Synthesis of 2-Methylcamalexin

Abstract

2-methylcamalexin, a novel derivative of the phytoalexin Camalexin, was synthesized for the first time, using a convenient two-step approach. The approach realizes coupling of two aromatic heterocyclic moieties (2-methylindole and thiazole) by sequential α-amidoalkylation/oxidative re-aromatization. The target product was obtained in a cost-effective manner, with 88% yield over two steps. The structure of the synthesized product was unequivocally determined on the basis of NMR, HRMS and FTIR spectral measurments.

1. Introduction

Phytoalexins are low-molecular-weight molecules synthesised by plants in reaction to infection or stress. They are part of the active defence mechanisms in plants. A metabolite must demonstrate an antimicrobial effect to be designated as a phytoalexin [1]. The prospective use of phytoalexins to enhance plant resistance to several diseases is attracting significant attention [2,3]. Camalexin and 1-methylcamalexin for example are notable for their antimicrobial activities [4,5,6,7,8,9,10,11,12] (Figure 1). Furthermore, some authors highlight Camalexin’s remarkable antiproliferative profile against various cancer cell lines, motivating its potential application as a chemotherapeutic agent [13,14,15,16,17]. 1-methylcalamexin also exhibits potent cytotoxic activity against the human breast cancer cell line SKBr3 with IC₅₀—3.8 µM [18].

In nature, phytoalexins are generally biosynthesised in small quantities (e.g., 1–5 mg per kilogramme of fresh plant tissue), rendering their extraction from stressed plant material difficult when greater quantities are necessary. Chemical synthesis can provide the quantities of phytoalexins required for extensive screening and assessment of their biological action. Chinese researchers have recently synthesised a range of camalexin derivatives to explore their antiviral and fungicidal effects. Included among the synthesised compounds is the methylated phytoalexin 1-methylcamalexin, isolated in a modest yield of 43%, which demonstrates significant phytotoxic and antiviral properties [19].

Despite the apparent structural simplicity of the 1- and 2-methylated analogues, their synthesis has posed considerable difficulties, and all published procedures of 1-methylcamalexin are limited with regard to yield, scalability, or the accessibility of necessary reagents [11,19]. 2-methylcamalexin has not been synthesized so far by any published method, although it is of considerable interest.

One-pot, three-component α-amidoalkylation reactions involving thiazole, benzothiazole, and imidazole are the basis of a novel method, developed by us, for the synthesis of the phytoalexin Camalexin and its analogues—benzocamalexin, azacamalexin, and various oxy-camalexins [20,21,22]. The scale-up potential of this two-step approach makes it particularly interesting for the synthesis of novel analogues in larger quantities for bioassays and field studies of possible agrochemical applications. In this note we describe the application of our method to the preparation of 2-methylcamalexin.

2. Results

Thiazole and 2,2,2-trichloroethyl chloroformate (Troc-Cl) were used as the starting materials. These compounds readily reacted at 0 °C in dichloromethane as the solvent to provide the active acyliminium electrophilic species 2. Without isolation of 2, 2-methylindole and triethylamine were immediately added to the same reaction vessel. The ensuing electrophilic substitution at position 3 of the indole ring furnished the intermediate 3, which was isolated in 92% yield (Scheme 1).

In the next stage, the thiazole moiety in compound 3 was re-aromatised by oxidation with equimolar amount of o-chloranil (3,4,5,6-tetrachloro-1,2-benzoquinone). This oxidative rearomatisation proceeded quickly in acetonitrile at room temperature, providing the targeted 2-methylcamalexin (4) with a quantitative 96% yield in only 15 min. Thus, the achieved overall yield of the final product 4 is 88% over two steps.

The structure of the obtained intermediate 3 and that of the final product 4 were determined on the basis of their NMR, FTIR, and HRMS spectra. A notable feature in the NMR spectra of 3 at 25 °C is the significant broadening of some signals (particularly those of the Troc CH₂ protons) due to slow rotameric interconversion with rotation around the N-CO bond of the Troc carbamate group. To resolve this, the NMR spectra were measured at 80 °C in DMSO-d₆, which helped to reveal a clearer signal for the AB spin system of the diastereotopic Troc CH₂ protons. The obtained data were in full agreement with the proposed structure of 3, with the sp³ C-2′ thiazole signal appearing at δ = 6.99/60.0 ppm (¹H/¹³C), as indicated by HSQC. The NMR spectra of the final product 4 were measured at 25 °C in two different solvents (CDCl₃ and DMSO-d₆) and indicated only aromatic sp^{2 13}C and ¹H signals, except for those corresponding to the NH and CH₃ groups in the indole moiety. Full NMR spectral assignment of 4 was done in DMSO-d₆, using 2D correlations (NOESY, COSY, HSQC, HMBC) and the data is presented in

Table 1. Full NMR assignment of 2-methylcamalexin (4) (600/150 MHz ¹H/¹³C, DMSO-d₆, δ ppm, J Hz). Heteronuclear multiple bond correlations (HMBC) are from proton(s) to the stated carbon.

Position	1H	13C	HMBC
1	11.64, s	-	2, 8, 9, 3
2	-	137.60
3	-	107.44
4	8.16, m	119.88	8, 6
5	7.16, m	120.83	7
6	7.16, m	122.00	4
7	7.40, m	111.57	9, 5
8	-	135.46
9	-	126.27
10	2.74, s	14.43	3, 2, 2′, 9
2′	-	163.29
4′	7.88, d, J = 3.3	142.67
5′	7.59, d, J = 3.3	116.35	2′, 4′

and Figure 2 (for full spectra see Supplementary Materials S1).

3. Materials and Methods

All reagents and solvents were obtained from commercial suppliers (Merck, Darmstadt, Germany) and used as supplied, with no additional purification. Melting points were determined on a Krüss M5000 device (A.Krüss Optronic GmbH, Hamburg, Germany). FTIR spectra were measured on an Alpha II FTIR spectrometer (Bruker Optics GmbH, Ettlingen, Germany) with an attenuated total reflection (ATR) accessory, with absorption frequencies given in inverse centimetres (cm⁻¹). HRMS measurements were performed on a Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were measured on a Bruker Avance NEO AV600 spectrometer (Bruker, Billerica, MA, USA) chemical shifts (δ, ppm) are reported downfield from tetramethylsilane (TMS) and J-constants are given in Hz. Thin-layer chromatography was done on Merck Silica gel 60, 0.2 mm aluminum backed plates. Neutral alumina 90 active (0.063–0.200 mm) was used for column chromatography.

3.1. Synthetic Procedure for the Synthesis of 2,2,2-Trichloroethyl 2-(2-methyl-1H-indol-3-yl)thiazole-3(2H)-carboxylate (3)

2,2,2-trichloroethyl chloroformate (Troc-Cl) (2.4 mmol, 0.33 mL) was slowly added with magnetic stirring to a solution of thiazole (2 mmol, 0.14 mL) in dry dichloromethane (CH₂Cl₂, 12 mL) at 0 °C (ice bath). Then, 2-methylindole (2 mmol, 0.262 g) was added to the reaction mixture. Next, triethylamine (Et₃N, 2 mmol, 0.28 mL) dissolved in dry dichloromethane (CH₂Cl₂, 2 mL) was gradually added in the course of 20 min. The reaction mixture was stirred for an additional 10 min. After completion of the reaction (monitored by TLC), the mixture was transferred to a separatory funnel with dichloromethane (30–40 mL), and then extracted consecutively with equal volumes of aqueous HCl (8%), Na₂CO₃ (3%) and water. The combined organic layer was dried over anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The product 2,2,2-trichloroethyl 2-(2-methyl-1H-indol-3-yl)thiazole-3(2H)-carboxylate (3) was isolated by column chromatography on neutral alumina, using a 2:1 mixture of petroleum/diethyl ether as the eluent and the increasing polarity to 1:1, yield: 92% (0.721 g), white solid, mp: 167–169 °C.

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 2.45 (s, 3H, -CH₃), 4.76 (d, J = 12.2 Hz, 1H, -COOCH₂CCl₃), 4.79 (d, J = 12.2 Hz, 1H, -COOCH₂CCl₃), 6.19 (d, J = 4.9 Hz, 1H), 6.71 (d, J = 4.9 Hz, 1H), 6.96 (app t, J = 7.5 Hz, 1H), 6.99 (s, 1H, *CH, thiazole), 7.03 (app t, J = 7.5 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 10.81 (s, 1H, -NH).

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 11.9, 60.0, 75.1, 95.9, 106.1(C-Ar), 111.2, 119.0, 119.3, 120.8, 121.0, 126.1, 134.0, 136.0, 151.1.

ATR-FTIR (cm⁻¹): ν(N-H) 3413; ν(C=O) 1703; ν(Csp2-H) 3089; ν(C=C) 1394, 1461, 1597; ν(C-O) 1055, 1129; ν(C-C-O) 1258; ν(C-O-C) 1322, 1340; ν(C-N) 787, 877; ν(C-S-C) 746.

HRMS m/z (ESI): calcd. for C₁₅H₁₃Cl₃N₂NaO₂S⁺ [M + Na]⁺ 412.9656; 414.9626, found 412.9647; 414.9612.

3.2. Synthetic Procedure for Oxidative Rearomatization of Compound 3 to 2-Methylcamalexin (4)

The N-alkoxycarbonyl compound 3 (0.5 mmol, 0.196 g) was dissolved in acetonitrile (CH₃CN, 10 mL), followed by the addition of the oxidant o-chloranil (0.5 mmol, 0.123 g). The reaction mixture was then stirred magnetically for 15 min at room temperature. Upon completion of the reaction, which was monitored by TLC, the solvent was evaporated under reduced pressure, and the mixture was subsequently dry-loaded onto 2 g neutral alumina. Column chromatography on a short column using petroleum/diethyl ether as the eluent (1:1, increasing polarity to diethyl ether) gave 2-methylcamalexin (4) as a pale yellow oil, yield 96% (0.103 g).

¹H-NMR (600 MHz, 25 °C, DMSO-d₆, δ ppm, J Hz): 2.74 (s, 3H), 7.16 (m, 2H), 7.40 (m, 1H), 7.59 (d, J = 3.3 Hz, 1H), 7.88 (d, J = 3.3 Hz, 1H), 8.16 (m, 1H), 11.64 (s, 1H, NH).

¹³C{¹H}-NMR (150 MHz, 25 °C, DMSO-d₆, δ ppm): 14.4, 107.4, 111.6, 116.3, 119.9, 120.8, 122.0, 126.3, 135.5, 137.6, 142.7, 163.3.

¹H-NMR (600 MHz, 25 °C, CDCl₃, δ ppm, J Hz): 2.56 (s, 3H), 7.08–7.11 (m, 1H), 7.13–7.16 (m, 2H), 7.19 (d, J = 3.4 Hz, 1H), 7.80 (d, J = 3.4 Hz, 1H), 8.12 (m, 1H), 8.46 (s, 1H, NH).

¹³C{¹H}-NMR (150 MHz, 25 °C, CDCl₃, δ ppm): 14.0, 108.3, 110.6, 115.9, 119.8, 121.1, 122.2, 126.5, 135.0, 136.2, 142.2, 163.6.

ATR-FTIR (cm⁻¹): ν(N-H) 3392; ν(Csp2-H) 3112, 3071; ν(C=C) 1461, 1498, 1556; ν(C-N) 867, 1248, 1299; ν(C-S-C) 740.

HRMS m/z (ESI): calcd. for C₁₂H₁₁N₂S⁺ [M + H]⁺ 215.0637, found 215.0629.