Methyl 6-(2,3-Dimethoxybenzamido)-2,3-dihydroxybenzoate

Abstract

Gymnaconitum gymnandrum, a Tibetan medicinal herb, is distributed across high-altitude regions of China. Despite its high toxicity, it exhibits analgesic, anti-inflammatory, antitumor, and other pharmacological activities. It is used topically for skin conditions and orally for rheumatism. Its main bioactive components are alkaloids. A new organic amine alkaloid has been isolated from the plant and structurally identified using HRMS, NMR, and X-ray diffraction.

1. Introduction

Gymnaconitum gymnandrum Maxim (syn. Aconitum gymnandrum Maxim), the sole species of the genus Gymnaconitum [1], is known in Tibetan medicine as “Gaoudiluo” or “Silanao Manba”, and also as “Luotieba” and “Zelan”. This species is predominantly distributed in the Tibet Autonomous Region, Qinghai, Southern Gansu, and Western Sichuan, where it commonly grows on montane grassy slopes, field margins, and riverside sandy soils at altitudes of 1550–3800 m [2]. The entire herb is used in Tibetan medicine, being pungent and warm in property but highly toxic. Modern pharmacological investigations have revealed its diverse activities, including analgesic, anti-inflammatory, antitumor, cardiotonic, immunomodulatory, and antimicrobial/insecticidal effects [3,4]. Phytochemical studies have shown that diterpenoid alkaloids (DAs) are regarded as the characteristic secondary metabolites of the genera Aconitum and Delphinium. So far, more than 1500 diterpenoid alkaloids have been isolated and structurally characterized from these plants, exhibiting a wide range of pharmacological activities, including anti-inflammatory [5], analgesic [6], and antiarrhythmic [7] effects. However, the biological activities attributed to DAs alone do not adequately explain the full spectrum of efficacy observed for Aconitum- and Delphinium-derived medicinal materials, especially those of G. gymnandrum. In recent years, many other ingredients have been isolated and identified from Aconitum and Delphinium, including terpenoids [8], flavonoids [8], and phenolic acids [9] and non-diterpenoid alkaloids such as amides and isoquinolines [10]. Non-diterpenoid alkaloids have demonstrated significant bioactivities, encompassing antibacterial [11], antitumor [12], and antioxidant effects [13]. As part of our ongoing investigation into the chemical constituents of G. gymnandrum, the isolation and structural identification of these compounds from this plant were carried out. This work has led to the discovery of an unreported benzamine. The typical structure of such a component is an amide formed by the amino group in anthranilate (A ring) replacing the carboxyl group of another benzoic acid (B ring). The structure of the new benzamine was determined by HRMS, NMR spectroscopy, and unequivocally confirmed by single-crystal X-ray diffraction. Herein, we disclose the isolation and full spectroscopic characterization of this compound.

2. Results

Compound 1 was obtained as pale yellow crystals, which exhibited strong fluorescence under UV light (254 nm) and gave a reddish-brown colour upon spraying with modified bismuth potassium iodide solution, suggesting that it might be an alkaloid. The molecular formula of compound 1 was established as C₁₇H₁₇NO₇ on the basis of HR-ESI-MS, which afforded a protonated molecular ion at m/z 348.1087 [M + H]⁺ (calcd. for C₁₇H₁₈NO₇, 348.1078; Δ = 2.6 ppm) (Supplementary Materials Figure S1). The IR spectrum (Figure S2) revealed characteristic absorptions for amino/hydroxyl groups (3486, 3330 cm⁻¹), conjugated aromatic C=C bonds (1646 cm⁻¹), and C–O linkages (1253 cm⁻¹). The ¹H NMR spectrum (Figure S3) of 1 displayed resonances for three protons in an aromatic moiety appearing as an ABC spin system at δ_H 7.52 (dd, J = 7.6, 1.8 Hz, H-6′), 7.23 (dd, J = 8.2, 1.8 Hz, H-4′), and 7.19 (dd, J = 8.2, 7.6 Hz, H-5′) (

Table 1. ¹H NMR and ¹³C NMR data of compound 1 (δ in ppm, J in Hz).

Position	1H NMR	13C NMR
1		108.9, C
2		150.1, C
3		144.4, C
4	7.01, d, (8.8)	120.1, CH
5	7.64, d, (8.8)	116.0, CH
6		130.7, C
7		171.0, C
1′		128.8, C
2′		148.8, C
3′		154.4, C
4′	7.23, dd, (8.2, 1.8)	117.1, CH
5′	7.19, dd, (8.2, 7.6)	125.6, CH
6′	7.52, dd, (7.6, 1.8)	123.0, CH
7′		166.0, C
7-OCH3	3.98, s	53.3, CH3
2′-OCH3	3.93, s	62.0, CH3
3′-OCH3	3.92, s	56.6, CH3

). The other spin system contained two ortho-coupled protons at δ_H 7.64 (d, J = 8.8 Hz, H-5) and 7.01 (d, J = 8.8 Hz, H-4). In addition, three methoxy protons appeared as singlet signals at (δ_H 3.98, 3.93, 3.92, each 3H, s). The ¹³C NMR spectrum (Figure S4) displayed 17 carbon resonances, including two carbonyl carbons (δ_C 171.0, 166.0), 12 aromatic carbons (δ_C 154.4, 150.1, 148.8, 144.4, 130.7, 128.8, 125.6, 123.0, 120.1, 117.1, 116.0, 108.9), and three methoxy carbons (δ_C 62.0, 56.6, 53.3).

These data (Table 1) suggested that compound 1 might be composed of two substituted aromatic units linked through an amide bond. The ¹H–¹H COSY spectrum (Figure S6) revealed two independent spin-coupling systems, which were identified as H-4/H-5 and H-4′/H-5′/H-6′, indicating the presence of two separate aromatic rings with different substitution patterns (Figure 1). The HMBC correlations (Figure S7) from H-4 to C-2 and C-6, and from H-5 to C-1 and C-3 defined the substitution pattern of the first aromatic ring as a 1,2,3-trisubstituted benzene bearing a methyl ester at C-7 and hydroxy groups at C-2 and C-3. For the second aromatic ring, the HMBC correlations from H-4′ to C-2′, H-5′ to C-1′ and C-3′, and H-6′ to C-7′ established a 1,2,3-trisubstitution pattern with two methoxy groups at C-2′ and C-3′, and a carbonyl-bearing carbamoyl substituent at C-1′. The HMBC correlation from the exchangeable amide proton (δ_H 10.28, br s) to the carbonyl carbon C-7′ (δ_C 166.0) confirmed the presence of a benzamide unit. Furthermore, the connection between the two aromatic moieties was established by the HMBC correlation from the amide N–H to C-6 (δ_C 130.7) of the methyl dihydroxybenzoate ring, which unambiguously demonstrated that the two units are linked via a –NH–CO– bridge between the C-6 position of methyl 2,3-dihydroxybenzoate and the 2,3-dimethoxybenzoyl carbonyl group. The locations of the three methoxy groups were determined by HMBC correlations from 7-OCH₃ to C-7, 2′-OCH₃ to C-2′, and 3′-OCH₃ to C-3′. The hydroxy groups at C-2 and C-3, on the other hand, were assigned based on the distinctive chemical shifts in their oxygen-bearing aromatic carbons (δC 148.8 and 154.4).

The NMR data of 1 were similar to those of delphiniumine E [14], with the exception of the difference in¹³CNMR chemical shift in a more deshielded methoxy group (δ_C 53.3 in 1 vs. δ_C 61.9 in delphiniumine E), suggesting that the methoxy group was at C-7 in 1 instead of at C-2 in delphiniumine E. This deduction was supported by the HMBC correlations from the methoxy protons (δ_H3.98) to C-7 (δ_C171.0). The structure of 1 was further supported by single-crystal X-ray diffraction analysis using CuKα radiation (Figure 2, CCDC 2548037). Thus, compound 1 was determined and named as methyl 6-(2,3-dimethoxybenzamido)-2,3-dihydroxybenzoate, an unreported benzamine.

3. Discussion

In this study, a benzylamine alkaloid featuring a 2,3-dihydroxybenzoate methyl ester moiety linked to a 2,3-dimethoxybenzamido unit via an amide bond was isolated from Gymnaconitum gymnandrum. The chemical structure was unequivocally established by IR, HRMS, NMR, and single-crystal X-ray diffraction analyses. This structural type is relatively uncommon among natural products and is characterized by multiple functional groups including phenolic hydroxyls, a methyl ester, and an amide, which confer potential bioactive properties. Notably, single-crystal X-ray diffraction not only corroborated the above spectroscopic assignments but also unambiguously revealed the absolute configuration and the intramolecular hydrogen-bonding network of the compound. The crystal structure disclosed that an intramolecular hydrogen bond (O–H···O=C; phenyl–H···O=C; N–H···OCH₃ ) is formed between the ortho-phenolic hydroxyl and the ester carbonyl oxygen within the 2,3-dihydroxybenzoate methyl ester moiety, which may provide a protective effect on the methyl ester group under specific conditions. Meanwhile, the amide bond exhibits good planarity, indicating a certain degree of conformational rigidity, which likely serves as the structural basis for the compound’s enhanced stability. To further define the stability boundaries of this compound under varying pH conditions, we conducted a series of controlled experiments in which the pure compound was individually exposed to buffered solutions at pH 2, 5, 7, 9, and 10 at 25 °C for 24 h. The results demonstrated that the compound was most stable within the pH 5–7 range, with recovery exceeding 95%, consistent with the protective effects conferred by the intramolecular hydrogen bond and the rigid amide conformation, wherein the ester group receives optimal protection within this pH interval. In contrast, at pH 2 and pH 10, minor degradation products (relative peak area < 5%) were detected after 24 h.

4. Materials and Methods

4.1. General Experimental Procedures

The melting points of the crystals were measured by a micro melting point apparatus featuring an X-4 digital display system (Beijing Tech Instrument Co., Ltd., Beijing, China). The IR data were recorded on a VERTEX 70V spectrometer (Bruker Optics, Ettlingen, Germany). The HRESIMS were obtained using an APEX II instrument (Bruker, Karlsruhe, Germany); the NMR data were acquired on a Bruker 500 MHz AVANCE NEO spectrometer (Bruker, Karlsruhe, Germany). Single-crystal X-ray diffraction data were collected on a ROD Synergy Custom system, HyPix diffractometer with Cu Kα radiation. Sephadex LH-20 (Amersham Pharmacia Biotech, Buckinghamshire, UK), macroporous resin HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), and silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). Thin-layer chromatography (TLC) was performed on fluorescent silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Compound spots on the plates were visualized in situ under UV irradiation at 254 nm, or alternatively, by spraying with a 5% ethanolic sulfuric acid solution followed by heating to effect carbonization for detection.

4.2. Plant Material

The whole plant of Gymnaconitum gymnandrum Maxim. was collected in August 2021 from Guide County (36°2′46.1″ N, 101°25′46.8″ E), Hainan Tibetan Autonomous Prefecture, Qinghai Province, China. The botanical identity was authenticated by Prof. Dr. Quesheng, a third-level senior professor at the College of Ethnic Minorities, Qinghai Normal University. A voucher specimen (No. LRWT-20210917) has been deposited in the Institute of Natural Product Development, Lanzhou Jiaotong University.

4.3. Extraction and Isolation

The air-dried whole plants of Gymnaconitum gymnandrum Maxim. (16.7 kg) were pulverized to a coarse powder using a mechanical grinder and were subsequently subjected to maceration with 95% EtOH (50 L) at room temperature for three cycles, each of 7 days’ duration, with intermittent stirring being applied to ensure adequate mass transfer. The combined macerates were filtered under vacuum to remove insoluble particulate matter, and the resulting filtrate was concentrated to dryness in vacuo on a rotary evaporator at 50 °C, by which a dark brown crude ethanol extract (1.54 kg) was afforded. This crude residue was then suspended in distilled water (2 L) at 60 °C with vigorous stirring to obtain a homogeneous suspension. Under continuous agitation, the pH of the suspension was adjusted to 2.0 with 5% aqueous HCl, by which protonation of the alkaloidal constituents was effected, and they were consequently converted into their water-soluble salt forms and transferred into the aqueous phase. The acidified aqueous layer was successively partitioned against petroleum ether (bp 60–90 °C, 3 × 2.0 L) and ethyl acetate (3 × 2.0 L) to sequentially remove lipophilic and moderately polar non-alkaloidal impurities, respectively. Following complete removal of these interfering components, the purified aqueous phase was basified with 10% NH₄OH to pH 9–10, whereby the alkaloids were liberated as free bases. The liberated bases were then exhaustively extracted with CH₂Cl₂ (4 × 2.0 L), and the combined organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure, yielding the total alkaloid fraction as a yellowish-brown amorphous powder (142.0 g).

The total alkaloid fraction (142.0 g) was subjected to column chromatography (CC) over pretreated macroporous resin (D101 type, 10 × 120 cm) and eluted stepwise with MeOH–H₂O gradients (0%, 30%, 50%, 80%, and 100% MeOH, each 10.0 L) at a flow rate of 30 mL/min. The resulting eluates were concentrated under reduced pressure to afford five main fractions, designated Fr.1–Fr.5, corresponding to 0%, 30%, 50%, 80%, and 100% MeOH, with weights of 27.8, 16.2, 29.6, 17.1, and 14.6 g, respectively. Among these, Fr.4 (17.1 g), which displayed a complex alkaloidal profile with multiple well-resolved spots on TLC, was selected for further purification. It was applied to a normal-phase silica gel column (200–300 mesh, 5 × 90 cm) and eluted with a stepwise gradient of petroleum ether–acetone (80:1, 50:1, 20:1, 10:1, 5:1, and 0:1, v/v), with 1% diethylamine added to each eluent as a modifier to improve peak symmetry and minimize tailing of the basic alkaloids. Fractions of 250 mL were collected and monitored by TLC using petroleum ether–acetone–diethylamine (10:2:0.1) as the mobile phase. On the basis of TLC profiles, fractions sharing identical Rf values and comparable staining characteristics were pooled to yield six subfractions (D1–D6). Subfraction D3 (2.3 g), which showed a major alkaloid spot on TLC, was re-chromatographed over a second normal-phase silica gel column (300–400 mesh, 2.5 × 60 cm) eluted with petroleum ether–acetone gradients (30:1 and 20:1, each containing 1% diethylamine) to achieve finer separation. The desired fractions were combined according to TLC monitoring and concentrated to afford compound 1 (35.0 mg), which was obtained as pale yellow crystals from acetone.

Compound 1, mp 240−242 °C, IR (KBr) νmax 3486, 3330, 2960, 2839, 1646, 1533, 1432, 1253, 992, 746 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) and ¹³C NMR data (126 MHz, CDCl₃), see Table 1; HRESIMS: m/z 348.1087, [M + H]⁺ (calcd for C₁₇H₁₈NO₇, 348.1078).

Crystal Data for 1: C₁₇H₁₇NO₇ (M = 347.31 g/mol): monoclinic, space group P2₁/n (no. 14), a = 17.4691(3) Å, b = 10.23868(14) Å, c = 18.1176(2) Å, β = 108.0055(15)°, V = 3081.82(8) ų, Z = 8, T = 149.8(7) K, μ(Cu Kα) = 0.997 mm⁻¹, Dcalc = 1.497 g/cm³, 31,593 reflections measured (8.458° ≤ 2θ ≤ 152.08°), 6165 unique (R_int = 0.0278, R_sigma = 0.0167) which were used in all calculations. The final R₁ was 0.0534 (I > 2σ(I)), and wR₂ was 0.1478 (all data). The crystallographic data have been deposited at the Cambridge Crystallographic Data Centre with deposition number CCDC 2548037 (Table S1). These data can be obtained free of charge via https://www.ccdc.cam.ac.uk (accessed on 21 April 2026).