Xile Deng, Wenna Zheng, Can Jin, Qingcai Zhan, Lianyang Bai*
Keywords:Metolachlor;Hydrazone;Rice;Herbicide safeners;Glutathione S-transferase
ABSTRACT
One strategy for solving the phytotoxicity of herbicides is to apply herbicide safeners that can efficiently alleviate the injuries of agricultural crops caused by herbicides. When metolachlor, a chloroacetamide herbicide, is applied with paddy rice, for example, the mechanisms associated with metolachlor and its residue negatively impact on the growth and yields of rice. To identify novel high-activity herbicide safener candidates for meto- lachlor, a series of (E)-4-(2-substituted hydrazinyl)-6-chloro-2-phenyl pyrimidines were synthesized and their structures were confirmed using IR (infrared radiation), 1H NMR, 13C NMR, and HRMS (high resolution mass spectrometry). The herbicide safener activities were then evaluated via primary tests. Compounds 3i and 3t were found to have the best herbicide activity on plant height. These compounds were then further screened for their activities at lower concentrations and showed better or similar activities compared to the positive control fen- clorim, a commercial herbicide safener. The compounds 3i and 3t significantly enhanced glutathione S-trans- ferase (GST) activity related with the herbicide safener activity in both shoots and roots tissues. Moreover, a qPCR (Real-time quantitative polymerase chain reaction) analysis found that the 3i and 3t treatments enhanced the expressions of OsGSTU3, OsGsTU39, and OsGSTF5. Finally, the results of an acute toxicity assessment with zebrafish (Danio rerio) embryos using treatments 3i and 3t indicated they are relatively safe to aquatic organisms.
1.Introduction
Competition between weeds and crop plants (e.g. rice, maize, wheat, and soybean) can significantly affect the production of these agricultural crops; as such, infesting weeds have become a major global challenge to agriculture [1-6]. To date, herbicides have been generally used to control weeds to ensure that food crops can meet adequate production levels to meet population growth requirements [7,8]. Meanwhile, the internal mechanisms of herbicides and their residues negatively impact the growth and yields of agricultural crops when applied to fields [9-11]. Among all the methods that protect agricultural crops from potential injuries caused by herbicides, herbicide safeners have emerged as an effective, straightforward, and cost-effective strategy [12-15]. Herbicide safeners are generally added to seeds by pre-sowing seed treatments or are applied in combination with herbicides. As such, they effectively enhance the herbicide resistance of agricultural crops to reduce herbicide-induced toxicity to crops and improve herbicide selectivity for crops [16]. In 1970, the Gulf Oil Company developed the first commercialized herbicide safener (1,8-naphthalic anhydride, NA) to protect maize from thiocarbamate herbicide injury [17]. Almost twenty synthetic herbicide safeners, e.g., dichlormid, benoxacar, fen- clorim, cloquintocet-mexyl, and flurazole, have been developed and launched on the market to protect crops (Fig. 1) [18]. It is reported the main action mechanisms of herbicide safeners alleviating herbicide injury are involved with the metabolism and detoxification in crop plants, for example, the enhancement of activities of glutathione S- transferases (GSTs), glutathione (GSH), polyphenol oxidase (PPO), UDP- glucuronosyltransferases (UGTs),and cytochrome P450 mono- oxygenases (CYPs) to detoxify herbicides [19-21].
Of the commercial herbicide safeners, fenclorim (4,6-dichloro-2- phenyl-pyrimidine) is a heterocyclic herbicide safener used for pretila- chlor, a pre-emergent chloroacetanilide herbicide, which is applied to control annual grasses, broadleaf weed, and some sedges [22-24]. It also shows certain safener activity with other chloroacetanilide herbicides, such as metolachlor for rice plants [12]. When fenclorim is soaked with rice seeds in pre-sowing applications or in combination with chlor- oacetanilide herbicides on rice seedlings, it improves the tolerance of rice seedlings to chloroacetanilide herbicides. It detoxifies herbicides by improving the glutathione S-transferases (GSTs) expression in rice, catalyzing the conjugation of chloroacetanilide herbicides with gluta- thione (GSH) [25,26]. Meanwhile, it should be mentioned that fen- clorim was found to have a high acute toxicity rainbow trout (Oncorhynchus mykiss) with an LC50 (median lethal concentration) of 0.6 mg/kg (96 h), indicating it has a
potential risk to aquatic organisms [27].
A few studies have investigated the structure-activity relationship (SAR) of fenclorim on herbicide safener activity. This research offers guidance for further screening new pyrimidine herbicide safeners [12,28,29]. These studies examined the safener activities of phenyl and pyrimidine ring substituted fenclorim derivatives. These studies also found that the position on the chlorine atom in the pyrimidine (3-po- sition) offenclorim is a possible modification site for screening high- activity safener compounds. A variety of fenclorim derivatives, substituted with groups such as methyl moiety, bromine atom, and phenoxy moiety, have been reported with respect to their safener ac- tivity. However, there are many opportunities to assess other modifi- cations at this position using other new active groups or active structures.Many novel herbicide safeners have been discovered using random screening methods, such as active substructure combination [30-32]. Some commercial safeners, such as fenchlorazole, have been found to bind to the herbicide target acetyl-CoA carboxylase (ACCase) to show herbicide safener activity [32]. However, no studies to date have demonstrated the virtual screening and rational design of safeners using a computer-assisted drug design method based on binding modes during modeling. The Intermediate Derivatization Methods (IDMs) approach has recently emerged as a novel method used to discover pesticides with a novel skeleton. This method applies a series of synthetic methodolo- gies to key intermediates to efficiently obtain innovative chemical structures with excellent biological activity. IDMs can be divided into three main methods: the Common Intermediate Method (CIM), Terminal Group Replacement Method (TRM), and Active Compound Derivatiza- tion Method (ADM) [33]. Among these methods, ADM uses known active compounds, with functionally amenable groups, as the immediate compounds for the derivation. The discovery of IDMs has led to their use to screen insecticides, fungicides, and herbicides with new structures and higher activity. Although the reported reviews related to IDMs have not yet noted their potential use to identify novel herbicide safeners [33,34], current research revealed that IDMs are efficacious ways to identify novel safeners with higher activity [19,35].
Compounds containing a hydrazone structure have been demon- strated as having widespread pharmacological activities, such as anti- tumor activity, herbicidal activity, insecticide activity, and fungicidal activity [36-39]. The hydrazone structure is considered an active structure in drug design. To date, we do not believe that any commercial herbicide safener has included this structure, and hydrazone moiety has rarely been used as an active group to design novel herbicide safeners.Based on the discussions above, this study synthesized a series of (E)- 4-(2-substituted hydrazinyl)-6-chloro-2-phenypyrimidines using ADM to identify novel high active herbicide safeners. The fenclorim was used as the immediate, and its position on the chlorine atom at the 3-position in the pyrimidine was chosen as a reaction site. Further, a condensation reaction was applied as the synthetic methodology to derivatize the hydrazone core structure into the fenclorim (Scheme 1). The structures were verified using IR (infrared radiation), NMR spectra, and HRMS (high resolution mass spectrometry) data, and were evaluated using a single crystal structure. The study also assessed the bioactivities that serve as herbicide safeners formetolachlorin rice; assessed the safener mechanism that improves the glutathione S-transferases (GSTs) activity and GSTs expression in rice seedlings (shoots and roots tissues); and evaluated the toxicity of the highly active compound to zebrafish (Danio rerio) embryos. These results provide useful guidance for using IDMs to discover new herbicide safeners that are safer for aquatic organisms based on the structure of fenclorim.
2.Results and discussion
2.1.Synthesis and characterization of target compounds 3a-y
Target compounds 3a-y were synthesized using the synthetic route in Scheme 2. Intermediate 2 and target compounds 3a-y were synthesized using the method reported in the reference with modifications [40,41]. Intermediate 2 was first synthesized in ethanol using a condensation reaction of fenclorim with hydrazine hydra in a 49% yield. Then, target compounds 3a-y were obtained using condensation reactions of Inter- mediate 2, with 43-92% yields.
Fig. 1. Structures of selected commercial herbicide safeners.
Scheme 1. Design strategy of target compounds 3a-y.
Scheme 2. Synthetic route of target compounds 3a-y.
The chemical structures of the target compounds were characterized using 1H NMR spectra data, 13C NMR spectra data, and HRMS spectra data. In particular, the compound 3k (4-chloro-6-(2-(2-methyl- benzylidene)hydrazinyl)-2-phenylpyrimidine) was chosen as a model compound. IR spectra of 3k showed strong absorption band peaks at 1569 and 3188 cm− 1, respectively. The latter is attributed to the presence of the C-(-)N and N-H stretching vibration of the hydrazine moiety. 1H NMR spectra of 3k showed the proton of imino group as a singlet at 9.27 ppm, and the protons of the phenyl ring as doublets at 7.94 ppm and 7.80 ppm, respectively, and multiplicities at 7.48-7.50 ppm, 7.26-7.32 ppm, and 7.20-7.22 ppm, respectively. In the 13C NMR spectra of compound 3k, the resonance values of the pyrimidine ring were 101.0 ppm and 161.3-164.5 ppm, while the C-(-)N double bond is 142.5 ppm and the phenyl and substituted phenyl ring are at 126.3-136.7 ppm.To determine the presence of primary, secondary, and tertiary car- bon atoms, DEPT (Distortionless enhancement by polarization transfer) spectra of compounds 3a-y were evaluated. 13C NMR and DEPT spectra of compounds 3a-y confirmed the absence of carbons in the hydrazone C-(-)N double bonds, which appeared at 136.6-152.8 ppm Unused medicines (upward in DEPT). 13C NMR spectra of compounds 3k-p showed signals for the primary carbons at 。20.0-21.4 ppm (upward in DEPT) corresponding to the methyl group and at 。56.2-55.8 ppm (upward in DEPT) corre- sponding to the methoxyl group. 13C NMR and DEPT spectra of com- pounds 3a-g confirmed the presence of straight or branched carbon chains that are saturated, by signals at 。10.7-41.3 ppm (downward or upward in DEPT). In the case of 3h and 3i, 13C NMR spectra of com- pounds 3hand 3i showed the appearance of signals at 126.8-142.7 ppm (upward in DEPT) corresponding to the presence of C-(-)C double bonds in unsaturated alkyl chains.
2.2.Crystal structure of compound 3k
The chemical structures of target compounds 3a-y were further identified using X-ray single crystallography, using the representative compound 3k. Fig. 2 shows that the skeleton of (E)-4-chloro-6-(2-(2-
Fig. 2. Molecular structure of 3kmethylbenzylidene)hydrazinyl)-2-phenylpyrimidine (3k) consisted of a chlorine-substituted pyrimidine ring, a phenyl ring, and a 2-chloro substituted phenyl ring. A substituted phenyl ring was connected to pyrimidine by a hydrazone group, with a bond length of N(3)-N(4) (1.390 Å). This was similar to the C–N bonds (1.350–1.382 Å) re- ported in previous studies [42–44]. The C–N double bond can be observed as trans-conformation. The bonds in the two phenyl rings and pyrimidine ring were similar to the reported C–C and C–N bonds in the phenyl ring and pyrimidine ring [12,45,46]. The non-substituted ben- zene ring was connected to the pyrimidine ring through a C(5)–C(4) bond,with a bond length of 1.476 Å. The packing view of compound 3k revealed that the molecules were linked through intermolecular hydrogen bond interactions such as N–H⋅⋅⋅N (Fig. 3) and the hydrogen bond geometry of 3k is listed in Table S1.
2.3. Evaluation of safener activity
Before testing the target compounds 3a-y for their herbicide safener activity, we evaluated their phytotoxicity to rice seedlings at a concen- tration of 1 mg/L (7 days). Table 1 shows that the relative plant height, root length, and fresh weight of fenclorim (F) were 92.5%, 97.5%, and 93.1%, respectively. The relative plant height, root length, and fresh weight values of 3a-yvaried from 88.6% to 99.1%, 80.9% to 99.1%, and 87.3% to 99.1%, respectively. These results revealed that 3a-y and fenclorim had few inhibitive effects on rice seedlings, and were rela- tively safe to the rice seedlings at a concentration of 1 mg/L.Table 2 illustrates the herbicide safener activities of target com- pounds 3a-y and fenclorim for rice seedlings. Metolachlor had a sig- nificant inhibitive effect on the growth (plant height, root length, and fresh weight) of rice seedlings at a concentration of 0.25 µM; this was similar to the results reported in the references [12,13]. When rice seedlings were treated with 1 mg/L 3a-y, and 0.25 µM metolachlor, 3a-y showed herbicide safener activities on plant height, root length, and fresh weight in various degrees. The relative plant height, root length, and fresh weight varied from 44.6 to 71.1%, 53.3–86.7%, and 74.3–90.8%, respectively. The treatments 3i and 3t represented the best herbicide safener activity on plant height, with relative plant heights of 71.1% and 70.3%, respectively. These values were similar to when using the positive control fenclorim (70.6%). In addition, when the R group was substituted with an alkyl, compounds 3 g-i bearing moieties with five carbon atoms provided better herbicide safener activities compared
Fig. 3. Packing view of compound 3k (hydrogen bonds are described black dashed lines).
Mcl refers to 0.25 µM metolachlor. 3a-y + Mcl refers to the combined treatment of 1 mg/L compounds 3a-y and 0.25 µM metolachlor. F + Mcl refers to the combined treatment of 1 mg/L fenclorim and 0.25 µM metolachlor to the treatments (3a-f) with shorter carbon atoms and (1E, 3E)-1,3- pentadienyl substitution, prior to the amyl and (E)-3 pentenyl sub- stitutions. Compared to 3j-q, the unsubstituted phenyl moiety showed lower herbicide safener activity on plant height compared to the substituted phenyl moieties. This may be due to the substitution effects of phenyl ring substitutions. Particularly, for plant height, ortho-phenyl substitutions displayed more activity compared to the meta- and para- phenyl substituents, with the exception of F atom substitution. The 3t treatment, with a chlorine atom at the ortho-position of the phenyl ring, showed the most impact on plant height.
For root length, while the R group indicated an alkyl substitute, the 3a treatment with the methyl substitution showed a higher herbicide safener activity compared to the other 3b-i compounds with substitutes with longer carbon atoms. While R moiety indicated the substitution of phenyl or gut microbiota and metabolites substituted phenyl, the herbicide safener activity exhibited the following pattern: ortho- phenyl substitution was higher than meta- phenyl substitution, which was higher than the para-phenyl substitu- tion. The exception was the bromine atom on the phenyl ring. The 3t treatment displayed very similar safener activity (85.4%) compared to the fenclorim (85.6%).
For fresh weight, all the alkyl substituted target compounds 3a-i had a similar level of herbicide safener activity (83.8–90.3%) and were more active than with fenclorim (82.1%). In particular, the 3c treatment with n-propyl moiety had the best biological activity. Compared with the phenyl or substituted phenyl substitutions 3j-y, when the methyl, methoxy, and fluorine atoms were substituted for the phenyl moieties, with a meta-substitution, there was more activity compared to an ortho- substitution and para-substitution. When a chlorine and bromine atom was substituted for the phenyl moieties, ortho-substitution was more active compared to that of meta-substitution and para-substitution.
The results described above indicate there were no significant dif- ferences between the alkyl-substituent and phenyl- or substituted phenyl-substitution in the herbicide safener activities. However, it can be concluded that different substitutes affected the herbicide safener activity that protected rice seedlings from injury caused by metolachlor. However, it was difficult to identify an explicit relationship between the length of alkyl substituents and the safening effects. Besides, while R group is the substitution of phenyl or substituted phenyl, the fluorine atom was a similar size compared to the hydrogen atom, however, it has a strong induction effect. This may be explained by the fact that when the fluorine atom was substituted with phenyl, the activity was higher compared to when there was no substituted phenyl substitution. In particular, with respect to the herbicide safener activity on the fresh weight, methyl substitution on the phenyl ring displayed more activity compared to the methoxy moiety. It might be the smaller steric hin- drance resulted in the stronger activity of methyl substitution. Mean- while, the electron withdraw group showed the following pattern: the fluorine atom with the strongest induction effect and the smallest steric hindrance was greater than the chlorine atom, which was greater than the bromine atom. In all, the 3i treatment bearing a (1E, 3E)-1,3-pen- tadienyl substitution and 3t bearing an ortho-chlorine atom substituted with a phenyl moiety showed the highest herbicide safener activity on plant height,and also had positive impacts on plant root length and fresh weight.To better understand the activity associated with the 3i and 3t treatments, the herbicide safener activity of 3i, 3t, and fenclorim were further evaluated in different samples at concentrations of 0.5, 0.25, 0.1, 0.05, and 0.025 mg/L. When the test concentrations decreased, 3i and 3t were generally more active compared to fenclorim with respect to plant height and fresh weight (Fig. 4a and b) and showed similar activities compared to fenclorim (Fig. 4c). These results indicated that 3i and 3t showed excellent safener activity, even at low concentrations.
2.4. Evaluation of GST activities of 3i, 3t and fenclorim
GST is associated with the protective effect filled by safener func- tions, alleviating the plant cells from oxidative injury by enhancing the detoxification with the addition of GSH [19]. Based on the preliminary bioassay experiments results above, the treatments 3i and 3t and the commercial safener fenclorim were selected as representative com- pounds for a further enzyme GST assay in vivo. Among these, 3i, 3t, and fenclorim had similar effects on GST activity in rice seedlings. It should be noted that the GST activities of 3i, 3t, and fenclorim treatments were enhanced in both shoots and roots tissues, and exceeded levels in both themetolachlor treated rice seedlings and in the control check sample (CK) (Fig. 5). In the shoots, the 3i and 3t treatments were associated
Fig.5.Comparison on GSTs activities for the treatments of 3i, 3t, or fenclorim (F) and metolachlor (M), metolachlor, and control check (CK) sample in rice seedlings.
Fig.4.The plant height relative values (a), root length relative values (b) and fresh weight relative values (c) of the combined treatments 3i, 3t and fenclorim under 0.5 mg/L, 0.25 mg/L, 0.1 mg/L1, 0.05 mg/L, and 0.025 mg/L with 0.25 µM metolachlor on the rice seedlings with GST activity levels that were approximately 3.18 and 3.01 times, respectively, compared to themetolachlor treatment, and showed about 4.99 and 3.01 times GST activity levels, compared to CK, respectively. Meanwhile, in the root, 3i and 3t were also associated with approxi- mately 1.14 and 1.10 times GST activities, respectively, compared to the metolachlor-treated group, and exhibited about 1.48 and 1.44 times GST activities, respectively, comparing CK. According to these results, the compounds 3i and 3t displayed good protective effects against Mcl and enhanced the tolerance of rice seedlings by improving the GST activities both in shoots and roots tissues.
2.5.Evaluation of GST gene relative expression of 3i, 3t, and fenclorim
To verify the difference in the GST activity, three related GST genes were selected and were further analyzed to assess their relative expression levels under different treatments. In shoot tissues, the rela- tive mRNA expression levelsofOsGSTU3 (1.26-fold, 1.26-fold, and 1.98- fold), OsGSTU39 (2.55-fold, 1.78-fold, and 2.38-fold), and OsGSTF5 (2.24-fold, 2.16-fold, and 2.11-fold) were greatly enhanced in the 3i, 3t, and fenclorim treatments, compared to themetolachlor-treated control l (Fig. 6a). Furthermore, in root tissues, the relative mRNA expression levels of OsGSTU3 (5.29-fold, 4.42-fold, and 5.26-fold), OsGSTU39 (10.06-fold, 6.64-fold, and 14.13-fold), and OsGSTF5 (2.99-fold, 5.99- fold, and 1.56-fold) were enhanced in the 3i, 3t, and fenclorim treat- ments, comparing the metolachlor-treated control (Fig. 6b). The un- regulated related GST genes might contribute to the improvement of the GST activities in the shoots and roots tissues.
2.6.Acute toxicities of 3i, 3t, and fenclorim to zebrafish embryos
Fenclorim was proved to pose a potential risk to aquatic organisms, such as fish [27]. As such, we further evaluated the acute toxicity of the 3i, 3t, and fenclorim treatment to zebrafish embryos (96 h, Table 3). which is used as a model to evaluate the toxicity of aquatic organisms. Results showed that treatments 3i and 3t showed very low toxicities, with LC50 values of 29.96 mg/L and 13.80 mg/L, respectively. These levels were significantly lower compared to that of fenclorim (1.37 mg/ L), indicating they are generally safe to aquatic organisms as fish.
3.Experimental procedure
3.1.Chemicals
Fenclorim (98%), hydrazine monohydrate (98%), and substituted aldehydes (37-99%) were purchased commercially from Jilin Chinese Academy of Sciences-Yanshen Technology Co., Ltd. (Changchun, Jilin, China). Ethanol (EtOH), ethyl acetate (EA), and petroleum ether (PE) were bought commercially from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and were used without further purification. Melting points were recorded using a Hanon MP100 automatic melting point apparatus (Jinan Hanon Instruments Co., Ltd., Jinan, Shangdong, China) and autocorrected. The1H NMR and 13C NMR spectra were obtained using a Bruker Avance-DMX 500 Spectrometer (Bruker Optics, Inc., Ettlingen,BW, Germany) operating at 500 MHz (1H NMR) and 125 MHz (13C NMR), respectively. The DEPT-135 spectra were recorded on a BRUKER 400 MHZ-NMR spectrophotometer. Tetramethylsilane (TMS) was used as the internal standard. The IR spectra were collected using an ATR-method (attenuated total reflection) with a TENSOR II-Bruker FT- IR spectrometer (Bruker Optics, Inc., Ettlingen, BW, Germany) or a pellet KBr method with a Nicolet iS50 FT-IR Spectrometer (Thermo Fisher Scientific, Ettlingen, Waltham, MA, America). HRMS data were collected using an FTICR-MS Varian 7.0T FTICR-MS instrument (Varian IonSpec, Lake Forest, IL, USA). Single-crystal X-ray data were collected using a Rigaku SuperNova, Dual, Cu at zero, AtlasS2 diffractometer (Agilent Technologies Inc., Santa Clara, CA, USA).
3.2.Experimental procedures for the synthesis of target compounds 3a-y
3.2.1.Synthesis of 4-chloro-6-hydrazinyl-2-phenylpyrimidine (2)
A mixture of 0.44 mL 98% Hydrazine monohydrate (8.8 mmol, 0.45 g) in 10 mL EtOH was slowly added into a mixture of fenclorim (4.4 mmol, 1.00 g) and 40 mL EtOH. Next, this mixture solution was stirred at 60 ◦ C for 5 h, cooled, and filtered to obtain precipitates as the crude
Fig. 6. Comparison of relative mRNA expression levels of GSTs in shoots (a) and root (b) of rice seedlingsproduct. The crude product was further purified using column chro- matography (PE/EA = 1/10) to obtain the pure target immediate 2 (0.48 g, yield: 49%).
3.2.2.Synthesis of target compounds 3a-y
A mixed solution of substituted aldehydes (2.4 mmol) in 10 mL EtOH was slowly added into a mixture of immediate 2 (2.2 mmol, 0.48 g) in 30 mL EtOH. The mixture solution was then stirred overnight at room temperature, and filtered to obtain filter residue as the crude products. The crude product was purified using column chromatography (PE/EA = 1/6) to generate the pure target compounds 3a-y:
3.2.2.1.(E)-4-chloro-6-(2-ethylidenehydrazinyl)-2-phenylpyrimidine (3a). Yellow solid, yield 89%, m.p. 120-121 ◦ C; IR (ATR) ν: 3193, 1579 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.74 (s, 1H, NH), 8.33-8.36 (m, 2H, ArH), 7.46-7.52 (m, 3H,ArH), 7.06-7.09 (m, 2H, CH + PyH), 1.98 (d, J = 5.4 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.6, 161.1, 143.6, 136.6, 131.0, 128.5, 128.3, 100.5, 18.2; DEPT (100 MHz, DMSO-d6): δ 146.0, 131.6, 129.0, 128.2, 99.5, 18.9; HRMS calcd. for C12H11ClN4 ([M + H]+), 247.0745; found, 247.0744.
3.2.2.2. (E)-4-chloro-2-phenyl-6-(2-propylidenehydrazinyl)pyrimidine (3b). White solid, yield 85%, m.p. 114-115 ◦ C; IR (KBr) ν: 3110, 1670 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.36 (dd, J = 7.8, 3H, 1.9 Hz,ArH), 7.46-7.50 (m, 3H, CH + ArH), 7.25 (t, J = 4.9 Hz, 1H,ArH), 7.06 (s, 1H, PyH), 2.39 (qd, J = 7.5, 5.0 Hz, 2H, CH2), 1.18 (t, J = 7.5 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.5, 161.0, 148.4, 136.6, 131.0, 128.5, 128.3, 100.6, 25.7, 10.7; DEPT (100 MHz, CDCl3): δ 148.4, 131.0, 128.5, 128.3, 100.6, 25.7, 10.7; HRMS calcd. for C13H13ClN4 ([M + H]+), 261.0902; found, 261.0901.
3.2.2.3.(E)-4-(2-butylidenehydrazinyl)-6-chloro-2-phenylpyrimidine (3c). White solid, yield 50%, m.p. 104-105 ◦ C; IR (ATR) ν: 3194, 1580 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.35 (dd, J = 8.0, 1.7 Hz, 3H, NH + ArH), 7.48-7.50, (m, 3H, ArH), 7.06 (s, 1H, PyH), 6.93 (t, J = 7.5 Hz, 1H, CH), 2.34 (td, J = 7.4, 5.4 Hz, 2H, CH2), 1.58-1.66 (m, 2H, CH2), 0.94 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.6, 161.0, 147.7, 136.6, 131.1, 128.5, 128.3, 100.6, 34.2, 19.8, 13.7; DEPT (100 MHz, CDCl3): δ 147.3, 131.0, 128.4, 128.3, 100.6, 34.2, 19.9, 13.7; HRMS calcd. for C14H15ClN4 ([M + H]+), 275.1058; found, 275.1057.
3.2.2.4. (E)-4-chloro-6-(2-(2-methylpropylidene)hydrazinyl)-2-phenyl- pyrimidine (3d). Yellow solid, yield 84%, m.p. 133-134 ◦ C; IR (ATR) ν: 3190, 1586 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.41 (s, 1H, NH), 8.34-8.37 (m, 2H, ArH), 7.44-7.52 (m, 3H, ArH), 7.11 (d, J = 5.0 Hz, 1H, CH), 7.05 (s, 1H,PyH), 2.60 (pd, J = 6.9, 5.0 Hz, 1H, CH), 1.66 (d, J = 6.9 Hz, 6H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.9, 161.0, 152.8, 136.5, 131.2, 128.6, 128.4, 100.6, 31.3, 19.6; DEPT (100 MHz, CDCl3): δ 154.0, 131.6, 129.0, 128.3, 99.6, 31.4, 20.0; HRMS calcd. for C14H15ClN4 ([M + H]+), 275.1058; found, 275.1057.
3.2.2.5.(E)-4-chloro-6-(2-pentylidenehydrazinyl)-2-phenylpyrimidine (3e). White solid, yield 59%, m.p. 120-121 ◦ C; IR (ATR) ν: 3322, 1551 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.53 (s, 1H, NH), 8.32-8.40 (m, 2H, ArH), 7.44-7.54 (m, 3H,ArH), 7.14 (t, J = 5.4 Hz, 1H, CH), 7.06 (s, 1H, PyH), 2.29-2.37 (m, 2H, ArH), 1.48-1.59 (m, 2H, ArH), 1.40 (dt, J = 14.5, 7.4 Hz, 2H, ArH), 0.97 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.6, 161.0, 147.9, 136.5, 131.1, 128.5, 128.3, 100.6, 31.9, 28.5, 22.2, 13.8; DEPT (100 MHz, CDCl3): δ 147.8, 131.1, 128.5, 128.5, 128.3, 100.6, 31.9, 28.5, 22.2, 13.9; HRMS calcd. for C15H17ClN4 ([M + H]+), 289.1215; found, 289.1212.
3.2.2.6. (E)-4-chloro-6-(2-(3-methylbutylidene)hydrazinyl)-2-phenyl- pyrimidine (3f). White solid, yield 57%, m.p. 125-126 ◦ C; IR (KBr) ν: 3310,1600 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.36 (dd, J = 8.0, 1.7 Hz, 3H, ArH + NH), 7.46-7.52 (m, 3H,ArH), 7.22 (t, J = 5.8 Hz, 1H, CH3), 7.07 (s, 1H,PyH), 2.26 (dd, J = 6.9, 5.8 Hz, 2H, CH2), 1.92 (dq, J = 13.5, 6.8 Hz, 1H, CH), 1.01 (d, J = 6.7 Hz, 6H, CH3). 13C NMR (125 MHz, CDCl3): δ 164.5, 162.6, 161.1, 147.2, 136.5, 131.1, 128.5, 128.3, 100.6, 41.0, 26.7, 22.4; DEPT (100 MHz, DMSO-d6): δ 149.1, 131.6, 129.0, 128.2, 99.6, 41.3, 26.7, 22.7; HRMS calcd. for C15H17ClN4 ([M + H]+),289.1215; found, 289.1214.
3.2.2.1.(E)-4-chloro-6-(2-hexylidenehydrazinyl)-2-phenylpyrimidine (3 g). White solid, yield 63%, m.p. 219-220 ◦ C; IR (KBr) ν: 3190, 1590 cm− 1; 1H NMR (500 MHz, CDCl3): δ 8.83 (s, 1H, NH), 8.33-8.36 (m, 2H, ArH), 7.46-7.52 (m, 3H,ArH), 7.06 (s, 1H,PyH), 7.04 (t, J = 5.4 Hz, 1H, CH), 2.25-2.29 (m, 2H, CH2), 1.49 (p, J = 7.5 Hz, 2H, CH2), 1.29-1.41 (m, 4H, CH2), 0.92 (t, J = 7.0 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.8, 161.1, 148.2, 136.6, 131.2, 128.6, 128.4, 100.7, 32.3, 31.4, 26.1, 22.5, 14.1; DEPT (100 MHz, CDCl3): δ 147.8, 131.1, 128.5, 128.5, 128.3, 100.6, 31.9, 28.5, 22.2, 13.9; HRMS calcd. for C16H19ClN4 ([M + H]+), 303.1371; found, 303.1369.
3.2.2.7. 4-chloro-6-(2-((1E,2E)-hex-2-en-1-ylidene)hydrazinyl)-2-phe- nylpyrimidine (3h). White solid, yield 72%, m.p. 121-122 ◦ C; IR (KBr) ν: 3190, 1590 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.52 (s, 1H, NH), 8.33-8.38 (m, 2H, ArH), 7.46-7.52 (m, 3H, ArH), 7.42 (d, J = 9.8 Hz, 1H, CH), 7.07 (s, 1H, PyH), 6.28 (ddt, J = 15.6, 9.1, 1.4 Hz, 1H, CH), 6.07 (dt, J = 14.8, 7.0 Hz, 1H, CH), 2.22 (qd, J = 7.2, 1.5 Hz, 2H, CH2), 1.52 (h, J = 7.4 Hz, 2H, CH2), 0.97 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.4, 161.2, 146.3, 142.9, 136.5, 131.2, 128.6,128.3, 126.7, 100.7, 34.8, 21.9, 13.7; DEPT (100 MHz, DMSO-d6): δ 147.5, 142.7, 131.6, 129.0, 128.3, 127.9, 99.8, 34.8, 21.9, 14.0; HRMS calcd. for C16H17ClN4 ([M + H]+), 301.1215; found, 301.1212.
3.2.2.8.4-chloro-6-(2-((1E,2E,4E)-hexa-2,4-dien-1-ylidene)hydra- zinyl)-2-phenylpyrimidine(3i).White solid, yield 50%,m.p. 168-169 ◦ C; IR (KBr) ν: 3060, 1650 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.23 (s, 1H, NH), 8.34-8.35 (m, 2H,ArH), 7.47-7.49 (m, 3H,ArH), 7.15 (d, J = 9.6 Hz, 1H, CH), 7.08 (s, 1H,PyH), 2.17 (q, J = 6.8, 6.2 Hz, 2H, CH2), 1.48 (q, J = 7.3 Hz, 2H, CH2), 0.95 (d, J = 14.6 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.4, 161.3, 146.2, 139.5, 136.6, 134.5, 131.3, 131.1, 128.7, 128.5, 125.5, 100.9, 18.7; DEPT (100 MHz, DMSO-d6): 147.4, 139.5, 134.4, 131.8, 131.7, 129.1, 128.3, 126.8, 99.9, 18.8; HRMS calcd. for C16H15ClN4 ([M + H]+), 299.1058; found, 299.1055.
3.2.2.9. (E)-4-(2-benzylidenehydrazinyl)-6-chloro-2-phenylpyrimidine (3j). White solid, yield 44%, m.p. 203-204 ◦ C; IR (KBr) ν: 3180, 1610 cm− 1; 1H NMR (500 MHz, CDCl3): δ 9.22 (s, 1H, NH), 8.37-8.41 (m, 2H, ArH), 7.62-7.64 (m, 3H, CH + ArH), 7.49-7.52 (m, 3H,ArH), 7.40-7.45 (m, 3H, ArH), 7.24 (s, 1H, PyH); 13C NMR (125 MHz, CDCl3): δ 164.7, 162.6, 161.4, 143.6, 136.6, 133.8, 131.4, 130.2, 128.9, 128.7, 128.5, 127.1, 101.1. DEPT (100 MHz, DMSO-d6): δ 144.8, 131.7, 130.3, 129.3, 129.1, 128.3, 127.4, 100.3; HRMS calcd. for C17H13ClN4 ([M + H]+), 309.0902; found, 309.0899.
3.2.2.10.(E)-4-chloro-6-(2-(2-methylbenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3k). White solid, yield 62%, m.p. 187-188 ◦ C; IR (ATR) ν: 3188, 1569 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.27 (s, 1H, NH), 8.40-8.43 (m, 2H,ArH), 7.94 (d, J = 5.3 Hz, 1H,ArH), 7.80 (d, J = 7.3 Hz, 1H, ArH), 7.48-7.50 (m, 3H, CH + ArH), 7.26-7.32 (m, 2H, ArH), 7.20-7.22 (m, 2H, ArH + PyH), 2.40 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.5, 161.3, 142.5, 136.7, 136.4, 131.7, 131.3, 131.0, 129.8 128.6 128.4 127.0, 126.3 101.0, 19.9; DEPT (100 MHz, CDCl3): δ 142.3, 131.2, 131.1, 129.8, 128.5, 128.3, 127.2, 126.3, 100.9, 20.0; HRMS calcd. for C18H15ClN4 ([M + H]+), 323.1058; found, 323.1054.
3.2.2.11.(E)-4-chloro-6-(2-(3-methylbenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3l). White solid, yield 56%, m.p. 204-205 ◦ C; IR (ATR) ν: 3199, 1566 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.02 (s, 1H, NH), 8.38-8.40 (m, 2H,ArH) 7.66 (s, 1H, CH), 7.49-7.50 (m, 4H,ArH), 7.43 (d, J = 7.0 Hz, 1H,ArH), 7.32 (t, J = 7.6 Hz, 2H,ArH), 7.24 (s, 1H,PyH), 2.43 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.4, 161.2, 143.7, 138.6, 136.5, 133.6, 131.2, 131.0, 128.7, 128.5, 128.4, 127.4, 124.4, 101.0, 21.4; DEPT (100 MHz, DMSO-d6): δ 145.0, 131.7, 131.0, 129.2, 129.0, 128.3, 127.7, 124.7, 100.3, 21.4; HRMS calcd. for C18H15ClN4 ([M + H]+), 323.1058 found, 323.1055.
3.2.2.12. (E)-4-chloro-6-(2-(4-methylbenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3m). White solid, yield 57%, m.p. 215-216 ◦ C; IR (ATR) ν: 3177, 1573 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.91 (s, 1H, NH),8.38-8.40 (m, 2H,ArH), 7.68 (s, 1H, CH), 7.56 (d, J = 8.1 Hz, 2H,ArH), 7.50 (dd, J = 5.2, 1.9 Hz, 3H,ArH), 7.22-7.25 (s, 3H,PyH + ArH), 2.42 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.5, 161.2, 143.8, 140.4, 136.5, 131.2, 130.9, 129.5, 128.6, 128.4, 127.0, 100.9, 21.5; DEPT (100 MHz, CDCl3): δ 131.7, 131.4, 126.9, 126.0, 116.3, 110.4, 20.2; HRMS calcd. for C18H15ClN4([M + H]+), 323.1058; found, 323.1055.
3.2.2.13. (E)-4-chloro-6-(2-(2-methoxybenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3n). White solid, yield 68%, m.p. 159-160 ◦ C; IR (ATR) ν: 3310, 1589 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.90 (s, 1H, NH), 8.38-8.40 (m, 2H, ArH), 8.20 (s, 1H, CH), 7.98-8.00 (m, 1H, ArH), 7.46-7.50 (m, 3H, ArH), 7.36-7.40 (m, 1H, ArH), 7.20 (s, 1H, PyH), 7.02-7.05 (d, J = 7.5 Hz, 1H,ArH), 6.92 (d, J = 6.1 Hz, 1H,ArH), 3.88 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.4, 161.1, 157.7, 139.6, 136.5, 131.3, 131.1, 128.4, 128.3, 126.2, 122.2, 120.9, 111.1, 100.9, 55.5; DEPT (100 MHz, DMSO-d6): δ 140.4, 131.8, 131.7, 129.1, 128.3, 126.1, 121.3, 112.2, 100.2, 56.2; HRMS calcd. for C18H15ClN4O ([M + H]+), 339.1007; found, 339.1005.
3.2.2.14. (E)-4-chloro-6-(2-(3-methoxybenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3o). White solid, yield 43%, m.p. 211-212 ◦ C; IR (ATR) ν: 3199, 1569 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.29 (s, 1H, NH), 8.38-8.40 (m, 2H,ArH), 7.72 (m, 1H, CH), 7.48-7.52 (m, 3H,ArH), 7.36 (t, J = 7.9 Hz, 1H,ArH), 7.26 (dd, J = 2.4, 1.5 Hz, 1H,ArH), 7.21-7.23 (m, 2H, ArH + PyH), 6.98 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H,ArH), 3.91 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.5, 161.3, 159.9, 143.5, 136.4, 135.0, 131.3, 129.8, 128.6, 128.4, 120.1, 116.1, 111.4, 101.0, 55.4; DEPT (100 MHz, DMSO-d6): δ 144.7, 131.7, 130.4, 129.1, 128.3, 120.1, 116.3, 111.9, 100.4, 55.7; HRMS calcd. for C18H15ClN4O ([M + H]+), 339.1007; found, 339.1006.
3.2.2.15.(E)-4-chloro-6-(2-(4-methoxybenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3p). White solid, yield 72%, m.p. 204-205 ◦ C; IR (ATR) ν: 3183, 1573 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.29 (s, 1H, NH), 8.39 (dd, J = 6.3, 2.9 Hz, 2H, ArH), 7.48-7.53 (m, 6H, CH + ArH), 7.20 (s, 1H, ArH), 6.93 (d, J = 8.7 Hz, 2H, ArH), 3.86 (s, 3H, OCH3); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.5, 161.2, 161.1, 143.5, 136.5, 131.2, 128.6 128.5, 128.4 126.4, 114.2, 100.8, 55.4; DEPT (100 MHz, DMSO-d6): 144.8, 131.7, 129.1, 129.0, 128.3, 114.8, 100.1, 55.8; HRMS calcd. for C18H15ClN4O ([M + H]+), 339.1007; found, 339.1006.
3.2.2.16.(E)-4-(2-(2-bromobenzylidene)hydrazinyl)-6-chloro-2-phe- nylpyrimidine (3q). White solid, yield 55%, m.p. 183-184 ◦ C; IR (KBr) ν: 3180, 1610 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.04 (s, 1H, NH), 8.39-8.41 (m, 2H,ArH), 8.18 (s, 1H, CH), 8.06 (dd, J = 7.9, 1.5 Hz, 1H, ArH), 7.58-7.60 (m, 1H,ArH), 7.47-7.50 (m, 3H,ArH), 7.39 (t, J = 7.5 Hz, 1H,ArH), 7.26 (td, J = 8.0, 1.7 Hz, 1H,ArH), 7.21 (s, 1H,PyH); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.3, 161.3, 142.1, 136.3, 133.2, 132.6, 131.3, 131.1, 128.5, 128.4, 127.7, 127.5, 123.8, 101.0; DEPT (100 MHz, DMSO-d6): 143.0, 133.6, 131.8, 131.8, 129.1, 128.5, 128.3, 127.8, 100.6; HRMS calcd. for C17H12BrClN4 ([M + H]+), 387.0007; found, 387.0006.
3.2.2.17.(E)-4-(2-(3-bromobenzylidene)hydrazinyl)-6-chloro-2-phe- nylpyrimidine (3r). White solid, yield 69%, m.p. 216-217 ◦ C; IR (KBr) ν: 3190, 1610 cm− 1;1H NMR (500 MHz, DMSO-d6): δ 11.99 (s, 1H, NH), 8.29-8.31 (m, 2H,ArH), 8.16 (s, 1H, CH), 7.99 (d, J = 1.8 Hz, 1H,ArH), 7.77 (d, J = 7.8 Hz, 1H, ArH), 7.59 (dd, J = 8.3, 1.4 Hz, 1H, ArH), 7.51-7.56 (m, 3H,ArH), 7.40 (t, J = 7.8 Hz, 1H,ArH), 7.25 (s, 1H,PyH); 13C NMR (125 MHz, DMSO-d6): δ 164.0, 163.2, 160.5, 143.0, 137.2, 136.6, 132.7, 131.7, 131.3, 129.5, 129.1, 128.3, 126.5, 122.7, 100.6; DEPT (100 MHz, DMSO-d6): δ 143.0, 132.7, 131.7, 131.3, 129.5, 129.1, 128.3, 126.5, 100.6; HRMS calcd. for C17H12BrClN4 ([M + H]+), 387.0007 found, 387.0008
3.2.2.18.(E)-4-(2-(4-bromobenzylidene)hydrazinyl)-6-chloro-2-phe- nylpyrimidine (3 s). White solid, yield 84%, m.p. 240-241 ◦ C; IR (ATR) ν: 3190, 1593 cm− 1;1H NMR (500 MHz, DMSO-d6): δ 8.85 (s, 1H, NH), 8.38-8.45 (m, 2H,ArH), 8.26 (s, 1H, CH), 8.09 (dd, J = 7.9, 1.3 Hz, 1H, ArH), 7.61 (d, J = 7.8 Hz, 1H,ArH), 7.48-7.52 (m, 3H,ArH), 7.41 (t, J = 7.5 Hz, 1H,ArH), 7.25-7.30 (m, 1H,ArH), 7.22 (s, 1H, PyH); 13C NMR(125 MHz, DMSO-d6): δ 164.0, 163.2, IDE397 160.4, 143.4, 136.6, 134.0, 132.2, 131.7, 129.2, 129.0, 128.3, 123.4, 100.4; DEPT (100 MHz, DMSO-d6): δ 143.5, 132.2, 131.7, 129.2, 129.1, 128.3, 100.4; HRMS calcd. for C17H12BrClN4 ([M + H]+), 387.0007; found, 387.0008.
3.2.2.19.(E)-4-chloro-6-(2-(2-chlorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3t). White solid, yield 55%, m.p. 172-173 ◦ C; IR (ATR) ν: 3177, 1545 cm− 1;1H NMR (500 MHz, CDCl3): δ 9.06 (s, 1H, NH), 8.38-8.41 (m, 2H,ArH), 7.68 (d, J = 2.0 Hz, 1H,ArH), 7.66 (s, 1H, CH), 7.48-7.53 (m, 4H,ArH), 7.35-7.40 (m, 2H,ArH), 7.24 (s, 1H,PyH); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.3, 161.3, 139.8, 136.3, 133.8, 131.3, 131.2, 130.8, 129.9, 128.5, 128.4, 127.1, 127.1, 101.0; DEPT (100 MHz, DMSO-d6): δ 143.0, 132.7, 131.7, 131.3, 129.5, 129.1, 128.3, 126.5, 100.8; HRMS calcd. for C17H12Cl2N4 ([M + H]+), 343.0512; found, 343.0511.
3.2.2.20.(E)-4-chloro-6-(2-(3-chlorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3u). White solid, yield 65%, m.p. 213-214 ◦ C; IR (ATR) ν: 3193, 1563 cm− 1;1H NMR (500 MHz, CDCl3): δ 11.98 (s, 1H, NH), 8.29-8.30 (m, 2H,ArH), 8.17 (s, 1H, CH), 7.86 (d, J = 2.2 Hz, 1H,ArH,), 7.71-7.72 (m, 1H, ArH), 7.51-7.57 (m, 3H, ArH), 7.45-7.48 (m, 2H, ArH), 7.25 (s, 1H, PyH); 13C NMR (125 MHz, DMSO-d6): δ 163.99, 163.24, 160.46, 143.06, 136.93, 136.60, 134.18, 131.68, 131.06, 129.80, 129.03, 128.28, 126.54, 126.11, 100.58; DEPT (100 MHz, CDCl3): δ 139.8, 131.3, 130.9, 129.9, 128.5, 128.4, 127.1, 127.9, 101.0; HRMS calcd. for C17H12Cl2N4 ([M + H]+), 343.0512; found, 343.0509.
3.2.2.21.(E)-4-chloro-6-(2-(4-chlorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3v). White solid, yield 92%, m.p. 241-242 ◦ C; IR (ATR) ν: 3187, 1552 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.73 (s, 1H, NH), 8.38-8.40 (m, 2H,ArH), 7.78 (s, 1H, CH), 7.65 (d, J = 8.4 Hz, 2H,ArH), 7.47-7.52 (m, 3H,ArH), 7.43 (d, J = 8.4 Hz, 2H,ArH), 7.21 (s, 1H,PyH); 13C NMR (125 MHz, DMSO-d6) δ 164.0, 163.2, 160.4, 143.3, 136.6, 134.6, 133.6, 131.7, 129.3, 129.0, 128.9, 128.3, 100.4; DEPT (100 MHz, DMSO-d6): δ 143.4, 131.7, 129.3, 129.1, 129.0, 128.3, 100.4; HRMS calcd. for C17H12Cl2N4 ([M + H]+), 343.0512; found, 343.0509.
3.2.2.22.(E)-4-chloro-6-(2-(2-fluorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3w). Yellow solid, yield 46%, m.p. 181-182 ◦ C; IR (ATR) ν: 3322, 1551 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.87 (s, 1H, NH), 8.44-8.45 (m, 2H,ArH), 8.06 (s, 1H, CH), 8.02 (td, J = 7.6, 1.8 Hz, 3H, ArH), 7.55-7.45 (m, 2H,ArH), 7.40 (dddd, J = 8.4, 7.3, 5.3, 1.8 Hz, 1H, ArH), 7.29 (s, 1H, PyH), 7.23-7.26 (m, 1H, ArH); 13C NMR (125 MHz, CDCl3): δ 164.5, 162.4, 161.3, 160., 136.6, 136.3, 131.4, 131.2, 128.5, 128.4, 126.6, 124.5, 121.6, 115.8, 101.0; DEPT (100 MHz, CDCl3): δ 136.4, 131.5, 131.2, 128.5, 128.4, 126.6, 124.5, 116.0, 101.0; HRMS calcd. for C17H12ClFN4 ([M + H]+), 327.0807; found, 327.0804.
3.2.2.23.(E)-4-chloro-6-(2-(3-fluorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3x). White solid, yield 69%, m.p. 223-224 ◦ C; IR (ATR) ν: 3189, 1549 cm− 1;1H NMR (500 MHz, DMSO-d6): δ 11.98 (s, 1H, NH), 8.29-8.31 (m, 2H, ArH), 8.19 (s, 1H, CH), 7.67-7.69 (m, 1H, ArH), 7.46-7.59 (m, 5H,ArH), 7.22-7.27 (m, 2H, ArH + PyH); 13C NMR (126 MHz, DMSO-d6): δ 163.99, 163.27, 162.00, 160.47, 143.28, 137.33, 136.60, 131.69, 131.27, 129.05, 128.28, 123.92, 116.99, 113.28, 100.59; DEPT (100 MHz, DMSO-d6): δ 137.3, 132.0, 131.9, 131.7, 129.0, 128.3, 126.9, 125. 3, 116.4, 100.5; HRMS calcd. for C17H12ClFN4 ([M + H]+), 327.0804; found, 327.0807.
3.2.2.24.(E)-4-chloro-6-(2-(4-fluorobenzylidene)hydrazinyl)-2-phe- nylpyrimidine (3y). White solid, yield 43%, m.p. 221-222 ◦ C; IR (ATR) ν: 3191, 1573 cm− 1;1H NMR (500 MHz, CDCl3): δ 8.96 (s, 1H, NH), 8.38-8.40 (m, 2H, ArH), 7.70 (s, 1H, CH), 7.65-7.68 (m, 2H, ArH), 7.48-7.52 (m, 3H,ArH), 7.21 (s, 1H,PyH), 7.11-7.16 (m, 2H,ArH); 13C NMR (125 MHz, CDCl3): δ 164.6, 162.5, 161.3, 142.3, 136.4, 131.3, 128.8, 128.8, 128.6, 128.4, 116.0, 115.9, 101.0; DEPT (100 MHz, DMSO-d6): δ 143.5, 131.7, 129.5, 129.0, 128.3, 116.3, 100.3; HRMS calcd. for C17H12ClFN4 ([M + H]+), 327.0807; found, 327.0805.
3.3. Crystal structural determination
The single-crystal structure of compound 3k was recrystallized from the dilute methanol solution of 3k to form colorless single crystals. Then, the suitable crystal was selected and X-ray single-crystal structure data of compound 3k were collected with a Rigaku SuperNova, Dual, Cu at zero, AtlasS2 diffractometer equipped with MoKα radiation (λ = 1.54184 Å). The crystal was kept at 100.01(10) K during data collection. The structure was further solved using the ShelXS structure solution program by Direct Methods and refined using a ShelXL refinement package via Least Squares minimization using Olex2 [47–49]. Selected crystallographic data and molecular structure parameters of compound 3k are described in Tables S2 and S3, respectively. The crystallographic data of compound 3k were deposited at the Cambridge Crystallographic Data Centre (CCDC) (CCDC number: 1966186). These data are available at https://www.ccdc.cam.ac.uk/structures.
3.4.Evaluation of herbicide safener activity
The germination method reported in the literature was applied for the rice seed experiments (Oryza sativa L.) to evaluate the phytotoxicity and herbicide safener activity [12]. Rice seeds with full grains and uniform sizes were sterilized using 5% sodium hypochlorite solution and were washed using deionized water. Next, the sterilized rice seeds were soaked in deionized water for 24 hand then germinated for another 36 h in a growth cabinet at 28 ◦ C in the dark.The phytotoxicity and herbicide safener activity were evaluated under laboratory conditions using the agar medium method [50]. Uni- formly germinated paddy rice seedlings were selected before emergence and were transplanted into a 0.3% agar media containing 0.25 μM metolachlor (Mcl), 1 mg/Lof compounds 3a-y, combined fenclorim (F) with 1 mg/L and 0.25 μM Mcl, and combined compounds 3a-y with 1 mg/L and 0.25 μM Mcl. Agar media without compounds were used as the non-treated controls. Fifty seeds were placed into each plate and were then incubated for 14 h under a grow light (at intensity 110– 130 μE m− 2 s− 1 and 30 ◦ C). This was followed by a 10 h dark photoperiod (at 25 ◦ C). The indices of plant height, root length, and fresh weight related to herbicide safener activity were measured after 7 days. The herbicide safening effects of the relative plant height, root length, and fresh weight were used to support a structure–activity relationship analysis, calcu- lated by the following equations (1) to (3):Relative plant height= (plant height under each treatment)/(the average height of untreated control plants) × 100% (1) Relative root length = (root length under each treatment)/(the average root length of the untreated control) × 100% (2) Relative fresh weight = (fresh weight under each treatment)/(the average fresh weight of the untreated control) × 100% (3)The compounds with good herbicide safener activities on plant height were screened using additional experiments at lower concentra- tions (0.5 mg/L, 0.25 mg/L, 0.1 mg /L, 0.05 mg/L, and 0.025 mg/L). All experiments of testing herbicide safener activity were performed in triplicate.
3.5. Evaluation of GST activity and GST gene relative expression
A GST activity assay of rice-seedling (7 days) treatment with 0.25 μM metolachlor, the combination of 1 mg/L compounds 3i, 3t, and dichlormid, 0.25 μMmetolachlor, and no compounds (control check, no- treatment) was performed using a method with modifications reported in the references [49]. GST activity was tested with a 0.1 g sample of rice seedling shoots and roots. Shoots and roots were first flash frozen in liquid nitrogen and ground into a powder, and 1 mL of extraction so- lution containing phosphate buffer (PB, 100 mM, pH 7.8), polyvinyl pyrrolidone (5%, w/v), and sodium pyrosulfite (1 mM) was added. After centrifugation at 12000 rpm for 15 min at 4 ◦ C, the supernatant was collected to test the GST activity. The GST activity was further assessed by the amount of conjugate constituted by glutathione (GSH) and 1- chloro-2,4-dinitrobenezene (CDNB) catalyzed by GST per minute per gram per tissue sample (nmol/min/g FW). The protein content of the rice seedlings was measured at 340 nm using bovine serum albumin as a standard.Total RNA isolated from 0.1 g shoots or roots of rice seedlings was prepared using the reagent Trizol (Takara). The first-strand comple- mentary DNA (cDNA) was synthesized from total RNA (1 µg) by the High RNA Tissue Kit (12033674001) for a quantitative real-time polymerase chain reaction (qPCR) with gDNA removal. To make sure equal amounts of cDNA for each reaction, the gene encoding eEF-1a (AK061464) was applied as the internal control. In order to confirm the differential GST activity, several representative GST genes as OsGSTU3 (AF309379), OsGSTF5 (AF309382), and OsGSTF39 (NM_192151) in different treat- ment combinations were selected, and the qPCR analysis was performed via gene-specific primers [13,51–52]. The qPCR data were analyzed using the 2-ΔΔCt method [53]. Table S4 lists the corresponding primer GST gene sequences and Table S5 depicts the qPCR program system settings. Each sample is independently repeated three times for the qPCR analysis.
3.6.Acute toxicity of treatments 3i, 3t and fenclorim to zebrafish embryos.
Zebrafish embryos at an age of 2 h post-fertilization were randomly transferred into 2 mL of test solutions with varying 3i, 3t, and fenclorim concentrations in 24-well plates. Experiments were conducted to determine the acute toxicity (median lethal concentration, LC50 value) of 3i, 3t, and fenclorim treatments, using a similar method in the reference [54]. Concentrations of 3i and 3t were 2.5, 5, 10, 20, 40, and 80 mg/L and concentrations of fenclorim were 0.625, 1.25, 2.5, 5, 10, and 20 mg/L, respectively. For each concentration, 60 embryos were used, and all experiments were performed in triplicate. Plates were covered with transparent lids to prevent evaporation and were incu- bated at 27 ◦ C with a 14:10 h light/dark photoperiod. The number of dead individuals was assessed daily. Reconstituted water (0.75 mmol/L Na+, 0.074 mmol/L K+, 0.5 mmol/L Mg2+, and 2 mmol/L Ca2+) was used to prepare all test solutions and a solvent control of 0.01% acetone dissolved in reconstituted water (control check, ck, v/v) was
used.
3.7.Statistical analysis
All data for the target compound 3a-y and their positive control fenclorim were analyzed using a one-way analysis of variance with the SPSS 22.0 software package (IBM, NY, USA). The differences were compared using a Duncan ’s range at a significance level of p < 0.05.
4.Conclusions
In this research, a series of (E)-4-(2-substituted hydrazinyl)-6-chloro- 2-phenypyrimidines were synthesizedand their structures were confirmed using 1H NMR, 13C NMR, and HRMS using the IDM method. Herbicide safener activities were evaluated using a primary test and compounds 3i and 3t were observed as having the best herbicide activity on plant height. These two compounds were further screened at lower concentrations to assess their activity compared to the positive control fenclorim, a commercial herbicide safener. The treatments 3i and 3t significantly enhanced glutathione S-transferase (GST) activity related with the herbicide safener activity in both shoots and roots tissues. Further, the qPCR (Real-time quantitative polymerase chain reaction) analysis showed that the treatments 3i and 3t also enhanced the ex- pressions of OsGSTU3, OsGsTU39, and OsGSTF5. Finally, the results of acute toxicity to zebrafish (Danio rerio) when treated by 3i and 3t indicated they are relatively safe to aquatic organisms. This research offers novel guidance to design high-activity new herbicide safeners that are also eco-friendly.
