,2H, J= 4.2 Hz,ArH),6.90 (s,1H,pyrH),2.54 (s,3H,CH3). N-(3-Fluorophenyl)-N'-(2-amino-4-hydroxy-6-methylpyrimidl) urea (40): White powder,yield 75.8%,mp 271-273°C,IR (KBr,cm-1):n3431 (N-H),3223 (O-H),3116 (Ar-H),1707 (C55O). 1H NMR (600 MHz,DMSO-d6):δ8.13 (d,1H,J= 5.4 Hz,ArH),7.74 (m,2H,J= 7.8 Hz,ArH),7.37 (s,1H,ArH),5.97 (s,1H,pyrH),2.20 (s, 3H,Me).

N-(4-Fluorophenyl)-N'-(2-amino-4-hydroxy-6-methylpyrimidl) urea (41): White powder,yield 68.3%,mp 225-226°C,IR (KBr,cm-1):n3415 (N-H),3230 (O-H),3070 (Ar-H),1712 (C55O). 1H NMR (600 MHz,DMSO-d6):δ7.54 (m,2H,J= 5.4 Hz,ArH),7.16 (m,2H,J= 7.8 Hz,ArH),5.92 (s,1H,pyrH),2.22 (s,3H,Me).

The IR and 1H NMR spectral data of the other target compounds and all the 1H NMR spectra can be found in the Supporting information. 2.2. Herbicidal activity testing

Herbicidal activity testing of compounds 3-47 againstARand SVwere evaluated according to the standard protocol [18]. All compounds were formulated as 10000 mg/L emulsified concentrates by using dimethyl sulfoxide (DMSO) as the solvent and TW-80 as an emulsification reagent. Then,they were diluted with distilled water to the desired concentration (10,25,50,100,and 200 mg/L). Twenty seedlings were each grown in a 9 cm Petri dish containing two pieces of filter paper and 10 mL solution at 25±1°C,relative humidity (RH) (60±5) % in a greenhouse. Distilled water and bensulfuron,a commercially available herbicide,were used as the controls. For the entire bioassay test,each treatment was repeated twice. Herbicidal activity was evaluated by measuring the lengths of the roots and hypocotyls. Then,Predictive Analytics Software (PASW) 18.0 was used to perform the regression analysis in order to obtain the IC50values. 2.3. 3D-QSAR analysis

The 45 target compounds were divided into a training set and a testing set which included 38 and 7 compounds,respectively. The testing set compounds were randomly chosen. The IC50 values have been changed into the minus logarithmic scale [pIC50] for the QSAR study [19, 20]. The three-dimensional structures of all compounds were built using the SYBYL 7.3 software [21]. Partial atomic charges were calculated by the Gasteiger-Hu¨ckel method, and energy minimizations were performed using the Tripos force field and the Powell conjugate gradient algorithm with a convergence criterion of 0.05 kcal/(mol Å ) [22, 23].

The steric and electrostatic interaction fields for CoMFA were calculated using the SYBYL default parameters: 2.0 Å grid points spacing,a sp3carbon probe atom with +1 charge and a van der Waals radius of 1.52 Å,and column filtering of 2.0 kcal/mol [24]. The descriptors calculated from the CoMFA analysis were used as independent variables,and the experimental pIC50 values were used as dependent variables in partial least squares (PLS) analysis to derive 3D-QSAR model. Leave-one-out (LOO) cross-validated and the SAMPLS program [25] were performed to obtain the optimal number of components (n) and cross-validated coefficient (q2). After the optimal number of components was determined,a non-cross-validated analysis was performed without column filtering to obtain regression coefficients (r2) which determine the external predictive ability [26, 27, 28]. 3. Results and discussion

3.1. Synthesis ofN-fluorinated phenyl-N' -pyrimidyl urea derivatives 3-47

The synthetic route forN-fluorinated phenyl-N'-pyrimidyl urea derivatives is depicted in Scheme 1. Fluorinated phenyl isocyanates were prepared by reacting BTC with respective amines in toluene at 0°C for 1 h and then stirred at 80°C for 2 h [16, 17]. The intermediate 1,without further isolation,reacted with the corresponding amine to produce the target compounds 3-47 with yields of 46-79%. All the compounds were purified by crystallization,and all the compound structures were assigned by IR and 1H NMR spectral data,which were reported in the experimental section and Supporting information.

Scheme 1. Synthetic route for target compounds 3-47.
3.2. Herbicidal activity

All synthesized compounds 3-47 and bensulfuron,a commercially available herbicide,were evaluated for their herbicidal activity againstARandSV. Bensulfuron was employed as a positive control. Herbicidal activity was evaluated by measuring the lengths of roots and hypocotyls. The biological testing results showed that all of compounds showed better herbicidal activity against the roots ofARandSV. The results were expressed as concentrations of IC50and listed in Table 1.

Table 1
Structures and biological activities of the compounds used in the training and testing sets.

As shown in Table 1,compounds 10,25,40,and 41 exhibit high activities with IC50below 50 mg/L againstSV,and compound 25 (11.67 mg/L) showed slightly better activity than bensulfuron (27.45 mg/L). For AR,compounds 20,37,39,40,41,44 and 46 showed slightly weaker activity,and the IC50 are less than 100 mg/L. In terms of the structure-activity relationship,the different position of the substituent on the benzene ring displayed different activities. ForAR,compounds where the substituent-F lies in thepara-position display the best herbicidal activity,such as 14 (4-F)>13 (3-F),12 (2-F) and 32 (4-F)>31 (3-F),30 (2-F). However,the substituent-F in themeta-position led to the highest activity againstSV,for example,4(3-F)>3(2-F),5(4-F) and 40 (3-F)>39(2-F),41(4-F). The substituent -CF3 on the metaposition and -2,4-di-F led to higher activity in bothARandSV,for instance,16(3-CF3)>15(2-CF3),17(4-CF3) and 46(2,4-di-F)>45 (2,6-di-F),47(2,5-di-F). In addition,the compounds with optimal activity are 41(35 mg/L) forARand 25(11 mg/L) for SV. 3.3. Quantitative structure-activity relationship analysis

Compared toAR,the biological testing results showed that most of the synthesized compounds showed higher herbicidal activity against SV. Subsequently,we performed QSAR studies on the biological testing data forSV. The accuracy of the prediction of the QSAR model and reliability of the contour maps are directly dependent on the structural alignment rule. So,the molecular alignment is considered one of the most sensitive parameters in QSAR analysis. In this study,compound 25 with the best activity was selected as a reference molecule,and the common fragment (shown in Fig. 1A) was used as a template for all compound alignments,as shown in Fig. 1B.

Fig. 1. Structure of the urea derivatives: (A) general structure for title compounds, (B) 3D view of all the aligned molecules in training and testing sets.

The statistical results of the 3D-QSAR model showed that the cross-validated correlation coefficients (q2) and the regression coefficients (r2) for SV are 0.869 and 0.989,respectively. The obtained results indicated that the CoMFA model has good prediction capability. The predicted pIC50values are generally in good agreement with the experiment data,and the residuals are all small as shown in Fig. S46 and Table S1 in Supporting information.

The contribution of the steric field and electrostatic field are 34.8% and 65.2% in the 3D-QSAR model,respectively. The steric and electrostatic contribution contour maps of the model are displayed in Fig. 2. The 3D contour maps showed that the changes of molecular fields are associated with the differences of the biological activity. The steric fields are in green and yellow. The region of green contour suggests that more bulky substituents in these positions will improve the biological activity,while the yellow region indicates that an increased steric bulk is unfavorable for the inhibitory activity. The CoMFA electrostatic fields in blue indicate the regions where the occurrence of positive charge would enhance the activity,while the red contours regions suggest that negatively charged groups would be favorable.

Fig. 2. CoMFA contour maps: (A) electrostatic contour map,(B) steric contour map.

As show in the electrostatic contour map (Fig. 2A),a large blue contour around R3 indicates that electropositive groups at the position would be able to increase the activity. The bulky blue contour around the 2-position and 4-position on the benzene ring indicates that electropositive groups will significantly increase the activity. In addition,a red contour in the 4-position indicates that electronegative groups are also favorable here.

In the steric map (Fig. 2B),on the R3 position,there are bulky green contours above and behind it,which indicate that bulky groups here would benefit activity. For example,compounds9(- OMe),36(-OMe) and 45(-CH3) indicate better herbicidal activity than 18(-H). The yellow contours near 2-position suggest that the smaller groups in this region would be advantageous,such as 3(2-F)>6(2-CF3),39(2-F)>42(2-CF3),while green contours around the 3-position indicate that bulky groups would be favorable here. Moreover,a yellow contour around the 5,6-position indicates that small groups here can promote activity.

As the most active compound,25 has a strong electronwithdrawing group (-CF3) in the 3-position,an electropositive group (-CH3)on R2 ,and an electronegative group (-Cl) on R3 , which all conform to the steric and electrostatic contribution contour maps. On the other hand,compound 6,with a large electronegative group (-CF3) in the 2-position,an electronegative group (-Cl) on R2 ,and an electropositive group (-OMe) on R3 ,did not match the model,and had the weakest activity. Based on the CoMFA model,we suppose that an electropositive group in the 4-position and a small group added to 5,6-position would be able to increase the activity. Subsequently,compound optimization work will be undertaken in our research group. 4. Conclusion

Through this study,a series of N-fluorinated phenyl-N'-pyrimidyl urea derivatives were obtained by a convenient BTC one-pot synthetic method. Compounds 20,37,39,40,41,44,and 46 exhibited good herbicidal activities againstAR. Compounds 10,25, 39,40,41,and 44 displayed excellent herbicidal activities against SV. 3D-QSAR study has been carried out to understand the structural features responsible for the potency of the derivatives againstSV. CoMFA model withq2(0.869) andr2(0.989) was obtained. The low standard error of estimation and the highFvalues confirm that the obtained CoMFA model would have significant prediction ability. A small and electropositive group in the 2-position,bulky groups in the 3-position,and small groups in the 5,6-position would be able to enhance the herbicidal activity effectively. Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 30900935),and the Fundamental Research Funds for the Central Universities (Nos. 2011PY086,2011PY039). Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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