Synthesis of 3-indolylacetamide derivatives and evaluation of their plant growth regulator activity

Abstract

In this paper, the 3-indolylacetamide derivatives were synthesised via the coupling of indole-3-acetic acid (IAA) with aminobenzene derivatives; and were screened for their plant growth regulator activities on rice seedling. The results show that some of the compounds synthesised possess considerable growth promoting activities in the concentration range of 1.7-0.17 ppm compared to IAA.

Headnote

Abstract: Some 3-indolylacetamide derivatives were synthesised via the coupling of indole-3-acetic acid (IAA) with aminobenzene derivatives and were screened for their plant growth regulator activities on rice seedling. The results show that some of the compounds synthesised possess considerable growth promoting activities in the concentration range of 1.7-0.17 ppm compared to IAA.

Keywords: 3-indolylacetamides, indole-3-acetic acid, plant growth regulator, rice seedling

INTRODUCTION

Plant growth regulators comprise a large number of structurally diverse compounds capable of regulating many biological processes including cell division, differentiation and enlargement, chloroplast development, and senescence. Their wide use in agriculture and plant biotechnology gives them a relevant role in science and technology. Among them, auxins play an important role in plant growth regulation, especially the ability to promote cell division, hasten root initiation, and increase the number and quality of roots [1-4]. The most widely occurring natural auxin, indole-3- acetic acid (IAA), is found in both the free form and the covalently bound via its carboxyl group to yield esters, glycosides and/or peptides of surprising structural diversity [5-6]. Other compounds such as derivatives of indole, naphthalenes, phenoxyacetic acids and some benzoic acids are known as synthetic growth regulators that elicit auxin-type responses in cell growth and cell division [7]. A halogen substituted on the benzene ring of benzoic acid was reported to increase the growth regulator activity quite markedly [8]. In order to access compounds with improved growth regulating properties in comparison to the original auxin and also to establish their structure-activity relationship, the search for potential plant growth regulators is of particular interest. Indoleacetic acid incorporated with aminobenzene derivatives via an amide link have been brought to our attention since both moieties possess plant growth promoting properties. Thus, they might reinforce the plant growth regulating properties of the resulting compounds. In this study some of such compounds, 3-indolylacetamide derivatives, obtained from indole-3-acetic acid conjugated with aminobenzene derivatives are reported and their plant growth regulator activities on rice seedling are investigated.

SYNTHESIS OF 3-INDOLYLACETAMIDE DERIVATIVES

Discussion

The indole derivatives (3a-b, 4a-c) were synthesised via the coupling of indole-3-acetic acid (1) and phenols or aromatic amines at room temperature (Scheme 1). The benzene derivatives (2a and 2b) were prepared [9] for use as starting materials. Reductive amination [10] of o-bromoaniline (2c) using formaldehyde or benzaldehyde with sodium cyanoborohydride rapidly afforded the corresponding N-substituted-o-bromoanilines (2d and 2e) in moderate yields. The benzene derivatives were then acylated by indole-3-acetic acid (1) at room temperature in the presence of DCC in dichloromethane to give 3a-b and 4a-c (Scheme 1). The structures of these synthesised compounds were characterised by their spectral data.

In order to study the structure-activity relationship of the synthesised compounds, N- alkylation on the indole ring of compounds 4a-c was performed using alkyl halides in the presence of potassium hydroxide to give compounds 5a-d in moderate to high yields (Scheme 2).

Experimental

Melting points were determined on a Stuart Scientific SMP 2 melting point apparatus and were uncorrected. Infrared spectra were recorded as CH2Cl2-film with a Perkin-Elmer GX FT-IR spectrophotometer. 1H- and 13C-NMR spectra were recorded in CDCl3 at 300 MHz for 1H and 75 MHz for 13C with a Bruker Avance 300 spectrometer. TMS was used as internal standard. Mass spectra were recorded on a Polaris Q or Hewlett Packard 5973 mass spectrometer. Reagents were purchased from Sigma-Aldrich or Fluka and were used as received. All solvents used were of analytical reagent grade except dichloromethane, which was distilled over CaH2 under nitrogen prior to use.

Methyl 3-(2-(1H-indol-3-yl)acetoxy)-2,4,6-tribromobenzoate (3a)

N,N'-dicyclohexylcarbodiimide (DCC) (0.10 g, 0.48 mmol) was added to a stirred solution of 3-indolyl acetic acid (0.07 g, 0.40 mmol) and methyl 2,4,6-tribromo-3-hydroxybenzoate (2a) (0.20 g, 0.53 mmol) in dry CH2Cl2 (5.0 mL) at 0o under argon atmosphere. The reaction mixture was stirred at room temperature for 24 hr. The precipitate was filtered and washed with CH2Cl2. The filtrate was evaporated to give a crude product which was purified by column chromatography (silica gel; hexane: EtOAc = 4:1) to give a yellow oil (0.14 g, 65%): IR 1079, 1095, 1152, 1222, 1271, 1570, 1739, 3407 cm-1; 1H-NMR d 3.94 (s, 3H), 4.13 (s, 2H), 7.11-7.22 (m, 3H), 7.33 (d, J=7.8 Hz, 1H), 7.66 (d, J=7.5 Hz, 1H), 7.76 (s, 1H), 8.20 (br s, 1H, NH) ppm; 13C-NMR d 30.7, 53.2, 106.6, 111.2, 116.0, 118.8, 119.3, 119.8, 122.3, 123.7, 127.1, 135.5, 136.0, 138.1, 146.2, 165.4, 167.7 ppm; HRES-MS m/z calulated for [M+H]+ C18H12Br3NO4= 565.8214, found 565.8214.

General Procedure for Preparation of N,N-Disubstituted-2-(1H-indol-3-yl)acetamides (3b, 4a-c) [11]

To a solution of 3-indolylacetic acid (1 eq.) and methyl 2,4,6-tribromo-3-aminobenzoate or 2-bromoaniline derivative (1 eq.) in dry CH2Cl2 was added N,N'-dicyclohexyl carbodiimide (DCC) (1 eq.). The reaction mixture was stirred for 5 hr at room temperature under argon atmosphere. The reaction mixture was then filtered and the filtrate was evaporated in vacuo to give the crude product which was purified by column chromatography (silica gel; hexane: EtOAc= 4:1) to afford a pure N- (substituted phenyl)-2-(1H-indol-3-yl)acetamide.

Methyl 3-(2-(1H-indol-3-yl)acetamido)-2,4,6-tribromobenzoate (3b)

According to the general procedure, treatment of 3-indolylacetic acid (0.090 g, 0.52 mmol) and 2,4,6-tribromo-3-amino benzoate (2b) (0.20 g, 0.52 mmol) in dry CH2Cl2 (26.0 mL) with DCC (0.10 g, 0.52 mmol) under argon atmosphere gave 3b as yellow oil (0.034 g, 12%): IR 1010, 1095, 1163, 1624, 1730, 2850, 2925, 3057, 3047 cm-1; 1H-NMR d 3.69 (s, 3H), 3.78 (s, 2H), 7.10-7.21 (m, 3H), 7.24 (s, 1H), 7.33 (d, J=7.5 Hz, 1H), 7.60 (d, J=7.8 Hz, 1H), 8.14 (br s, 1H, NH) ppm; 13C-NMR d 31.1, 51.9, 108.4, 111.2, 111.3, 118.6, 119.7, 119.8, 120.1, 122.1, 122.2, 122.5, 123.0, 123.4, 127.2, 136.1, 172.5 ppm; HRES-MS m/z calculated for [M+H]+ C18H13Br3N2O3= 564.8374, found 564.8304.

N-(2-Bromophenyl)-2-(1H-indol-3-yl)acetamide (4a) [11]

According to the general procedure, treatment of 3-indolylacetic acid (0.10 g, 0.57 mmol) and 2-bromoaniline (2c) (0.10 g, 0.58 mmol) in dry CH2Cl2 (25.0 mL) with DCC (0.24 g, 1.16 mmol) under argon atmosphere gave 4a as a yellow solid (0.078 g, 42%), mp. 95.9-104.5o: IR 1023, 1302, 1434, 1520, 1589, 1672, 3332 cm-1; 1H-NMR d 3.94 (s, 2H), 6.88 (td, J=1,2 Hz, 6.0 Hz, 1H), 7.16 (td, J=0.9 Hz, 6.9 Hz, 1H), 7.22-7.28 (m, 3H), 7.35 (dd, J=1.2 Hz, 8.1 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 8.03 (br s, 1H, NH), 8.37 (dd, J=1.2 Hz, 8.4 Hz, 1H), 8.51 (br s, 1H, NH) ppm; 13C-NMR d 34.66, 108.36, 111.39, 113.17, 118.82, 120.31, 121.34, 122.90, 124.01, 124.97, 126.95, 128.22, 132.09, 135.58, 136.45, 169.81 ppm.

N-(2-Bromophenyl)-N-methyl-2-(1H-indol-3-yl)acetamide (4b)

According to the general procedure, treatment of 3-indolylacetic acid (0.082 g, 0.47 mmol) and N-methyl-2-bromoaniline (2d) (0.085 g, 0.45 mmol) in dry CH2Cl2 (23.0 mL) with DCC (0.10 g, 0.48 mmol) under argon atmosphere gave 4b as a pale yellow solid (0.083 g, 53%), mp. 152.0- 154.5o: IR 564, 1383, 1432, 1582, 1651, 2882, 3301 cm-1; 1H-NMR d 3.22 (s, 3H), 3.51 (br s, 1H), 3.52 (br s, 1H), 6.95 (s, 1H), 7.03 (t, J=6.9 Hz, 1H), 7.11-7.37 (m, 6H), 7.69 (d, J=8.1 Hz, 1H), 8.03 (s, 1H, NH) ppm; 13C-NMR d 31.1, 36.1, 109.3, 110.9, 119.4, 121.9, 123.0, 124.0, 127.5, 128.8, 129.7, 130.1, 133.8, 135.9, 142.6 ppm; HRES-MS m/z calculated for [M+H]+ C17H16BrN2O= 365.0265, found 365.0260.

N-(2-Bromophenyl)-N-benzyl-2-(1H-indol-3-yl)acetamide (4c)

According to the general procedure, treatment of 3-indolylacetic acid (0.18 g, 1.01 mmol) and N-benzyl-2-bromoaniline (2e) (0.27 g, 1.0 mmol) in dry CH2Cl2 (50.0 mL) with DCC (0.20 g, 1.0 mmol) under argon atmosphere gave 4c as a pale yellow solid (0.293 g, 69%), mp. 146.3-148.3o: IR 699, 1030, 1583, 1654, 2850, 2928, 3059, 3313 cm-1; 1H-NMR d 3.51 (br s, 1H), 3.52 (br s, 1H), 4.06 (d, J=14.4 Hz, 1H), 5.66 (d, J=14.4 Hz, 1H), 6.67 (dd, J=1.5 Hz, 9.0 Hz, 1H), 6.94 (s, 1H), 7.00-7.37 (m, 11H), 7.68 (dd, J=1.5 Hz, 9.0 Hz, 1H), 8.08 (br s, 1H, NH) ppm; 13C-NMR d 31.3, 51.7, 109.1, 109.6, 110.9, 118.8, 119.3, 121.9, 123.1, 124.0, 127.4, 128.1, 128.2, 128.3, 129.3, 129.7, 131.7, 133.8, 134.136.0, 137.0, 140.8, 171.1 ppm; HRES-MS m/z calculated for [M+H]+ C23H19BrN2O= 441.0578, found 441.0578.

General Procedure for Preparation of N,N-Disubstituted-2-(1-substituted-1H-indol-3-yl)acetamides (5a-d)

A solution of N,N-disubstituted-2-(1H-indol-3-yl)acetamide (1 eq.) and KOH (1.5 eq.) in DMSO or DMF was stirred for 10-20 min. at room temperature under argon atmosphere. Benzyl chloride or methyl iodide (2 eq.) was then added at 0o. The reaction mixture was stirred at room temperature for 2-3 hr and then added to ice water and diluted with EtOAc or Et2O. The organic layer was washed with saturated NaCl solution, dried with anhydrous Na2SO4 and concentrated in vacuo to give the crude product which was purified by column chromatography (silica gel; hexane: EtOAc = 4:1) to afford a pure N,N-disubstituted-2-(1-substituted-1H-indol-3-yl)acetamide.

N-(2-Bromophenyl)-N-methyl-2-(1-methyl-1H-indol-3-yl)acetamide (5a)

According to the general procedure, treatment of N-(2-bromophenyl)-2-(1H-indol-3- yl)acetamide (4a) (0.088 g, 0.27 mmol) and KOH (0.090 g, 1.6 mmol) in DMSO (4.0 mL) with methyl iodide (0.20 g, 1.5 mmol) under argon atmosphere gave (5a) [11] as a colourless oil (0.033 g, 45%): IR 129, 740, 1373, 1476, 1583, 1651, 1666, 3024, 3054 cm-1; 1H-NMR d 3.21 (s, 3H), 3.49 (br s, 1H), 3.50 (br s, 1H), 3.70 (s, 3H), 6.84 (s, 1H), 6.99 (d, J=1.2 Hz, 1H), 7.01 (t, J=1.2 Hz, 1H), 7.14-7.33 (m, 5H), 7.68 (dd, J=1.2 Hz, 7.8 Hz, 1H) ppm; 13C-NMR d 30.85, 32.57, 36.10, 107.50, 108.96, 118.79, 118.88, 121.39, 123.58, 127.78, 128.78, 129.63, 130.14, 133.80, 136.80, 142.92, 166.10 ppm; HRES-MS m/z calculated for [M+H]+ C18H17BrN2O= 356.0524, found 356.0524.

N-(2-Bromophenyl)-N-methyl-2-(1-benzyl-1H-indol-3-yl)acetamide (5b)

According to the general procedure, treatment of N-(2-bromophenyl)-N-methyl-2-(1H-indol- 3-yl)acetamide (4b) (0.24 g, 0.69 mmol) and KOH (0.090 g, 1.6 mmol) in DMF (7.0 mL) with benzyl chloride (0.20 mL, 1.73 mmol) under argon atmosphere gave 5b as a pale yellow oil (0.10 g, 34%): IR 1464, 1664, 2923 cm-1; 1H-NMR d 3.21 (s, 3H), 3.51 (br s, 1H), 3.52 (br s, 1H), 5.22 (s, 2H), 6.83 (s, 1H), 7.01 (t, J=0.8 Hz, 1H), 7.02 (d, J=0.8 Hz, 1H), 7.12 (td, J=2.3 Hz, 7.8 Hz, 4H), 7.17-7.27 (m, 5H), 7.39 (d, J=7.8 Hz, 1H), 7.62-7.66 (m, 1H) ppm; 13C-NMR d 31.2, 36.1, 49.9, 77.2, 108.3, 109.4, 119.1, 119.2, 121.6, 123.6, 126.9, 127.1, 127.5, 128.0, 128.6, 128.7, 129.6, 130.2, 133.8, 136.4, 137.6, 142.8, 171.1 ppm; HRES-MS m/z calculated for [M+H]+ C24H21BrN2O = 455.0735, found 455.0732.

N-Benzyl-N-(2-bromophenyl)-2-(1-methyl-1H-indol-3-yl)acetamide (5c)

According to the general procedure, treatment of N-(2-bromophenyl)-N-benzyl-2-(1H-indol- 3-yl)acetamide (4c) (0.29 g, 0.69 mmol) and KOH (0.10 g, 1.78 mmol) in DMSO (6.0 mL) with methyl iodide (0.10 mL, 1.60 mmol) under argon atmosphere gave 5c as a yellow oil (0.15 g, 51%): IR 561, 1474, 1583, 1661, 2925, 3058 cm-1; 1H-NMR d 3.51 (br s, 1H), 3.52 (br s, 1H), 3.70 (s, 3H), 4.05 (d, J=14.4 Hz, 1H), 5.68 (dd, J=1,2 Hz, 7.2 Hz, 1H), 6.85 (S, 1H), 7.01 (t, J=7.2 Hz, 1H), 7.10 (tt, J=1.2 Hz, 1H), 7.16-7.32 (m, 9H), 7.68 (d, J=7.8 Hz, 1H) ppm; 13C-NMR d 31.2, 32.6, 51.7, 107.4, 107.9, 118.8, 118.9, 121.4, 124.0, 124.9, 127.4, 127.8, 127.9, 128.9, 128.1, 128.3, 129.3, 129.4, 129.6, 133.7, 133.8, 137.0, 140.8, 171.1 ppm; HRES-MS m/z calculated for [M+H]+ C24H21BrN2O= 455.0735, found 455.0748.

N-(2-Bromophenyl)-N-benzyl-2-(1-benzyl-1H-indol-3-yl)acetamide (5d)

According to the general procedure, treatment of N-(2-bromophenyl)-N-benzyl-2-(1H-indol- 3-yl)acetamide (4c) (0.10 g, 0.24 mmol) and KOH (0.020 g, 0.36 mmol) in DMF (2.4 mL) with benzyl chloride (0.060 g, 0.48 mmol) under argon atmosphere gave 5d as a pale yellow oil (0.10 g, 86%): IR 698, 1585, 1613, 1660, 2849, 2917, 3060 cm-1; 1H-NMR d 3.51 (br s, 1H), 3.52 (br s, 1H), 4.04 (d, J=14.4 Hz, 1H), 5.22 (s, 2H), 5.64 (d, J=14.4 Hz, 1H), 6.62 (dd, J=1.5 Hz, 7.8 Hz, 1H), 6.86 (s, 1H), 6.98-7.27 (m, 15H), 7.37 (d, J=8.1 Hz, 1H), 7.63 (dd, J=1.2 Hz, 7.8 Hz, 1H) ppm; 13C-NMR d 31.5, 49.9, 51.6, 108.2, 109.4, 119.1, 121.7, 124.0, 126.9, 127.1, 127.4, 127.5, 128.0, 128.3, 128.6, 129.3, 129.6, 131.7, 133.7, 136.4, 137.0, 137.6, 140.8, 171.0 ppm; HRES-MS m/z calculated for [M+H]+ C30H25BrN2O= 531.1048, found 531.1053.

PLANT GROWTH PROMOTING ACTIVITIES

Methods

The methods of Vargas et al. [12] and Fulbright et al. [13] were employed. Solutions of the synthesised compounds or IAA in 1.7, 0.17, 0.017 and 0.0017 ppm concentrations were prepared in H2O-MeOH (H2O: MeOH = 99.95: 0.05). Rice (Oryza sativa) seeds were surface disinfected by 70% ethanol for 1 min., followed by 1% sodium hypochlorite for 10 min. They were then washed five times with sterile distilled water. The sterilised seeds were immersed in sterile distilled water overnight. Sterilised Petri dishes (9.0-cm diameter), each containing 20 seeds, were used for germination experiments. Each dish contained two sheets of filter paper which were moistened with 4.0 mL of water or a solution of a test compound. The Petri dishes were placed under artificial light for 24 hr at 30°. The shoot growth and root growth were determined on day 5. Each experiment was done in triplicate.

Results and Discussion

Plant growth regulator activities of the selected compounds (3a-b, 4a, 4c, 5a-b and 5d) are shown in Table 1 and Figures 1-2 in comparison to the commercial plant growth regulator (IAA). Compounds 3b and 5b exhibit higher activities than IAA on both shoot and root growth in the concentration range of 1.7-0.017 ppm, while compounds 3b, 5b and 5d exhibit better activities than IAA on root growth in the same range of concentration. Maximum shoot growth was observed in the presence of 4a at the concentration of 1.7 ppm and maximum root growth was observed in the presence of 5b at the same concentration.

From Table 1, it can be seen that the methyl ester group and a number of bromine atoms on the benzene ring have no effect on the growth promoting activities. Compounds 3a and 3b show slight difference in activities on both root and shoot while the substituents on the nitrogens of acetamide and indole affect the activities. Compound 4a, which has no N-substituents, shows better growth promoting activities at low concentration than compound 4c, which carries a benzyl group at the amide nitrogen. However, compound 4c can stimulate the root growth at high concentration (1.7 ppm). The benzyl substituent on the indole nitrogen (5d) shows fluctuation in activities compared to compound 4c. The methyl substituent on the acetamide nitrogen (5a) imparts better growth promoting activities on both shoot and root than the benzyl substituent (5b) in the concentration range of 1.7-0.17 ppm while the reverse is the case in the concentration range of 0.017-0.0017 ppm.

CONCLUSIONS

Although the plant growth regulator activities of some of the synthesised 3-indolylacetamide derivatives are higher than those of IAA in the concentration range of 1.7-0.017 ppm, the overall activities of the derivatives are considered comparable to those of IAA.

ACKNOWLEDGEMENTS

We are grateful to the Department of Chemistry, Faculty of Science, Silpakorn University and the Thailand Toray Science Foundation for financial support. We also thank Kasetsart University Research and Development Institute and the National Research Council of Thailand for financial support to W.P.

References

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