Exploration of a Library of Triazolothiadiazines as Potent Plant Growth Promoters: Design, Synthesis, X-ray Diffraction Analysis and Bioactivity Studies①

DING Qi-Chun CAI Yi-Min WANG Si-Lei YAO Pei-Pao YANG Ru-Ya

(Department of Pharmacy, Zhangzhou Health Vocational College, Zhangzhou363000, China)

ABSTRACT A series of novel 3-methyl-6-aryl-7-aroyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were designed, synthesized and tested for their antiproliferative activity against HepG2 cell lines in vitroby the standard SRB assayandplant growth regulation activities on wheat (amonocotyledon) and radish (adicotyledon).The results indicated all the title compounds exhibited a very weak antiproliferative activity against HepG2 cell lines in vitrounexpectedly, while showed potent plant growth-regulating activities on both wheat and radish. The crystal structure of trans-4d was obtained from X-ray diffraction: C18H13N4OSCl3, Mr= 439.75, monoclinic system,space group P21/n, a= 5.3224(7), b= 14.3578(18), c= 24.442(3) Å, β= 94.180(2)°, V= 1862.8(4) Å3, F(000) =899, Z= 4, Dc= 1.5679 g/cm3, λ= 0.71073 Å, μ= 0.621 mm-1 and the final R= 0.0382 for 3274 unique reflections with 2851 observed ones (I> 2σ(I)).

Keywords: synthesis, 1,2,4-triazole, triazolothiadiazine, crystal structure, antiproliferative, plant growth regulation, bioactivity; DOI: 10.14102/j.cnki.0254–5861.2011–3225

1 INTRODUCTION

1,2,4-Triazoles and their numerous derivatives are one of the most prevalent class of nitrogen heterocyclic cores, which span a wide range in organic and medicinal chemistry, owing to their excellent broad spectrum biological activities,chemotherapeutic potential and diagnostic applications[1-6].Among them, 1,2,4-triazolothiadiazines (1,2,4-TTDs) attract more and more attention as they are shown to be more distinctive features[7-12]. However, the relationship between the structures of 3,6-disubstituted-7-aroyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and their bioactivities is rarely reported, which has been mentioned in our recent paper[13]. Therefore, as a part of our ongoing research on developing novel 1,2,4-TTD compounds with more effective bioactivities, we attempted to introduce an alkyl group into the 3-position of this kind of compounds, which might be an effective approach, since a class of similar compounds like 3-alkyl-6-disubstituted-7H-[1,2,4]triazolo-[3,4-b][1,3,4]thiadiazines have been claimed to exhibit numerous bioactivities by many papers[14-20].

As a result, in the present work a number of 3-methyl-6-aryl-7-aroyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]-thiadiazines were designed and prepared, and the antiproliferative and plant growth regulation effects of them were investigated respectively. The test results surprised us that all the title compounds exhibited a very weak antiproliferative activity against HepG2 cell linesin vitro, while showed potent plant growth-regulating activities on both wheat and radish.These results afford a new scaffold for the investigation of potent plant growth promoters potentially harmless to humans(further experiments are needed). The study on further structure modifications and structure-activity relationships will be carried out in the future.

2 EXPERIMENTAL

2. 1 Reagents and apparatus

All reagents were of analytical grade and directly used as obtained from commercial sources and used without further purification. The melting points were determined on an XT-4A apparatus and uncorrected. The FT-IR spectra were recorded on a Thermo Scientific NICOLET 5700 FT-IR Spectrometer in ATR mode in the range of 4000～400 cm-1.NMR spectra were recorded on a Burker 500 MHz Nuclear Magnetic Resonance Spectrometer with TMS as an internal standard and DMSO-d6as the solvent. MS spectra were recorded on a Q-Exactive LC-MS/MS. Crystal structure determination was carried out on a Bruker Smart APEX II CCD diffractometer.

2. 2 Synthesis

2. 2. 1 Preparation of 1,2,4-triazole (1)

4-Amino-5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione(1), whose tautomer is 4-amino-5-methyl-4H-1,2,4-triazole-3-thiol, was prepared from acetic acid and thiocarbohydrazide.The mixture was refluxed for 4 h under stirring. After concentration under reduced pressure, the crude product was recrystallized from alcohol to afford compound 1 (Scheme 1).

Scheme 1. Synthetic route of compound 1

2. 2. 2 General procedure for the synthesis of(E)-3-substituted-4-arylideneamino-1H-1,2,4-triazole-5(4H)-thione (2)

To a solution of compound 1 (2 mmol) dissolved in absolute alcohol (20 mL), the appropriate benzaldehyde (2 mmol) was added, and the pH was adjusted to 5～6 with dilute hydrochloric acid. The mixture was refluxed on an oil-bath for about 4 h with stirring. The solid that was obtained upon cooling was filtered, washed with cold water,dried, and recrystallized from alcohol to give the Schiff base 2(Scheme 2).

Scheme 2. Synthetic route of the title compounds

2. 2. 3 General procedure for the synthesis of the title compounds

The title compound was prepared by the reaction of(E)-3-substituted-4-arylideneamino-1H-1,2,4-triazole-5(4H)-thione (2, 2 mmol) and appropriate substitutedω-bromoacetophenone (2 mmol) in the mixture of ethanol (40 mL) and triethylamine (1 mL) at room temperature for about 30 min.After concentration under reduced pressure, the crude products were recrystallized from ethanol to afford the compounds 4a～j (Scheme 2).

(3-Methyl-6-phenyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(phenyl)methanone (4a)

Yield: 80%. m.p.: 193～195 °C. IR (cm-1): 3423 (NH),3062 (ArH), 2966 (CH3), 1677 (C=O), 1631 (C=N), 702(C-S-C).1H NMR (500MHz, DMSO-d6)δ: 2.34 (s, 3H, CH3),5.05 (t,J= 5.8 Hz, 1H, N-CH), 5.76 (d,J= 5.2 Hz, 1H,S-CH), 7.04 (d,J= 6.7 Hz, 1H, NH), 7.28 (t,J= 7.2 Hz, 1H,ArH), 7.34 (t,J= 7.6 Hz, 2H, ArH), 7.50 (d,J= 7.6 Hz, 2H,ArH), 7.58 (t,J= 7.8 Hz, 2H, ArH), 7.71 (t,J= 7.4 Hz, 1H,ArH), 8.06 (d,J= 7.5 Hz, 2H, ArH);13C NMR (125 MHz,DMSO-d6)δ: 10.15, 42.67, 57.83, 127.71, 128.46, 129.02,129.30, 129.56, 134.72, 134.96, 137.61, 139.80, 150.92,195.68. MS-ESI (m/z): 335.10 ([M-H]-), 371.07 ([M+Cl]-).

(4-Methoxyphenyl)(3-methyl-6-phenyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)methanone (4b)

Yield: 85%. m. p.: 212～214 °C. IR (cm-1): 3415 (NH),2978 (ArH), 2839 (CH3), 1672 (C=O), 1598 (C=N), 694(C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.33 (s, 3H, CH3),3.87 (s, 3H, OCH3), 4.97 (t,J= 6.3 Hz, 1H, N-CH), 5.71 (d,J= 5.6 Hz, 1H, S-CH), 7.00 (d,J= 7.2 Hz, 1H, NH), 7.09 (d,J= 8.8 Hz, 2H, ArH), 7.27 (t,J= 7.2 Hz, 1H, ArH), 7.33 (t,J=7.4 Hz, 2H, ArH), 7.49 (d,J= 7.6 Hz, 2H, ArH), 8.03 (d,J=8.8 Hz, 2H, ArH).13C NMR (125 MHz, DMSO-d6)δ: 10.14,42.32, 56.21, 58.30, 114.84, 127.68, 127.78, 128.46, 129.00,131.77, 137.62, 140.02, 150.87, 164.49, 193.90.

(6-(3-Chlorophenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(phenyl)methanone (4c)

Yield: 82%. m.p.: 203～205 °C. IR (cm-1): 3427 (NH),3053 (ArH), 2927 (CH3), 1678 (C=O), 1595 (C=N), 775(C-Cl), 686 (C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.35(s, 3H, CH3), 5.16 (t,J= 5.2 Hz, 1H, N-CH), 5.78 (d,J= 4.5 Hz, 1H, S-CH), 7.13 (d,J= 6.1 Hz, 1H, NH), 7.35～7.39 (m,2H, ArH), 7.46 (d,J= 7.1 Hz, 1H, ArH). 7.60 (t,J= 7.6 Hz,2H, ArH), 7.65 (s, 1H, ArH), 7.72 (t,J= 7.4 Hz, 1H, ArH),8.10 (d,J= 7.9 Hz, 2H, ArH);13C NMR (125 MHz,DMSO-d6)δ: 10.15, 41.90, 56.70, 126.40, 127.69, 128.42,129.42, 129.50, 130.90, 133.71, 134.71, 134.85, 139.42,140.18, 150.86, 195.58.

(6-(3-Chlorophenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(2,4-dichlorophenyl)-methanone (4d)

Yield: 78%. m.p.: 191～193 °C. IR (cm-1): 3392 (NH),3076 (ArH), 2916 (CH3), 1705 (C=O), 1585 (C=N), 756(C-Cl), 698 (C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.36(s, 3H, CH3), 5.15 (t,J= 5.7 Hz, 1H, N-CH), 5.56 (d,J= 4.8 Hz, 1H, S-CH), 7.13 (d,J= 6.5 Hz, 1H, NH), 7.35～7.39 (m,3H, ArH), 7.55 (s, 1H, ArH), 7.58 (dd,J1= 1.9 Hz,J2= 8.4 Hz,1H, ArH), 7.77 (d,J= 1.9 Hz, 1H, ArH), 7.79 (d,J= 8.5 Hz,1H, ArH);13C NMR (125 MHz, DMSO-d6)δ: 10.17, 46.56,56.76, 126.34, 127.59, 128.01, 128.55, 130.58, 130.94, 131.79,132.96, 133.74, 135.57, 137.58, 139.16, 139.69, 150.86,196.35.

(2,4-Dichlorophenyl)(3-methyl-6-phenyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)methanone (4e)

Yield: 90%. m.p.: 165～167 °C. IR (cm-1): 3396 (NH),3051 (ArH), 2916 (CH3), 1705 (C=O), 1583 (C=N), 763(C-Cl), 694 (C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.35(s, 3H, CH3), 5.03 (t,J= 6.2 Hz, 1H, N-CH), 5.53 (d,J= 5.3 Hz, 1H, S-CH), 7.06 (d,J= 7 Hz, 1H, NH), 7.28 (t,J= 7 Hz,1H, ArH), 7.34 (t,J= 6.9 Hz, 2H, ArH), 7.43 (d,J= 7.4 Hz,2H, ArH), 7.54 (dd,J1= 1.8 Hz,J2= 8.4 Hz, 1H, ArH), 7.63(d,J= 8.4 Hz, 1H, ArH), 7.76 (d,J= 1.7 Hz, 1H, ArH);13C NMR (125 MHz, DMSO-d6)δ: 10.17, 47.33, 58.04, 127.30,127.62, 128.07, 129.09, 130.54, 131.70, 132.54, 135.79,137.09, 137.56, 139.45, 151.02, 196.75.

(4-Methoxyphenyl)(6-(4-methoxyphenyl)-3-methyl-6,7-idhydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)methanone (4f)

Yield: 84%. m.p.: 187～189 °C. IR (cm-1): 3400 (NH),2997 (ArH), 2937 (CH3), 1666 (C=O), 1597 (C=N), 700(C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.32 (s, 3H, CH3),3.71 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.87 (t,J= 6.7 Hz,1H, N-CH), 5.69 (d,J= 5.9 Hz, 1H, S-CH), 6.89 (d,J= 8.7 Hz, 2H, ArH), 6.93 (d,J= 7.5 Hz, 1H, NH), 7.10 (d,J= 8.8 Hz, 2H, ArH), 7.42 (d,J= 8.6 Hz, 2H, ArH), 8.02 (d,J= 8.8 Hz, 2H, ArH);13C NMR (125 MHz, DMSO-d6)δ: 10.12,42.44, 55.53, 56.21, 58.10, 114.35, 114.86, 127.74, 129.10,129.41, 131.73, 140.15, 150.91, 159.34, 164.50, 193.87.

(6-(4-Methoxyphenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]-triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(phenyl)methanone (4g)

Yield: 88%. m.p.: 184～186 °C. IR (cm-1): 3410 (NH),3068 (ArH), 2947 (CH3), 1654 (C=O), 1612 (C=N), 692(C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.39 (s, 3H, CH3),3.66 (s, 3H, OCH3), 4.92 (dd,J1= 1.7 Hz,J2= 11 Hz, 1H,N-CH), 5.77 (d,J= 2.4 Hz, 1H, S-CH), 6.82 (d,J= 8.7 Hz,2H, ArH), 7.16 (d,J= 11 Hz, 1H, NH), 7.27 (d,J= 8.6 Hz,2H, ArH), 7.48 (t,J= 7.7 Hz, 2H, ArH), 7.63 (t,J= 7.4 Hz,1H, ArH), 7.86 (d,J= 7.7 Hz, 2H, ArH);13C NMR (125 MHz,DMSO-d6)δ: 10.28, 40.56, 55.53, 59.31, 114.23, 128.47,128.64, 128.91, 129.41, 134.60, 135.77, 139.05, 150.59,159.24, 197.25.

(2,4-Dichlorophenyl)(6-(4-methoxyphenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)methanone (4h)

Yield: 85%. m.p.: 186～188 °C. IR (cm-1): 3431 (NH),3070 (ArH), 2924 (CH3), 1701 (C=O), 1606 (C=N), 752(C-Cl), 694 (C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.33(s, 3H, CH3), 3.71 (s, 3H, OCH3), 4.90 (t,J= 6.5 Hz, 1H,N-CH), 5.49 (d,J= 5.7 Hz, 1H, S-CH), 6.86 (d,J= 8.7 Hz,2H, ArH), 6.97 (d,J= 7.4 Hz, 1H, NH), 7.33 (d,J= 8.7 Hz,2H, ArH), 7.53 (dd,J1= 1.7 Hz,J2= 8.4 Hz, 1H, ArH), 7.57(d,J= 8.4 Hz, 1H, ArH), 7.76 (d,J= 1.7 Hz, 1H, ArH);13C NMR (125 MHz, DMSO-d6)δ: 10.13, 47.71, 55.61, 58.01,114.45, 128.55, 128.97, 130.49, 131.66, 132.96, 135.32,135.88, 137.52, 139.95, 151.07, 159.05, 196.84.

(6-(4-Hydroxyphenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]-triazolo[3,4-b][1,3,4]thiadiazin-7-yl)(phenyl)methanone (4i)

Yield: 84%. m.p.: 187～189 °C. IR (cm-1): 3425 (NH),3254 (OH), 3005 (ArH), 2910 (CH3), 1682 (C=O), 1612(C=N), 686 (C-S-C).1H NMR (500 MHz, DMSO-d6)δ: 2.33(s, 3H, CH3), 4.86 (t,J= 8.0 Hz, 1H, N-CH), 5.72 (d,J= 7.1 Hz, 1H, S-CH), 6.71 (d,J= 10.7 Hz, 2H, ArH), 6.92 (d,J=9.2 Hz, 1H, NH), 7.31 (d,J= 10.7 Hz, 2H, ArH), 7.58 (t,J=9.7 Hz, 2H, ArH), 7.70 (t,J= 9.2 Hz, 1H, ArH), 8.02 (d,J=9.3 Hz, 2H, ArH), 9.52 (s, 1H, OH);13C NMR (125 MHz,DMSO-d6)δ: 10.13, 43.09, 58.13, 115.75, 127.52, 129.05,129.20, 129.55, 134.71, 135.03, 140.19, 151.02, 157.61,195.65.

(2,4-Dichlorophenyl)(6-(4-hydroxyphenyl)-3-methyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-7-yl)methanone (4j)

Yield: 88%. m.p.: 200～202 °C. IR (cm-1): 3410 (NH),3240 (OH), 3015 (ArH), 2895 (CH3), 1688 (C=O), 1612(C=N), 771 (C-Cl), 694 (C-S-C).1H NMR (500 MHz,DMSO-d6)δ: 2.32 (s, 3H, CH3), 4.79 (t,J= 8.7 Hz, 1H,N-CH), 5.46 (d,J= 7.6 Hz, 1H, S-CH), 6.69 (d,J= 10.8 Hz,2H, ArH), 6.93 (d,J= 9.8 Hz, 1H, NH), 7.21 (d,J= 10.8 Hz,2H, ArH), 7.47 (dd,J1= 2.3 Hz,J2= 10. 6 Hz, 1H, ArH), 7.60(d,J= 10. 6 Hz, 1H, ArH), 7.75 (s, 1H, ArH), 9.55 (s, 1H,OH);13C NMR (125 MHz, DMSO-d6)δ: 10.12, 48.04, 58.73,115.81, 128.05, 129.04, 130.48, 131.59, 132.38, 135.30,135.97, 137.56, 139.91, 151.13, 157.24, 196.94.

2. 3 Crystal data and structure determination

Purified product 4d was dissolved in ethanol and kept at r.t.for 5 d, and single crystals were formed. A colorless block single crystal of compound 4d (0.28mm × 0.2mm × 0.2mm)was placed on an APEX II CCD area detector equipped with a graphite-monochromatic MoKαradiation (λ= 0.71073 Å) at 296.15 K. The structure was solved with the olex2.solve structure solution program using Charge Flipping[21]and refined with the olex2.refine refinement package using Gauss-Newton minimisation[21]. A total of 14591 reflections were collected in the range of 2.84<θ<25.00°, of which 3274 were independent withRint= 0.0260 and 2851 were observed withI> 2σ(I). All non-hydrogen atoms were located with successive difference Fourier syntheses and refined by using anisotropic thermal parameters. All hydrogen atoms were located in the calculated positions and refined according to theoretical models. The final full-matrix least-squares refinement gaveR= 0.0382,wR= 0.0929 (w= 1/[σ2(Fo2) +(0.0434P)2+ 1.5692P], whereP= (Fo2+ 2Fc2)/3),S= 1.054,(Δρ)max= 0.677, (Δρ)min= -0.653 e/Å3and (Δ/σ)max= 0.0013.

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2. 4 Biological activity

To this day, there are very few reports on the relationship between 3,6-disubstituted-7-aroyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines and their bioactivities. Only Zhanget alreported some similar compounds showed moderate to potent antiproliferative activities against four cancer cell lines, PC-3, HepG2, A549, and MCF-7[12]and we discovered PTM can promote the growth of radish and inhibit that of wheat in a dose-dependent manner[13]. Therefore, in order to develop novel 1,2,4-TTD compounds with more effective bioactivity, the activities of the title compounds on antiproliferative and the growth regulation of plant were investigated.

2. 4. 1 Antiproliferative activity

The antiproliferative activityin vitroagainst HepG2 cell lines of the title compounds by the standard SRB assay was tested by the National Center for Drug Screening of China.HepG2 cells during the logarithmic phase according to appropriate density (90 μL per well) were seeded to 96-well plates for attachment overnight. Then the cells were treated with different concentrations of the title compounds for 72 hours. The test was carried out in triplicate. The control group and blank group (no cells) were set with corresponding concentrations. At the end of the action, the culture medium was removed and 10% trichloroacetic acid (w/v) was added with 100 μL per well to adherent cells at 4 °C. The cells were fixed for 1 hour, and then washed by distilled water for five times. After being dried at room temperature, the cells were stained with 100 μL per well Sulforhodamine B solution (4 mg/mL Sulforhodamine B (w/v) in 1% acetic acid (v/v)) at room temperature for 15 minutes and washed with 1% acetic acid to remove any unbound dye. Then after drying at room temperature, 100 μL of 10 mmol/L tris solution was added per well. The absorbance values (the optical density, OD) of the plates were measured using a Microplate reader (SpectraMax 190, Molecular Devices) at a wavelength of 560 nm, and the inhibition rate was calculated by the formula:inhibition rate of proliferation(%)(Acontrol–Acompounds)/Acontrol× 100%. The data were expressed as mean ±SD from three independent experiments. For comparison, the inhibitory value of adriamycin against HepG2 cell at similar concentrations under the same conditions is provided.

2. 4. 2 Activity of regulating the growth of plant

The biological activity on regulating the growth of plant of the title compounds was also investigated. Wheat and radish were selected to represent monocotyledonous and dicotyledonous plants, respectively. After treating with culture solution of 20, 50, and 100 ppm of the title compounds 4a～j for 5 days, the growth regulating percentage has been calculated. The equations of the growth regulating percentage(the stalk and radicel of the plant) are: [the average of sample length (cm) – the average of the controls (cm)]/the average of the controls (cm) × 100%. ―+‖ suggests a positive result, and―-‖ means an inhibitory effect. The degree of promotion or inhibition is represented by the magnitude of a numerical value.

3 RESULTS AND DISCUSSION

3. 1 Synthesis

The infrared (IR) spectra of the title compounds showed the absence of C=S, S–H and NH2absorption bands has confirmed that they were obtained via cyclocondensation. The broad bands around 3400 cm-1are due to the symmetric stretching vibrations of N–H, while aromatic C–H stretching vibrations are observed at approximately 3000 cm-1. The C–H stretching peaks of the CH3group are at about 2900 cm-1.Sharp bands observed at 1650 cm-1or so are obviously attributed to C=O stretching vibrations. In the1H NMR spectra, there are a double and a triple peaks in the ranges of aboutδ5.60 and 5.00, corresponding to S–C–H and N–C–H,respectively, consistent with the thiadiazine ring-closure. In the13C NMR spectra, the peaks at aroundδ195, 150 and 140 assigned to be C=O, N=C–S and N=C–N respectively, also proving the formation of the title compounds.

3. 2 Molecular structure

The crystal structure of the target compound 4d was confirmed by X-ray diffraction analysis and only that of the puretrans-isomer was obtained. The molecular structure oftrans-4d is shown in Fig.1. All bond lengths and bond angles are in normal ranges. The intermolecular interactions are shown in Fig.2, and in the crystal structure there is no typical hydrogen bonding orπ-πinteractions between adjacent molecules, and only one weak non-classical intermolecular hydrogen bond C(10)–H(10)···N(1) exists in the structure. The intermolecular interactions cause molecules to form a one-dimensional chain-like structure. Crystal data fortrans-4d: C18H13N4OSCl3,Mr= 439.75, monoclinic system,space groupP21/n,a= 5.3224(7),b= 14.3578(18),c=24.442(3) Å,β= 94.180(2)°,V= 1862.8(4) Å3,F(000) = 899,Z= 4,Dc= 1.5679 g/cm3,λ= 0.71073 Å,μ= 0.621 mm-1and the finalR= 0.0382 for 3274 unique reflections with 2851 observed ones (I> 2σ(I)). The selected bond lengths and bond angles are shown in Table 1. The triazole and thiadiazine rings are almost coplanar with a very small dihedral angle of 0.42(0.13)°. However, the difference be- tween the dihedral angles formed by the thiadiazine ring and two benzene rings respectively, 83.58(0.06)° and 44.20(0.08)°, is a little large,which may be due to the fact that the benzene ring (C(13)～C(18)) is directly connected to the thiadiazine ring, while the benzene ring (C(1)～C(6)) is indirectly con- nected. The C(10)–C(13) and C(7)–C(8) bands are on both sides of the thiadiazine ring, perpendicular to the thiadiazine ring respectively and approximately parallel to each other, which should be as much as possible to minimize the steric hindrances among the three rings.

Fig.1. Molecular structure of trans-4d

Fig.2. Packing diagram in a unit cell of trans-4d

Table 1. Selected Bond Lengths (Å) and Bond Angles (°) for trans-4d

Table 2. Hydrogen Bond Lengths (Å) and Bond Angles (°)

3. 3 Biological activity

3. 3. 1 Antiproliferative activity

Thein vitroantiproliferative activity of the title compounds against HepG2 cell lines (human hepatoma cells) was assayed by SRB method. For comparison, adriamycin was used as the reference drug. The data are summarized in Tables 3 and 4,respectively. All the title compounds showed very weak cytotoxic activity against HepG2 cell lines unexpectedly,which greatly puzzled us because some compounds of the same type synthesized by Zhanget alhad been shown to exhibit moderate to potent antiproliferative activity against HepG2 cell lines[12]. After careful consideration, we presumed that it might be because their substituent types at the 3-, 6-,7-positions of the 1,2,4-TTD parent nucleus are very different from ours, respectively. However, since the activity of a compound is a generally broad spectrum, these compounds may not also be cytotoxic to humans, so they may be developed as drugs used in other fields that are harmless to humans (further experiments are needed). Further researches on the mechanisms of these compounds and modification are underway.

Table 3. Inhibition Rate of the Title Compounds on the HepG2 Cell Proliferation (%)

Table 4. Inhibition Rate of Adriamycin on HepG2 Cell Proliferation (%)

3. 3. 2 Activity of regulating the growth of plant

The title compounds were investigated in regulating the growth of wheat (monocotyledon) and radish (dicotyledon)with reference of distilled water. After treating with culture solutions of 20, 50 and 100 ppm of the title compounds for 5 days, the growth regulating percentages have been calculated.The data of the test are presented in Table 5. The results indicated that the overwhelming majority of the title compounds had significant promoting effects on the growth of wheat and radish for both stalks and radicels. Especially, the promoting effects on the growth of radish were generally stronger than those on the growth of wheat. Furthermore, the promoting effects on the growth of the radicels of radish were much greater than those on the growth of stalks of radish,while the differences between those on the stalks and radicels of wheat were not obvious. Most of the title compounds showed promoting effects on the radicels of radish were more than 200%, of which 4a (20 ppm) actually reached 390.1%.The promoting effects of the title compounds on the stalks of radish were weaker than those on the radicels of radish, but the promoting rates were still around 100%. By comparison,the promoting growth effects on wheat were generally below 100% for both stalks and radicels. However, it was gratifying that 4h had the potent growth promoting effects on both wheat and radish for both stalks and radicels, and most of the promoting growth rates were above 100%. Only 4g had the least promoting effects on wheat growth. Nevertheless, a similar structural compound withp-tolyl at the 3-position,PTM, has been shown to promote the growth of radish but inhibit that of radish in a dose-dependent manner in our recent paper[13]. The above also indicated that the difference of substituents has great influence on the bioactivity of the compounds. Moreover, some 3,6-disubstituted-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines, without substituents at the 7-position, have been shown to be potent inhibition on the growth of wheat and radish basically[22-25]. Therefore, the structure and bioactivity relationship is worth studying further.

4 CONCLUSION

A series of novel 3-methyl-6-aryl-7-aroyl-6,7-dihydro-5H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines derived from 4-amino-5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-thione were designed, synthesized and characterized by spectroscopic methods. The crystal structure of compound 4dwas determined by single-crystal X-ray diffraction. All the title compounds exhibited a very weak antiproliferative activity against HepG2 cell linesin vitrounexpectedly,while showed potent plant growth-regulating activities on both wheat and radish.These results afford a new scaffold for the investigation of potent plant growth promoters potentially harmless to humans(further experiments are needed). The study on further structure modifications and structure-activity relationships will be carried out in the future.

Table 5. Effect of 4a-j on the Plant Growth-regulating of Wheat and Radish