Novel, unnatural benzo-1,2,3-thiadiazole-7-carboxylate elicitors of taxoid biosynthesis

Article abstract of DOI:10.1021/jf0618574

In order to establish the chemical biological technology for production of valuable secondary metabolites, a novel family of unnatural elicitors derived from the plant activator benzo-1,2,3-thiadiazole-7-carboxylic acid were designed and synthesized. New synthetic elicitors that showed powerful eliciting activities upon taxoid biosynthesis by Taxus chinensis suspension cells were obtained. For example, benzo-1,2,3-thiadiazole-7-carboxylic acid 2-(2-hydroxybenzoxyl)ethyl ester was more effective and resulted in nearly 40% increase in taxuyunnanine C content and production in comparison with methyl jasmonate, which was previously reported as the most powerful chemical elicitor for taxoid biosynthesis. The novel class of elicitors was found to induce plant defense responses, including promotion of H₂O₂ levels originating from oxidative burst and activation of phenylalanine ammonia lyase. Interestingly the plant defense responses induced corresponded well to the superior stimulating activity in T. chinensis cell cultures. The work indicates that the newly synthesized benzothiadiazoles can act as a new family of elicitors for taxoid biosynthesis in plant cells.

Full text of DOI:10.1021/jf0618574

J. Agric. Food Chem. 2006, 54, 8793−8798 8793
Novel, Unnatural Benzo-1,2,3-thiadiazole-7-carboxylate Elicitors
of Taxoid Biosynthesis
YUFANG XU,† ZHENGJIANG ZHAO,† XUHONG QIAN,*,† ZHIGANG QIAN,‡
WENHONG TIAN,† AND JIANJIANG ZHONG*,‡
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, and State Key Laboratory of
Bioreactor Engineering, East China University of Science and Technology, P.O. Box 544,
130 Meilong Road, Shanghai 200237, China
In order to establish the chemical biological technology for production of valuable secondary
metabolites, a novel family of unnatural elicitors derived from the plant activator benzo-1,2,3-
thiadiazole-7-carboxylic acid were designed and synthesized. New synthetic elicitors that showed
powerful eliciting activities upon taxoid biosynthesis by Taxus chinensis suspension cells were
obtained. For example, benzo-1,2,3-thiadiazole-7-carboxylic acid 2-(2-hydroxybenzoxyl)ethyl ester
was more effective and resulted in nearly 40% increase in taxuyunnanine C content and production
in comparison with methyl jasmonate, which was previously reported as the most powerful chemical
elicitor for taxoid biosynthesis. The novel class of elicitors was found to induce plant defense
responses, including promotion of H2O2 levels originating from oxidative burst and activation of
phenylalanine ammonia lyase. Interestingly the plant defense responses induced corresponded well
to the superior stimulating activity in T. chinensis cell cultures. The work indicates that the newly
synthesized benzothiadiazoles can act as a new family of elicitors for taxoid biosynthesis in plant
cells.
KEYWORDS: Benzo-1,2,3-thiadiazole-7-carboxylate derivatives; chemically synthesized elicitors; plant
defense; plant cell culture; taxoid induction; secondary metabolites
INTRODUCTION
Plant cell culture is a promising alternative for mass produc-
tion of valuable plant products. Among the manipulative
techniques available to promote the productivity of useful
secondary metabolites from plant cell cultures, the use of
elicitors has been one of the best approaches for dramatically
increasing product yields (1-3). Jasmonic acid and its methyl
ester (methyl jasmonate) (Figure 1) are important members of
the family of jasmonates that are linolenic acid-derived cyclo-
pentanone-based compounds of wide distribution in higher
plants. Exogenously applied methyl jasmonate was shown to
result in enhanced production of secondary metabolites by a
variety of plant species, and it was especially demonstrated as
the most effective abiotic elicitors in Taxus cell cultures to
produce the effective anticancer drug taxol and the neuron
growth factor (NGF) active material taxuyunnanine C (4-8).
Our recent work showed that new methyl jasmonate derivatives
were more efficient than methyl jasmonate (9-13). However,
it is very difficult to isolate, purify, and synthesize methyl
jasmonate because of the two chiral carbons in the methyl
jasmonate molecule (14, 15). Therefore, the design and synthesis
Figure 1. Three inducers of plant SAR (systemic acquired resistance).
of novel unnatural elicitors are a great challenge to chemists.
As a long-term interest, it is very important to establish the
chemical biological technology for valuable secondary metabo-
lites through the design and application of elicitors with new
backbone structure.
Salicylic acid (Figure 1) is also an important signal molecule
to cause systemic acquired resistance (SAR) in plants to
pathogens and pests (16). Salicylic acid and its derivatives could
also elicit secondary metabolism in Taxus cell cultures as with
methyl jasmonate (17, 18). It was noticed that S-methyl benzo-
1,2,3-thiadiazole-7-carboxylate, the first commercial SAR ac-
tivator for plants, could induce the same set of defense responses
as salicylic acid in signal transduction (19). Therefore, it was
interesting to prove whether or not the S-methyl benzo-1,2,3-
thiadiazole-7-carboxylate derivatives could also induce second-
ary metabolism in plant cell cultures besides their effect on crop
protection as reported.
* Corresponding author: Tel: +86 21 64253589. Fax: +86 21
64252603. E-mail: xhqian@ecust.edu.cn.
† Shanghai Key Lab. of Chemical Biology.
‡ State Key Laboratory of Bioreactor Engineering.
10.1021/jf0618574 CCC: $33.50
© 2006 American Chemical Society
Published on Web 10/17/2006

8794 J. Agric. Food Chem., Vol. 54, No. 23, 2006
Xu et al.
Systemic acquired resistance occurs during stress environment
stimuli, including addition of elicitors, and leads to the increased
amount of secondary metabolites such as phytoalexins (20). It
is reasonable to assume that identical characteristics for defense
responses exist in cells in vitro as in plants. Enhanced levels of
H2O2 production and phenylalanine ammonia lyase (PAL)
activity are two typical events related to defense responses. The
production of H2O2 originating from oxidative burst is consid-
ered to be one important event in plant cell defense response
(21). Our previous work proved that eliciting efficiency of
methyl jasmonate derivatives in Taxus chinensis cell cultures
corresponded well to the H2O2 level (22). As a key enzyme
required for biosynthesis of phenolic defense compounds, PAL
was reported to be a plant cell defense response marker caused
by specific external stimuli, including additions of elicitors (23,
24). S-Methyl benzo-1,2,3-thiadiazole-7-carboxylate as a SAR
activator enhanced the elicitation of PAL mRNA and the
induction of coumarin secretion in parsley cells (25). However,
few reports have been published about S-methyl benzo-1,2,3-
thiadiazole-7-carboxylate as elicitor in the production of second-
ary metabolites. When S-methyl benzo-1,2,3-thiadiazole-7-
carboxylate was added to medium on which Amni majus cultures
were planted, the growth rates of the elicited and the nonelicited
tissues were not significantly different, suggesting that S-methyl
benzo-1,2,3-thiadiazole-7-carboxylate was an activator of SAR
but not an elicitor (26). However, a mixture of S-methyl benzo-
1,2,3-thiadiazole-7-carboxylate and yeast enhanced rosmarinic
acid production in a suspension culture of Agastache rugosa
(27).
In this study of the important anticancer drug taxol and other
taxoids, a T. chinensis cell system was selected to evaluate the
eliciting efficiency of new synthetic S-methyl benzo-1,2,3-
thiadiazole-7-carboxylate derivatives and the relationship be-
tween the defense response and metabolite production. A series
of novel benzo-1,2,3-thiadiazole-7-carboxylate derivatives was
designed and synthesized, and their eliciting activities in
suspension cultures of T. chinensis were evaluated. In addition,
two early and important events in plant defense responses,
oxidative burst and activation of PAL, were also evaluated to
investigate the relationship between the defense response and
the metabolite production.
Figure 2. Preparation of S-methyl benzo-1,2,3-thiadiazole-7-carboxylate
derivatives.
The benzo-1,2,3-thiadiazole-7-carboxylic acid chloride was dissolved
in methylene chloride (20 mL) and cooled by ice-water bath, 15%
methyl methyl mercaptan sodium solution (3 mL) was added dropwise
with stirring during 0.5 h, and the mixture stirred at room temperature
for another 5 h. The mixture was washed with water three times and
dried with anhydrous MgSO4 overnight. The filtrate was concentrated
by vacuum distillation and the brown oil obtained was purified by silica
gel column chromatography and eluted with ethyl acetate/petroleum
(1:4). Light yellow crystals (0.12 g, 34% yield) were obtained. Mp:
134-135 °C. 1H NMR (500 MHz, DMSO-d6): δ 2.59 (s, 3H, SCH3),
7.98 (dd, 1H, J ) 7.26 and 7.89 Hz, 5-Ar-H), 8.60 (d, 1H, J ) 7.26
Hz, 4-Ar-H), 9.08 (d, 1H, J ) 7.88 Hz, 6-Ar-H). IR (KBr): ν 3000,
1625, 1524, 1470, 1398, 1087, 1071, 908, 780, 730 cm-1
.
Synthesis of 2,2,2-Trifluoroethyl Benzo-1,2,3-thiadiazole-7-car-
boxylate (3d) (General Procedures for 3e and 3f) (28). Benzo-1,2,3-
thiadiazole-7-carboxylic acid chloride prepared as above was dissolved
in dry toluene (4 mL) and then dropped into to a mixture of
trifluoroethanol (2 mL), toluene (4 mL), and triethylamine (0.8 mL)
with stirring at room temperature. After 4 h, the mixture was poured
into water (20 mL), extracted with ethyl acetate, washed with water,
and dried with anhydrous MgSO4 overnight. The filtrate obtained was
concentrated by vacuum distillation and the brown solid obtained was
purified by silica gel column chromatography and eluted with benzene.
Light yellow crystals (0.24 g, 54%) were obtained. Mp: 119-121 °C.
1H NMR (500 MHz, DMSO-d6): δ 5.16 (q, 2H, J ) 9.0 Hz, CH2CF3),
7.98 (dd, 1H, J ) 8.35 and 7.36 Hz, 5-Ar-H), 8.49 (d, 1H, J ) 7.37
Hz, 4-Ar-H), 9.10 (d, 1H, J ) 8.35 Hz, 6-Ar-H). IR (KBr): ν 3074,
MATERIALS AND METHODS
Triethylamine was dried over KOH and distilled. Methyl jasmonate
was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan).
Trifluoroethanol, pentafluoropropanol, and pentafluorobenzyl alcohol
were purchased from Fluka Chem. Co. All other solvents and chemicals
were reagent grade and used without further purification. Melting Points
were recorded by an electrothermal digital apparatus and are uncor-
1
rected. The H NMR spectra were recorded with a Brucker AM-500
2978, 1718, 1558, 1410, 1305, 1180, 1135, 1050, 980, 820, 753 cm-1
.
spectrometer. The MS spectra were measured with a HRMS Micromass
GCT CA 055 spectrometer. The elemental analysis data was measured
by an Elementar vario EL III analyzer.
HRMS (EI): m/z: 262.0026 [M+], C9H5F3N2O2S requires 262.0024.
3e: 1H NMR (500 MHz, DMSO-d6): δ 5.25 (t, 2H, J ) 9.0 Hz,
CH2CF2CF3), 8.00 (dd, 1H, J ) 8.13 Hz and 7.32 Hz, 5-Ar-H), 8.45
(d, 1H, J ) 7.32 Hz, 4-Ar-H), 9.11 (d, 1H, J ) 8.12 Hz, 6-Ar-H). IR
(KBr): ν 3081, 2963, 1710, 1558, 1454, 1298, 1190, 1130, 1070, 870,
830, 764 cm-1. HRMS (EI): m/z 311.9613 [M+], C10H5F5N2O2S
requires 311.9963.
Syntheses of New Elicitors. The compound 3c (Figure 2) was
prepared from benzo-1,2,3-thiadiazole-7-carboxylic acid chloride 2 and
CH3SNa solution instead of CH3SH as in the literature (28-30). 3d-f
were obtained by the acylation of fluorine-containing alcohols and 2.
5a-d were prepared from benzo-1,2,3-thiadiazole-7-carboxylic acid
chloride 2 and corresponding monoesterification of aromatic acids and
diols (4) (31). Specific detailed data for each of the new compounds
are given below.
Synthesis of S-Methyl Benzo-1,2,3-thiadiazole-7-carboxylate (3c)
(28). A mixture of 0.3 g (1.7mmol) of benzo-1,2,3-thiadiazole-7-
carboxylic acid and thionyl chloride (5 mL) was heated and maintained
at refluxing temperature of 90 °C for 8 h. The resulting oil was solidified
when the excess thionyl chloride was removed by vacuum distillation.
3f: 1H NMR (500 MHz, DMSO-d6): δ 5.62 (s, 2H, CH2), 7.93 (dd,
1H, J ) 7.35 and 8.11 Hz, 5-Ar-H), 8.40 (d, 1H, J ) 7.35 Hz, 4-Ar-
H), 9.04 (d, 1H, J ) 8.10 Hz, 6-Ar-H). IR (KBr): ν 3059, 2978, 1707,
1506, 1410, 1291, 1135, 1040, 935, 870, 770, 615 cm-1. HRMS (EI):
m/z 359.9983 [M+], C14H5F5N2O2S requires 359.9992.
Synthesis of Benzo-1,2,3-thiadiazole-7-carboxylic Acid 2-Benzoyl-
oxyethyl Ester (5a) (General Procedures for 5b, 5c and 5d) (28).
Sulfuric acid (98%, 0.5 mL) was added to a mixture of benzoic acid

Novel, Unnatural Elicitors of Taxoid Biosynthesis
J. Agric. Food Chem., Vol. 54, No. 23, 2006 8795
(2.4 g, 0.02 mol) and ethylene glycol (6 mL). The mixture was heated
to 120 °C with stirring for 2.5 h. After cooling, water (40 mL) was
added and the mixture was neutralized with NaHCO3. The separated
oil was washed with NaHCO3 solution and water and then dissolved
in ethyl acetate and dried with MgSO4. The 2-hydroxyethyl benzoate
was obtained as a colorless oil after concentration (87%) (31).
Benzo-1,2,3-thiadiazole-7-carboxylic acid chloride was dissolved in
dry toluene (4 mL) and then dropped into a mixture of 2-hydroxyethyl
benzoate (0.3 g, 1.7 mmol), toluene (6 mL), and triethylamine (0.36
mL) with stirring at room temperature. After 16 h of stirring, the mixture
was poured into water (20 mL), extracted with ethyl acetate, washed
with water, and dried with anhydrous MgSO4 overnight. The filtrate
was concentrated by vacuum distillation and the brown solid obtained
was purified by silica gel column chromatography, eluted with ethyl
acetate/hexane (2:1) as white crystals (0.24 g, 55%). Mp: 79-82 °C.
1H NMR (500 MHz, DMSO-d6): δ 4.67-4.78 (m, 4H, OCH2CH2O),
7.48 (dd, 2H, J ) 7.77 Hz and 7.38 Hz, 3′,5′-Ar-H), 7.63 (t, 1H, J )
7.39 Hz, 4′-Ar-H), 7.92 (dd, 1H, J ) 7.35 and 7.87 Hz, 5-Ar-H), 7.95
(d, 2H, J ) 7.78 Hz, 2′, 6′-Ar-H), 8.42 (d, 1H, J ) 7.30 Hz, 4-Ar-H),
9.01 (d, 1H, J ) 7.99 Hz, 6-Ar-H). IR (KBr): ν 3070, 2945, 1720,
1570, 1460, 1270, 1120, 1070, 860, 760, 720 cm-1. HRMS (EI): m/z
328.0503 [M+], C16H12N2O4S requires 328.0518. Elemental analysis
(%) found: C 58.66, H 3.54, N 8.49. Requires: C 58.53, H 3.68, N
8.53.
were evaporated to dryness at 25 °C. The residue was dissolved in 2
mL of dichloromethane and 2 mL of distilled water. After sufficient
mixing, the mixture was centrifuged at 4000g for 10 min. The organic
phase was collected and evaporated to dryness at 25 °C. The residue
was dissolved in 1 mL of methanol and filtered through a 0.22 µm
PVDF syringe filter (Millipore); 20 µL was analyzed by reverse phase
HPLC, using a Hewlett-Packard series 1100 HPLC system (Agilent,
Palo Alto, CA). A 250 × 4.6 mm i.d. 5 µm Zorbax Phenyl column
(Agilent) with a Zorbax Phenyl guard column was used at 25 °C. The
mobile phase consisted of acetonitrile and water (58:42, v/v), and the
flow rate was 1 mL/min. Taxane was monitored at the wavelength of
227 nm by using authentic standards as the reference.
Assay of Oxidative Burst. H2O2, associated with the so-called
oxidative burst, originates from superoxide generated by a plasma
membrane-associated NADH oxidase in challenged plant cells. H2O2
produced by the cells and released into the medium was determined
by the scopoletin fluorescence oxidative quenching method (excitation
wavelength, 350 nm; emission, 460 nm) according to the literature (32).
To measure H2O2 accumulation, samples were taken at various intervals
over the 180-min period following elicitation. Aliquots of 4 mL of
extracellular medium were mixed with 40 µL of 5 mM stock solution
of scopoletin in DMSO and 40 µL of 1 mg/mL stock solution of
peroxidase (Sino-American Biotechnology Co., Shanghai), respectively.
The concentration of H2O2 in the medium was calculated from the
fluorescence decrease using a calibration curve established in the
presence of H2O2. A standard curve by adding scopoletin to the solutions
at different H2O2 concentrations was prepared by using cell-free
medium.
5b: 1H NMR (500 MHz, DMSO-d6): δ 4.75-4.80 (m, 4H, OCH2-
CH2O), 7.96 (dd, 1H, J ) 7.31 and 8.37 Hz, 5-Ar-H), 8.42 (d, 1H, J
) 7.30 Hz, 4-Ar-H), 9.0 (d, 1H, J ) 8.37 Hz, 6-Ar-H). HRMS (EI):
m/z 418.0373 [M+], C16H7F5N2O4S requires 418.0358.
5c: 1H NMR (500 MHz, DMSO-d6): δ 4.69-4.77 (m, 4H, OCH2-
CH2O), 7.40-7.44 (m, 1H, Ar-H), 7.53-7.55 (m, 2H, Ar-H), 7.82 (d,
1H, J ) 8.53 Hz, Ar-H), 7.94 (dd, 1H, J ) 7.29 and 7.88 Hz, 5-Ar-H),
8.43 (d, 1H, J ) 7.28 Hz, 4-Ar-H), 9.03 (d, 1H, J ) 7.90 Hz, 6-Ar-H).
IR (KBr): ν 3067, 2956, 1703, 1558, 1436, 1284, 1135, 1060, 860,
749, 695 cm-1. HRMS (EI): m/z 362.0092 [M+], C16H11ClN2O4S
requires 362.0128. Elemental analysis (%) found: C 53.20, H 3.06, N
7.59. Requires: C 52.97, H 3.06, N 7.72.
5d: 1H NMR (500 MHz, DMSO-d6): δ 4.71-4.80 (m, 4H, OCH2-
CH2O), 6.88-6.91 (m, 1H, Ar-H), 6.95-6.97 (m, 1H, Ar-H), 7.48-
7.51 (m, 1H, Ar-H), 7.77-7.80 (m, 1H, Ar-H), 7.94 (dd, 1H, J ) 7.34
and 8.34 Hz, 5-Ar-H), 8.45 (d, 1H, J ) 7.33 Hz, 4-Ar-H), 9.04 (d, 1H,
J ) 8.34 Hz, 6-Ar-H), 10.38 (s, 1H, OH). IR (KBr): ν 3280 (OH),
3059, 2963, 1722, 1666, 1606, 1580, 1480, 1400, 1290, 1250, 1161,
1080, 860, 756, 700 cm-1. HRMS (EI): m/z 344.0451 [M+], C17H12-
Cl2N2O5S requires 344.0467. Elemental analysis (%) found: C 55.88,
H 3.53, N 7.99. Requires: C 55.81, H 3.51, N 8.14.
Cell Subculture Conditions. The T. chinensis cell culture was
maintained on Murashige and Skoog medium supplemented with 0.5
mg/L of 6-benzyladenine, 0.2 mg/L of 2,4-dichlorophenoxy acetic acid,
0.5 mg of naphthalene acetic acid, 100 mg/L of ascorbic acid, and 30
g/L of sucrose. The pH was adjusted to 5.8 before autoclaving. The
cells were subcultured at an interval of 2 weeks in a 500-mL Erlenmeyer
flask containing 200 mL of medium on a rotary shaker at 110 rpm and
25 °C in the dark.
Elicitation Study. For elicitation experiments, ca. 5 g of fresh cell
aggregates were incubated into a 250-mL Erlenmeyer flask containing
50 mL of medium with the same culture conditions as in subcultures.
All elicitors were added to the cultures in 1 µL of ethanol per 1 mL of
culture medium, sterilized by filtering through 0.22 µm polyvinylidene-
difluoride (PVDF) syringe filters (Millipore) and finally added to the
culture medium at day 7 of the subculturing period. The experiments
were performed in triplicate.
Effects of various jasmonate elicitors on peroxidase-dependent assay
for H2O2 determination were tested. Various jasmonates were added
to cell-free medium to obtain final concentrations of 10, 50, or 100
µM, respectively. In the assay conditions, an addition of jasmonate
elicitors had no obvious effect on the decrease of scopoletin fluorescence
because of H2O2 addition.
Enzyme Extraction and PAL Activity Analysis. Cells were
harvested as described above, and then samples of 1 g of fresh cells
were frozen in liquid nitrogen. After grinding the cells with a mortar
and pestle, crude enzymes in the frozen powder were extracted by
adding 50 mg of polyvinypyrrolidone and 2 mL of prechilled buffer
of pH 7.2 (0.1 M phosphate buffer, 2 mM ethylenediaminetetraacetic
acid, 4 mM dithiothreitol), and then the mixture was homogenized at
4 °C. The mixture was centrifuged at 10 000 g for 30 min at 4 °C. The
supernatant was used directly for PAL assay using a method slightly
modified from a previous report (33): 200 µL of the protein extracts
was incubated with 120 µL of 0.1 M L-phenylalanine dissolved in 280
µL of 0.1 M borate buffer of pH 8.8 at 30 °C for 60 min. The reaction
was stopped by adding 50 µL of 5 N trichloroacetic acid. After
centrifugation at 10 000g for 30 min, the supernatant was analyzed by
HPLC under the following conditions: solvent, water:methanol:acetic
acid (40:60:1, v/v/ v); detection, 280 nm; flow, 1 mL/min; column,
250 × 4.6 mm i.d., 5 µm Zorbax ODS (Agilent); injection volume, 20
µL. Genuine trans-cinnamic acid (Sigma) was used as an external
standard. One unit (U) of enzyme activity is defined as the amount of
enzyme forming 1 pmol of trans-cinnamic acid from the substrate
L-phenylalanine per min.
RESULTS AND DISCUSSION
Preparation of S-Methyl Benzo-1,2,3-thiadiazole-7-car-
boxylate and Its Derivatives (Figure 2). S-Methyl benzo-1,2,3-
thiadiazole-7-carboxylate derivatives (1, 3a, and 3b) were
synthesized according to the literature (28). Target compounds
3c-f and 5a-d were prepared as in Figure 2. Compounds 3d-
5d have not been previously reported. All compounds were
separated and purified by recrystallization or silica gel chro-
matography. Their structures were identified by 1H NMR,
HRMS, and elemental analyses.
Measurement of Cell Mass. The samples from flasks were filtered
under vacuum and washed with several volumes of distilled water to
remove residual medium. The cells were weighed to obtain the fresh
weight, and 5 g was dried at 50 °C to a constant weight for the
measurement of cell dry weight (DW).
Taxane Extraction and Analysis. For taxane extraction, 100 mg
of powdered dry cells was soaked in 2 mL of methanol for 2 days and
then the mixture was ultrasonicated twice for 40 min. After centrifuga-
tion at 4000g for 10 min, the extract was removed and the cell debris
was extracted once more with 2 mL of methanol. The combined extracts
Compound 3c was prepared by acylation of benzothiadiazole-
7-carboxylic acid chloride and CH3SNa solution instead of
CH3SH (29, 30), which avoids the odor and volatility of CH3SH.

8796 J. Agric. Food Chem., Vol. 54, No. 23, 2006
Xu et al.
Table 1. Effects on the Maximum Cell Concentration of Compound 3a
on the Taxuyunnanine C Content and Production in Cell Cultures of T.
chinensis
taxuyunnanine C (21 d)a
cell concn
concn of
3a ( M)
(g DW/L)
(14 d)
content
(mg/g DW)
production
(mg/L)
µ
0
1
10
100
200
15.7
15.5
16.1
10.9
8.5
±
±
±
±
±
0.5
0.6
0.6
0.6
0.3
15.3
16.2
18.5
18.7
19.4
±
±
±
±
±
0.1
0.3
1.1
0.5
0.1
160
226
240
255
151
±
±
±
±
±
3.8
7
15
8
14
a Data are the means with standard deviations of three flasks.
Figure 3. Eliciting activity of S-methyl benzo-1,2,3-thiadiazole-7-carboxylate
derivatives on taxuyunnanine C content (control, methyl jasmonate, 3b,
3d, and 5d).
Table 2. Compounds Prepared and Their Eliciting Activity Comparison
with Controls, Methyl Jasmonate, and Salicylic Acid in Taxus chinensis
Cell Suspension Culture
taxuyunnanine C
As shown in Figure 3, the S-methyl benzo-1,2,3-thiadiazole-
7-carboxylate derivatives showed different dynamic profiles of
taxuyunnanine C content from methyl jasmonate. Generally, an
increase in taxuyunnanine C content was observed after the
addition of methyl jasmonate at 7 day, but it did not appear in
the case of S-methyl benzo-1,2,3-thiadiazole-7-carboxylate
derivatives. After 15 days, there was an obvious increase in
taxuyunnanine C content for S-methyl benzo-1,2,3-thiadiazole-
7-carboxylate derivatives. During the cultivation, at 15 days the
taxuyunnanine C content for 5d was 16.2 mg/g dry weight
(DW), and 6 days later, their taxuyunnanine C content increased
rapidly to 39.4 mg/g DW.
By comparison of 3d with 3b, it was found that the presence
of a trifluoromethyl group (3d) produced nearly a 40% increase
in taxuyunnanine C content, but the eliciting activities of 3e
and 3f, which have a pentafluoroethyl or pentafluorophenyl
group, were lower than that of 3b. Fluorine-containing com-
pounds usually have higher lipophilicity, and it seemed that
too high a lipophilicity (A log P98 of 3d, 3e and 3f is esti-
mated to be 2.82, 3.31, and 4.42, respectively), might be not
beneficial to their interaction with their receptors in plant cell
membranes.
Introduction of aromatic acids to benzo-1,2,3-thiadiazole-7-
carboxylates gave better results. Activities of 5a, 5c and 5d
seemed to relate to 2-position substituents on the benzoic acid
moiety, and the introduction of 2-Cl and 2-OH contributed
remarkably to the eliciting activity. The compound 5d is the
only one of the benzo-1,2,3-thiadiazole-7-carboxylates whose
eliciting activity surpassed that of methyl jasmonate, which has
been considered to be one of the most effective abiotic elicitors
in T. chinensis cell cultures. Its bioactivity was also more than
that of two parts of benzo-1,2,3-thiadiazole-7-carboxylic acid
and salicylic acid combined.
To find some biological evidence for our results, 3c and 3d
were selected to demonstrate typical examples of the investiga-
tion of the elicitor-induced plant defense responses, while the
cultures with nonelicitor addition was used as controls.
Figure 4 shows that increased levels of H2O2 production
followed by increased PAL activity and taxoid overproduction
was observed in T. chinensis suspension cultures elicited with
S-methyl benzo-1,2,3-thiadiazole-7-carboxylate derivatives. There
is an increasing body of evidence that H2O2 can act as a
diffusible signal to activate defense genes and the biosynthesis
of plant secondary metabolites (37-39). The increased PAL
activity provided additional evidence for occurrence of the
oxidative burst in elicited cell cultures. In addition, the time
courses of H2O2 production and PAL activity of cells elicited
cell growth
(elicitor/
control)
content
(elicitor/
control)
production
(elicitor/
control)
activity
score
compd
R
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
MJA
SA
OH
OCH3
SCH3
0.81
0.86
0.85
0.96
0.83
1.01
1.07
0.95
1.11
1.04
0.95
0.98
1.40
1.48
1.44
2.07
1.39
1.28
1.26
1.38
1.96
3.37
2.44
1.48
1.52
1.72
1.62
1.87
1.35
1.25
1.13
1.34
1.90
3.36
2.44
1.68
1.00
1.13
1.07
1.23
0.89
0.82
0.74
0.88
1.25
2.21
1.60
1.10
OCH2CF3
OCH2CF2CF3
OCH2C6F5
C6H5
C6F5
C6H4Cl(2-)
C6H4OH(2-)
−
−
However, esterification of benzothiadiazole-7-carboxylic acid
and trifluoroethanol did not work in the preparation of 3d under
some conditions, because of the acidity of trifluoroethanol, so
condensation of benzothiadiazole-7-carboxylic acid chloride and
trifluoroethanol in the presence of triethylamine was adopted.
In the process of preparing compound 5d, the protection of
hydroxyl group with acetic anhydride was not required. Because
of steric factors and intramolecular H-bond (34), the free
phenolic hydroxyl group would not participate in the reaction
as long as no excess acid chloride was added.
Biological Activity of S-Methyl Benzo-1,2,3-thiadiazole-
7-carboxylate and Its Derivatives. For the evaluation of the
newly synthesized derivatives of S-methyl benzo-1,2,3-thiadia-
zole-7-carboxylate, a suspension culture of T. chinensis cell was
used. Under the same experiment conditions, no elicitor was
introduced in the culture system as control. To compare the
elicitation efficiency of S-methyl benzo-1,2,3-thiadiazole-7-
carboxylate derivatives, methyl jasmonate, which was the best
chemical elicitor reported earlier, was also used (35, 36). The
optimal concentration and culture time experiments of com-
pound 3a as example are shown in Table 1. The optimal
concentration was 100 µm and the maximal taxuyunnanine C
content was reached in 21 days. The taxuyunnanine C content
and production of all the new elicitors are listed in Table 2.
Compounds 3a-c are known or commercial SAR activators
for plants. It was shown in our experiments that although
S-methyl benzo-1,2,3-thiadiazole-7-carboxylate (3c) was the
most effective in causing SAR in plants (30), it was not the
most effective elicitor in the cell culture among its derivatives.
Its eliciting activity was a little lower than that of methyl benzo-
1,2,3-thiadiazole-7-carboxylate (3b).

Novel, Unnatural Elicitors of Taxoid Biosynthesis
J. Agric. Food Chem., Vol. 54, No. 23, 2006 8797
provide another method to evaluate efficiency of new elicitors
in plant cell cultures.
Compounds 3d, 5c, and 5d had excellent eliciting activities.
By comparison with methyl jasmonate, 5d was more effective
and resulted in 38% increase in taxuyunnanine C content and
production. It is much easier to synthesize these compounds
than to synthesize methyl jasmonate derivatives (40-45). To
the best of our knowledge, this is the first report that new
S-methyl benzo-1,2,3-thiadiazole-7-carboxylate derivatives ef-
fectively enhanced both taxuyunnanine C content and produc-
tion. In addition, S-methyl benzo-1,2,3-thiadiazole-7-carboxylate
derivatives were found to induce plant defense responses as
effectively as methyl jasmonate, including oxidative burst and
activation of PAL. The work indicates that the newly synthesized
S-methyl benzo-1,2,3-thiadiazole-7-carboxylate derivatives may
act as powerful inducing signals for secondary metabolite
biosynthesis in plant cell cultures.
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Received for review July 3, 2006. Revised manuscript received
September 11, 2006. Accepted September 12, 2006. This work was done
under the auspices of The National Basic Research Program of China
(2003CB114400), National Natural Science Foundation of China, the
National Key Technologies R&D Program (grant 2005BA711A04), and
The Science and Technology Foundation of Shanghai.
JF0618574