Control of Bacterial Wilt by a Derivative of 3-Indolepropionic Acid
J. Agric. Food Chem., Vol. 46, No. 10, 1998 4417
and 5 mL of 5% aqueous NaOH solution, and the solution was
heated under reflux for 2 h. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in water.
After acidification with 2 N HCl solution, white precipitates
were yielded. Recrystallization from hexane containing a
small portion of ethyl acetate gave colorless fine needles (160
mg, 60.7%): mp 130-131 °C; 1H NMR δ (CD3OD) 1.16 (3H, d,
J ) 6.5), 2.81 (2H, d, J ) 9.5), 3.08-3.19 (1H, m), 6.95-7.55
(5H, m). Anal. Calcd for C12H13NO2: C, 70.92; H, 6.45; N, 6.89.
Found: C, 70.92; H, 6.47; N, 6.77.
4. An tiba cter ia l Activity. Antibacterial activity of the
compounds toward R. solanacearum was measured as de-
scribed (Matsuda et al., 1993). Test solution was prepared by
adding methanol stock of the compound in distilled water and
mixing with an equal volume of 2 × PCG medium (20 g of
casamino acid and 20 g of glucose in 1 L of water). Three
milliliters of the test solution was inoculated with 60 mL of
the bacterial suspension (5 × 105 cells/mL) and cultured with
shaking for 24 h at 30 °C. The minimum inhibitory concentra-
tion (MIC, µg/mL), required to suppress the bacterial numbers
to <5% of control, was determined for each compound.
5. Effects on Gr ow th of Tom a to Seed lin gs. Seeds of
tomato (Lycopersicon esculentum Mill cv. Poderosa) were
placed on moistened paper in a Petri dish and incubated at
26 °C for 2-3 days in the dark for germination and root
initiation. Germinated seeds with 1-2 mm roots were trans-
ferred to a filter paper supplied with different concentrations
of test compounds and subsequently incubated for a further 3
days. The auxinic effect of test compounds was examined by
measuring the length of radicles and hypocotyls. Alterna-
tively, the auxinic effect of the compounds was examined using
1-month-old tomato seedlings. That is, germinated seeds were
hydroponically cultured for 1 month in a growth chamber
controlled at 26 °C and then transferred to an aqueous solution
containing the test compounds. After 10 days of incubation,
the appearance of stem winding was recorded and scored from
1 to 3 for slight, moderate, and great windings, respectively.
6. Con tr ol of th e Ba cter ia l Gr ow th by 3-IBA in a
Hyd r op on ic Cu ltu r e of Tom a to P la n ts. The antibacterial
action of 3-IBA on the pathogen was examined in a hydroponic
culture of the tomato plants. The pathogen was first inocu-
lated at a concentration of ∼1 × 108 cells/mL into a hydroponic
culture medium containing 4.0 mM KNO3, 1.5 mM Ca(NO3)2,
1.0 mM MgSO4, 0.66 mM NH4H2PO4, 0.057 mM FeEDTA,
0.048 mM H3BO3, and 0.009 mM MnSO4 on day 0, followed
by the addition of 3-IBA on the next day (day 1). Certain
numbers of the tomato plants grown for 1 month were
transferred to the culture on day 2, and numbers of the wilted
plants were counted every day for 6 days (n ) 3).
F igu r e 1. Indolepropanoids and related compounds tested for
their antibacterial activity against R. solanacearum.
(Campaigne and Knapp, 1970), 3-indazolepropionic acid, and
2-methyl-(3-indolyl)propionic acid were synthesized from the
formyl derivatives of benzo[b]thiophene, indazole, and indole,
respectively. IPA derivatives having an alkyl branch on the
â-carbon were synthesized according to the literature (Oikawa
et al., 1978; Kato et al., 1993). Of these compounds, 3-inda-
zolepropionic acid and 2-methyl-(3-indolyl)propionic acid have
not yet been described; thus, their syntheses are shown below.
IPA derivatives having the substituent in the propionic acid
moiety have an asymmetric carbon. We separated optical
isomers of 3-IBA according to the method of Kato et al. (1993)
using lipase AK for selectively hydrolyzing the ethyl ester of
the S isomer, followed by porcine liver esterase. Optical
purities of the isomers were confirmed to be >98% ee by HPLC
using a Chiralcel OD column (4.6 × 250 mm, Daicel Chemical
Industries, Tokyo, J apan) with a mixture of hexane, 2-pro-
panol, and trifluoroacetic acid (9:1:0.5) at a flow rate of 1 mL/
min.
3.1. 3-Indazolepropionic Acid. 3-(3-Indazolyl)acrylic acid
(100 mg, 531 nmol; lit. Dikopolova and Suvorov, 1979) in 5
mL of methanol was hydrogenized by stirring with 10 mg of
5% palladium carbon under 1 atm of hydrogen at room
temperature for 5 h. The catalyst was removed by filtration,
and the filtrate was concentrated in vacuo. Recrysallization
from hexane containing a small portion of ethyl acetate
afforded colorless fine needles (67.7 mg, 67.0%): mp 170-171
1
°C; H NMR δ (CD3OD) 2.79 (2H, t, J ) 7.5), 3.25 (2H, t, J )
7.5), 7.08-7.77 (4H, m). Anal. Calcd for C10H10N2O2: C, 63.15;
H, 5.30; N, 14.73. Found: C, 62.95; H, 5.30; N, 14.61.
3.2. 2-Methyl-3-(3-indolyl)propionic Acid (2-MIPA). 3.2.1.
Ethyl 2-Methyl-3-(3-indolyl)acrylate. To a solution of indole-
3-carbaldehyde (300 mg, 2.07 mmol) in 20 mL of anhydrous
benzene was added 1.34 g (3.70 mmol) of phosphorane
prepared from triphenylphosphine and ethyl 2-bromopropi-
onate, and the solution was heated under reflux for 4 h. The
reaction mixture was concentrated in vacuo, and the residue
was purified by silica gel column chromatography with hex-
ane/ethyl acetate (2:1); subsequent recrystallization from
hexane containing a small amount of ethyl acetate afforded
colorless plates: yield 450 mg (95%); mp 135-136 °C; HRMS
RESULTS AND DISCUSSION
1. R ole of In d ole R in g in t h e An t ib a ct er ia l
Activity. Indazole has been shown to mimic indole for
the compounds exhibiting 5-HT3 antagonist activity
(Bermudez et al., 1990), probably due to resemblance
of the physicochemical properties with those of indole
moiety (Palmer and Kennedy, 1994; Palmer et al., 1975).
However, in the antibacterial activity, replacement of
the indole moiety of IPA for indazole (compound III)
abolished the antibacterial activity, as with benzo[b]-
thiophene (compound II) (data not shown). Similar
results were obtained with aromatic moieties of naph-
thalene or quinoline, regardless of whether the propionic
acid moiety extends from position 3 or 4 of these
aromatic rings (data not shown), suggesting an impor-
tant role of the five-membered ring in the antibacterial
action.
1
229.1095, calcd for C14H15NO2 229.1102; H NMR δ (CDCl3)
1.38 (3H, t, J ) 7.0), 2.19 (3H, s), 4.30 (2H, q, J ) 7.0), 7.18-
7.84 (5H, m), 8.06 (1H, s), 8.64 (1H, s).
3.2.2. Ethyl 2-Methyl-3-(3-indolyl)propionate. To a solution
of the acrylate (300 mg, 1.31 mmol) in 10 mL of methanol was
added 220 mg of 5% palladium-coated carbon, and the mixture
was stirred under hydrogen (1 atm) at room temperature for
1 h. The reaction mixture was filtered, and the filtrate was
concentrated in vacuo. The residue was purified by a silica
gel column eluting with hexane/ethyl acetate (4:1) to yield
290 mg of colorless oil: yield 95.8%; HRMS 231.1257, calcd
1
for C14H17NO2 231.1259; H NMR δ (CDCl3) 1.19 (3H, t, J )
2. Su b st it u en t E ffect s on t h e P r op ion ic Acid
Moiety. Even though it was previously found that
neither the carboxylic acid function nor the length of
the side-chain moiety could be modified to obtain greater
7.0), 1.21 (3H, s), 2.83 (2H, d, J ) 9.5), 3.18 (1H, m), 4.09 (2H,
q, J ) 7.0), 6.99-7.63 (5H, m), 8.00 (1H, s).
3.2.3. 2-MIPA. Ethyl 2-methyl-3-(3-indolyl)propionate (300
mg, 1.30 mmol) was dissolved in a mixture of 5 mL of ethanol