Synthesis and biological evaluation of trifluoromethyl-containing auxin derivatives

Abstract

This study focused on the chemical synthesis of auxin analogs, wherein a trifluoromethyl group was introduced near the carboxyl group in the side chain of natural and synthetic auxins, including IAA, NAA, IBA, 2,4-D, and 4-Cl-IAA. The effects of these synthetic compounds and natural auxins on plant growth regulation and callus growth were evaluated. In experiments with black gram, CF₃-IAA and 4-Cl-CF₃-IAA exhibited comparable effects to the parent compound, IAA. Meanwhile, CF₃-NAA, CF₃-2,4-D, CF₃-IBA-1, and CF₃-IBA-2 displayed effects that differed considerably from those of their respective parent auxins. In experiments with lettuce, CF₃-IAA, 4-Cl-CF₃-IAA, CF₃-NAA, CF₃-2,4-D, and CF₃-IBA-1 showed effects comparable to the corresponding parent auxins. However, at low concentrations, these analogs induced hypocotyl and root elongations, a response distinct from that observed with their parent compounds. Furthermore, CF₃-IBA-2 considerably promoted hypocotyl and root elongations across all concentrations relative to the control. The addition of synthetic compounds to callus cultures revealed that CF₃-IAA, 4-Cl-CF₃-IAA, CF₃-NAA, and CF₃-2,4-D promoted callus proliferation, whereas CF₃-IBA-1 and CF₃-IBA-2 did not enhance callus growth.

Introduction

Fluorine has played an important role in diverse research fields, including pharmaceuticals and pesticides, and its significance has been increasing. Although fluorine is relatively abundant in nature, naturally occurring fluorine-containing organic compounds are rare.¹⁾ This scarcity presents opportunities for artificial structural modifications that incorporate fluorine. Particularly, since the discovery of fludrocortisone in 1953—the first fluorine-containing drug approved by the U.S. Food and Drug Administration (FDA)—the strategy of incorporating fluorine into biologically active compounds has attracted the close attention of the pharmaceutical industry. Nowadays, more than 30% of marketed drugs contain at least one fluorine atom.²⁾ Fluorine incorporation is expected to enhance drug efficacy through improved stability against oxidative metabolism owing to its strong electron-withdrawing properties, enhanced lipophilicity that facilitates drug absorption, and increased binding affinity for target enzymes through hydrogen bonding, etc.

Auxins are a vital class of plant growth regulators, known as phytohormones, that play essential roles in numerous developmental and physiological processes throughout a plant’s life cycle. The four naturally occurring auxins (produced by plants) are indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 4-chloroindole-3-acetic acid (4-Cl-IAA), and phenylacetic acid (PAA). In the 1940s, various synthetic derivatives of IAA were developed that exhibited auxin-like properties, including 1-naphthaleneacetic acid (NAA), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 2,4-dichlorophenoxyacetic acid (2,4-D). Although numerous IAA derivatives with auxin activity have been synthesized, derivatives featuring substitutions to the side chain of IAA remain limited.^3,4) Kato et al. synthesized 3,3,3-trifluoro-2-(3-indolyl)propionic acid (CF₃-IAA) containing a trifluoromethyl group at the α-position of the carbonyl group and demonstrated that the compound exhibited high auxin activity with enhanced stability against enzymatic oxidation (Fig. 1).^5,6) However, we hypothesized that, in addition to stability against enzymatic oxidation, the electronic and steric effects arising from the introduction of the trifluoromethyl group must be considered. Accordingly, we incorporated a trifluoromethyl group at the α-position of the carbonyl group or at the allylic position in other auxins and evaluated the auxin activities of the synthesized compounds.

Fig. 1. Structures of natural and synthetic auxins.

Herein, we present the synthesis of trifluoromethyl-containing auxin derivatives, including NAA, IBA, 2,4-D, and 4-Cl-IAA, in which a trifluoromethyl group was introduced near the carbonyl group (Fig. 2).

Fig. 2. Structures of the synthesized trifluoromethyl-containing auxin derivatives.

Materials and methods

1. General

¹H- and ¹³C-NMR spectra were acquired with Bruker-Biospin Avance III 500 MHz NMR spectrometer and taken in CDCl₃, unless otherwise noted. Chemical shift values are expressed in ppm relative to internal tetramethylsilane. Coupling constants J values are presented in Hz. Abbreviations are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. IR spectra were recorded with a Shimadzu IRAffinty-1S spectrometer. IR spectroscopy of oil sample was measured as neat liquid film. The wave-numbers of maximum absorption peaks of IR spectroscopy are presented in cm⁻¹. MS (ESI) is presented in m/z. Extracts were washed with brine and then dried over sodium sulfate. Silica gel column chromatography was used for purification. IAA, NAA, IBA, 2,4-D were purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan).

2. Plant materials and biological assays on black gram and lettuce

Black gram (Vigna mungo L.) and lettuce (Lactuca sativa L.) seeds were stored at 5°C and used in the auxin activity tests. Seed germination experiments were carried out according to the literature method with slight modifications.⁷⁾ Black gram and lettuce seeds were germinated on cotton soaked with distilled water in Petri dishes at 28°C in the dark for 24 hr. Sterilized Petri dishes, each containing 5.0 mL of distilled water or test solutions (0.5, 5, or 50 ppm) on filter paper, were prepared. 5.0 mg of the test compound was dissolved in 100 µL of DMSO to prepare a 100 ppm stock solution, which was then diluted with distilled water to obtain the desired test solutions. Ten germinated seeds were evenly distributed in each dish. After a 72-hr dark incubation at 28°C, the hypocotyl and main root lengths of three average-growing seeds were measured, and hypocotyl swelling and lateral root formation were recorded.

3. Callus Culture

Taheebo calluses were obtained from the leaves of Tabebuia avellanedae and prepared according to the reported procedure with modifications.⁸⁾ Briefly, Taheebo leaf segments, consisting of the midrib only, were cut into approximately 1 cm pieces. These pieces were sterilized, bleached, and then placed on a slanted Murashige and Skoog (MS) medium solidified with 0.2% gellan gum, supplemented with 1.0 ppm naphthaleneacetic acid (NAA), 1.0 ppm kinetin (K), and 2% sucrose. After incubation at 25°C for 3 weeks in the dark, the resulting calluses were sectioned into 3–4 pieces, each approximately 5 mm in size, and transferred to fresh MS medium solidified with 0.2% gellan gum in Petri dishes. The medium contained 1.0 ppm test solution, 1.0 ppm kinetin (K), and 2% sucrose. This fresh medium contained 1.0 ppm of the test solution, 1.0 ppm kinetin, and 2% sucrose. Following a 5-week incubation at 25°C in the dark, callus induction and growth were assessed visually.

4. Synthesis

4.1. Ethyl 3,3,3-trifluoro-2-hydroxy-2-(1H-indol-3-yl)propanoate (2a)

To a stirred solution of indol (1a) (586 mg, 5.0 mmol) in CHCl₃ (10 mL) was added Ethyl Trifluoropyruvate (1.36 g, 8.0 mmol). After stirred for 5 hr at 50°C under Ar atmosphere, the solvent was removed in vacuo. The crude product was chromatographed on silica gel. Yield 99% (1.42 g). White solid. rf (hexane/EtOAc=3/1)=0.35. ¹H-NMR: δ 8.30 (brs, 1H), 7.91 (d, J=8.2 Hz, 1H), 7.49 (d, J=2.6 Hz, 1H), 7.39 (d, J=8.1, 1H), 7.22 (ddd, J=8.2, 7.7, 1.1 Hz, 1H), 7.16 (ddd, J=7.9, 8.1, 1.2 Hz, 1H), 4.5–4.33 (m, 2H), 1.35 (t, J=7.2 Hz, 3H).

4.2. Ethyl 3,3,3-trifluoro-2-(1H-indol-3-yl)propanoate (3a)

To a stirred solution of 2a (1.66 g, 5.78 mmol) in dry DMF (10 mL) was added Thionyl Chloride (1.04 mL, 14.5 mmol) at 0°C. After stirred for 0.5 hr at 0°C, sodium borohydride (722 mg, 19.1 mmol) was added to the reaction mixture. After stirred for additional 0.5 hr at rt, the reaction mixture was filtered through a pad of Celite. The Celite was washed with EtOAc, and the combined organics were washed with H₂O, brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 76% (1.19 g). White solid. rf (hexane/EtOAc=2/1)=0.49. ¹H-NMR: δ 8.30 (brs, 1H), 7.68 (d, J=7.9 Hz, 1H), 7.43–7.39 (m, 2H), 7.24 (ddd, J=8.2, 7.5, 1.2 Hz, 1H), 7.19 (ddd, J=8.0, 7.6, 1.3 Hz, 1H), 4.67 (q, J=8.4 Hz, 1H), 4.23–4.15 (m, 2H), 1.26 (t, J=7.1 Hz, 3H).

4.3. 3,3,3-Trifluoro-2-(1H-indol-3-yl)propanoic acid (CF₃-IAA)⁵⁾

To a stirred solution of 3a (499 mg, 1.84 mmol) in 1,4-dioxane (2.0 mL) was added conc. HCl (4.0 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 89% (398 mg). ¹H-NMR: δ 8.33 (brs, 1H), 7.67 (d, 1H), 7.42 (d, 2H), 7.22 (m, 2H), 4.72 (q, J=8.4 Hz, 1H), 4.12(q, J=7.1 Hz, 1H).

4.4. Ethyl 2-(4-chloro-1H-indol-3-yl)-3,3,3-trifluoro-2-hydroxypropanoate (2b)⁹⁾

To a stirred solution of indole 1b (151 mg, 1.0 mmol) in 1,4-dioxane (2.0 mL) was added ethyl trifluoropyruvate (187 mg, 1.1 mmol). After the mixture was refluxed for 16 hr under Ar atmosphere, the solvent was removed in vacuo. The crude product was chromatographed on silica gel. Yield 62% (200 mg). White solid. rf (hexane/EtOAc=2/1)=0.40. ¹H-NMR: δ 8.44 (brs, 1H), 7.43 (dd, J=5.1, 2.6 Hz, 1H), 7.28–7.25 (m, 1H), 7.16–7.09 (m, 2H), 4.54 (s, 1H), 4.42–4.26 (m. 2H), 1.23 (t, J=7.0 Hz, 3H).

4.5. Ethyl 2-(4-chloro-1H-indol-3-yl)-3,3,3-trifluoropropanoate (3b)

To a stirred solution of 2b (94 mg, 0.29 mmol) in dry DMF (1.5 mL) was added thionyl chloride (53 µL, 0.73 mmol) at 0°C. After stirred for 0.5 hr at 0°C, sodium borohydride (36 mg, 0.96 mmol) was added to the reaction mixture. After stirred for additional 0.5 hr at rt, the reaction mixture was filtered through a pad of Celite. The Celite was washed with EtOAc, and the combined organics were washed with H₂O, brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 87% (77.5 mg). White solid. rf (hexane/EtOAc=2/1)=0.45. ¹H-NMR: δ 8.55 (brs, 1H), 7.41 (s, 1H), 7.18 (dd, J=7.6, 1.9 Hz, 1H), 7.06–6.99 (m, 2H), 5.63 (q, J=8.8 Hz, 1H), 4.24–4.09 (m, 2H), 1.20 (t, J=7.4 Hz, 3H). ¹⁹F-NMR: δ −68.1 (s, CF₃). ¹³C-NMR: δ 167.7 (q, J=2.7 Hz), 137.0 (C), 126.8 (CH), 126.8 (C), 125.5 (C), 124.2 (q, J=280 Hz, C), 123.1 (CH), 121.8 (CH), 110.6 (CH), 104.1 (q, J=2.0 Hz, C), 62.3 (CH₂), 46.5 (q, J=29.4 Hz, CH), 14.0 (CH₃). IR (KBr): 2986, 1736, 1350, 1157, 111, 740 cm⁻¹. HRMS (ESI) m/z: [M–H]⁻ calcd for [C₁₃H₁₀ClF₃NO₂]⁻, 304.0352, Found, 304.0352.

4.6. 2-(4-Chloro-1H-indol-3-yl)-3,3,3-trifluoropropanoic acid (4Cl-CF₃-IAA)

To a stirred solution of 3b (907 mg, 2.97 mmol) in 1,4-dioxane (15 mL) was added conc. HCl (15 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 90% (741 mg). White solid. rf (hexane/EtOAc=1/1)=0.05. ¹H-NMR: δ 8.49 (brs, 1H), 7.56 (d, J=2.3 Hz, 1H), 7.32 (dd, J=6.8, 2.3 Hz, 1H), 7.17–7.11 (m, 2H), 5.77 (q, J=8.8 Hz, 1H), 5.51–3.03 (brs, 1H). ¹⁹F-NMR: δ −70.1 (s, CF₃). ¹³C-NMR: δ 172.2 (C), 136.9 (C), 126.1 (CH), 125.5 (C), 123.8 (q, J=280 Hz, C), 123.3 (CH), 123.0 (C), 122.1 (CH), 110.5 (CH), 103.6 (C), 46.1 (q, J=30 Hz, CH). IR (KBr): 2904, 1720, 1427, 1258, 1165, 1119, 934, 741 cm⁻¹. HRMS (ESI) m/z: [M–H]⁻ calcd for [C₁₁H₆ClF₃NO₂]⁻, 276.0039, Found, 276.0035.

4.7. Ethyl 3,3,3-trifluoro-2-hydroxy-2-(naphthalen-1-yl)propanoate (4)¹⁰⁾

To a stirred solution of ethyl trifluoropyruvate (2.55 g, 15 mmol) in THF (30 mL) was added portionwise 1-naphthylmagnesium bromide (1.0 M slurry in THF, 18 mL, 18 mmol) at −78°C. After stirring for at 0°C 2 hr under an Ar atmosphere, the solvent was removed in vacuo. The crude product was chromatographed on silica gel. Yield 92% (4.1 g). ¹H-NMR: δ 8.17–8.10 (m, 1H), 7.94–7.84 (m, 2H), 7.81–7.75 (d, J=7.9 Hz, 1H), 7.54–7.42 (m, 3H), 4.46 (s, 1H), 4.38–4.17 (m, 2H), 1.11 (t, J=7.0 Hz, 3H).

4.8. Ethyl 3,3,3-trifluoro-2-((methylsulfonyl)oxy)-2-(naphthalen-1-yl)propanoate (5)¹¹⁾

To a stirred solution of 4 (209 mg, 0.70 mmol), Et₃N (195 µL, 3.46 mmol) and catalytic amount of DMAP in dry CH₂Cl₂ (5.0 mL) was added methanesulfonyl chloride (82 µL, 1.1 mmol) at 0°C. After stirred for 0.5 hr at rt, the reaction mixture was quenched by addition of H₂O. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was used in the next step without further purifications. Under H₂ atmosphere, a mixture of the crude product (200 mg) and 10% Pd/C (28 mg, 0.03 mmol) in MeOH (2.5 mL) was stirred for 1 hr at rt. The mixture was filtered through a pad of Celite. The Celite was washed with MeOH, and the combined organics were concentrated. The crude product was chromatographed on silica gel. Yield 71% in 2 steps (141 mg). ¹H-NMR: δ 8.05 (d, J=8.65 Hz, 1H), 7.92 (d, J=8.22 Hz, 2H), 7.78 (d, J=7.35 Hz, 1H), 7.64–7.59 (m, 1H), 7.57–7.48 (m, 2H), 5.24 (q, 1H), 4.31–4.15 (m, 2H), 1.21 (t, 3H).

4.9. 3,3,3-Trifluoro-2-(naphthalen-1-yl)propanoic acid (CF₃-NAA)

To a stirred solution of 8 (240 mg, 0.85 mmol) in 1,4-dioxane (6.0 mL) was added conc. HCl (6.0 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 78% (169 mg). ¹H-NMR (MeOD): δ 8.20 (d, J=8.6 Hz, 1H), 7.92 (d, J=8.3 Hz, 2H), 7.75 (d, J=7.3 Hz, 1H), 7.62–7.48 (m, 3H), 5.53 (q, J=8.5 Hz, 1H), 5.02 (brs, 1H). ¹⁹F-NMR (MeOD): δ −68.3 (CF₃). ¹³C-NMR (MeOD): δ 168.5 (q, J=2.7 Hz, C), 134.2 (C), 132.0 (C), 129.4 (CH), 128.7 (CH), 127.0 (CH), 127.0 (C), 126.7 (CH), 125.7 (CH), 124.8 (CH), 124.5 (q, J=280 Hz, C), 122.7 (CH), 49.3 (q, J=28 Hz, CH). IR (KBr): 2900, 1728, 1435, 1358, 1258, 1157, 1119, 779 cm⁻¹. HRMS (ESI) m/z: [M–H]⁻ calcd for [C₁₃H₈F₃O₂]⁻, 253.0482, Found, 253.0483.

4.10. 3,3,3-Trifluoro-2-(1H-indol-3-yl)propanal (6)

To a stirred solution of 3a (200 mg, 0.74 mmol) in dry toluene (3.0 mL) was added diisobutylaluminum hydride (1.0 M in toluene, 0.90 mL, 0.90 mmol) at −78°C. After stirred for 1 hr at −78°C under Ar atmosphere, the reaction was quenched by addition of CH₃OH, and then concentrated. The crude product was chromatographed on silica gel. Yield 55% (92 mg). Yellow solid. rf (hexane/EtOAc=3/1)=0.39. ¹H-NMR: δ 9.72 (d, J=1.8 Hz, 1H), 8.39 (brs, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.1 Hz, 1H), 7.29–7.17 (m, 3H), 4.60 (q, J=9.1 Hz, 1H). ¹⁹F-NMR: δ −73.0 (CF₃). ¹³C-NMR: δ 190.5 (q, J=55 Hz, C), 135,8 (C), 126.2 (C), 124.6 (q, J=8.0 Hz, 1H), 124.1 (q, J=280 Hz, C), 123.0 (CH), 120.6 (CH), 118.2 (CH), 111.4 (q, J=42 Hz, CH), 101.2 (q, J=9.0 Hz, C), 53.4 (q, J=27 Hz, CH). IR (KBr): 3410, 1735, 1334, 1257, 1165, 1118, 748 cm⁻¹. HRMS (ESI) m/z: [M+H]⁺ calcd for [C₁₁H₉F₃O₃NO]⁺, 228.0631, Found, 228.0646.

4.11. Methyl (E)-5,5,5-trifluoro-4-(1H-indol-3-yl)pent-2-enoate (7)

To a stirred solution of 6 (112 mg, 0.49 mmol) in dry CH₂Cl₂ (3.0 mL) was added methyl (Triphenylphosphoranylidene)acetate (327 mg, 0.97 mmol) at rt. After stirred for 1 hr at rt under Ar atmosphere, the reaction mixture was concentrated. The crude product was chromatographed on silica gel. Yield 76% (105 mg, trans : cis=6 : 4) as mixture of cis/trans isomers. The mixture of stereoisomers was used in the next reaction without further purification.

4.12. Methyl 5,5,5-trifluoro-4-(1H-indol-3-yl)pentanoate (8)

Under H₂ atmosphere, a mixture of 7 (270 mg, 0.96 mmol) and Pd/C (16 mg, 0.15 mmol) in MeOH (2.0 mL) was stirred for 12 hr at rt. The mixture was filtered through a pad of Celite. The Celite was washed with EtOAc, and the combined organics were concentrated. The crude product was chromatographed on silica gel. Yield 92% (252 mg). Brown oil. rf (hexane/EtOAc=3/1)=0.35. ¹H-NMR: δ 8.22 (s, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.38 (d, J=8.1 Hz, 1H), 7.22 (ddd, J=8.1, 7.5, 1.0 Hz, 1H), 7.17–7.13 (m, 2H), 3.72 (qt, J=5.2, 4.0 Hz, 1H), 2.45–2.20 (m, 4H). ¹⁹F-NMR: δ −72.1 (CF₃). ¹³C-NMR: δ 173.2 (C), 136.0 (C), 127.1 (q, J=280 Hz, C), 126.9 (C), 123.3 (CH), 122.4 (CH), 120.0 (CH), 118.9 (CH), 111.3 (CH), 108.7 (q, J=18 Hz, C), 51.5 (CH), 40.7 (q, J=28 Hz, CH), 31.0 (CH₂), 24.1 (CH₂). IR (KBr): 3410, 1728, 1265, 1157, 1111, 740 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₁₄H₁₄F₃NO₂Na]⁺, 308.0869, Found, 308.0885.

4.13. 5,5,5-Trifluoro-4-(1H-indol-3-yl)pentanoic acid (CF₃-IBA-1)

To a stirred solution of 8 (110 mg, 0.38 mmol) in 1,4-dioxane (3.0 mL) was added conc. HCl (3.0 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 31% (32 mg). White solid. rf (hexane/EtOAc=1/1)=0.31. ¹H-NMR (MeOD): δ 10.5 (brs, 1H), 7.45 (d, J=8.1 Hz, 1H), 7.28 (d, J=8.2 Hz, 1H), 7.12 (s, 1H), 7.02 (ddd, J=8.2, 7.6 Hz, 1.0 Hz, 1H), 6.93 (ddd, J=8.1, 7.5, 0.9 Hz, 1H), 3.71 (qt, J=9.7, 4.1 Hz, 1H), 2.31–2.05 (m, 4H). ¹⁹F-NMR (MeOD): δ −71.6 (CF₃). ¹³C-NMR (MeOD): δ 176.6 (C), 138.0 (C), 128.8 (q, J=270 Hz, CH), 128. 4 (C), 125.2 (C), 122.7 (CH), 120.3 (CH), 119.6 (CH), 112.5 (C), 108.6 (CH), 41.8 (q, J=27 Hz, CH), 31.8 (CH₂), 25.1 (CH₂). IR (KBr): 3417, 1705, 1265, 1157, 1111, 748 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₁₃H₁₂F₃NO₂Na]⁺, 294.0712, Found, 294.0714.

4.14. Ethyl 4-(1H-indol-3-yl)butanoate (9)⁷⁾

To a stirred solution of 3-Indolebutyric Acid (1.0 g, 4.92 mmol) in EtOH (15.0 mL) was added conc. H₂SO₄ (few drops). After the mixture was refluxed for 3 hr, the reaction was quenched by addition of H₂O. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 95% (1.08 g). White solid. rf (hexane/EtOAc=3/1)=0.74. ¹H-NMR: δ 8.00 (s, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.20 (m, 1H), 7.13 (m, 1H), 6.99 (d, J=1.9 Hz, 1H), 4.14 (q, J=7.1 Hz, 2H), 2.83 (t, J=7.4 Hz, 2H), 2.40 (t, J=7.5 Hz, 2H), 2.07 (q, J=7.5 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H).

4.15. tert-Butyl 3-(4-ethoxy-4-oxobutyl)-1H-indole-1-carboxylate (10)⁷⁾

To a stirred solution of 9 (1.61 g, 6.99 mmol), 4-Dimethylaminopyridine (171 mg, 1.4 mmol) in CH₂Cl₂ (15.0 mL) was added di-tert-butyl dicarbonate (1.83 g, 8.39 mmol). After the mixture was stirred for 0.5 hr at rt, the reaction was quenched by addition of H₂O. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 71% (1.64 g). White solid. rf (hexane/EtOAc=4/1)=0.76. ¹H-NMR: δ 8.12 (brs, 1H), 7.53 (d, J=7.9 Hz, 1H), 7.36 (brs, 1H), 7.31 (ddd, J=8.2, 7.6, 1.1 Hz, 1H), 7.23 (ddd, J=7.9, 7.5, 1.0 Hz, 1H), 4.13 (q, J=7.2 Hz, 2H), 2.73 (t, J=7.8 Hz, 2H), 2.39 (t, J=7.5 Hz, 2H), 2.04 (tt, J=7.5, 7.8 Hz, 2H), 1.6 (s, 9H), 1.25 (t, J=7.2 Hz, 3H). ¹³C-NMR: δ 173.4 (C), 149.8 (C), 135.5 (C), 130.5 (C), 124.3 (CH), 122.6 (CH), 122.3 (CH), 120.1 (CH), 119.0 (CH), 115.2 (CH), 83.3 (CH), 60.3 (CH₂), 33.8 (CH₂), 28.2 (CH₃), 24.4 (CH₂), 24.2 (CH₂), 14.2 (CH₃). IR (KBr): 2978, 1728, 1450, 1373, 1257, 1157, 1087, 748 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₁₉H₂₅NO₄Na]⁺, 354.1676, Found, 354.1653.

4.16. tert-Butyl 3-(4-oxobutyl)-1H-indole-1-carboxylate (11)⁷⁾

To a stirred solution of 10 (815 mg, 2.46 mmol) in dry toluene (10 mL) was added diisobutylaluminum hydride (1.0 M in toluene, 2.95 mL, 2.95 mmol) at −78°C. After stirred for 1 hr at −78°C under Ar atmosphere, the reaction was quenched by addition of CH₃OH, and then concentrated. The crude product was chromatographed on silica gel. Yield 44% (310 mg). White oil. rf (hexane/EtOAc=5/1)=0.44. ¹H-NMR: 9.75 (t, J=1.6 Hz, 1H), 8.10 (d, J=8.0 Hz, 1H), 7.53 (ddd, J=7.7, 1.2, 0.8 Hz, 1H), 7.38 (s, 1H), 7.29 (m, 1H), 7.21 (m, 1H), 2.72 (td, J=7.6, 1.0 Hz, 2H), 2.51 (td, J=7.3, 1.5 Hz, 2H), 2.01 (d, J=7.4 Hz, 2H), 1.65 (s, 9H).

4.17. tert-Butyl 3-(3-chloro-4-oxobutyl)-1H-indole-1-carboxylate (12)¹²⁾

To a stirred solution of 11 (267 mg, 0.93 mmol) in dry CH₂Cl₂ (15 mL) was added N-Chlorosuccinimide (105 mg, 0.79 mmol) and DL-Proline (9.0 mg, 0.08 mmol) at −78°C. After stirred for 1 hr at −78°C under Ar atmosphere, the reaction was quenched by addition of CH₃OH, and then concentrated. The crude product was chromatographed on silica gel. Yield 44% (131 mg). yellow oil. rf (hexane/EtOAc=8/1)=0.25. ¹H-NMR: δ 9.55 (d, J=1.8 Hz, 1H), 8.13 (d, J=7.5 Hz, 1H), 7.54 (d, J=7.2 Hz, 1H), 7.42 (s, 1H), 7.28 (m, 2H), 4.24 (m, 1H), 3.03–2.83 (m, 2H), 2.41 (m, 1H), 2.20 (m, 1H), 1.67 (s, 9H).

4.18. tert-Butyl 3-(4,4,4-trifluoro-3-(methoxycarbonyl)butyl)-1H-indole-1-carboxylate (13)¹²⁾

To a stirred solution of 12 (163 mg, 0.51 mmol) in dry CH₂Cl₂ (10 mL) was added NHC-1 (36 mg, 0.1 mmol) and Togni Reagent II (604 mg, 1.91 mmol) at 0°C. After stirred for 10 hr at 0°C under Ar atmosphere, DBN (130 µL, 1.02 mmol) was added to the reaction mixture. After stirred for additional 2 hr at 0°C, the reaction was quenched by addition of H₂O. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 22% (43 mg). Brown oil. rf (hexane/EtOAc=5/1)=0.46. ¹H-NMR: δ 8.13 (d, J=8.3 Hz, 1H), 7.50 (d, J=8.3 Hz, 1H), 7.39 (s, 1H), 7.36–7.21 (m, 2H), 3.79 (s, 3H), 3.30–3.14 (m, 1H), 2.89–2.65 (m, 2H), 2.42–2.12 (m, 2H), 1.67 (s, 9H).

4.19. Methyl 4-(1H-indol-3-yl)-2-(trifluoromethyl)butanoate (14)

To a stirred solution of 13 (75 mg, 0.19 mmol) in dry CH₂Cl₂ (0.3 mL) was added trifluoroacetic acid (0.3 mL) at 0°C. After stirred for 0.5 hr at rt under Ar atmosphere, the reaction was quenched by addition of H₂O. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 78% (78 mg). Brown oil. rf (hexane/EtOAc=5/1)=0.28. ¹H-NMR: δ 7.98 (brs, 1H), 7.57 (d, J=7.9 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.21 (ddd, J=8.1, 7.6, 1.0 Hz, 1H), 7.13 (ddd, J=7.9, 7.6, 1.0 Hz, 1H), 7.00 (d, J=2.3 Hz, 1H), 3.74 (s, 3H), 3.20 (qt, J=8.8, 4.3 Hz, 1H), 2.95–2.74 (m, 2H), 2.39–2.16 (m, 2H). ¹⁹F-NMR: δ −73.6 (CF₃). ¹³C-NMR: δ 168.0 (C), 136.2 (C), 127.0 (C), 124.6 (q, J=280 Hz, C), 122.7 (CH), 121.1 (CH), 119.4 (CH), 118.5 (CH), 113.9 (C), 111.1 (CH), 52.5 (CH₃), 49.6 (q, J=27 Hz, CH), 26.4 (CH₂), 22.3 (CH₂). IR (KBr): 3417, 1743, 1265, 1157, 1111, 740 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₁₄H₁₄F₃NO₂Na]⁺, 308.0869, Found, 308.0865.

4.20. 4-(1H-Indol-3-yl)-2-(trifluoromethyl)butanoic acid (CF₃-IBA-2)

To a stirred solution of 14 (42 mg, 0.15 mmol) in 1,4-dioxane (3.0 mL) was added conc. HCl (3.0 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 56% (23 mg). Brown solid. rf (hexane/EtOAc=1/1)=0.08. ¹H-NMR (MeOD): δ 7.47 (d, J=7.9 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.05 (ddd, J=8.2, 7.6, 1.1 Hz, 1H), 7.00 (s, 1H), 6.96 (ddd, J=7.9, 7.6, 0.9 Hz, 1H), 3.18 (qt, J=9.1, 4.1 Hz, 1H), 2.85–2.65 (m, 2H), 2.20–1.99 (m, 2H). ¹⁹F-NMR (MeOD): δ −69.7 (CF₃). ¹³C-NMR (MeOD): δ 173.2 (C), 140.7 (C), 130.9 (C), 129.1 (q, J=278 Hz, C), 125.8 (CH), 124.9 (CH), 122.9 (CH), 121.6 (CH), 116.7 (CH), 113.7 (CH), 114.8 (CH), 53.2 (q, J=27 Hz, CH), 30.6 (CH₂), 25.9 (CH₂). IR (KBr): 3417, 1728, 1265, 1157, 1118, 740 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₁₃H₁₂F₃NO₂Na]⁺, 294.0712, Found, 294.0708.

4.21. Ethyl 2-diazo-3,3,3-trifluoropropanoate (15)¹³⁾

Ethyl trifluoropyruvate (850 mg, 5.0 mmol) was dissolved in anhydrous dichloromethane (6.0 mL), followed by the addition of p-toluenesulfonyl hydrazide (930 mg, 5.0 mmol) to form the tosylhydrazone. The resulting tosylhydrazone was dissolved in pyridine (2.5 mL). To this solution, POCl₃ (610 µL, 6.5 mmol) was added, and the reaction mixture was stirred overnight. The resulting mixture was extracted with hydrochloric acid. The organic layer was washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford compound 14 (213 mg, 23%). ¹H-NMR: δ 4.32 (q, J=7.1 Hz, 2H), 1.32 (t, J=7.1 Hz, 3H).

4.22. Ethyl 2-diazo-3,3,3-trifluoropropanoate (16)

Compound 15 (600 mg, 3.3 mmol) was dissolved in dry toluene (10.0 mL) under an argon atmosphere at room temperature. Then, 2,4-dichlorophenol (485 mg, 2.98 mmol) and copper trifluoroacetate (109 mg, 0.29 mmol) were added, and the mixture was stirred at 80°C for 1 hr under an argon atmosphere. Subsequently, the mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography, yielding compound 16 (352 mg, 34%). White solid. rf (hexane/EtOAc=8/1)=0.76. ¹H-NMR: δ 7.42 (d, J=2.5 Hz, 1H), 7.19 (dd, J=2.5, 8.8 Hz, 1H), 6.91 (d, J=8.8 Hz, 1H), 4.93 (q, J=6.3 Hz, 1H), 4.34 (qq, J=7.1, 1.7 Hz, 2H), 1.31 (t, J=7.1 Hz, 3H). ¹⁹F-NMR: δ −73.6 (CF₃). ¹³C-NMR: δ 164.4 (C), 152.6 (C), 131.3 (CH), 129.5 (C), 129.0 (CH), 125.8 (C), 123.3 (q, J=282 Hz, C), 117.7 (CH), 116.4 (CH), 76.8 (q, J=32 Hz, CH), 64.3 (CH₂), 14.2 (CH₃). IR (KBr): 3718, 1743, 1519, 1219, 1141 cm⁻¹. HRMS (ESI) m/z: [M+H]⁺ calcd for [C₁₁HCl₂F₃O₃]⁺, 316.9954, Found, 316.9958.

4.23. 2-(2,4-Dichlorophenoxy)-3,3,3-trifluoropropanoic acid (CF₃-2,4-D)

To a stirred solution of 16 (352 mg, 1.11 mmol) in 1,4-dioxane (3.0 mL) was added conc. HCl (3.0 mL) at rt. After the mixture was refluxed for 16 hr, the reaction was quenched by addition of NH₄Cl. The mixture was extracted with EtOAc. The organic extracts were washed with brine, dried over Na₂SO₄, and then concentrated. The crude product was chromatographed on silica gel. Yield 44% (138 mg). White solid. rf (hexane/EtOAc=1/1)=0.23. ¹H-NMR (MeOD): δ 7.45 (d, J=2.5 Hz, 1H), 7.25 (dd, J=2.5 Hz, 8.9 Hz, 1H), 7.03 (dd, J=8.9 Hz, 1H), 5.48 (q, J=6.9 Hz, 1H). ¹⁹F-NMR (MeOD): δ −75.1 (CF₃). ¹³C-NMR (MeOD): δ 152.8 (C), 131.2 (CH), 129.1 (CH), 128.9 (CH), 125.6 (C), 123.5 (q, J=280 Hz, C), 117.3 (CH), 77.7 (q, J=32 Hz, CH). IR (KBr): 3078, 1728, 1481, 1357, 1249, 1141, 1111 cm⁻¹. HRMS (ESI) m/z: [M+Na]⁺ calcd for [C₉H₆Cl₂F₃O₃Na]⁺, 310.9460, Found, 310.9472.

Results and discussion

1. Chemistry

The synthetic routes for CF₃-IAA, 4-Cl-CF₃-IAA, and CF₃-NAA were developed by modifying previously reported methods.⁵⁾ Scheme 1 shows the synthesis of CF₃-IAA and 4-Cl-CF₃-IAA. Indoles 1 were reacted with ethyl trifluoropyruvate to afford compounds 2 in yields ranging from 62 to 99%. Chlorination of the hydroxy group with thionyl chloride, followed by reduction using sodium borohydride, afforded the 3-trifluoroethyl ester 3 in 76–87% yield. Subsequent acidic hydrolysis in 1,4-dioxane overnight gave the target compounds, CF₃-IAA or 4-Cl-CF₃-IAA, in 89–90% yield.

Scheme 1. Synthesis of CF₃-IAA and 4-Cl-CF₃-IAA.

Scheme 2 shows the synthesis of CF₃-NAA. 1-Naphthylbromomagnesium was reacted with ethyl trifluoropyruvate in tetrahydrofuran (THF) at −78°C for 4 hr, producing compound 4 in a 92% yield. Mesylation of the hydroxy group with methanesulfonyl chloride afforded the mesylate in an 88% yield. Palladium-on-carbon reduction then yielded the 3-trifluoroethyl ester 5 in a 94% yield. Finally, acidic hydrolysis in 1,4-dioxane overnight gave the target CF₃-NAA in a 78% yield.

Scheme 2. Synthesis of CF₃-NAA.

Scheme 3 outlines the synthetic route for CF₃-IBA-1, which contains a trifluoromethyl group adjacent to the indole group. The aldehyde reduction of compound 3a using diisobutylaluminum hydride in dichloromethane (DCM) at −78°C for 1 hr afforded compound 6 in a 55% yield. Compound 7 was then obtained in a 76% yield by reacting 6 with phosphorus ylide (methyl 2-(triphenyl-λ-phosphaneylidene) acetate) at room temperature for 4 hr. The inseparable cis–trans isomer mixture was used in subsequent palladium-on-carbon reduction, producing compound 8 in a 92% yield. Finally, the hydrolysis of compound 8 under acidic conditions and refluxing in 1,4-dioxane for 12 hr gave CF₃-IBA-1 in a 31% yield.

Scheme 3. Synthesis of compound CF₃-IBA-1 from compound 3a.

Subsequently, with minor adaptations to reported procedures,¹⁴⁾ we synthesized CF₃-IBA-2, which contained a trifluoromethyl group at the α-position of the carbonyl group (Scheme 4). Using IBA as the starting material, the reaction was performed at 100°C for 24 hr in a sulfuric acid/ethanol solution, yielding compound 9 in a 95% yield. N-Boc protection of the indole ring was then followed by reduction with diisobutylaluminum hydride at −78°C, producing compound 11 in a 31% yield over two steps. α-Chlorination of aldehydes was achieved using DL-proline and N-chlorosuccinimide, yielding a 44% yield. Subsequently, N-heterocyclic carbene–catalyzed trifluoromethylation of the generated α-chloro aldehydes was performed to synthesize compound 13.¹²⁾ Ultimately, Boc group deprotection using trifluoroacetic acid and hydrolysis of the resultant ester produced CF₃-IBA-2.

Scheme 4. Synthesis of CF₃-IBA-2 from 3-indolebutyric acid.

Scheme 5. Synthesis of compound CF₃-2,4-D from ethyl trifluoropyruvate.

The synthesis of CF₃-2,4-D was accomplished through the selective insertion of a CF₃-substituted carbene, generated catalytically from methyl 3,3,3-trifluoro-2-diazo propionate, into the hydroxyl group of chlorophenol using Cu(CF₃-acac)₂ as the catalyst.¹⁵⁾ The reaction of ethyl trifluoropyruvate with tosylhydrazine in DCM at room temperature for 18 hr resulted in the formation of the tosylhydrazone intermediate. This intermediate was then treated with phosphorus oxychloride and pyridine, affording compound 15 in a 23% yield. The incorporation of the trifluoromethyl group was confirmed using ¹⁹F-nuclear magnetic resonance (NMR) spectroscopy. Next, a copper-mediated coupling reaction with 2,4-dichlorophenol in toluene produced compound 16 in a 34% yield. Finally, the acid-catalyzed hydrolysis of the resulting ester afforded CF₃-2,4-D.

2. Biology

2.1. Evaluation of plant growth–regulating activity on black gram or lettuce

The plant growth–promoting effects of the synthesized fluorine-containing auxins as well as natural auxins—IAA, NAA, 2,4-D, and IBA—were evaluated on black gram and lettuce at doses of 0.5, 5, and 50 ppm (Tables 1–2 and Fig. S1–S2). IAA is well known to induce hypocotyl swelling and lateral root formation.^16,17) Other natural auxins—NAA, IBA, and 2,4-D—exhibited similar effects to IAA (Tables 1–2, entries 1–12, and Figs. S1–S2, entries 2–5).

Table 1. Effects of natural auxins and synthesized auxin derivatives on black gram hypocotyl and root lengths

Entry	Compound	Concentration (ppm)	Hypocotyl elongation	Root elongation
1	IAA	0.5	1.14	0.76
2		5	0.78	0.75
3		50	0.52	0.35
4	NAA	0.5	0.13	0.28
5		5	0.22	0.22
6		50	0.55	0.18
7	2,4-D	0.5	0.60	0.37
8		5	0.23	0.20
9		50	0.14	0.16
10	IBA	0.5	0.89	0.79
11		5	0.50	0.45
12		50	0.23	0.21
13	CF3-IAA	0.5	1.08	1.08
14		5	0.61	0.54
15		50	0.41	0.16
16	4-Cl-CF3-IAA	0.5	1.12	0.62
17		5	0.88	0.22
18		50	0.53	0.11
19	CF3-NAA	0.5	0.79	0.98
20		5	1.19	0.69
21		50	1.06	0.64
22	CF3-2,4-D	0.5	0.72	1.31
23		5	1.03	1.17
24		50	0.83	1.15
25	CF3-IBA-1	0.5	0.94	2.16
26		5	0.56	1.81
27		50	0.97	2.32
28	CF3-IBA-2	0.5	0.79	2.11
29		5	1.06	1.87
30		50	0.74	2.24

^a Hypocotyl and root elongations are presented as relative ratios to the control (water). Values represent the means of at least three independent experiments. In the control sample, the hypocotyl height and root length are 36 and 20 mm, respectively.

Table 2. Effects of natural auxins and synthesized auxin derivatives on lettuce hypocotyl and root lengths

Entry	Compound	Concentration (ppm)	Hypocotyl elongation	Root elongation
1	IAA	0.5	0.54	0.28
2		5	0.52	0.16
3		50	0.59	0.13
4	NAA	0.5	0.39	0.13
5		5	0.52	0.13
6		50	0.26	0.08
7	2,4-D	0.5	0.2	0.12
8		5	0.2	0.08
9		50	0.13	0.08
10	IBA	0.5	0.54	0.42
11		5	0.52	0.15
12		50	0.46	0.11
13	CF3-IAA	0.5	1.25	1.15
14		5	0.83	0.62
15		50	0.38	0.37
16	4-Cl-CF3-IAA	0.5	1.13	1.21
17		5	0.52	0.82
18		50	0.12	0.09
19	CF3-NAA	0.5	1.19	1.26
20		5	1.05	0.90
21		50	0.17	0.16
22	CF3-2,4-D	0.5	1.29	1.35
23		5	1.24	1.18
24		50	0.44	0.33
25	CF3-IBA-1	0.5	1.15	2.01
26		5	0.98	1.51
27		50	0.31	0.81
28	CF3-IBA-2	0.5	1.35	1.27
29		5	1.37	1.51
30		50	1.05	1.85

^a Hypocotyl and root elongation are presented as relative ratios to the control (water). Values represent the means of at least three independent experiments. In the control sample, hypocotyl height and root length are 12 and 10 mm, respectively.

First, we will discuss the results obtained for black gram. CF₃-IAA and 4-Cl-CF₃-IAA at 50 and 5 ppm induced hypocotyl swelling similar to IAA but did not promote lateral root formation. At 0.5 ppm, no hypocotyl swelling was observed (Table 1, entries 13–18 and Fig. S1, entries 6–7). Compared with NAA, CF₃-NAA did not induce hypocotyl swelling, but it stimulated lateral root formation at all concentrations (Table 1, entries 19–21 and Fig. S1, entry 8). Compared with 2,4-D, CF₃-2,4-D did not induce hypocotyl swelling, but it promoted lateral root formation and root elongation at all concentrations (Table 1, entries 22–24 and Fig. S1, entry 9). CF₃-IBA-1 and CF₃-IBA-2 did not induce hypocotyl swelling observed with IBA but demonstrated substantial root elongation compared to the control at all concentrations. Additionally, CF₃-IBA-1 and CF₃-IBA-2 promoted lateral root formation (Table 1, entries 25–30 and Fig. S1, entries 10–11).

Next, we will discuss the results obtained for lettuce. At 50 ppm, CF₃-IAA and 4-Cl-CF₃-IAA induced mild hypocotyl swelling similar to IAA but did not considerably promote lateral root formation. However, at 0.5 ppm, they exhibited root elongation effects (Table 2, entries 13–18 and Fig. S2, entries 6–7). Compared with NAA, CF₃-NAA showed a weaker hypocotyl swelling effect but promoted root elongation at low concentrations (Table 2, entries 19–21 and Fig. S2, entry 8). Compared with 2,4-D, CF₃-2,4-D exhibited a weaker hypocotyl swelling effect but moderately promoted root elongation at low concentrations (Table 2, entries 22–24 and Fig. S2, entry 9). CF₃-IBA-1 and CF₃-IBA-2 demonstrated a weak hypocotyl swelling effect but considerably enhanced root elongation compared to the control at low concentrations. This effect was more pronounced for CF₃-IBA-1 than that for CF₃-IBA-2 (Table 2, entries 25–30 and Fig. S2, entries 10–11). Katayama et al. reported similar findings regarding the elongation of lettuce hypocotyls and roots using 4,4,4-trifluoro-3-(indole-3-)butyric acid.¹⁸⁾

2.2. Evaluation of growth activity on plant callus derived from Tabebuia avellanedae

Next, we examined the effect of the synthesized trifluoromethyl-containing plant hormones on the growth of plant callus derived from Tabebuia avellanedae (commonly known as Taheebo), cultivated in our laboratory.⁸⁾ We prepared solid media, each containing 1.0 ppm of the six synthesized hormones—CF₃-NAA, CF₃-2,4-D, CF₃-3,5-D, CF₃-IBA1, CF₃-IBA2, and 4-Cl-CF₃-IAA—along with kinetin (cytokinin k). Callus that had been subcultured under standard conditions with the addition of NAA as a plant hormone was then transplanted and cultured for one month (Fig. 3). As a result, growth was promoted in calli cultured under conditions with CF₃-IAA, 4-Cl-CF₃-IAA, CF₃-NAA, and CF₃-2,4-D. In contrast, calli cultured under conditions with CF₃-IBA-1 and CF₃-IBA-2 exhibited browning, and no growth promotion was observed. Our previous studies have demonstrated that Taheebo callus grows well in the presence of NAA, shows no growth in the presence of IAA, and withers and dies in the presence of 2,4-D (data not shown, except for the IAA results). Our findings indicate that synthetic auxins containing the CF₃ group exhibit a similar promoting effect on callus culture as natural auxins. We are currently investigating their effect on the metabolic products of callus cultured in liquid medium.

Fig. 3. Development of callus culture of Tabebuia avellanedae with IAA or synthesized auxins.

Conclusion

Herein, we chemically synthesized auxin analogs, wherein a trifluoromethyl group, a strong electron-withdrawing group, was introduced near the carboxyl group on the side chain of natural auxins. We then evaluated the effects of the synthesized compounds and natural auxins on plant growth regulation and callus growth. In experiments with black gram, CF₃-IAA and 4-Cl-CF₃-IAA exhibited similar results to the parent compound IAA, while CF₃-NAA, CF₃-2,4-D, CF₃-IBA-1, and CF₃-IBA-2 displayed effects that considerably differed from those of the corresponding parent auxins. Notably, CF₃-IBA-1 and CF₃-IBA-2 induced considerable root elongation and lateral root formation compared to the control at all tested concentrations. Further, in experiments with lettuce, CF₃-IAA, 4-Cl-CF₃-IAA, CF₃-NAA, CF₃-2,4-D, and CF₃-IBA-1 exhibited similar results to the corresponding parent auxins. However, at low concentrations, they induced hypocotyl and root elongations, which differed from the effects observed with their parent auxin compounds. CF₃-IBA-2 showed a markedly different effect, with substantial hypocotyl and root elongations observed at all concentrations compared to the control. In an experiment examining the effect of synthesized trifluoromethyl-containing plant hormones on Taheebo callus growth, growth was promoted in callus cultures supplemented with CF₃-IAA, 4-Cl-CF₃-IAA, CF₃-NAA, and CF₃-2,4-D. Conversely, growth was not promoted in callus cultures supplemented with CF₃-IBA-1 and CF₃-IBA-2. Previous studies have shown that callus growth is not observed when natural auxins IAA and 2,4-D are used. Therefore, it is clear that introducing the trifluoromethyl group into these auxins promotes callus culture. In summary, the incorporation of the trifluoromethyl group near the carbonyl group of auxins endows them with growth-regulating properties, which are distinct from those of the parent auxins. Further research is necessary to elucidate the effects of these compounds on callus metabolites and their action mechanisms.

Acknowledgments

The authors are grateful to Taheebo Japan Co., Ltd. and late Tetsuro Fujita, professor emeritus of Kyoto University, for their generous financial support to this project.

Conflict of interests

The authors declare no conflicts of interest associated with this manuscript.

Electronic supplementary materials

The online version of this article contains supplementary materials, which are available at https://www.jstage.jst.go.jp/browse/jpestics/.

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