(4R,4aS,6bR,8aR,12bS,14aS)-2-((E)-2-Bromo-4-chlorobenzylidene)-4,4a,6b,8a,11,11,12b,14a-octamethylicosahydropicen-3(2H)-one

Abstract

Friedelin, a pentacyclic triterpene, has been reported to inhibit potential reactive oxygen species (ROS)-scavenging activity. Accordingly, we modified the structure of this compound with the aim of obtaining derivatives. A new derivative (compound 4), with an α,β-unsaturated ketone moiety, was synthesized via an aldol condensation. Structural characterization of this compound was performed using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry.

1. Introduction

Natural products (NPs) and their derivatives account for a significant proportion of pharmaceuticals that successfully achieve regulatory approval [1]. Plant-derived NPs, ranging from aspirin to taxol, continue to be a source of novel molecules for medicinal chemistry, providing ongoing inspiration for researchers. Triterpenes constitute a vast and structurally diverse class of NPs and exhibit remarkable pharmaceutical activities. With four triterpenes presently approved for clinical use, three of them have been developed via semi-synthesis [2,3].

ROS are critical mediators, functioning as crucial signaling molecules in normal physiology and are also being implicated in disease etiology [4]. And ROS are integral to numerous physiological processes; their targeted scavenging represents a rational treatment approach for ROS-related diseases. A diverse array of inhibitors, including nonselective scavengers, site-specific ROS inhibitors, and ferroptosis inhibitors, have been reported [5]. Compounds 1 [6] and 2 [7] (Figure 1) share certain structural similarities, notably halogen-substituted phenyl groups that readily access hydrophobic pockets, coupled with a relatively rigid region. Among these, compounds 1 and 2 have previously been identified as monoamine oxidase (MAO) inhibitors.

Friedelin (compound 3), a pentacyclic triterpene (Figure 2) first isolated in 1807 [8], has a diverse range of pharmacological activities, including antioxidant [9], antimicrobial [10], and anticancer [11] activities. Christstudas [9] reported that friedelin exhibited potent ROS scavenging activity in vitro and significant hepatoprotective effects in vivo, indicating its potential as a candidate for further structural optimization. Inspired by the structures of the two reported ROS inhibitors and friedelin, we designed an alkali-catalyzed aldol condensation as an efficient method to construct derivatives featuring an α,β-unsaturated ketone moiety in one step.

2. Results and Discussion

Compound 4 was synthesized via aldol condensation (Scheme 1).

Data Analysis

Column chromatography on silica gel (R_f = 0.6 petroleum ether/ethyl acetate = 15/1) provided 4 (12 mg, yielding 67%). Compound 4 was isolated as a white amorphous powder, with the molecular formula C₃₇H₅₂BrClO confirmed by HR-ESI-MS at m/z 627.2970 [M + H]⁺ (calc. 627.2963). The IR spectra exhibited a characteristic absorption band at 1690 cm⁻¹, which was consistent with the presence of a carbonyl group (δ_C 204.3). The ¹H NMR spectra indicated the presence of 2,4-disubstituted benzene [δ_H 7.63 (d, J = 2.1 Hz, 1H), 7.30 (dd, J = 8.3, 2.1 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H)], as well as eight methyl groups [δ_H 1.17 (s, 3H), 1.05 (m, 3H), 1.01 (s, 3H), 0.99 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H)]. In contrast to those of friedelin, the signal for H-2 on ring A was absent, which can be attributed to the formation of a new bond. The major variations in the chemical shifts of H-1 (+0.77, +0.71) and H-23 (+0.17) were consistent with a decrease in the electron density around these protons. The chemical shift of C-2 shifted downfield as a result of a new bond formation. Concurrently, seven carbon signals were observed in the range from 125.4 to 134.7 ppm, indicating the introduction of benzene into the molecular structure. The presence of these characteristic structural features was also confirmed through analysis of the 2D NMR analysis. In the ¹H-¹H COSY spectra (Figure 3), correlations were observed between 2.74 (H-1α), 2.40 (H-1β) with 1.81 (H-6α), and 1.27 (H-6β), between δ_H 2.28 (H-4) with δ_H 1.05 (H-23), and between δ_H 7.30 (H-5′) with δ_H 7.21 (H-6′). The relative configuration of compound 4 was elucidated by conformational analysis, supported by ROESY spectra (Figure S5). The E-isomer was identified on basis of the ROESY correlations from H-1α (δ_H 2.74) and H-1β (δ_H 2.40) to H-6′ (δ_H 7.21). Additionally, we carried out chemical calculations to obtain further evidence (Figures S11 and S12). The final results were then generated through a DP4+ analysis (Table S1). All results confirmed the E-configuration of compound 4, which was consistent with the evidence from the ROESY spectra.

In the HMBC spectra, the correlations of δ_H 2.74/2.40 (H-1 α/β) to δ_C 132.6 (C-7′) and δ_C 204.3 (C-3), of δ_H 7.17 (H-7′) to δ_C 25.4 (C-1) and δ_C 204.3 (C-3), of δ_H 7.21 (H-6′) to δ_C 139.6 (C-2), along with the correlations from δ_H 2.74 (H-1α) and δ_H 2.40 (H-1β) to δ_C 204.3 (C-3) collectively verified that the α,β-unsaturated ketone presented in compound 4 (Figure 3). The remaining chemical shift values were largely unchanged from those of friedelin. All the ¹H and ¹³C NMR data are listed in

Table 1. ¹³C NMR and ¹H NMR data of compound 4 [δ_H, ppm, (mult, J in Hz)].

No.	13C	1H	No.	13C	1H	No.	13C	1H
1	25.4	2.74 (m), 2.40 (m)	14	38.3	-	27	18.6	0.99 (s)
2	139.6	-	15	32.3	1.47 (m), 1.19 (m)	28	32.0	1.17 (s)
3	204.3	-	16	35.9	1.52 (m), 1.36 (m)	29	34.8	0.93 (s)
4	58.9	2.28 (m)	17	30.0	-	30	31.9	0.98 (s)
5	39.5	-	18	42.7	1.53 (m)	1′	134.3	-
6	41.3	1.81 (m), 1.27 (m)	19	35.0	1.33 (m), 1.14 (m)	2′	125.4	-
7	18.1	1.49 (m), 1.41 (m)	20	28.1	-	3′	131.1	7.63 (d, 2.1)
8	53.0	1.38 (m)	21	32.8	1.45 (m), 1.25 (m)	4′	134.7	-
9	37.4	-	22	39.1	1.50 (m), 0.91 (m)	5′	127.1	7.30 (dd, 8.3, 2.1)
10	57.2	1.56 (m)	23	7.6	1.05 (d)	6′	131.0	7.21 (d, 8.3)
11	35.2	1.35 (m) 1.15 (m)	24	14.8	0.85 (s)	7′	132.6	7.17 (m)
12	30.1	1.29 (m)	25	17.3	0.92 (s)
13	39.6	-	26	20.2	1.01 (s)

.

3. Experimental

3.1. General Experimental Conditions

Infrared spectra were recorded on a Bio-Rad FTS-135 spectrometer (Hercules, CA USA) with samples prepared as KBr pellets. NMR spectra were obtained on Bruker spectrometers (Billerica, MA USA) operating at 600 MHz, using CDCl₃ as the solvent. Splitting patterns were denoted using the following conventional abbreviations: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). HR-ESI-MS data were collected on an Agilent triple quadrupole mass spectrometer.

For chromatographic separations, various mesh sizes of silica gel were used: silica gels with mesh sizes of 300–400 mesh (Qingdao Marine Chemical Inc., Qingdao, China); and precoated silica gel GF₂₅₄ plates (Qingdao Puke Abruption Materials Co., Ltd., Qingdao, China).

3.2. Synthesis of Friedelin Derivative

Friedelin derivative 4 with an α,β-unsaturated ketone was successfully synthesized via an aldol condensation (Scheme 1). Friedelin (20 mg, 46.87 µmol) and potassium hydroxide (5 mg, 89.11 µmol) were added to a mixed solution of dichloromethane (5 mL) and ethanol (5 mL). Then, 2-bromo-4-chlorobenzaldehyde (21 mg, 95.70 µmol) was added into the mixture and stirred it at room temperature. The resulting mixture was concentrated under reduced pressure and purified by flash chromatography to obtain compound 4 (12 mg).

4. Conclusions

Compound 4 is a friedelin derivative that was synthesized via aldol condensation. This reaction can yield products with two distinct configurations, including the Z- and E-isomers [12,13,14]. We successfully determined the structure of compound 4 by HR-ESI-MS and NMR and established its configuration as the E-isomer.