Synthesis, X-ray Crystallographic Analysis and Bioactivities of α-Aminophosphonates Featuring Pyrazole and Fluorine Moieties①

HONG Yn-Ping SHANGGUAN Xin-Chen IQBAI Zfr YIN Xio-Li

a (Jiangxi Key Laboratory of Natural Product and Functional Food, College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China)

b (PCSIR Laboratories Complex, Feroze Pur Road Lahore 54000, Pakistan)

c (Library of Jiangxi Agricultural University, Nanchang 330045, China)

1 INTRODUCTION

As the phosphorus structural analogues of natural amino acids, α-aminophosphonic acids and their ester derivatives have attracted tremendous research interest ranging from chemistry, medicinal and agricultural sciences by virtue of their broad spectrum of biological and pharmacological activities. In the last two decades, a large number of literatures have shown that many α-aminophosphonates can serve not only as fungicide[1], herbicide[2,3]and plant virucide[4], but also as antitumor agents[5], antibacterial agents[6], HIV inhibitors[7]and so on. It is well known that the pyrazole ring is an important heterocycle which has been utilized as a synthon widely in agrochemicals and pharmaceuticals[8]. The introduc-tion of pyrazole into the parent compounds may remarkably improve the biological properties of the compounds. Some pyrazole-containing pesticides and medicines have already been commercialized.For example, pyraclostrobin[9], pyrazosulfuronethyl[10]and fipronil[11]were developed as fungicide,herbicide and insecticide, respectively, whereas zaleplon[12]has been used as a hypnotic drug. In addition, fluorine compounds have been widely applied to synthesize new agrochemicals[13-15].Keeping in view the above facts, an attempt has been made to design and synthesize novel α-aminophosphonate derivatives with high bioactivity and low toxicity. Pyrazole and fluorine moieties were incorporated in α-aminophosphonate 4 (Scheme 1).

Scheme 1. General procedure of preparing compound 4

2 EXPERIMENTAL

2.1 Reagents, instruments and activity bioassay

All chemicals and reagents were commercially available and used without further purification. The melting point was determined with Tektronix X4 microscopic melting point apparatus and uncorrected.Infrared spectra were obtained on a Nicolet FTIR-5700 instrument with the compound in a KBr disc matrix. The1H NMR and13C NMR spectra were carried out with a Bruker DRX-400 (400 MHz)spectrometer, using TMS as internal standard. The elemental analysis was performed by Elemento EL-III. Column chromatography was performed on silica gel (200～300 mesh). Sonication was performed on a KQ3200DB ultrasonic cleaner (with frequencies of 40 KHz and a nominal power of 150 W). X-ray diffraction analysis was carried out with a Rigaku Saturn 724 CCD X-ray diffraction instrument.

The antifungal activities against two pathogenic fungi, namely Fusarium graminearum Schw. and Sclerotium rolfsii Sacc., were carried out by the plate growth rate method. Compounds 4a～4h were dissolved in acetone before mixing with Potato Dextrose Agar (PDA: 20% potato extract, 2%dextrose, 2% agar). The final concentration of compounds 4a～4h in the medium was fixed at 50 and 200 μg/mL, respectively. Two kinds of fungi were incubated in PDA at 25±1 ℃ for 3 days to get new mycelium for antifungal assay, then a mycelia disk of approximately 5 mm diameter cut from the culture medium was picked up with a sterilized inoculation needle and inoculated in the center of PDA plate. The inoculated plates were incubated at 25±1 ℃ for 74 h. Acetone in aseptic water served as control, and three replicates were carried out. The in vitro inhibition rates of the compound were then calculated according to the following formula (A and B represent the diameters of fungi growth on PDA treated with control and the compound,respectively).

The assay against cancer cell proliferation was evaluated by MTT method. All compounds tested were dissolved in DMSO (1～100 μM solution) and subsequently diluted in the culture medium before treatment of the cultured cells. Tested cells were plated in 96-well plates at a density of 2 × 103cells/well/100 μL of the proper culture medium and treated with the compounds at concentration of 1 and 10 μM for 72 h. In parallel, the cells were treated with 0.1% of DMSO as control. An MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide] assay (Roche Molecular Biochemicals)was performed according to the instructions provided by Roche. This assay is based on the cellular cleavage of the tetrazolium salt, MTT, into a formazan that is soluble in cell culture medium and measured at 550 nm directly in 96-well assay plates.Absorbance is directly proportional to the number of living cells in culture. In this study, PC3 (prostate cancer) cell (provided by Cell Bank of Committee on Type Culture Collection of Chinese Academy of Science) was cultivated in RPMI 1640 and supplemented with 10% fetal bovine serum. Tissue culture reagents were obtained from Gibco Co.Inhibition rates of the compound were calculated according to the following formula (A1and A2mean the optical densities of untreated cells and drug treated cells, respectively).

The antiviral activity against tobacco mosaic virus(TMV) was carried out by the conventional half-leaf method (Ningnanmycin was used as the reference antiviral agent). TMV (concentration of 6.0 × 10-3mg/mL) was dipped and inoculated on the whole leaves of the same ages of tobacco; 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 counted and recorded 3～4 days after inoculation. There are three replicates for the title compound and Ningnanmycin. The in vivo inhibition rates of the compound were then calculated according to the following formula (“av”means average, and controls were not treated with compound).

2.2 Syntheses

3,5-Difluorobenzaldehyde or 4-trifluoromethylbenzaldehyde 1 (5 mmol), 3-amino-4-cyanopyrazole 2 (5 mmol), para-toluenesulfonic acid (0.10 mmol)and anhydrous ethanol (30 mL) were taken in an oven-dried round-bottomed flask. The mixture was heated to 80 ℃ in the ultrasonic cleaning bath and irradiated for 40～50 minutes. The reaction progress was monitored by TLC (V(acetone)∶V(petroleum)= 2∶3)analysis. After completion of the reaction, the ethanol was removed in vacuum. Then dialkylphosphite (15 mmol) was added into the resulted imine intermediate 3 and refluxed under free-solvent condition for 3～4 h until the reaction was completed (monitored by TLC). The residue was purified by silica gel column chromatography eluting with petroleum ether/acetone to afford 4 as pure products.

Diethyl[(4-cyano-1H-pyrazol-3-ylamino)(3,5-difluorophenyl)methyl]phosphonate (4a) Colorless block solid; yield: 79.7%; m.p.: 206～207 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ: 1.13～1.27 (m,6H, 2OCCH3), 3.92～3.96 (m, 1H, POCH), 4.01～4.07 (m, 1H, POCH), 4.10～4.16 (m, 2H, POCH2),5.26 (d, J3P-H= 23.2, 1H, PCHN), 6.93～6.97 (m,1H, Ar-H), 7.29～7.34 (m, 2H, Ar-H), 8.07 (s, 1H,C=CHpyrazole).13C NMR (DMSO-d6, 100Hz, ppm) δ:16.0, 16.2, 55.1, 62.5, 62.9, 102.9, 111.4, 111.7,114.8, 141.8, 155.2, 160.7, 163.2. IR (KBr,vmax/cm-1): 3337(w), 3194(w), 2983(m), 2227(s),1624(m), 1601(m), 1550(s), 1463(w), 1313(w),1222(s), 1034(s), 987(s), 795(w). Anal. Calcd. (%)for C15H17F2N4O3P: C, 48.51; H, 4.65; N, 15.16.Found (%): C, 48.65; H, 4.63; N, 15.13.

Di-n-propyl[(4-cyano-1H-pyrazol-3-ylamino)(3,5-difluorophenyl)methyl]phosphonate (4b)Colorless block solid; yield: 81.2%; m.p.: 177～178 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:0.82～0.86 (t, 3H, CCH3), 0.88～0.92 (t, 3H, CCH3),1.53～1.58 (m, 2H, OCCH2), 1.62～1.67 (m, 2H,OCCH2), 3.84～3.85 (m, 1H, POCH2), 3.94～3.96(m, H, POCH2), 4.04～4.08 (m, 2H, POCH2), 5.29(d, J3P-H= 23.6, 1H, PCHN), 6.92～6.98 (m, 1H,Ar-H), 7.30～7.32 (m, 2H, Ar-H), 8.10 (s, 1H,C=CHpyrazole).13C NMR (DMSO-d6, 100Hz, ppm) δ:9.7, 9.8, 23.2, 23.4, 55.1, 67.7, 68.3, 102.8, 111.5,111.8, 114.8, 141.7, 155.2, 160.7, 163.3. IR (KBr,vmax/cm-1): 3350(w), 3188(w), 2974(m), 2223(s),1623(m), 1601(m), 1550(s), 1466(w), 1310(m),1222(s), 1013(s), 992(s), 751(w). Anal. Calcd. (%)for C17H21F2N4O3P: C, 51.21; H, 5.36; N, 14.09.Found (%): C, 51.26; H, 5.31; N, 14.06.

Diisopropyl[(4-cyano-1H-pyrazol-3-ylamino)(3,5-difluorophenyl)methyl]phosphonate (4c)[18]Colorless block solid; yield: 84.3%; m.p.: 191～192 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:1.01～1.05 (m, 3H, OCCH3), 1.20～1.22 (m, 3H,OCCH3), 1.26～1.32 (m, 6H, 2OCCH3), 4.56～4.57(m, 1H, POCH), 4.72～4.75 (m, 1H, POCH), 5.16(d, J3P-H= 23.6, 1H, PCHN), 6.91～6.95 (m, 1H,Ar-H), 7.28～7.30 (m, 2H, Ar-H), 8.08 (s, 1H,C=CHpyrazole).13C NMR (DMSO-d6, 100Hz, ppm) δ:23.1, 23.3, 23.7, 23.9, 55.7, 71.1, 71.5, 102.8, 111.6,111.9, 114.8, 141.8, 155.2, 160.8, 163.2. IR (KBr,vmax/cm-1): 3363(w), 3194(w), 2988(m), 2224(s),1621(m), 1599(m), 1552(s), 1465(w), 1307(m),1220(s), 1012(s), 993(s), 779(w). Anal. Calcd. (%)for C17H21F2N4O3P: C, 51.19; H, 5.29; N, 14.13.Found (%): C, 51.26; H, 5.31; N, 14.06.

Di-n-butyl[(4-cyano-1H-pyrazol-3-ylamino)(3,5-difluorophenyl)methyl]phosphonate (4d)Colorless needle solid; yield: 85.1%; m.p.: 102～103 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:0.82～0.89 (m, 6H, 2CCH3), 1.25～1.36 (m, 4H,2CCH2), 1.48～1.62 (m, 4H, 2OCCH2), 3.86～4.01(m, 2H, POCH2), 4.07～4.12 (m, 2H, POCH2), 5.28(d, J3P-H= 23.2, 1H, PCHN), 6.93～6.98 (m, 1H,Ar-H), 7.29～7.31 (m, 2H, Ar-H), 8.09 (s, 1H,C=CHpyrazole).13C NMR (DMSO-d6, 100Hz, ppm) δ:13.3, 13.3, 18.0, 18.1, 31.8, 31.9, 55.1, 65.7, 66.4,102.8, 111.5, 111.7, 114.7, 142.4, 155.1, 160.8,163.3. IR (KBr, vmax/cm-1): 3426(w), 3215(m),2963(s), 2222(s), 1603(s), 1559(w), 1513(w),1462(m), 1315(m), 1219(s), 1028(s), 992(s), 765(w).Anal. Calcd. (%) for C19H25F2N4O3P: C, 51.66; H,5.93; N, 13.17. Found (%): C, 53.52; H, 5.91; N,13.14.

Diethyl[(4-cyano-1H-pyrazol-3-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate(4e) Colorless needle solid; yield: 78.9%; m.p.:140～141 ℃.1H NMR (CD3COCD3, 400Hz, ppm)δ: 1.11～1.15 (t, 3H, OCCH3), 1.12～1.27 (t, 3H,OCCH3), 3.88～3.90 (m, 1H, POCH), 3.99～4.02(m, H, POCH), 4.08～4.17 (m, 2H, POCH2), 5.35 (d,J3P-H= 23.2, 1H, PCHN), 7.69～7.84 (m, 4H, Ar-H),8.06 (s, 1H, C=CHpyrazole).13C NMR (CDCl3, 100Hz,ppm) δ: 16.0, 16.4, 55.0, 63.6, 64.3, 99.9, 114.5,122.5, 125.4, 125.7, 128.7, 130.2, 130.5, 134.2,136.3, 140.1. IR (KBr, vmax/cm-1): 3426(w), 3214(w),2963(m), 2223(s), 1609(s), 1562(w), 1461(w),1329(s), 1217(s), 1029(s), 938(w), 737(w). Anal.Calcd. (%) for C16H18F3N4O3P: C, 47.83; H, 4.53; N,13.97. Found (%): C, 47.77; H, 4.51; N, 13.93.

Di-n-propyl[(4-cyano-1H-pyrazol-3-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (4f)Colorless needle solid; yield: 82.7%; m.p.: 155～156 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:0.78～0.81 (t, 3H, CCH3), 0.87～0.91 (t, 3H, CCH3),1.49～1.66 (m, 4H, 2OCCH2), 1.49～1.54 (m, 2H,OCCH2), 1.61～1.66 (m, 2H, OCCH2), 3.76～3.80(m, 1H, POCH), 3.90～3.94 (m, 1H, POCH),4.01～4.05 (m, 2H, POCH2), 5.35 (d, J3P-H= 23.2,1H, PCHN), 7.69～7.85 (m, 4H, Ar-H), 8.07 (d, 1H,C=CHpyrazole).13C NMR (DMSO-d6, 100Hz, ppm) δ:9.7, 9.8, 23.2, 23.4, 55.4, 67.7, 68.2, 99.9, 114.8,118.5, 122.9, 124.7, 124.8, 125.6, 127.9, 128.2,129.1, 141.7. IR (KBr, vmax/cm-1): 3427(w), 3212(w),2972(m), 2224(s), 1610(s), 1563(w), 1458(w),1328(s), 1217(s), 1016(s), 937(m), 741(w). Anal.Calcd. (%) for C18H22F3N4O3P: C, 51.32; H, 5.13; N,13.07. Found (%): C, 50.24; H, 5.15; N, 13.02.

Diisopropyl[(4-cyano-1H-pyrazol-3-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (4g)Colorless needle solid; yield: 84.4%; m.p.: 214～215 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:0.95～0.99 (m, 3H, OCCH3), 1.20～1.21 (m, 3H,OCCH3), 1.24～1.31 (m, 6H, 2OCCH3), 4.51～4.56(m, 1H, POCH), 4.70～4.75 (m, 1H, POCH), 5.24(d, J3P-H= 23.6, 1H, PCHN), 7.69～7.83 (m, 4H,Ar-H), 8.06 (s, 1H, C=CHpyrazole).13C NMR(DMSO-d6, 100Hz, ppm) δ: 23.0, 23.3, 23.8, 23.9,55.9, 71.0, 71.4, 114.8, 122.9, 124.7, 125.6, 127.9,128.2, 128.3, 129.3, 130.7, 141.8. IR (KBr,vmax/cm-1): 3429(w), 3209(w), 2987(m), 2225(s),1606(s), 1561(w), 1455(w), 1328(s), 1216(s),1011(s), 937(m), 769(w). Anal. Calcd. (%) for C18H22F3N4O3P: C, 51.27; H, 5.16; N, 12.99. Found(%): C, 50.24; H, 5.15; N, 13.02.

Di-n-butyl[(4-cyano-1H-pyrazol-3-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (4h)Colorless needle solid; yield: 83.9%; m.p.: 162～163 ℃.1H NMR (CD3COCD3, 400Hz, ppm) δ:0.79～0.83 (t, 3H, CCH3), 0.85～0.89 (t, 3H, CCH3),1.21～1.35 (m, 4H, 2CCH2), 1.44～1.61 (m, 4H,2OCCH2), 3.82～3.86 (m, 1H, POCH), 3.94～3.96(m, 1H, POCH), 4.08～4.11 (m, 2H, POCH2), 5.35(d, J3P-H= 23.6, 1H, PCHN), 7.69～7.85 (m, 4H,Ar-H), 8.07 (s, 1H, C=CHpyrazole).13C NMR(DMSO-d6, 100Hz, ppm) δ: 12.1, 12.2, 26.4, 26.4,44.1, 50.3, 71.1, 80.7, 99.5, 114.9, 122.9, 125.3,125.4, 125.6, 127.6, 127.9, 128.2, 128.3, 128.6,140.3. IR (KBr, vmax/cm-1): 3427(w), 3216(w),2991(w), 2224(s), 1613(s), 1562(w), 1419(w),1329(s), 1217(s), 1029(s), 977(w), 752(w). Anal.Calcd. (%) for C20H26F3N4O3P: C, 51.55; H, 5.74; N,12.17. Found (%): C, 52.40; H, 5.72; N, 12.22.

2.3 Crystal structure determination

A colorless prism of compound 4a (0.20mm ×0.18mm × 0.12mm) was used for data collection with a Rigaku Saturn 724 CCD diffractometer equipped with a multilayer-monochromatic MoKα radiation (λ = 0.71073 ?) using a φ-ω scan mode in the ranges of 1.87°≤θ ≤25.01°, –9≤h≤7,–12≤k≤12, and –13≤l≤13 at 113(2) K. A total of 7401 reflections including 3046 unique ones with Rint= 0.0678 were collected, of which 1582 observed reflections with I > 2σ(I) were employed in the structure determination and refinements. The structure was solved by direct methods and refined by full-matrix least-squares on F2using SHELXS-97 and SHELXL-97 software packages[16,17]. All non-hydrogen atoms were refined anisotropically,and all hydrogen atoms were located on difference Fourier maps and treated as riding atoms with C-H distances of 0.95～1.00 ? for aryl, methyl, methine and tertiary alkyl, respectively. The final refinement converged at R = 0.0487 and wR = 0.0823 (w =1/[σ2(Fo2) + (0.0000P)2+ 0.0000P], where P = (Fo2+2Fc2)/3), S = 1.064 and (Δ/σ) = 0.000. The max and min difference peaks and holes on the final difference Fourier map are 0.303 and -0.396 e·?-3,respectively.

3 RESULTS AND DISCUSSION

The dialkylphosphite was prepared according to the literature[19]. Ultrasonic irradiation has been successfully employed in performing the synthetic reactions due to its chemical effects caused by the cavitations. The cavitations induce very high local temperature and pressure inside the bubbles(cavities), which would lead to a turbulent flow in the liquid and enhance the mass transfer quickly[20].Up to now, many organic reactions have been reported in higher yields, shorter reaction time and milder conditions under the ultrasonic irradiation[21,22].In this paper, the target compounds were synthesized via a two-step method. The imine intermediate 3 was conveniently prepared under ultrasonic irradiation in ethanol solution without separation process. As to the addition reaction, we used three times amount of dialkylphosphite as compared to the starting materials and heated in solvent-free conditions. The advantages of the procedure lie in the shorter reaction period and high yield towards the target compounds.

The structures of the target compounds 4 were characterized by1H NMR,13C NMR, IR and elemental analysis. All spectra and analytical data were consistent with the assigned structures. In the IR spectrum, the absorption band at 3400～3300 cm-1corresponds to the stretching vibrations of N-H,while the absorption near 3200 cm-1was ascribed to the O-H of strong intermolecular hydrogen bonding[23]in the title compounds, which can be confirmed by the hydrogen-bond information of 4a(Table 3). The C=C and C=N of the aromatic ring were characterized by absorption in the range of 1601～1419 cm-1, while the absorption at 1222～1216 cm-1was assigned to the P=O stretching absorption bands and the absorption 1034～1011 cm-1to the C-O stretching absorption bands in the P-O-C group. The characteristic absorption band for P-C and CN was at 737～795 and 2222～2227 cm-1, respectively. In the1H NMR spectrum, all of the title compounds exhibited a double at 5.16～5.35 ppm indicative of the H atom at the α-C coupling with the adjacent phosphorus (3JP-H= 23.2～23.6 Hz). The pyrazole ring exists in atom N(1) which is linked to the α-C to hinder the free rotation of P(1)-C(7) bond, which causes the chemical shift of H atom affiliated to the CH or CH2or CH3in the two alkyl groups to appear at different shifts respectively due to their magnetically nonequivalent surroundings.

The molecular structure and crystal packing diagram of 4a are shown in Figs. 1 and 2, respectively. The bond lengths and bond angles (Table 1)reveal that the P atom adopts a distorted tetrahedral configuration[24,25], which can be deduced from the fact that the O(1)-P(1)-O(2) (115.99(19) ?) and O(1)-P(1)-C(7) (110.45(13) ?) bond angles are significantly larger than O(2)-P(1)-O(3) (103.12(12)?), and also from the fact that the P(1)-O(1) bond(1.4700(19) ?) is considerably shorter than P(1)-O(2) (1.566(2) ?) and P(1)-O(3) (1.568(2) ?).The interatomic distances of N(1)-C(8) (1.388(3) ?)and N(3)-C(11) (1.314(3) ?) are remarkably shorter than the length of normal C-N (1.47 ?), but very close to the typical C=N (1.34 ?)[26,27], which suggests that the electron is delocalized in N(1) and the pyrazole ring. The single bond lengths of C(8)-C(9) (1.419(4) ?), C(9)-C(10) (1.415(4) ?)and C(9)-C(11) (1.395(4) ?) are shorter than that of the typical C-C bond (1.54 ?), but exhibit obvious C=C bond characteristic (1.35 ?), indicating the electron densities of the pyrazole ring and CN group are considerably delocalized. Meanwhile, the sum of C(11)-N(3)-N(2), C(11)- N(3)-H(3) and N(2)-N(3)-H(3) bond angles is 359.6o, so the atom N(3) is of sp2hybridization.

Fig. 1. Crystal structure of 4a with atomic labeling scheme at 50% probability displacement ellipsoids. One disordered component is shown

Fig. 2. Packing diagram of 4a showing the hydrogen bonding interactions and π-π interaction

Table 1. Selected Bond Lengths (?) and Bond Angles (o) for Compound 4a

It can be seen that there are two planes in the molecule of compound 4a (Fig. 1). The benzene ring system with its conjunction atoms C(7), F(1) and F(2)is essentially planar with a maximum deviation of-0.0221 ? for atom C(3), and the pyrazole ring with the connected nitrogen atom N(1) and C(11), N(4) of CN group are nearly coplanar, with the largest deviation from the least-squares plane to be 0.0346 ? for atom N(2) which is slightly twisted from the plane with the N(3)-N(2)-C(8)-N(1) and N(2)-C(8)-C(9)-C(11) torsion angles of -177.0(3)° and 0.6(3)° (Table 2), respectively (plane equation is-0.9026x + 0.2469y - 0.3526z = -10.1699). The dihedral angle between the two planes is 71.51°. As shown in Fig. 2, two strong intermolecular hydrogen bonds O(1)-H(1)···N(4) and N(3)-H(3)···O(1), and a face-to-face π···π stacking interaction among 3,5-difluorophenyl are found in compound 4a (Tables 3～4). In the solid state, the hydrogen bonding interactions play an important role in constructing the one-dimensional chains, and such chains further form a two-dimensional grid via π···π interaction and intermolecular hydrogen bonds. The above interactions are effective in stabilizing the crystal structure.

Table 2. Selected Torsional Angles (°) for Compound 4a

Table 3. Intermolecular Hydrogen Bonds of Compound 4a

Table 4. π···π Interaction of Compound 4a

The results of bioassay in vitro against two fungi,Tobacco Mosaic Virus (TMV) and PC3 cell, are given in Table 5. From Table 5 it may be observed that the title compounds 4a～4h possess weak bioactivities towards the two fungi at 50 μg/mL. At 200 μg/mL, however, compounds 4b, 4c, 4g and 4h exhibited good activities on Sclerotium rolfsii Sacc at 54.42%, 61.82%, 65.95% and 53.35%, respectively. The results against TMV indicated that the antiviral activity depended on the substituents present. At 500 μg/mL, in the case of R1is the same alkyl group. When R is a 4-trifluoromethyl group,the compound showed better curative effect against TMV relatively, slightly higher than that of the compound in which R is 3,5-difluoro group; when R1is propyl or isopropyl group, compounds 4b, 4c,4f and 4g exhibited good anti-TMV activities at 33.13%, 43.35%, 41.68% and 50.02% respectively,slightly lower than that of Ningnamycin (58.69%).The antitumor activities in vitro for title compounds 4a～4h were evaluated against PC3 cells by the MTT method. It can be seen that all of the compounds exhibited weak antitutor activities at 1 and 10 μmol/L, respectively.

Table 5. Antifungical, Antiviral and Antitumor Activities of Compounds 4a～4h

4 CONCLUSION

In summary, a series of α-aminophosphonates 4 containing pyrazole ring and fluorine moieties was synthesized through condensation and addition reactions. The structures of them were verified by spectroscopic method. Their bioactivities against two fungi, TMV and PC3 cells, were evaluated. It was found that compounds 4b, 4c and 4g possessed good curative effect against TMV and good antifungal activity towards Sclerotium rolfsii Sacc.The bioassay results indicated that all of the compounds exhibited a weak antitumor activity.

ACKNOWLEDGEMENT

We are grateful to the group of Professor Song Baoan (Key Laboratory of Green Pesticide and Agricultural Bioengineering, Guizhou University) for their assistance in antiviral, antifungal and antitumor activities bioassay.

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