Synthesis and biological activities of 4-trifluoromethylindole-3-acetic acid: A new fluorinated indole auxin

Article abstract of DOI:10.1271/bbb.80138

In our studies on the development of new promoters for the root formation of tree cuttings, 4-trifluoromethylindole-3-acetic acid (4-CF₃-IAA), a new fluorinated auxin, was synthesized via 4-trifluoromethylindole and 4-trifluoromethylindole-3-acetonitrile by using 2-methyl-3-nitrobenzotrifluoride as the starting material. As a control compound for comparing biological activities, 4-methylindole-3-acetic acid (4-CH₃-IAA) was also synthesized by using 2,3-dimethylnitrobenzene as the starting material. The biological activities of these compounds were compared by three bioassays with those of indole-3-acetic acid and 4-chloroindole-3-acetic acid (4-Cl-IAA), which, like 4-CF₃-IAA and 4-CH₃-IAA, has a substituent at the 4-position of the indole nucleus. 4-CF₃-IAA showed strong root formation-promoting activity with black gram cuttings which was 1.5 times higher than that of 4-(3-indole)butyric acid at 1 × 10^-4 M. 4-CH ₃-IAA, however, only weakly promoted root formation in spite of its strong inhibition of hypocotyl growth in Chinese cabbage and promotion of hypocotyl swelling and lateral root formation in black gram. On the other hand, 4-CF₃-IAA demonstrated weaker activities than 4-CH₃-IAA and 4-Cl-IAA in these two bioassays.

Full text of DOI:10.1271/bbb.80138

Biosci. Biotechnol. Biochem., 72 (8), 2025–2033, 2008
Synthesis and Biological Activities of 4-Trifluoromethylindole-3-acetic Acid:
A New Fluorinated Indole Auxin
y
Masato KATAYAMA,1; Yuko MASUI,2 Eiji KAGEYAMA,2
4
Youichi KAWABATA,3 and Kozo KANAYAMA
1Cooperative Research Laboratory for Plant Growth Regulator Development,
National Institute of Advanced Industrial Science and Technology (AIST),
2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan
2Tokai-Kasei Company Ltd., 66 Sodai, Mino, Gifu 501-3714, Japan
3Nippon Pharmaceutical Chemicals Company Ltd., 2-8-18 Choda, Higashiosaka, Osaka 577-0056, Japan
4Advanced Wood-based Material Technology Group, Materials Resaerch Institute for Sustainable Development,
AIST, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan
Received March 5, 2008; Accepted May 19, 2008; Online Publication, August 7, 2008
[doi:10.1271/bbb.80138]
In our studies on the development of new promoters
for the root formation of tree cuttings, 4-trifluorometh-
ylindole-3-acetic acid (4-CF3-IAA), a new fluorinated
auxin, was synthesized via 4-trifluoromethylindole and
4-trifluoromethylindole-3-acetonitrile by using 2-methyl-
3-nitrobenzotrifluoride as the starting material. As a
control compound for comparing biological activities,
4-methylindole-3-acetic acid (4-CH3-IAA) was also
synthesized by using 2,3-dimethylnitrobenzene as the
starting material. The biological activities of these
compounds were compared by three bioassays with
those of indole-3-acetic acid and 4-chloroindole-3-acetic
acid (4-Cl-IAA), which, like 4-CF3-IAA and 4-CH3-IAA,
has a substituent at the 4-position of the indole nucleus.
4-CF3-IAA showed strong root formation-promoting
activity with black gram cuttings which was 1.5 times
higher than that of 4-(3-indole)butyric acid at 1 Â
10À4 M. 4-CH3-IAA, however, only weakly promoted
root formation in spite of its strong inhibition of
hypocotyl growth in Chinese cabbage and promotion
of hypocotyl swelling and lateral root formation in black
gram. On the other hand, 4-CF3-IAA demonstrated
weaker activities than 4-CH3-IAA and 4-Cl-IAA in these
two bioassays.
Pisum sativum.1,2) Subsequently, 5,6-dichloroindole-3-
acetic acid (5,6-Cl2-IAA), the most active of the known
natural and synthetic auxins, was synthesized.3) The
synthesis and biological activities of 4-Cl-IAA and its
esters have also been reported.4,5) Soaking and spraying
methods employing these compounds are potentially
useful for the mass production of tree saplings in
reforestation.6,7) In a recent report describing the syn-
thesis and auxin activities of the L-lactic acid derivatives
of 4-Cl-IAA, the root formation activities of some esters
with black gram cuttings were greater than or the same
as that of 4-Cl-IAA.8)
Biologically active substances that contain fluorine
have been previously synthesized in the medical and
agricultural fields.9–12) The introduction of fluorine
atoms into biologically important molecules has a
dramatic effect on the properties of those molecules.
In studies of fluorinated plant growth regulators, the
fluoroindole auxins, 5,6-difluoroindole-3-acetic acid
(5,6-F2-IAA) and 2-(5,7-difluoro-3-indolyl)propionic
acid (5,7-F2-IPA), as well as the fluoroindole antiauxin,
2-(5,7-difluoro-3-indolyl)isobutyric acid (5,7-F2-IIBA),
have been synthesized.13,14) The introduction of fluorine
atom(s) into the benzene moiety of indole-3-acetic acid
(IAA) resulted in an increase in the auxin activity and
enhanced the growth of the lateral roots of mung beans.
Furthermore, we have reported that such fluorinated root
growth promoters as the nonsubstituted and substi-
tuted 4,4,4-trifluoro-3-(3-indolyl)butyric acids (TFIBAs)
showed strong root growth-promoting activities in
Chinese cabbage, lettuce, and rice seedlings, whereas
the nonfluorinated compound, 3-(3-indolyl)butyric acid,
only showed weak promoting activity.15–17) Thus,
replacement of the methyl moiety of 3-(3-indolyl)bu-
Key words: 4-trifluoromethylindole-3-acetic acid; 4-
methylindole-3-acetic acid; fluoroindole
auxin; 4-chloroindole-3-acetic acid; root
formation-promoting activity
Our past studies of halogenated plant growth regu-
lators, especially of root formation promoters, originated
from the initial isolation of 4-chloroindole-3-acetic acid
(4-Cl-IAA), a natural auxin, from immature seeds of
y
To whom correspondence should be addressed. Fax: +81-52-736-7611; E-mail: m.katayama@aist.go.jp

2026
M. KATAYAMA et al.
tyric acid with a trifluoromethyl group caused a dramatic
increase in root growth promotion in these three plants.
Some other interesting activities of TFIBAs have also
been recently reported.18–22) We are continuously devel-
oping new compounds that induce roots in the cuttings
of trees that often do not easily produce roots. 4-
Methylindole-3-acetic acid (4-CH3-IAA) has been re-
ported to have strong growth promoting activity toward
pea pericarp as well as 4-Cl-IAA.23) We therefore
focused on the synthesis of the new fluorinated auxin,
4-trifluoromethylindole-3-acetic acid (4-CF3-IAA, 1),
replacing the methyl group of 4-CH3-IAA with a
trifluoromethyl group for the development of new
compounds with strong root formation-promoting activ-
ity. We report here the synthesis and biological activities
of 4-CF3-IAA (1).
washed with a 0.1 N hydrochloric acid solution, water, a
saturated sodium bicarbonate solution and saturated
brine, dried over anhydrous sodium sulfate, decolorized
with activated charcoal (1 g), and evaporated in vacuo to
give 76.8 g of a brown oil. This oil was distilled under
reduced pressure (0.5 mm of Hg at 63–68 ꢀC) to afford
56.4 g (67.0% yield from the trifluoride) of 4-trifluo-
romethylindole (3) as a pale purple oil. NMR (300 MHz)
ꢀH (acetone-d6): 6.64 (1H, dq, J ¼ 3:2, 1.5 Hz), 7.26
(1H, ddq, J ¼ 8:1, 7.4, 0.8 Hz), 7.40 (1H, dq, J ¼ 7:4,
0.9 Hz), 7.57 (1H, dd, J ¼ 3:2, 1.4 Hz), 7.73 (1H, dq,
J ¼ 8:1, 0.8 Hz), 10.80 (1H, broad s); MS (relative
intensity, %) m=z: 185 (Mþ, 100), 166 (29), 158 (7), 138
(10), 135 (29), 116 (5), 107 (6), 89 (12), 68 (9), 63 (11).
IR ꢁmax(KBr) cmꢁ1: 3442, 1624, 1586, 1508, 1442,
1419, 1368, 1346, 1318, 1272, 1191, 1164, 1117, 1078,
1053, 939, 897, 830, 800, 755, 726, 609, 487. Found: C,
58.39; H, 3.06; N, 7.68%. Calcd. for C9H6F3N: C,
58.38; H, 3.27; N, 7.57%.
Materials and Methods
Instrumentation. Infrared spectra were measured with
a Shimadzu FTIR-8600PC spectrometer. 1H-NMR spec-
tra were recorded with a Varian INOVA-300 spectrom-
eter, with tetramethylsilane in acetone-d6 used as the
internal standard. Mass spectra were recorded with a
Jeol DX-705L spectrometer. Elemental analyses were
conducted with a Perkin-Elmer 2400 CHN elemental
analyzer.
3-Dimethylaminomethyl-4-trifluoromethylindole (4).
A portion of 37% aqueous formaldehyde (26.7 g,
0.32 mol) was added dropwise at below 7 ꢀC to a mixture
of 31.3 g (0.35 mol) of a 50% dimethylamine solution
and 40 ml of acetic acid that had been prepared below
20 ꢀC. A solution of 4-trifluoromethylindole (3, 55.4 g,
0.299 mol) in acetic acid (15 ml) was added to the
mixture over about 1.5 h at 5 ꢀC, and the mixture was
stirred overnight at room temperature. The reaction
mixture was acidified with a 4 N-hydrochloric acid
solution and treated three times with ethyl acetate. Water
(330 ml) was added to the reaction mixture, and the
mixture was treated twice with a mixed solvent (110 ml)
of n-hexane and tert-butyl methyl ether (MTBE; 1:1).
The aqueous layer was alkalized with a 24% sodium
hydroxide solution and treated twice with MTBE. The
combined organic layer was successively washed with
water and saturated brine, dried over anhydrous magne-
sium sulfate, and evaporated in vacuo to give pale brown
crystals. These crystals were disperse-washed with a
mixed solvent (100 ml) of n-hexane-MTBE (2:1) to give
57.0 g (78.6% yield) of 3-dimethylaminomethyl-4-
trifluoromethylindole (4). Mp 134–135 ꢀC; NMR ꢀH
(acetone-d6): 2.27 (6H, s), 3.59 (2H, dq, J ¼ 1:1, 1.1 Hz),
7.23 (1H, ddq, J ¼ 8:1, 7.4, 0.8 Hz), 7.44 (1H, dq,
J ¼ 7:4, 0.8 Hz), 7.53 (1H, s), 7.72 (1H, dq, J ¼ 8:1,
0.8 Hz), 10.73 (1H, broad s); IR ꢁmax(KBr) cmꢁ1: 3313,
2954, 2830, 1468, 1429, 1373, 1318, 1280, 1199, 1162,
1112, 1073, 996, 942, 860, 821, 796, 763, 725; MS
(relative intensity, %) m=z: 242 (Mþ, 43), 198 (100), 178
(11), 169 (13), 151 (20), 147 (12), 129 (6), 101 (4), 75
(6), 58 (7). Found: C, 59.51; H, 5.28; N, 11.64%. Calcd.
for C12H13F3N2: C, 59.50; H, 5.41; N, 11.56%.
Synthesis of 4-trifluoromethylindole-3-acetic acid (1)
4-trifluoromethylindole (3). A mixture of 2-methyl-3-
nitrobenzotrifluoride (2, 93.3 g, 0.455 mol), dimethyl-
formamide dimethyl acetal (108 g, 0.91 mol) and di-
methylformamide (149 ml) was heated at 110 ꢀC under
nitrogen as methanol produced in the reaction was
removed with Dean-Stark apparatus. After stirring for
22 h, the reaction mixture was cooled to room temper-
ature, and the solvent was removed in vacuo to give a
dark brown oil (139 g) which was dissolved in diiso-
propyl ether (300 ml), the ether layer being washed with
water and the aqueous layer treated twice with diiso-
propyl ether. The combined ether layer was successively
washed with water and saturated brine, dried over
anhydrous sodium sulfate, and evaporated in vacuo to
give 128 g of a styrene derivative as a dark brown oil
which was used in the next reaction without purification.
Raney-nickel (50 ml) was added to a stirred solution
of the styrene (128 g) in methanol while cooling, and the
styrene was reduced under a pressure of 3 kgf/cm2 of
hydrogen as the temperature was raised to room
temperature. To complete the reduction, additional
Raney-nickel (5 ml) was added to the solution while
cooling. After this reductive cyclization, the reaction
solution was filtered through Celite, and the filtrate was
evaporated in vacuo to give a brown oil which was
dissolved in a mixture (300 ml) of n-hexane and ethyl
acetate, before the mixture was treated with water. The
aqueous layer was treated with the mixed solvent
(200 ml). The combined organic layer was successively
4-Trifluoromethylindole-3-acetonitrile (5). A solution
of 4 (57.0 g, 0.235 mol) in methanol (740 ml) was added
to a solution of potassium cyanide (33.0 g, 0.51 mol) in
water (57 ml). Methyl iodide (110.5 g, 0.78 mol) was

4-Trifluoromethylindole-3-acetic Acid
2027
added dropwise to the solution while cooling with ice,
and the mixture was stirred overnight as the temperature
was raised to room temperature. Additional potassium
cyanide (3.3 g, 0.051 mol) was added to the mixture
which was then stirred overnight. Water (1 liter) was
added to the reaction solution, before treating with
MTBE (600 ml) and a mixed solvent (400 ml) of MTBE
and n-hexane (1:1). The combined organic layer was
successively washed water and saturated brine, treated
with activated charcoal (1 g) and sodium sulfate, and
evaporated in vacuo to give a nitrile which was
recrystallized from ethyl acetate-n-hexane to afford
51.3 g (97.6% yield) of 4-trifluoromethylindole-3-aceto-
nitrile (5). Mp 121–122 ꢀC; NMR ꢀH (acetone-d6): 4.01
(2H, dq, J ¼ 1:4, 0.6 Hz), 7.33 (1H, ddq, J ¼ 8:2, 7.6,
0.8 Hz), 7.53 (1H, dq, J ¼ 7:6, 0.8 Hz), 7.72 (1H, s),
7.81 (1H, dq, J ¼ 8:2, 0.8 Hz), 11.01 (1H, broad s); IR
n-hexane (1:1). The aqueous layer was treated with
activated charcoal (2.5 g) and then stirred for half an
hour, before the charcoal was filtered off. The pH value
of the aqueous filtrate was adjusted to 8–9, and activated
charcoal (2.5 g) was again added to the solution which
was then stirred for half an hour. After removing the
charcoal by filtration, the aqueous solution was acidified
with a hydrochloric acid solution, and the resulting
powder was collected by filtration. This powder was
thoroughly washed with water and then dried to give an
indoleacetic acid which was recrystallized from IPA-
H2O and dried at 60 ꢀC to yield 43.1 g (77.5% yield from
the nitrile) of 4-trifluoromethylindole-3-acetic acid. Mp
206–208 ꢀC; NMR ꢀH (acetone-d6): 3.86 (2H, dq,
J ¼ 1:0, 1.0 Hz), 7.24 (1H, ddq, J ¼ 8:2, 7.4, 0.8 Hz),
7.45 (1H, dq, J ¼ 7:4, 0.8 Hz), 7.57 (1H, s), 7.74 (1H,
dq, J ¼ 8:2, 0.8 Hz), 10.80 (1H, broad s); IR ꢁmax
(KBr) cmꢁ1: 3357, 1708, 1426, 1312, 1226, 1208, 1185,
1158, 1131, 1100, 1074, 778, 745, 630; MS (relative
intensity, %) m=z: 243 (Mþ, 100), 224 (4), 198 (100),
178 (36), 176 (18), 151 (72), 147 (19), 129 (23), 128
(13), 101 (12), 75 (12), 60 (10). Found: C, 54.33; H,
3.10; N, 5.83%. Calcd. for C11H8F3NO2: C, 54.33; H,
3.32; N, 5.76%.
ꢁ
max(KBr) cmꢁ1: 3346, 2256, 1430, 1365, 1314, 1212,
1188, 1158, 1133, 1113, 1069, 946, 823, 796, 755, 721,
629, 605, 577, 486; MS (relative intensity, %) m=z: 224
(Mþ, 100), 223 (79), 205 (11), 198 (67), 176 (17), 155
(47), 151 (9), 127 (6), 77 (7), 75 (6), 60 (6). Found: C,
58.83; H, 3.02; N, 12.58%. Calcd. for C11H7F3N2: C,
58.93; H, 3.15; N, 12.50%.
4-Trifluoromethylindole-3-acetic acid (1). Dry hydro-
gen chloride gas, which was produced from concen-
trated sulfuric acid (500 ml) and hydrochloric acid
(280 ml) and then passed through concentrated sulfuric
acid, was slowly bubbled into a solution of nitrile 5
(51.3 g, 0.229 mol) in dry methanol (205 ml) below 5 ꢀC.
After stirring for 5 h while raising the temperature to
room temperature, the reaction mixture was poured into
a mixture of methanol (820 ml) and water (4.1 g,
0.23 mol), and the resulting mixture was stirred over-
night at room temperature. After refluxing for 1 h,
methanol was removed in vacuo, and the residue was
poured into water (650 ml). The resulting crystals were
cooled, filtered, and washed with water to give 87.8 g of
a wet and pale yellow methyl ester (6) which was
recrystallized from ethyl acetate-n-hexane to give
methyl 4-trifluoromethylindole-3-acetate (6). Mp 121–
122 ꢀC; NMR ꢀH (acetone-d6): 3.65 (3H, s), 3.87 (2H, s),
7.25 (1H, ddq, J ¼ 8:2, 7.4, 0.8 Hz), 7.45 (1H, dq,
J ¼ 7:4, 0.8 Hz), 7.55 (1H, s), 7.75 (1H, dq, J ¼ 8:2,
0.8 Hz), 10.81 (1H, broad s); IR ꢁmax(KBr) cmꢁ1: 3301,
1728, 1440, 1364, 1346, 1305, 1218, 1201, 1159, 1138,
1108, 1072, 1052, 995, 948, 765, 746; MS (relative
intensity, %) m=z: 257 (Mþ, 72), 210 (8), 198 (100), 178
(12), 169 (7), 151 (28), 147 (10), 129 (12), 101 (6), 73
(8), 60 (9). Found: C, 55.84; H, 3.78; N, 5.43%. Calcd.
for C12H10F3NO2: C, 56.04; H, 3.92; N, 5.45%.
Synthesis of 4-methylindole-3-acetic acid (11)
4-methylindole (8). A mixture of 2, 3-dimethylnitro-
benzene (7, 500 g, 3.31 mol), dimethylformamide di-
methyl acetal (477 g, 4.00 mol), pyrrolidine (205 g,
2.88 mol) and dimethylformamide (488 ml) was heated
at 95 ꢀC for 3 h under nitrogen, the methanol produced in
the reaction being removed with Dean-Stark apparatus.
After a further addition of dimethylformamide dimethyl
acetal (79 g, 0.66 mol), the reaction temperature was
gradually raised from 95 ꢀC to 125 ꢀC, and the mixture
was heated. After 30 h, the reaction mixture was cooled
to room temperature, and the solvent was removed in
vacuo to give a dark red oil (866 g) of a styrene which
was used in the next reaction without purification.
Raney-nickel (100 g) was added to a stirred solution
of this styrene (736 g) in tetrahydrofuran (THF, 1.7-liter)
while cooling, before a hydrazine solution (666 g,
13.3 mol) was added dropwise to the THF solution and
an additional hydrazine solution (15 g, 0.3 mol) was
added at 40 ꢀC, and this mixture then being heated at
60 ꢀC. The reaction solution was filtered through Celite,
and the filtrate was condensed in vacuo to two-thirds of
its volume, before being treated with n-hexane (700 ml)
and water. The aqueous layer was treated with n-hexane
(300 ml). The combine organic layer was successively
washed with water, a hydrochloric acid solution, water,
a saturated sodium hydrogen bicarbonate solution and
saturated brine, dried over anhydrous sodium sulfate,
and evaporated in vacuo to give a dark brown oil
(318.0 g) which was distilled under reduced pressure
(0.4 mm of Hg at 94 ꢀC) to yield 280.4 g (76.0% yield
from 7) of 4-methylindole (8) as a pale yellow oil. NMR
(300 MHz) ꢀH (acetone-d6): 2.50 (3H, s), 6.49–6.51 (1H,
A 24% sodium hydroxide solution (38.1 g, 0.23 mol)
was added to a solution of wet 6 in methanol (510 ml).
After stirring overnight at room temperature, the
reaction mixture was condensed to an aqueous solution
and water was added. The solution was treated twice
with a mixed solvent (100 ml and 50 ml) of MTBE and

2028
M. KATAYAMA et al.
m), 6.80 (1H, ddq, J ¼ 7:1, 0.9, 0.9 Hz), 6.99 (1H, dd,
J ¼ 8:1, 0.9 Hz), 7.24 (1H, dd, J ¼ 8:1, 0.9 Hz), 7.27–
was stirred for 40 min, and additional potassium cyanide
(15.6 g, 0.24 mol) was added. After heating at 55 ꢀC,
water (800 ml) was added to the reaction mixture, and
the resulting nitrile was collected, washed with water
and dispersed into a 0.01 N hydrochloric acid solution (1
liter). The nitrile was filtered, washed with water, and
dried in vacuo at 60 ꢀC to give 254.0 g (93.6% yield) of
4-methylindole-3-acetonitrile (10). Mp 104–105 ꢀC;
1H-NMR ꢀH (acetone-d6): 2.73 (3H, s), 4.17 (2H, d,
J ¼ 1:0 Hz), 6.81 (1H, ddq, J ¼ 7:1, 1.0, 1.0 Hz), 7.01
(1H, dd, J ¼ 8:1, 7.1 Hz), 7.26 (1H, d, J ¼ 8:1 Hz), 7.35
7.31 (1H, m), 10.20 (1H, broad s); IR ꢁmax(KBr) cmꢁ1
:
3408, 3053, 2918, 1608, 1583, 1500, 1456, 1410, 1377,
1354, 1342, 1281, 1238, 1157, 1115, 1080, 955, 895,
835, 752, 721, 619, 517; MS (relative intensity, %) m=z:
131 (Mþ, 75), 130 (100), 103 (11), 89 (2), 77 (17), 65
(19), 51 (14). Found: C, 82.35; H, 6.73; N, 10.86%.
Calcd. for C9H9N: C, 82.40; H, 6.92; N, 10.68%.
3-Dimethylaminomethyl-4-methylindole (9). A portion
of 37% aqueous formaldehyde (187 g, 2.30 mol) was
added dropwise to a mixture of 218 g (2.41 mol) of a 50%
dimethylamine solution and 224 ml of acetic acid at
below 5 ꢀC, and the mixture was stirred for half an hour.
Fifty-six ml of THF was added to the Shiff base solution,
and a mixed solution of 4-methylindole (8, 280 g, 2.13
mol) and methanol (56 ml) was then added to the mixture
over 2.5 h at 5 ꢀC, before the temperature of the reaction
solution was raised to room temperature and the solution
stirred overnight. The reaction mixture was filtered, and
the filtrate was evaporated in vacuo, before water
(840 ml) was added to the residue, and the aqueous
solution was treated three times with a mixed solvent of
MTBE-n-hexane (1:1). After the aqueous solution had
been evaporated again in vacuo to remove the materials
with a low boiling point, the resulting solution was
alkalized with a 24% sodium hydroxide solution to give
a dimethylaminomethylindole which was thoroughly
washed with water. The resulting wet powder was
dissolved in ethyl acetate. The ethyl acetate solution was
washed with saturated brine, dried over anhydrous
sodium sulfate, and evaporated to give a crude dimethyl-
aminomethylindole which was recrystallized from
ethyl acetate-n-hexane to give 296.7 g (73.8% yield) of
3-dimethylaminomethyl-4-methylindole (9). Mp 134–
135 ꢀC; 1H-NMR ꢀH (acetone-d6): 2.18 (6H, s), 2.74 (3H,
s), 3.51 (2H, d, J ¼ 0:6 Hz), 6.74 (1H, ddq, J ¼ 7:1, 1.0,
1.0 Hz), 6.95 (1H, dd, J ¼ 8:1, 7.1 Hz), 7.14 (1H, broad
s), 7.18 (1H, d, J ¼ 8:1 Hz), 10.02 (1H, broad s); IR
(1H, broad s), 10.30 (1H, broad s); IR ꢁmax(KBr) cmꢁ1
:
3396, 3055, 2966, 2932, 2249, 1614, 1545, 1504, 1460,
1427, 1416, 1340, 1252, 1159, 1123, 1059, 1026, 961,
918, 827, 781, 768. 758, 746, 638, 581, 529, 498; MS
(relative intensity, %) m=z: 170 (Mþ, 100), 169 (77), 155
(25), 144 (43), 143 (80), 142 (25), 130 (22), 115 (47), 89
(8), 63 (9). Found: C, 77.60; H, 5.84; N, 16.61%. Calcd.
for C11H10N2: C, 77.62; H, 5.92; N, 16.46%.
4-Methylindole-3-acetic acid (11). 4-Methylindole-3-
acetonitrile (10, 247 g, 1.45 ml) was added to a mixed
solution of a 48% sodium hydroxide solution (300 g,
3.6 mol), methanol (370 ml) and water (210 ml). After
refluxing for 15.5 h, the methanol was removed in
vacuo, water (600 ml) was added to the residual solution,
and the aqueous solution was treated with activated
charcoal (2.5 g). After filtration, the filtrate was acidified
with hydrochloric acid, and the resulting crystals were
filtered and washed with water. The wet crude crystals
were dissolved in ethyl acetate (1.2-liter), and the ethyl
acetate layer was washed with saturated brine, dried
over anhydrous sodium sulfate, and evaporated in vacuo
to give a crude indoleacetic acid. This acid was
dispersed in a mixed solvent of ethyl acetate and
n-hexane (1:1), cooled, filtered and dried in vacuo at
60 ꢀC to give 261.9 g (95.4% yield) of 4-methylindole-3-
acetic acid (11). Mp 158–159 ꢀC; NMR ꢀH (acetone-d6):
2.65 (3H, s), 3.91 (2H, d, J ¼ 0:8 Hz), 6.73 (1H, ddq,
J ¼ 7:0, 1.0, 1.0 Hz), 6.95 (1H, dd, J ¼ 8:0, 7.0 Hz),
7.21 (1H, d, J ¼ 8:0 Hz), 7.22 (1H, broad s), 10.08 (1H,
broad s); IR ꢁmax(KBr) cmꢁ1: 3414, 3391, 2914, 1701,
1578, 1504, 1460, 1441, 1414, 1400, 1346, 1333, 1298,
1261, 1240, 1219, 1159, 1115, 1067, 934, 770, 748, 689,
656, 625, 527, 496; MS (relative intensity, %) m=z: 189
(Mþ, 88), 145 (29), 144 (100), 143 (37), 142 (21), 130
(7), 115 (38), 91 (8), 89 (8), 72 (6), 63 (7). Found: C,
69.72 H; 5.72; N, 7.51%. Calcd. for C11H11NO2: C,
69.83; H, 5.86; N, 7.40%.
ꢁ
max(KBr) cmꢁ1: 3168, 3119, 2997, 2964, 2936, 2855,
2816, 2772, 1541, 1508, 1472, 1439, 1414, 1340, 1275,
1234, 1167, 1126, 1069, 1036, 993, 961, 853, 820, 741,
579; MS (relative intensity, %) m=z: 188 (Mþ, 13), 144
(52), 143 (100), 142 (40), 129 (11), 115 (48), 89 (12), 73
(16), 60 (17). Found: C, 76.42; H, 8.48; N, 15.03%.
Calcd. for C12H16N2: C, 76.56; H, 8.57; N, 14.88%.
4-Methylindole-3-acetonitrile (10). Dimethyl sulfate
(241 g, 1.91 mol) was added to dimethylformamide
(450 ml), before the solution was cooled and 3-dimeth-
ylaminomethyl-4-methylindole (9, 300 g, 1.59 mol) was
added at below ꢁ10 ꢀC. After the mixture had been
stirred for 1.5 h, a solution of potassium cyanide (125 g,
1.92 mol) in water (210 ml) was added, and the mixture
stirred for 1 h under slightly reduced pressure. After
cooling the solution, additional dimethyl sulfate (30 g,
0.24 mol) was added to it at below 10 ꢀC. The solution
High-performance liquid chromatography (HPLC).
A lipophilicity analysis of 4-CF3-IAA, 4-CH3-IAA,
4-Cl-IAA, IAA, and 4-(3-indole)butyric acid (IBA)
was performed in a reversed-phase HPLC column
containing Develosil C30-UG-5 (Nomura Chemical Co.,
Japan) with a solvent of CH3CN:CH3OH:H2O:AcOH
(5:5:10:0.1) at a flow rate of 1.0 ml/min and a UV
detection wavelength of 280 nm.

4-Trifluoromethylindole-3-acetic Acid
2029
CH3
CF3
CF3
CF3
CH3
NO2
N
CH3
a
b
NO2
N
H
2
3
CF3
CF3
CF3
CH3
N
CN
c
d
f
CH3
N
H
N
H
5
4
CF3
OH
OCH3
e
O
O
N
H
N
H
6
4-CF3-IAA ( 1 )
Fig. 1. Synthetic Scheme for 4-CF3-IAA.
a, DMFDMA/DMF, 110 ꢀC; b, Raney-nickel, H2 (3 kgf/cm2)/MeOH, THF; c, HCHO, Me2NH, AcOH; d, CH3I; KCN, MeOH, H2O;
e, CH3OH, p-TsOH, H2O; f, NaOH, MeOH, H2O; H3Oþ.
Plant materials and bioassay. Chinese cabbage
(Brassica pekinensis cv. Kinshu; Takii Seed Co., Ltd.,
Japan) and black gram (Vigna mungo (L.) Hepper;
Sakata Seed Co., Japan) seeds, were stored at 5 ꢀC and
used in the auxin activity tests.
methanol to afford a trifluoromethylindole (3) which
was converted to 4-trifluoromethylindole-3-acetonitrile
(5) in a good yield. Since direct hydrolysis of the
trifluoromethylindole-3-acetonitrile (5) to 4-CF3-IAA
(1) with an aqueous 40% potassium hydroxide solution
gave overreacted products (not identified), the methyl
ester (6) was prepared by methanolysis of the nitrile
under acidic conditions and finally converted to 4-CF3-
IAA (1) by alkaline hydrolysis with 24% sodium
hydroxide. The overall yield from six steps was
approximately 40%. 4-CH3-IAA was also synthesized
by a procedure similar to that used in the synthesis of
4-CF3-IAA to allow comparisons of the biological
activities of 4-CF3-IAA and 4-CH3-IAA. Although
4-CH3-IAA had already been synthesized from 4-
methylindole as a starting material under severe con-
ditions by Reinecke et al., the overall yield was very low
(<32%). We synthesized it in five steps from 2,3-
dimethylnitrobenzene in about a 50% overall yield (66%
from 4-methylindole by slightly improved procedure)
(Fig. 2). The only difference in the synthetic procedure
between 4-CF3-IAA and 4-CH3-IAA was that, for
4-CH3-IAA, the final acid was obtained by alkaline
hydrolysis of the nitrile.
The biological activities of 4-CF3-IAA were deter-
mined by three bioassays: Chinese cabbage hypocotyl
growth inhibition, black gram hypocotyl swelling and
lateral root formation, and root formation with black
gram cuttings. The activities of 4-CF3-IAA were then
compared to those of 4-CH3-IAA, 4-Cl-IAA, IAA and
IBA.
The results of the assays for hypocotyl growth
inhibition using intact Chinese cabbage are shown in
Fig. 3. 4-CF3-IAA strongly inhibited the hypocotyl
growth in Chinese cabbage. The inhibition activity of
4-CF3-IAA was six times higher than that of IAA,
Hypocotyl growth inhibition test with Chinese cab-
bage, and hypocotyl swelling and lateral root formation
tests with black gram. These tests were as described in a
previous study.4) Duplicate bioassays were conducted
five times.
Root formation-promoting activity test with black
gram cuttings. An ethanol solution (500 ml) of the
sample (2 ꢂ 10ꢁ2 M) was placed in a deep Petri dish
(6 cm ꢂ 6 cm i.d.) and evaporated in vacuo in a
desiccator, after which 100 ml of distilled water con-
taining 20 ml of a spreading agent (Dain; Sumika-Takeda
Garden Co., Japan) was added. The aqueous solution
was then sonicated for 10 min. The test procedure was
conducted as described in a previous study.8) Duplicate
bioassays were conducted five times.
IBA and IAA were purchased from Kanto Kagaku Co.
(Tokyo, Japan) and Merck Co., respectively, for use as
standards.
Results and Discussion
The new fluoroindole auxin, 4-CF3-IAA (1), was
synthesized from 2-methyl-3-nitrobenzotrifluoride (2)
by a procedure similar to that employed for the synthesis
of 4-Cl-IAA and its ester;4) the reactant was heated with
N,N-dimethylformamide dimethyl acetal (DMFDMA) in
dimethylformamide (DMF) to produce a dimethylami-
nostyrene derivative (Fig. 1). The product was subjected
to reductive cyclization with Raney nickel-hydrogen in

2030
M. KATAYAMA et al.
CH3
CH3
CH3
CH3
NO2
N
a
b
NO2
N
H
7
8
CH3
CH3
CH3
N
c
d
CN
CH3
N
H
N
H
9
10
CH3
OH
e
O
N
H
4-CH3-IAA ( 11 )
Fig. 2. Synthetic Scheme for 4-CH3-IAA.
a, DMFDMA, pyrrolidine/DMF, 95 ꢀC ! 125 ꢀC; b, Raney-nickel, H2NNH2-H2O/THF; c, HCHO, Me2NH, AcOH, THF/MeOH;
d, (CH3)2SO4/DMF; KCN/H2O; e, NaOH, MeOH, H2O; H3Oþ.
20
Control
18
16
14
12
10
8
4-CF3-IAA
4-Cl-IAA
4-CH3-IAA
IAA
6
4
2
0
1x10-7
1x10-6
1x10-5
1x10-4
Concentration (M)
Fig. 3. Hypocotyl Growth Inhibition in Chinese Cabbage by 4-CF3-IAA, 4-CH3-IAA, 4-Cl-IAA and IAA.
Each value is shown as the mean ꢃ SD, n ¼ 5.
whereas it was 40% and 6% of that of 4-CH3-IAA and
ent shape.24,25) In the case of hypocotyl growth inhib-
ition, the decrease in inhibition activity by replacement
of the methyl group in 4-CH3-IAA with a trifluoro-
methyl moiety may have been caused by the increase in
steric hindrance to entering the active site of the
receptor. The increased lipophilicity may benefit the
inhibition activity of 4-Cl-IAA and 4-CH3-IAA which
can fit smoothly into the receptor site. The relative
lipophilicity was determined from the retention time in a
reversed-phase HPLC column as shown in Table 1; that
is, the HPLC analysis showed that the lipophilicity was
4-Cl-IAA, respectively. Thus, replacement of the methyl
group in 4-CH3-IAA with a trifluoromethyl moiety
significantly decreased the inhibition activity observed
with Chinese cabbage. Reinecke et al. have explained
that the weaker activity of 4-ethylindole-3-acetic acid
(4-Et-IAA) than the activity of 4-Cl-IAA was caused by
steric problems entering the active site of the receptor in
the elongation activity of pea pericarp.23) The van der
Waals volume of the trifluoromethylgroup is similar to
that of the ethyl group, although of significantly differ-

4-Trifluoromethylindole-3-acetic Acid
2031
Table 1. Retention Times for 4-CF3-IAA, 4-CH3-IAA, 4-Cl-IAA,
IAA and IBA in the HPLC Analysisa
Table 2. Root Formation-Promoting Activities of 4-CF3-IAA,
4-CH3-IAA, 4-Cl-IAA, IBA and IAA in Black Gram Cuttingsa
Compound
Retention time (min)
Ratio to IBA
(%)
Compound
Root number/cutting
IAAb
6.15
8.12
4-CH3-IAA
4-Cl-IAA
IBAc
4-CF3-IAA (1)
4-CH3-IAA (11)
4-Cl-IAA
IBAb
21:5 ꢃ 2:5
9:7 ꢃ 1:1
53:0 ꢃ 7:8
13:8 ꢃ 1:4
4:9 ꢃ 0:4
4:8 ꢃ 0:4
156 ꢃ 12
70 ꢃ 11
384 ꢃ 15
100 ꢃ 10
36 ꢃ 8
9.34
11.07
12.17
4-CF3-IAA
IAAc
aThe analytical conditions for HPLC were as follows: column, Develosil
C30-UG-5, 250 mm ꢂ 4.6 mm i.d.; eluent, CH3CN:CH3OH:H2O:AcOH =
5:5:10:0.1; flow rate, 1.0 ml/min; Detector, UV at 280 nm
bIAA, indole-3-acetic acid
Control
35 ꢃ 8
aAll compounds were used at 1 ꢂ 10ꢁ4 M. IBA was used as a standard
promoter of root formation. Each value is presented as the mean ꢃ standard
error of the mean.
cIBA, 4-(3-indole)butyric acid
bIBA, 4-(3-indole)butyric acid
cIAA, indole-3-acetic acid
greater than the activity of 4-CF3-IAA, respectively; that
is, equivalent degrees of hypocotyl swelling and lateral
root formation were observed with 3 ꢂ 10ꢁ4 M 4-CF3-
IAA, 1 ꢂ 10ꢁ6 M 4-Cl-IAA, and 3 ꢂ 10ꢁ6 M 4-CH3-IAA.
4-CF3-IAA induced calluses to form on the black gram
seedlings at concentrations of more than 3 ꢂ 10ꢁ3
M
after only three days, whereas 4-Cl-IAA and 4-CH3-IAA
caused callus formation at a concentration threshold of
3 ꢂ 10ꢁ5 M. The activity strengths of these four com-
pounds in black gram seedlings were similar to those
observed for the inhibition of hypocotyl growth in
Chinese cabbage. The difference in the hypocotyl
swelling and lateral root formation activities of these
auxins in intact black gram seedlings may have been for
the same reason as that of the hypocotyl growth
inhibition of Chinese cabbage.
The adventitious root formation-promoting activity of
4-CF3-IAA in black gram cuttings is detailed in Table 2.
4-CF3-IAA strongly promoted root formation at a
concentration of 1 ꢂ 10ꢁ4 M. Its activity was 1.5 times
greater than that of IBA, the active ingredient in
commercially available root formation agents such as
Seradix and Hormodin. It was also twice greater than the
activity of 4-CH3-IAA. Interestingly, however, 4-CH3-
IAA weakly promoted root formation, even though it
strongly inhibited hypocotyl growth in Chinese cabbage,
and induced hypocotyl swelling and lateral root for-
mation in black gram; this contrasts with the other
compounds that showed strong activity toward hypo-
cotyl growth inhibition in Chinese cabbage, like the
hypocotyl swelling and lateral root formation in black
gram seedlings and root formation in black gram
cuttings.4,8) At 1 ꢂ 10ꢁ4 M, 4-CF3-IAA and 4-Cl-IAA
induced a number of adventitious roots all over the
cutting stems that were soaked in solutions of these
compounds, whereas IBA induced root formation only
on the lower portions of the cutting stems; in particular,
among the roots induced by 4-CF3-IAA, a few adhered
to each other to create a single intertwined root
structure. 4-CF3-IAA appeared to strongly stimulate
the induction of root primordia all over the soaked
cuttings, as has been observed for 4-Cl-IAA in cuttings
Fig. 4. Hypocotyl Swelling and Lateral Root Formation in Black
Gram by 4-CF3-IAA, 4-CH3-IAA, 4-Cl-IAA and IAA (1 ꢂ 10ꢁ4
M
concentration).
4-CF3-IAA > IBA > 4-Cl-IAA > 4-CH3-IAA > IAA.
4-CF3-IAA is more lipophilic than IBA which has two
methylene groups in the side chain of IAA. Furthermore,
it shows that 4-Cl-IAA was more liphophilic than 4-
CH3-IAA as indicated in Table 1, although the increase
of only the lipophilicity did not maximize the inhibition
activity, since 4-CF3-IAA had the highest lipophilicity
and moderate inhibition activity. On the other hand the
electron-withdrawing inductive effect may not be as
important in the inhibition activity.
The results from the bioassay of hypocotyl swelling
and lateral root formation in intact black gram seedlings
are shown in Fig. 4. 4-CF3-IAA enhanced hypocotyl
swelling and lateral root formation. The activity of 4-
CF3-IAA was slightly higher than that of IAA, but much
weaker than the activities of 4-Cl-IAA and 4-CH3-IAA,
for which the activities were about 300 and 100 times

2032
M. KATAYAMA et al.
from immature seeds of Pisum sativum. Agric. Biol.
Chem., 32, 117–118 (1968).
3) Hatano, T., Katayama, M., and Marumo, S., 5,6-
Dichloroindole-3-acetic acid as a potent auxin: its
synthesis and biological activity. Experientia, 43,
1237–1239 (1987).
4) Katayama, M., Synthesis and biological activities of
4-chloroindole-3-acetic acid and its esters. Biosci. Bio-
technol. Biochem., 64, 808–815 (2000).
5) Katayama, M., Hattori, H., and Kamuro, Y., Plant
growth-regulating activities of 4-chloroindole-3-acetic
acid and its esters. J. Pesticide Sci., 27, 68–70 (2002).
6) Katayama, M., Development of root formation pro-
moters and their utilization. Green Age, 21, 12–13
(2000).
of gardenia, cosmos, Rhododendron oomurasaki, and
Serissa japonica.12,14) In regard to the root formation in
black gram, the introduction of fluorine atoms onto the
methyl group of 4-CH3-IAA caused a dramatic increase
in the promoting activity. It has been reported that the
high auxin activity of 4-Cl-IAA may be ascribed to its
low reactivity to peroxidase.26) The electron-withdraw-
ing effect caused a decrease of electron density in the
indole nucleus to resist oxidative degradation of indole-
3-acetic acid by the reaction at the 2 or 3 position of the
indole nucleus with electrophilic oxgen and IAA oxidase
or peroxidase.27) Indeed, 4-CF3-IAA was extremely
stable to exposure to air on a silica gel thin layer in
darkness at room temperature for 20 h, but most of
4-CH3-IAA easily decomposed and was coloured dark
yellow, the same as IAA did under the same conditions
used in 4-CF3-IAA (data not shown). 4-Cl-IAA was also
stable to oxidation by air. The introduction of fluorine
atoms and a chlorine atom strongly increased the
stability to oxidation. The increase in the root forma-
tion-promoting activity in black gram cuttings by the
introduction of fluorine atoms may have been due to the
stability to oxidation that was brought by the electron-
withdrawing inductive effect, but it did not maximize
the promoting activity, since 4-CF3-IAA had the highest
electron-withdrawing group and moderate root forma-
tion-promoting activity. 4-Cl-IAA, which could fit
smoothly into the active site of the receptor and also
had the electron-withdrawing substituent, has the stron-
gest root formation activity. On the other hand, 4-CH3-
IAA with weak root formation activity did not have the
electron-withdrawing group, and reversely had the
electron-donating one. To strengthen the root formation
activity, the smooth fit into the active site of the receptor
and the increased resistance to plant oxidation enzymes
by the group with an electron-withdrawing inductive
effect may have been important. The auxin activity of
these compounds can also be influenced by such factors
as the chemical stability and facility for transport and
uptake, as well as the metabolic stability.
7) Katayama, M., Overseas reforestation by means of root
formation promoters: reforestation in Thailand. AIST
Today International Edition, 18 (2004).
8) Kageyama, E., Katayama, M., Masui, Y., Kageyama, K.,
and Kawabata, Y., Synthesis and plant growth-regulating
activities of L-lactic acid derivatives of 4-chloroindole-3-
acetic acid. J. Pesticide Sci., 31, 130–138 (2006).
9) Filler, R., and Kobayashi, Y., ‘‘Biomedical Aspects of
Fluorine Chemistry,’’ Kodansha Ltd., Tokyo (1982).
10) Filler, R., Fluorine-containing drugs. In ‘‘Organofluorine
Chemicals and Their Industrial Application,’’ ed. Banks,
E., Ellis Horwood Ltd., Chichester, pp. 123–153 (1979).
11) Newbold, G. T., Fluorine-containing pesticides. In
‘‘Organofluorine Chemicals and Their Industrial Appli-
cation,’’ ed. Banks, E., Ellis Horwood Ltd., Chichester,
pp. 167–187 (1979).
12) Uneyama, K., ‘‘Organofluorine Chemistry,’’ Blackwell
Publishing Ltd., Oxford, pp. 206–222 (2006).
13) Katayama, M., Kato, Y., Hatano, T., Hatori, M., and
Marumo, S., Synthesis and biological activities of 5,6-
difluoroindole-3-acetic acid; a new fluoroindole auxin.
J. Pesticide Sci., 23, 289–295 (1998).
14) Kato, Y., Katayama, M., and Marumo, S., Synthesis of
2-(5,7-difluoro-3-indolyl)propionic acid (5,7-F2-IPA)
and 2-(5,7-difluoro-3-indolyl)isobutyric acid (5,7-F2-
IIBA) and their respective auxin and antiauxin activities.
J. Pesticide Sci., 26, 266–270 (2001).
15) Katayama, M., Kato, K., Kimoto, H., and Fujii, S., (S)-
(+)-4,4,4-Trifluoro-3-(indole-3-)butyric acid, a novel
fluorinated plant growth regulator. Experientia, 51,
721–724 (1995).
Acknowledgment
16) Katayama, M., and Gautam, R. K., Synthesis and
biological activities of substituted 4,4,4-trifluoro-3-
(indole-3-)butyric acids, novel fluorinated plant growth
regulators. Biosci. Biotechnol. Biochem., 60, 755–759
(1996).
17) Katayama, M., and Gautam, R. K., Synthesis and
biological activities of fluorinated plant growth regula-
tors, 4,4,4-trifluoro-3-(3-indolyl)butyric acids and 4,4,4-
We thank Mr. Shigeyuki Kitamura of the Graduate
School of Bioagricultural Sciences at Nagoya University
for his help with the mass spectral measurements and
elemental analyses. This research was partially supported
by grant-aid from Japan Science and Technology
Agency.
trifluoro-3-(2-indolyl)butyric acid bearing
group on the indole nucleus. J. Pesticide Sci., 22, 331–
337 (1997).
18) Kaldorf, M., and Ludwig-Muller, J., AM fungi might
¨
a methyl
References
1) Marumo, S., Hattori, H., Abe, H., and Munakata,
K., Isolation of 4-chloroindolyl-3-acetic acid from
immature seeds of Pisum sativum. Nature, 214, 959–
960 (1968).
affect the root morphology of maize by increasing
indole-3-butyric acid biosynthesis. Physiol. Plant., 109,
58–67 (2000).
2) Marumo, S., Abe, H., Hattori, H., and Munakata, K.,
Isolation of a novel auxin, methyl 4-chloroindoleacetate
19) Zhang, N., and Hasenstein, K. H., Haloganated auxins
affect microtubules and root elongation in Lactuca

4-Trifluoromethylindole-3-acetic Acid
2033
sativa. J. Plant Growth Regul., 19, 397–405 (2000).
20) Zhang, N., and Hasenstein, K. H., 4,4,4-Trifluoro-3-
(indole-3-) butyric acid promotes root elongation in
Lactuca sativa independent of ethylene synthesis and
pH. Physiol. Plant., 116, 383–388 (2002).
indole-3-acetic acids affecting pea pericarp elongation.
Plant Growth Regul., 27, 39–48 (1999).
24) Muller, K., Faeh, C., and Diederich, F., Fluorine in
¨
pharmaceuticals: looking beyond intuition. Science, 317,
1881–1886 (2007).
21) Zhang, H.-X., and Zhou, X., The effects of TFIBA and
IBA on seminal root length and initiation of lateral root
primordial of rice and lettuce seedlings. Plant Physiol.
Commun., 39, 453–454 (2003).
25) Purser, S., Moore, P. R., Swallow, S., and Gouverneur,
V., Fluorine in medicinal chemistry. Chem. Soc. Rev.,
37, 320–330 (2008).
26) Marumo, S., Hattori, H., and Yamamoto, A., Biological
activity of 4-chloroindolyl-3-acetic acid. In ‘‘Plant
Growth Substances 1973,’’ Hirokawa Publishing Co.,
Inc., Tokyo, pp. 419–428 (1974).
22) Fitze, D., Wiepning, A., Kaldorf, M., and Ludwig-
Muller, J., Auxins in the development of an arbuscular
¨
mycorrhizal symbiosis in maize. J. Plant Physiol., 162,
1210–1219 (2005).
23) Reinecke, D. M., Ozga, J. A., Ilic, N., Magnus, V., and
27) Marumo, S., Chapter 2 Auxins. In ‘‘Chemistry of Plant
Hormones,’’ ed. Takahashi, N., CRC Press Inc., London,
pp. 9–56 (1986).
´
Kojic-Prodic, B., Molecular properties of 4-substituted
´
´