Abstract
Alkyl 2-cyano-3-methylthio-3-phosphonylacrylates were synthesized by the reaction of alkyl 2-cyano-3,3-dimethylthioacrylates with dialkyl phosphites. The structures of the new compounds were characterized by elemental analyses, IR, 1H-, 13C- and 31P-NMR spectral data. These compounds were tested in vitro against pathogenic fungi, namely, Fusarium graminearum, Cytospora mandshurica and Fusarium oxysporum. Amongst all compounds, 2d and 2t were found to be effective against the tested fungi at 50 µg/mL. A half-leaf method was used to determine the in vivo protective, inactivation and curative efficacies of the title products against tobacco mosaic virus (TMV). Title compounds 2a and 2b were found to possess good in vivo curative, protection and inactivation effects against TMV with inhibitory rates at 500 mg/L of 60.0, 89.4 and 56.5 and 64.2, 84.2 and 61.2 %, respectively. To the best of our knowledge, this is the first report on the antiviral and antifungal activity of alkyl 2-cyano-3-methylthio-3- phosphonylacrylates.
Keywords: 2-Cyanoacrylate, phosphonyl moiety, antiviral activity, antifungal activity, synthesis
Introduction
Cyanoacrylates, a class of highly potent herbicidal compounds, are known to disrupt photosynthetic electron transportation at a common binding domain on the 32 kDa polypeptide of the photosystem II (PSII) reaction center [1,2]. A large number of reports on the synthesis of cyanoacrylate derivatives exist due to their wide range of biological activities [3,4,5,6]. Some derivatives can serve not only as agrochemicals such as herbicides, insecticides, fungicides and plant virucides, but also as medicines such as antitumor agents. In our previous work, we designed and synthesized some chiral cyanoacrylates with antiviral activity by replacing the methylthio moiety of some 2-cyano-3-methyl- thio-3-substituted-phenylacrylates with (R)- or (S)-1-phenylethylamine groups. The (E) configuration of the reported chiral products was confirmed by X-ray single-crystal structure analysis. The bioassays showed that a chiral compound containing a 4-nitrophenyl moiety [(E)-ethyl 3[(S)-1-phenylethyl- amino]-3-(4-nitrophenylamino)-2-cyanoacrylate] exhibited good protection activity against TMV in vivo [7]. On the other hand, phosphonyl compounds, in general, have received wide attention in modern medicinal and pesticide chemistry. They are ideal for use in drug design due to their good bioactivity [8,9], low toxicity, and the ease of substitution with conventional heterocyclic ring groups [10,11,12]. In 2001 Chen et al. reported an efficient method for the synthesis of ethyl 2-cyano-3-methyl- thio-3-(diethoxyphosphonyl)acrylate under microwave irradiation conditions [13]. No reports on the fungitoxicity and antiviral activity of alkyl 2-cyano-3-methylthio-3-phosphonylacrylates have been published in the chemical or biological literature. In order to extend our research work on cyanoacrylates as antiviral agents and fungicides, we have designed and synthesized some novel cyanoacrylate derivatives 2a-2t containing phosphonyl moieties. The synthetic route is shown in Scheme 1. Diethyl phosphite or higher homologues were employed in the reaction due to their ease of preparation. The structures of 2 were established by well defined IR, NMR and elemental analysis. The results of bioassay revealed that some compounds of the above series have good anti-TMV and antifungal activity.
Scheme 1.
Synthesis of compounds 2a-2t.
Results and Discussion
In order to optimize the reaction conditions for the syntheses of compounds 2a-2t, the effects of various solvents, molar reagent ratios, reaction times and reaction temperatures on the reaction synthesis of 2b were examined. The results are summarized in Table 1. Cyanoacrylate 2b was obtained from 1 in poor yield (Table 1, entries 1-3) in solvents such as DMF, acetone and CH3CN, but when tetrahydrofuran (THF) was chosen as solvent, the yield of 2b increased from 11.2 % to 55.0 % (Table 1, entries 1, 4). Next we also examined the effect on the reaction of the molar ratios of the reactants. When the molar ratio of 2-cyano-3,3-(dimethylthio)- acrylate to O, O’-di-i-propylphosphite to sodium hydride was increased from 1:1:1 to 1:3:1, 1:3:2, 1:3:3 and 1:3:4 eq, compound 2b was obtained in 0, 12.0, 20.0, 55.0 and 49.5 % yield, respectively (Table 1, entries 4-8). With regard to the reaction time 0, 23.0 and 55.0 % yields of 2b were noted after 4, 8 and 16 h, respectively (Table 1, entries 9, 10, 4). When the reaction time was prolonged further to 20 h, no significant improvement was obtained (56.0 %, Table 1, entry 11), as compared to that seen after 16 h (55.0 %, Table 1, entry 4). As for the reaction temperature, a lower yield was observed at lower temperature (Table 1, entry 12). It could also be seen that the yield was significantly lower when the reaction was performed at 40-45 °C (Table 1, entry 13), compared to that seen at room temperature (Table 1, entry 4).
Table 1.
Synthesis of 2b under different reaction conditions.
| Entry | Solvent | Ratio a | Time (h) | Reaction temperature | Yield (%) b |
|---|---|---|---|---|---|
| 1 | DMF | 1:3:3 | 16 | r.t | 11.2 |
| 2 | acetone | 1:3:3 | 16 | r.t | 19.0 |
| 3 | CH3CN | 1:3:3 | 16 | r.t | 34.9 |
| 4 | THF | 1:3:3 | 16 | r.t | 55.0 |
| 5 | THF | 1:3:1 | 16 | r.t | 12.0 |
| 6 | THF | 1:3:2 | 16 | r.t | 20.0 |
| 7 | THF | 1:1:1 | 16 | r.t | 0 |
| 8 | THF | 1:3:4 | 16 | r.t | 49.5 |
| 9 | THF | 1:3:3 | 4 | r.t | 0 |
| 10 | THF | 1:3:3 | 8 | r.t | 23.0 |
| 11 | THF | 1:3:3 | 20 | r.t | 56.0 |
| 12 | THF | 1:3:3 | 16 | 0-5°C | 23.0 |
| 13 | THF | 1:3:3 | 16 | 40-45°C | 13.0 |
a Ratio of 2-cyano-3,3-dimethylthioacrylate:O,O’-di-i-propylphosphite:sodium hydride
b Isolated yield based on 2-cyano-3,3-dimethylthioacrylate
Antifungal Bioassay: Inhibitory effects of cyanoacrylate derivatives on phytopathogenic fungi
The three fungi used in the fungicidal bioassay, Fusarium graminearum, Cytospora mandshurica and Fusarium oxysporum, were tested by the poison plate technique. The results of preliminary bioassays were compared with that of a commercial agricultural fungicide, hymexazol. As indicated in Table 2, the new compounds 2d and 2t exhibited promising antifungal activity, inhibiting growth of F. graminearum at 64.5 and 49.7 %, C. mandshurica at 60.4 and 51.3 % and F. oxysporum at 65.0 and 41.1 %, respectively, which is a little lower than that of hymexazole (73.2 % against F. graminearum, 58.9 % against C. mandshurica, and 65.5 % against F. oxysporum at 50 µg/mL). Marked loss of activity was observed with other compounds such as 2a-2c and 2e-2s.
Table 2.
Inhibition effects of the title compounds on fungi.
| Compd. | Conc. (μg/mL) | Inhibition rate (%) | ||||||
|---|---|---|---|---|---|---|---|---|
| Fusarium graminearum | Cytospora mandshurica | Fusarium oxysporum | ||||||
| 2a | 50 | 11.14±0.21 | 11.00±0.25 | 18.21±0.11 | ||||
| 500 | 37.30±0.56 | 24.00±0.77 | 26.03±0.99 | |||||
| 2b | 50 | 4.94±0.49 | 1.34±0.65 | 8.08±0.98 | ||||
| 500 | 31.40±0.78 | 35.03±0.84 | 36.83±1.24 | |||||
| 2c | 50 | 8.31±0.57 | 13.10±0.78 | 13.47±1.24 | ||||
| 500 | 37.10±1.08 | 31.82±1.45 | 41.02±1.30 | |||||
| 2d | 50 | 64.50±0.40 | 60.43±0.63 | 65.03±1.22 | ||||
| 500 | 93.30±4.28 | 90.59±1.59 | 92.75±2.52 | |||||
| 2e | 50 | 22.04±0.90 | 32.01±0.78 | 26.71±0.90 | ||||
| 500 | 41.44±1.21 | 36.77±1.45 | 32.13±1.09 | |||||
| 2f | 50 | 35.22±1.78 | 41.09±3.21 | 46.11±3.34 | ||||
| 500 | 55.64±3.01 | 66.09±2.55 | 68.9±1.19 | |||||
| 2g | 50 | 7.79±0.57 | -0.80±0.72 | 6.29±0.97 | ||||
| 500 | 40.0±2.15 | 49.47±1.01 | 48.80±1.33 | |||||
| 2h | 50 | 4.68±0.56 | -3.21±0.85 | 5.99±0.87 | ||||
| 500 | 25.41±0.73 | 23.26±0.84 | 36.53±1.27 | |||||
| 2i | 50 | 5.45±0.73 | -2.67±0.67 | 1.20±0.89 | ||||
| 500 | 16.3±0.64 | 8.29±0.76 | 20.06±0.97 | |||||
| 2j | 50 | 0.97±0.11 | 11.27±0.45 | 9.99±0.22 | ||||
| 500 | 20.00±0.21 | 26.78±0.44 | 30.99±1.09 | |||||
| 2k | 50 | 10.09±0.76 | 32.20±0.55 | 19.39±0.44 | ||||
| 500 | 31.03±0.90 | 36.38±1.32 | 40.22±1.45 | |||||
| 2l | 50 | 21.08±0.98 | 30.07±0.67 | 12.11±1.02 | ||||
| 500 | 34.01±1.67 | 29.51±1.02 | 32.00±0.69 | |||||
| 2m | 50 | 3.93±0.22 | 14.23±0.51 | 23.09±0.88 | ||||
| 500 | 27.31±0.99 | 16.08±0.55 | 41.90±2.01 | |||||
| 2n | 50 | 12.22±1.01 | 9.97±0.88 | 11.91±0.42 | ||||
| 500 | 24.04±0.62 | 36.09±0.55 | 35.69±1.90 | |||||
| 2o | 50 | 3.75±0.52 | 10.61±0.69 | 4.01±0.55 | ||||
| 500 | 21.08±0.65 | 48.28±0.96 | 29.07±0.65 | |||||
| 2p | 50 | 4.45±0.55 | 24.93±0.76 | 6.52±0.56 | ||||
| 500 | 28.10±0.69 | 47.21±0.95 | 35.59±0.69 | |||||
| 2q | 50 | 7.03±0.60 | 15.38±0.61 | 3.26±0.44 | ||||
| 500 | 42.15±0.79 | 57.82±1.11 | 33.58±0.68 | |||||
| 2r | 50 | 11.71±0.96 | 23.87±0.75 | 9.52±0.57 | ||||
| 500 | 44.03±0.81 | 64.72±1.26 | 52.13±0.84 | |||||
| 2s | 50 | 9.84±0.61 | 35.01±0.72 | 13.53±0.48 | ||||
| 500 | 53.40±0.92 | 65.78±1.29 | 56.64±0.90 | |||||
| 2t | 50 | 49.67±0.65 | 51.30±0.70 | 41.05±0.51 | ||||
| 500 | 81.97±2.91 | 75.33±1.71 | 69.42±1.23 | |||||
| Hymexazol a | 50 | 73.2±1.41 | 58.89±1.16 | 65.51±1.76 | ||||
| 500 | 100±0.66 | 95.33±1.87 | 93.18±3.76 | |||||
| DMSO b | 0 | 0 | 0 | |||||
a reference compound; b control.
Figure 1 shows the inhibition of mycelial growth of isolated hypha of F. oxysporum by compound 2d at different concentrations (100, 50, 25, 10, and 1 μg/mL) as compared to control, when tested in vitro. Almost complete inhibition of mycelial growth was observed at 100 and 50 μg/mL concentrations as compared to control (full growth).
Figure 1.
Effect of various concentrations of 2d on the growth of isolated hypha of F. oxysporum (100, 50, 25, 5 and 1 μg/mL).
Key (μg/mL): 1, 100; 2, 50; 3, 25; 4, 5; 5, 1; and C, control.
Micro-observation results
Microphotography of the hyphal morphology of F. oxysporum treated with 100 μg/mL of 2d (Figure 2) showed a series of changes, i.e. the cell of the hyphal divarication increased, hyphal knots appeared, and hypha bulged partially, compared with control.
Figure 2.
Microphotograph (800×) of hyphal morphology of F. oxysporum treated with 2d.
Preliminary antiviral activity assay
The results of the in vivo bioassay against TMV are given in Table 3. Ningnanmycin was used as reference antiviral agent. The data provided in the table indicate that the introduction of dialkylphosphonyl moieties in cyanoacrylates might improve their protective activities. The title compounds 2a-2t showed protection rates of 40.8-61.2 %. When R1 and R2 are Et, the resulting compound 2a displayed a lower protective activity (56.5 %) than that of the reference compound (62.6 %). The highest protective activity was achieved when R1 is Et and R2 is i-Pr (compound 2b). A protective rate of 61.2 %, equivalent to ningnanmycin, against TMV at 500 µg/mL was recorded in this case. From the data in Table 3, it may be observed that the title compounds 2a-2t possess significant potential inactivation bioactivities, with values of 89.4, 84.7, 76.3, 80.3, 74.2, 79.6, 73.8, 70.8, 80.0, 77.9, 79.0, 78.2, 81.2, 80.2, 68.9, 67.3, 60.0, 70.0, 80.2 and 61.0 % at 500 µg/mL, respectively. Among these compounds, 2a (R1 and R2 are Et) is much more active against TMV than the other ones, with an inactivation rate of 89.4 %, equivalent to ningnanmycin (92.6 %) against TMV at 500 µg/mL. The data also indicate that a change in the substituent might also affect the curative activity of the title compounds 2a-2t. Compound 2a (R1 and R2 are Et) and compound 2b could cure TMV up to 60.0 % and 64.2 % at 500 µg/mL. The other compounds have a relatively lower curative activity than 2a and 2b.
Table 3.
The Protective, inactivation and curative effects of the new compounds against TMV in vivo.
| Agents | Concentration (mg/L) |
Protective Effect (%) |
Inactivation Effect (%) |
Curative Effect(%) |
|||
|---|---|---|---|---|---|---|---|
| 2a | 500 | 56.5*±1.4 | 89.4**±2.0 | 60.0*±0.9 | |||
| 2b | 500 | 61.2*±3.2 | 84.7**±1.0 | 64.2*±1.9 | |||
| 2c | 500 | 53.0*±1.8 | 76.3*±2.4 | 45.8*±1.0 | |||
| 2d | 500 | 53.4*±0.7 | 80.3**±3.4 | 41.2*±2.1 | |||
| 2e | 500 | 45.0*±1.1 | 74.2*±3.4 | 54.7*±2.2 | |||
| 2f | 500 | 47.9*±1.7 | 79.6**±1.6 | 28.4±4.4 | |||
| 2g | 500 | 40.8*±5.4 | 73.8*±3.0 | 41.8*±5.5 | |||
| 2h | 500 | 46.5*±2.3 | 70.8*±1.4 | 31.4*±1.7 | |||
| 2i | 500 | 50.1*±3.0 | 80.0**±2.0 | 36.6*±4.4 | |||
| 2j | 500 | 43.2*±5.4 | 77.9*±0.9 | 42.1*±3.8 | |||
| 2k | 500 | 45.7*±1.9 | 79.0*±0.7 | 44.3*±6.4 | |||
| 2l | 500 | 51.2*±1.1 | 78.2*±0.9 | 42.0*±1.5 | |||
| 2m | 500 | 50.0*±2.0 | 81.2*±1.6 | 50.0*±1.0 | |||
| 2n | 500 | 49.7±8.0 | 80.2**±1.2 | 51.2*±1.0 | |||
| 2o | 500 | 48.9*±0.9 | 68.9*±5.3 | 33.3*±4.9 | |||
| 2p | 500 | 47.6*±1.0 | 67.3*±2.4 | 33.3±8.9 | |||
| 2q | 500 | 45.6*±0.4 | 60.0*±3.1 | 10.9±6.0 | |||
| 2r | 500 | 50.9*±1.1 | 70.0*±1.9 | 36.7*±6.7 | |||
| 2s | 500 | 51.2*±0.9 | 80.2**±2.0 | 41.4*±9.9 | |||
| 2t | 500 | 46.6*±5.5 | 61.0*±5.1 | 24.1±8.7 | |||
| Ningnamycin | 500 | 62.6*±2.1 | 92.6**±1.0 | 53.9*±2.3 | |||
All results are expressed as mean ± SD; n= 3 for all groups; * P<0.05, **P<0.01.
Conclusions
A series of novel cyanoacrylate derivatives 2a-2t containing phosphonyl moieties were synthesized by treatment of alkyl 2-cyano-3,3-dimethylthioacrylates and dialkyl phosphites with NaH in THF solvent. This method is easy and gields the title compounds in moderate yields. The structures were verified by spectroscopic data. In the antifungal bioassay, the title compounds 2d and 2t were found to possess the highest activity against three kinds of fungi in vitro. The bioassay results showed that these title compounds exhibited moderate to good anti-TMV bioactivity. Title compounds 2a and 2b showed better biological activity than their structurally related analogues 2c-2t.
Experimental
General
The melting points of the products were determined on an XT-4 binocular microscope (Beijing Tech Instrument Co., P.R. China) and were not corrected. The IR spectra were recorded on a Bruker VECTOR 22 spectrometer in KBr disks. 1H-, 13C- and 31P-NMR spectra (solvent CDCl3) were recorded on a JEOL-ECX 500 NMR spectrometer at room temperature using TMS as an internal standard. Elemental analysis was performed on an Elementar Vario-III CHN analyzer. Analytical TLC was performed on silica gel GF254. Column chromatographic purification was carried out using silica gel. All reagents either belonged to analytical reagent grade or were chemically pure. THF was dried, deoxygenated and redistilled before use. Dialkyl phosphites and alkyl 2-cyano-3,3-(dimethyl- thio)acrylates were prepared according to literature methods [14,15].
General procedure for the preparation of title compounds 2a-2t.
A dry 100 mL round-bottom flask equipped with a magnetic stirrer and nitrogen inlet was charged with sodium hydride (0.66 g, 55%, 15.2 mmol) in THF (15 mL) at 0~5 °C. Dialkyl phosphite (15.2 mmol) was than slowly added through a dropping funnel into the resulting solution over a period of 30 min. Alkyl 2-cyano-3,3-(dimethylthio)acrylate (5.0 mmol) in THF (20 mL) was then slowly added to the above solution. The mixture was stirred for 16 h at room temperature. After removal of the solvent under vacuum, the residue was dissolved in ice-cold water (40 mL) and acidified with dilute hydrochloric acid (10%), extracted with ethyl acetate (3 × 20 mL) and the combined organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated. The residue was subjected to column chromatography using a mixture of petroleum ether and ethyl acetate (4:1) as eluent to give the title compounds 2a-2t.
Yields and product characterization
Ethyl 2-cyano-3-methylthio-3-(diethoxyphosphonyl)acrylate (2a): yellow liquid; yield 52.0%; 1H- NMR δ: 1.37~1.45 (m, 9H, ester CH3 + 2 × phosphonyl CH3), 2.76 (d, J = 6.8 Hz, 3H, SCH3), 4.32~4.35 (m, 6H, ester CH2 + 2 × phosphonyl CH2); 13C-NMR δ: 14.1 (ester CH3), 16.2 (2×phosphonyl CH3), 19.4 (SCH3), 62.7 (ester OCH2), 64.5 (2 × phosphonyl CH2), 106.2 (C=C C-2), 114.5 (CN), 162.1 (C=C C-3), 165.7 (C=O); 31P-NMR δ: 5.1; IR ν: 2983, 2214, 1745, 1251, 1018 cm-1; Anal. Calcd. for C11H18NO5PS (307.5): C, 42.93; H, 5.58; N, 4.55. Found: C, 42.99; H, 5.90; N, 4.56.
Ethyl 2-cyano-3-methylthio-3-(di-i-propoxyphosphonyl)acrylate (2b): yellow liquid; yield 55.0%; 1H- NMR δ: 1.36~1.38 (m, 15 H, 4 × phosphonyl CH3 + ester CH3), 2.73 (d, J = 6.9 Hz, 3H, SCH3), 4.33 (q, J = 7.2 Hz, 2H, ester CH2), 4.82~4.89 (m, 2H, 2 × phosphonyl CH); 13C-NMR δ: 14.1 (ester CH3), 19.4 (SCH3), 23.9 (4 × phosphonyl CH3), 63.1 (ester OCH2), 64.5 (2 × phosphonyl CH2), 74.2 (2 × phosphonyl CH), 106.9 (C=C C-2), 114.7 (CN), 162.1 (C=C C-3), 165.3 (C=O); 31P-NMR δ: 3.5; IR ν: 2980, 2212, 1718, 1504, 1246, 987 cm-1; Anal. Calcd. for C13H22NO5PS (335.1): C, 46.55; H, 6.56; N, 4.18. Found: C, 46.56; H, 6.61; N, 4.10.
Ethyl 2-cyano-3-methylthio-3-(di-n-propoxyphosphonyl)acrylate (2c): yellow liquid; yield 50.0%; 1H- NMR δ: 0.98~1.02 (m, 6H, 2 × phosphonyl CH3), 1.37 (t, J = 7.5 Hz, 3H, ester CH3), 1.73~1.75 (m, 4H, 2 × phosphonyl CH2), 2.75 (d, J = 6.9 Hz, 3H, SCH3), 4.09~4.19 (m, 4H, 2 × phosphonyl CH2), 4.33 (q, J = 7.1Hz, 2H, ester CH2); 13C-NMR δ: 10.0 (2 × phosphonyl CH3), 14.1 (ester CH3), 19.5 (SCH3), 23.7 (2 × phosphonyl CH2), 63.3 (ester OCH2), 69.8 (2 × phosphonyl CH2), 106.5 (C=C C-2), 114.6 (CN), 162.1 (C=C C-3), 165.5 (C=O); 31P-NMR δ: 5.9; IR ν: 2970, 2214, 1735, 1463, 1251, 1006 cm-1; Anal. Calcd. for C13H22NO5PS (335.1): C, 46.55; H, 6.56; N, 4.18. Found: C, 46.56; H, 6.61; N, 4.18.
Ethyl 2-cyano-3-methylthio-3-(di-n-butoxyphosphonyl)acrylate (2d): yellow liquid; yield 45.6%; 1H- NMR δ: 0.94~0.97 (m, 6H, 2 × phosphonyl CH3), 1.37 (t, J =7.2 Hz, 3H, ester CH3), 1.39~1.46 (m, 4H, 2 × phosphonyl CH2), 1.73~1.77 (m, 4H, 2 × phosphonyl CH2), 2.74 (d, J =6.9 Hz, 3H, SCH3), 4.23~4.34 (m, 6H, 2 × phosphonyl CH2 + ester CH2); 13C-NMR δ: 13.5 (2 × phosphonyl CH3), 13.6 (ester CH3), 18.7 (2 × phosphonyl CH2), 18.8 (SCH3), 32.3 (2 × phosphonyl CH2), 62.8 (ester CH2), 68.0 (2 × phosphonyl CH), 106.4 (C=C C-2), 114.5 (CN), 162.1 (C=C C-3), 165.6 (C=O); 31P-NMR δ:5.5; IR ν: 2960, 2214, 1735, 1465, 1259, 1028 cm-1; Anal. Calcd. for C15H26NO5PS (363.1): C, 49.57; H, 7.16; N, 3.86. Found: C, 49.54; H, 7.21; N, 3.69.
Ethoxyethyl 2-cyano-3-methylthio-3-(diethoxyphosphonyl)acrylate (2e): yellow liquid; yield 49.7%; 1H-NMR δ: 1.22 (t, J = 6.9 Hz, 3H, ester O-C-CH3), 1.38~1.45 (m, 6H, 2 × phosphonyl CH3), 2.75 (d, J = 8.6 Hz, 3H, SCH3), 3.57 (q, J = 6.3 Hz, 2H, ester OCH2), 3.72~3.74 (m, 2H, ester CH2), 4.30~4.32 (m, 4H, 2 × phosphonyl CH2), 4.39~4.41 (m, 2H, ester CH2); 13C-NMR δ: 15.2 (ethoxy CH3), 16.3 (2 × phosphonyl CH3), 19.6 (SCH3), 64.6 (2 × phosphonyl CH2), 65.9 (ester OCH2), 66.9 (ethoxy OCH2), 67.8 (ester CH2O), 106.0 (C=C C-2 ), 114.5 (CN), 162.2 (C=C C-3), 164.9 (C=O); 31P-NMR δ: 5.15; IR ν: 2953, 2992, 2852, 2214, 1714, 1444, 1249, 1120, 1012 cm-1; Anal. Calcd. for C13H22NO6PS (351.1): C, 44.43; H, 6.27; N, 3.99. Found: C, 44.44; H, 6.31; N, 3.89.
Ethoxyethyl 2-cyano-3-methylthio-3-(di-i-propoxyphosphonyl)acrylate (2f): yellow liquid; yield 57.5%; 1H-NMR δ: 1.22 (t, J = 6.9 Hz, 3H, ester O-C-CH3), 1.41~1.45 (m, 12H, 4 × phosphonyl CH3), 2.72 (d, J = 14.3 Hz, 3H, SCH3), 3.57 (q, J = 6.9 Hz, 2H, ester OCH2), 3.72~3.73 (m, 2H, ester OCH2), 4.39~4.41 (m, 2H, ester CH2), 4.82~4.89 (m, 2H, 2 × phosphonyl CH); 13C- NMR δ: 15.1 (ethoxy CH3), 19.7 (SCH3), 23.8 (4 × phosphonyl CH3), 65.7 (ester CH2), 66.7 (ethoxy OCH2), 67.7 (ester CH2O), 74.2 (2 × phosphonyl CH), 106.6 (C=C C-2), 114.5 (CN), 162.0 (C=C C-3), 166.8 (C=O); 31P-NMR δ: 4.76; IR ν: 2978, 2933, 2214, 1732, 1454, 1251, 1118, 987 cm-1; Anal. Calcd. for C15H26NO6PS (379.4): C, 47.44; H, 6.85; N, 3.69. Found: C, 47.48; H, 6.91; N, 3.51.
Ethoxyethyl 2-cyano-3-methylthio-3-(di-n-propoxyphosphonyl)acrylate (2g): yellow liquid; yield 55.6%; 1H-NMR δ: 0.98~1.00 (m, 6H, 2 × phosphonyl CH3), 1.21 (t, J =6.9 Hz, 3H, ester O-C-CH3), 1.73~1.80 (m, 4H, 2 × phosphonyl CH2), 2.75 (d, J = 9.1 Hz, 3H, SCH3), 3.57 (q, J = 7.2 Hz, 2H, ester OCH2), 3.72~3.74 (m, 2H, ester CH2), 4.16~4.20 (m, 4H, 2 × phosphonyl CH2), 4.39~4.41 (m, 2H, ester CH2); 13C-NMR δ: 10.1 (2 × phosphonyl CH3), 15.2 (ethoxy OCH3), 19.6 (SCH3), 23.7 (2 × phosphonyl CH2COP), 65.9 (ester OCH2), 66.3 (ethoxy OCH2), 67.8 (ester CH2O), 70.0 (2 × phosphonyl CH2OP), 106.1 (C=C C-2), 114.5 (CN), 162.0 (C=C C-3), 164.9 (C=O); 31P-NMR δ: 5.43; IR ν: 2968, 2897, 2214, 1745, 1456, 1381, 1238, 1116, 995 cm-1; Anal. Calcd. for C15H26NO6PS (379.4): C, 47.44; H, 6.85; N, 3.69. Found: C, 47.40; H, 6.81; N, 3.55.
Ethoxyethyl 2-cyano-3-methylthio-3-(di-n-butoxyphosphonyl)acrylate (2h): yellow liquid; yield 54.7%; 1H-NMR δ: 0.94~0.96 (m, 6H, 2 × phosphonyl CH3), 1.22 (t, J = 6.9 Hz, 3H, ester O-C-CH3), 1.42~1.44 (m, 4H, 2 × phosphonyl CH2), 1.69~1.76 (m, 4H, 2 × phosphonyl CH2), 2.75 (d, J = 10.3Hz, 3H, SCH3), 3.57 (q, J = 7.5 Hz, 2H, ethoxy OCH2), 3.72~3.74 (m, 2H, ester OCH2), 4.23~4.25 (m, 4H, 2 × phosphonyl CH2), 4.39~4.41 (m, 2H, ester CH2); 13C-NMR (δ: 13.6 (2 × phosphonyl CH3), 15.2 (ethoxy OCH3), 18.8 (2 × phosphonyl CH2), 19.6 (SCH3), 32.4 (2 × phosphonyl CH2), 65.9 (ester CH2), 66.9 (ethoxy OCH2), 67.8 (2 × phosphonyl CH2), 68.3 (ester CH2O), 106.1 (C=C C-2), 114.5 (CN), 162.0 (C=C C-3), 164.8 (C=O); 31P-NMR δ: 5.44; IR ν: 2960, 2872, 2214, 1732, 1714, 1249, 1056 cm-1; Anal. Calcd. for C17H30NO6PS (407.1): C, 50.11; H, 7.37; N, 3.44. Found: C, 50.13; H, 7.42; N, 3.26.
Methoxyethyl 2-cyano-3-methylthio-3-(diethoxyphosphonyl)acrylate (2i): yellow liquid; yield 60.1%; 1H-NMR δ: 1.36~1.45 (m, 6H, 2 × phosphonyl CH3), 2.75 (d, J = 7.5 Hz, 3H, SCH3), 3.41 (s, 3H, methoxy CH3), 3.68~3.70 (m, 2H, ester CH2), 4.25~4.32 (m, 4H, 2 × phosphonyl CH2), 4.39~4.42 (m, 2H, ester CH2); 13C-NMR δ: 16.2 (2 × phosphonyl CH3), 19.5 (SCH3), 59.2 (methoxy), 64.6 (2 × phosphonyl CH2), 65.6 (ester CH2), 69.8 (ester CH2O), 105.8 (C=C C-2), 114.4 (CN), 161.9 (C=C C-3), 165.0 (C=O); 31P-NMR δ: 5.11; IR ν: 2916, 2214, 1732, 1444, 1249, 1122, 1012 cm-1; Anal. Calcd. for C12H20NO6PS (337.1): C, 42.71; H, 5.93; N, 4.15. Found: C, 42.73; H, 5.98; N, 4.05.
Methoxyethyl 2-cyano-3-methylthio-3-(di-i-propoxyphosphonyl)acrylate (2j): yellow liquid; yield 58.1%; 1H-NMR δ: 1.36~1.42 (m, 12H, 4 × phosphonyl CH3), 2.72 (d, J = 13.2 Hz, 3H, SCH3), 3.40 (s, 3H, ester OCH3), 3.68~3.70 (m, 2H, ester CH2), 4.39~4.40 (m, 2H, ester OCH2), 4.82~4.89 (m, 2H, 2 × phosphonyl CH); 13C-NMR δ: 19.7 (SCH3), 24.0 (4 × phosphonyl CH3), 59.0 (methoxy OCH3), 65.9 (ester CH2), 70.0 (ester CH2), 74.1 (2 × phosphonyl CH), 106.4 (C=C C-2), 114.4 (CN), 162.0 (C=C C-3), 165.8 (C=O); 31P-NMR δ: 5.10; IR ν: 2981, 2933, 2214, 1747, 1454, 1249, 1000 cm-1; Anal. Calcd. for C14H24NO6PS (365.1): C, 46.01; H, 6.57; N, 3.83. Found: C, 45.92; H, 6.39; N 3.83.
Methoxyethyl 2-cyano-3-methylthio-3-(di-n-propoxyphosphonyl) acrylate (2k): yellow liquid; yield 55.3%; 1H-NMR δ: 0.98~1.00 (m, 6H, 2 × phosphonyl CH3), 1.73~1.80 (m, 4H, 2 × phosphonyl CH2), 2.75 (d, J = 7.5 Hz, 3H, SCH3), 3.42 (s, 3H, methoxy OCH3), 3.69~3.70 (m, 2H, ester OCH2), 4.13~4.21 (m, 4H, 2 × phosphonyl CH2), 4.40~4.42 (m, 2H, ester CH2); 13C-NMR δ: 10.0 (2 × phosphonyl CH3), 19.5 (SCH3), 23.7 (2 × phosphonyl CH2), 59.2 (methoxy OCH3), 65.6 (ester CH2), 69.8 (2 × phosphonyl CH2OP), 69.9 (ester CH2), 105.9 (C=C C-2), 114.4 (CN), 162.1 (C=C C-3), 165.0 (C=O); 31P-NMR δ: 5.38; IR ν: 2968, 2897, 2214, 1724, 1465, 1247, 1000 cm-1; Anal. Calcd. for C14H24NO6PS (365.1): C, 46.01; H, 6.57; N, 3.83. Found: C, 45.82; H, 7.42; N 3.83.
Methoxyethyl 2-cyano-3-methylthio-3-(di-n-butoxyphosphonyl)acrylate (2l): yellow liquid; yield 51.0%; 1H-NMR δ: 0.94~0.97 (m, 6H, 2 × phosphonyl CH3), 1.41~1.44 (m, 4H, 2 × CH2), 1.69~1.76 (m, 4H, 2 × CH2-C-O-P=O), 2.74 (d, J = 8.0 Hz, 3H, SCH3), 3.41 (s, 3H, ester OCH3), 3.68~3.70 (m, 2H, ester CH2), 4.20~4.24 (m, 4H, 2 × phosphonyl CH2), 4.40~4.42 (m, 2H, ester CH2); 13C-NMR δ: 13.5 (2 × phosphonyl CH3), 18.6 (2 × phosphonyl CH2), 18.7 (SCH3), 32.2 (2 × phosphonyl CH2), 59.2 (methoxy OCH3), 65.6 (ester CH2), 68.2 (2 × phosphonyl CH2OP), 69.8 (ester CH2), 105.8 (C=C C-2), 114.4 (CN), 161.9 (C=C C-3), 165.0 (C=O); 31P-NMR δ: 5.39; IR ν: 2956, 2872, 2214, 1734, 1458, 1253, 1002 cm-1;Anal. Calcd. for C16H28NO6PS (393.1): C, 48.84; H, 7.12; N, 3.56. Found: C 48.80, H, 7.17; N, 3.43.
Ethyl 2-cyano-3-methylthio-3-[di-(2-methoxyethoxy)phosphonyl]acrylate (2m): yellow liquid; yield 60.0%; 1H-NMR δ: 1.36~1.38 (m, 3H, ester CH3), 2.78 (d, J =10.9 Hz, 3H, SCH3), 3.39 (s, 6H, 2 × phosphonyl CH3), 3.62~3.67 (m, 4H, 2 × phosphonyl CH2), 4.31~4.40 (m, 6H, 2 × phosphonyl CH2 + ester CH2); 13C-NMR δ: 14.0 (ester CH3), 19.2 (SCH3), 58.7 (phosphonyl OCH3), 62.7 (ester OCH2), 66.7 (2 × phosphonyl CH2OP), 71.0 (2 × phosphonyl CH2), 106.0 (C=C C-2), 114.7 (CN), 162.1 (C=C C-3), 164.6 (C=O); 31P-NMR δ: 6.16; IR ν: 2985, 2926, 2212, 1714, 1446, 1367, 1242, 1014 cm-1; Anal. Calcd. for C13H22NO7PS (367.1): C, 42.50; H, 5.99; N, 3.81. Found: C, 42.45; H, 6.04; N, 3.72.
Ethyl 2-cyano-3-methylthio-3-[di-(2-ethoxyethoxy)phosphonyl]acrylate (2n): yellow liquid; yield 57.6%; 1H-NMR δ: 1.20 (q, 6H, J = 7.1 Hz, 2 × phosphonyl CH3), 1.36~1.38 (m, 3H, ester CH3), 2.78 (d, J=10.8 Hz, 3H, SCH3), 3.52~3.55 (m, 4H, 2 × phosphonyl CH2), 3.65~3.70 (m, 4H, 2 × phosphonyl CH2), 4.31~4.40 (m, 6H, 2 x phosphonyl CH2 + ester CH2); 13C-NMR δ: 15.0 (SCH3), 66.5 (ester OCH2), 66.6 (2 × phosphonyl CH2), 66.9 (2 × phosphonyl CH2), 67.0 (2 × phosphonyl CH2), 106.0 (C=C C-2), 114.7 (CN), 162.2 (C=C C-3), 165.0 (C=O); 31P-NMR δ: 5.93; IR ν: 2970, 2868, 2212, 1720, 1446, 1367, 1244, 1016 cm-1; Anal. Calcd. for C15H26NO7PS (395.1): C, 45.56; H, 6.58; N, 3.39. Found: C, 45.51; H, 6.63; N, 3.54.
Ethoxyethyl 2-cyano-3-methylthio-3-[di-(2-methoxyethoxy)phosphoryl]acrylate (2o): yellow liquid; yield 53.5%; 1H-NMR δ: 1.21 (t, 3H, J = 6.9 Hz, CH3 of ethoxy), 2.77 (d, JPH = 17.0Hz, 3H, SCH3), 3.38 (s, 6H, 2×OCH3), 3.55~3.74 (m, 8H, 2×CH2C-O of phosphonyl + COOCCH2 of ester + CH2 of ethoxy), 4.24~4.41 (m, 6H, 2×CH2O of phosphonyl + COOCH2 of ester); 13C-NMR δ:15.1 (CH3 of ethoxy), 19.3 (SCH3), 58.7 (2×OCH3), 59.0 (CH2 of ethoxy), 66.1 (OCH2 of ester), 66.8 (2×CH2O of phosphonyl), 67.8 (OCCH2 of ester), 71.1 (2×CH2C-O of phosphonyl), 105.7 (C-2 of C=C), 114.6 (CN), 162.1 (C-3 of C=C), 165.0 (C=O); 31P-NMR δ: 6.11; IR ν: 2927, 2214, 1747, 1456, 1371, 1253, 1033 cm-1; Anal. Calcd. for C15H26NO8PS (411.1): C 43.79; H 6.37; N 3.40. Found: C 43.76, H 6.43, N 3.41.
Ethoxyethyl 2-cyano-3-methylthio-3-[di-(2-ethoxyethoxy)phosphoryl]acrylate (2p): yellow liquid; yield 56.2%; 1H-NMR δ: 1.21~1.39 (m, 9H, 3×CH3), 2.78 (d, JPH = 17.1 Hz, 3H, SCH3), 3.50-3.59 (m, 6H, 3 × OCH2), 3.63~3.73 (m, 6H, 2 × phosphonyl CH2C-O + ester COOCCH2), 4.27~4.41 (m, 6H, 2 × phosphonyl CH2O + ester COOCH2); 13C-NMR δ: 15.1 (3 × ethoxy CH3), 19.3 (SCH3), 66.5 (3 × ethoxy OCH2), 66.8, 67.0 (ester OCH2 + 2 × phosphonyl CH2O), 68.9, 69.0 (ester OCCH2 + 2 × phosphonyl CH2C-O), 105.8 (C=C C-2), 114.6 (CN), 162.1 (C=C C-3), 165.1 (C=O); 31P-NMR δ: 6.30; IR ν: 2974, 2214, 1747, 1583, 1485, 1253, 1035 cm-1; Anal. Calcd. for C17H30NO8PS (439.1): C 46.46; H 6.88; N 3.19. Found: C 46.60, H 6.95, N 3.26.
Methyl 2-cyano-3-methylthio-3-(diethoxyphosphonyl)acrylate (2q): yellow liquid; yield 61.0%; 1H- NMR δ: 1.33~1.45 (m, 6H, 2 × phosphonyl CH3), 2.77 (d, JPH= 13.7 Hz, 3H, SCH3), 3.88 (d, JPH = 7.5 Hz, 3H, OCH3), 4.01~4.34 (m, 4H, 2 × phosphonyl CH2); 13C NMR δ: 16.2 (2 × phosphonyl CH3), 19.5 (SCH3), 53.7 (OCH3), 64.5 (2 × phosphonyl CH2), 105.5 (C=C C-2), 114.6 (CN), 162.6 (C=C C-3), 165.3 (C=O); 31P-NMR δ: 5.0; IR ν: 2983, 2214, 1749, 1255, 1037 cm-1; Anal. Calcd. for C10H16NO5PS (293.1): C 40.95, H 5.50, N 4.78. Found: C 40.91, H 5.47, N 4.81.
Methyl 2-cyano-3-methylthio-3-(di-i-propoxyphosphonyl)acrylate (2r): yellow liquid; yield 58.2%; 1H- NMR δ: 1.35~1.44 (m, 12H, 4 × phosphonyl CH3), 2.74 (d, JPH = 6.9 Hz, 3H, SCH3), 3.87 (d, J = 8.1 Hz, 3H, OCH3), 4.83~4.89 (m, 2H, 2 × phosphonyl CH); 13C-NMR δ: 19.8 (SCH3), 23.9 (4 × phosphonyl CH3), 53.7 (OCH3), 74.4 (2 × phosphonyl CH), 106.3 (C=C C-2), 114.9 (CN), 162.7 (C=C C-3), 166.3 (C=O); 31P-NMR δ: 4.90; IR ν: 2980, 2214, 1753, 1454, 1251, 999 cm-1; Anal. Calcd. for C12H20NO5PS (335.1): C 44.85, H 6.27, N 4.36. Found: C 44.77, H 6.11, N 4.40.
Methyl 2-cyano-3-methylthio-3-(di-n-propoxyphosphonyl)acrylate (2s): yellow liquid; yield 56.3%; 1H-NMR δ: 0.98~1.00 (m, 6H, 2 × phosphonyl CH3), 1.72~1.80 (m, 4H, 2 × phosphonyl CH2C-O), 2.76 (d, JPH = 4.6 Hz, 3H, SCH3), 3.87 (d, J = 10.3Hz, 3H, OCH3), 3.99~4.21 (m, 4H, 2 × phosphonyl CH2O); 13C-NMR δ: 10.1 (2 × phosphonyl CH3), 19.6 (SCH3), 23.8 (2 × phosphonyl CH2C-O), 53.8 (OCH3), 70.0 (2 × phosphonyl CH2O), 105.7 (C=C C-2), 114.6 (CN), 162.5 (C=C C-3), 165.4 (C=O); 31P-NMR δ: 5.3; IR ν: 2968, 2214, 1751, 1462, 1253, 1002 cm-1; Anal. Calcd. for C12H20NO5PS (321.3): C 44.85, H 6.27, N 4.36. Found: C 45.01, H 6.18, N 4.38.
Methyl 2-cyano-3-methylthio-3-(di-n-butoxyphosphonyl)acrylate (2t): yellow liquid; yield 57.3%; 1H-NMR δ: 0.94~0.98 (m, 6H, 2 × phosphonyl CH3), 1.38-1.47 (m, 4H, 2 × phosphonyl CH2CCO), 1.65-1.77 (m, 4H, 2 × phosphonyl CH2CO), 2.75 (d, JPH = 4.6 Hz, 3H, SCH3), 3.87 (d, J = 10.3 Hz, 3H, OCH3), 4.08~4.25 (m, 2H, 4H, 2 × phosphonyl CH2O); 13C-NMR δ: 13.6 (2 × phosphonyl CH3), 18.7 (2 × phosphonyl CH2CCO), 18.8 (SCH3), 32.2 (2 × phosphonyl CH2C-O), 53.8 (OCH3), 68.2 (2 × phosphonyl CH2O), 105.6 (C=C C-2), 114.6 (CN), 162.6 (C=C C-3), 165.3 (C=O); 31P-NMR δ: 5.3; IR ν: 2960, 2214, 1743, 1462, 1255, 1020 cm-1; Anal. Calcd. for C14H24NO5PS (349.1): C 48.13, H 6.92, N 4.01. Found: C 48.46, H 7.04, N 3.91.
Antifungal bioassay
The antifungal activity of all synthesized compounds were tested against three pathogenic fungi. namely Fusarium graminearum, Cytospora mandshurica and Fusarium oxysporum, by the poison plate technique [16]. Compounds were dissolved in DMSO (1 mL) before mixing with potato dextrose agar (PDA, 90 mL). The final concentration of compounds in the medium were fixed at 50 and 500 µg/mL. All types of fungi were incubated in PDA at 25±1 °C for 4 days to get new mycelium for the antifungal assays, then a mycelia disk of approximately 4 mm diameter cut from culture medium was picked up with a sterilized inoculation needle and inoculated in the center of the PDA plate. The inoculated plates were incubated at 25±1 °C for 5 days. DMSO in sterile distilled water served as control, while hymexazole severed as positive control. For each treatment, three replicates were conducted. The radial growth of the fungal colonies were measured and the data were statistically analyzed. The inhibitory effects of the test compounds on these fungi in vitro were calculated by the formula I = (C-T/C) x 100, where C represents the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA while I represents the inhibition rate.
Hyphal morphology of F. oxysporum
Compound 2d (final conc. 100 µg/mL) was added to sterilized Czapek media (0.2 % NaNO3, 0.131 % K2HPO4·3H2O, 0.05 % KCl, 0.05 % MgSO4·7H2O, 0.00183 % FeSO4·7H2O, 3 % sucrose, pH 6.8) [17] in which F. oxysporum had incubated for a few days. After incubating together at 27 oC for 24 h, it was observed under microscope (Olympus 800 ×). Acetone (0.5 mL) served as the control.
Antiviral Biological Assays
Purification of tobacco mosaic virus: Using Gooding’s method [18], the upper leaves of Nicotiana tabacum L inoculated with TMV were selected and were ground in phosphate buffer, then filtered through double layer pledget. The filtrate was centrifuged at 10,000 g, treated twice with PEG and centrifuged again. The whole experiment was carried out at 4 °C. Absorbance values were estimated at 260 nm using an ultraviolet spectrophotometer. Virus concn = (A260 × dilution ratio)/
Protective effects of compounds on TMV in vivo: The compound solution was smeared on the left side while solvent was served as control on the right side of growing Nicotiana tabacum. L leaves of the same ages. The leaves were then inoculated with the virus after 12 hours. A brush was dipped in tobacco mosaic virus of 6×10-3 mg/mL to inoculate the leaves, which were previously scattered with silicon carbide. The leaves were then washed with water and rubbed softly along the nervature once or twice. The local lesion numbers appearing 3-4 days after inoculation were counted [7]. Three repetitions were conducted for each compound.
Inactivation effect of compounds on TMV in vivo: The virus was inhibited by mixing with the compound solution at the same volume for 30 minutes. The mixture was then inoculated on the left side of the leaves of Nicotiana tabacum. L., while the right side of the leaves was inoculated with the mixture of solvent and the virus for control. The local lesion numbers were recorded 3-4 days after inoculation [7]. Three repetitions were conducted for each compound.
Curative effect of compounds on TMV in vivo: Growing leaves of Nicotiana tabacum. L of the same ages were selected. The tobacco mosaic virus (concentration of 6×10-3mg/mL) was dipped and inoculated on the whole leaves. Then the leaves were washed with water and dried. The compound solution was smeared on the left side and the solvent was smeared on the right side for control. The local lesion numbers were then recorded 3-4 days after inoculation [7]. For each compound, three repetitions were conducted to ensure the reliability of the results, which were measured according to the following formula:
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Acknowledgments
The authors wish to thank the National Key Project for Basic Research (Grant No. 2003CB114404, 2005CCA01500) and Key Technologies R&D Program (Grant No. 2006BAE01A02-5) and the National Natural Science Foundation of China (Grant No. 20672024) for their financial support.
Footnotes
Sample Availability: Samples of the compounds are available from authors.
References
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