((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-Acetoxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)methyl 2-Bromo-3-methylbenzoate

Abstract

In this report, we discuss the synthesis of modified betulin through the chemical derivatization of a natural compound. The compound was fully characterized by proton (¹H), carbon-13 (¹³C), heteronuclear single quantum coherence (HSQC) and distortionless enhancement through polarization transfer (DEPT) NMR and elemental analysis. We investigated the optical properties through ultraviolet (UV) and Fourier-transform infrared (FTIR) spectroscopy.

1. Introduction

Cancer is a major disease that seriously jeopardizes human health, and attacking cancer is a common challenge all over the world. Traditional treatments for cancer, such as surgery, chemotherapy and radiotherapy, although effective, suffer from many side effects and other drawbacks [1].

Due to the problems arising from the use of synthetic drugs, there has been a renewed interest in natural substances with high efficacy and low cytotoxicity. More than 60% of the anticancer drugs currently in use are of natural origin. They come from natural sources such as plants, microorganisms, marine organisms and animals [2].

Betulin (1) is a pentacyclic triterpene of the lupane type, characterized by three highly reactive functional groups in its structure. Its mechanism of action primarily involves the inhibition of key signaling pathway components related to proliferation, migration, interleukins and other factors. Betulin exhibits several beneficial biological activities, including anti-inflammatory [3], hepatoprotective [4], neuroprotective [5] and antitumor effects [6]. However, its poor bioavailability necessitates chemical modifications to improve its pharmacological and pharmacokinetic properties. The method of synthesis and the choice of substituents play a crucial role in enhancing its effects on cells and cancers (Figure 1) [7].

By introducing new functional groups into the triterpene scaffold of 1, there is potential to create compounds with improved activity, increased selectivity, and more favorable physicochemical and pharmacokinetic characteristics [8]. Molecule 1 contains two reactive centers, located at the hydroxyl groups in positions 3 and 28. The most prevalent method for modifying this compound involves exploiting the reactivity of these functional groups to convert them into ester [9], ether, glycosidic [8], or amide derivatives [10] (Figure 1), and some previous studies on acid derivatives have shown effectiveness against cancer cells [11].

This study describes the synthesis of a betulin derivative in which the scaffold has been linked to benzoic acid derivatives (Scheme 1), which was designed and fully characterized by NMR, IR, UV and elementary analyses.

Figure 1. Example of betulin derivatives reported in the literature: (a–c) [2], (d,e) [12], (f) [13].

Figure 1. Example of betulin derivatives reported in the literature: (a–c) [2], (d,e) [12], (f) [13].

2. Results and Discussion

We modified betulin in accordance with Scheme 1. Following the protocol reported in the literature [14], betulin derivative 2 was prepared through the reaction of betulin 1, 2-bromo-3-methylbenzoic acid (1.1 eq) and 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.08 eq). Then, 0.48 eq./equivalent 4-N,N-dimethylaminopyridine (DMAP) was added to DCM (15 mL) containing triethylamine (TEA) (0.48 eq). The mixture was then stirred at room temperature for 12 h (Scheme 1a). The crude product was purified on a flash column with n-hexane/EtOAc (8:2, v/v to afford 2 in 22.5% yield). Betulin derivative 2 was used as a starting compound for a reaction using DMAP/TEA to create the activation conditions, with a modified protocol reported by Wang, X. Y [15]. After stirring at room temperature for 12 h, betulin derivative 3 was isolated by column chromatography in a 50% yield.

The structure of 3 was verified by ¹H- and ¹³C-NMR analyses (Supplementary Materials, Figures S10 and S11). The ¹H-NMR spectrum of 3 displayed three peaks above 7 ppm, corresponding to the aromatic protons of the aromatic ring, as well as signals for the methyl group on acetate at 2.04 ppm and a methyl group on the aromatic ring at 2.4 ppm.

Regarding the ¹³C-NMR signals, the aromatic carbon signals in the 122–138 ppm range were important for assessing product formation. The peak at 23 ppm, in the low-field region, indicates the presence of a methyl group on the aromatic ring. Additionally, the peaks at 170 and 166.8 ppm confirm the presence of an ester group, which is crucial for the analysis.

Moreover, heteronuclear single quantum coherence spectroscopy (HSQC) was used to assign the ¹³C signals of compound 3, as shown in Table S1 (see Supplementary Materials for 2D spectra, Figures S12 and S13). The ¹³C-NMR spectrum of 3 exhibited forty carbon signals, classified by DEPT experiments as eight methyl groups, twelve methylenes, six methines, six aromatic, and eight quaternary carbons.

The IR spectrum of compound 3 displayed characteristic C–H stretching of the alkene at 3110 cm⁻¹, C–H stretching at 2962 cm⁻¹, and C=O ester stretching at 1735 cm⁻¹ (Supplementary Materials, Figure S16). Additional peaks at 1257 and 1095 cm⁻¹ were attributed to C–O stretching of the ester groups, while an alkene bending vibration was observed at 802 cm⁻¹.

The UV spectra of 2 and 3 were also recorded for further characterization (Supplementary Materials, Figures S18), showing an absorption peak at 211 nm and another lower absorption peak at 229 nm. The results from analyzing specific elements of compounds 2 and 3 provide confirmation that the above identified structure is correct. This suggests that the elemental analysis aligns with the expected molecular structure.

3. Materials and Methods

3.1. Chemistry

Silica gel (FCP 230-400 mesh) was used for column chromatography. Thin-layer chromatography was carried out on Merck precoated silica gel 60 F₂₅₄ plates (Merck, Darmstadt, Germany) and visualized with phosphomolybdic acid, iodine, or a UV–visible lamp.

All chemicals were purchased from Bide Pharmatech., Ltd. (Shanghai, China). ¹H-NMR and ¹³C-NMR spectra were collected in CDCl₃ at 25 °C on a Bruker Ascend^®-600 (Magnet System 600′54 Ascend LH, San Jose, CA, USA) NMR spectrometer (600 MHz for ¹H and 150 MHz for ¹³C). All chemical shifts were reported in the standard δ notation of parts per million using the peak of the residual proton signals of CDCl₃ as an internal reference (CDCl₃, δ_C 77.2 ppm, δ_H 7.26 ppm).

UV analysis was performed by a Shimadzu UV–2600 (Osaka, Japan) with a 1 cm quartz cell and a slit width of 2.0 nm. The analysis was carried out using wavelengths in the range of 200–700 nm.

3.1.1. Synthesis of (9-Hydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)methyl 2-Bromo-3-methylbenzoate (2)

To a stirred solution of 1 (0.2 g, 0.45 mmol) in dichloromethane (20 mL) held at RT under a nitrogen atmosphere were added 2-Bromo-3-Methylbenzoic acid (0.11 g, 0.52 mmol) over 5 min and EDC (0.1 g, 0.52 mmol), DMAP (0.026 g, 0.22 mmol), and TEA (31.4 µL, 0.22 mmol). The mixture was then stirred at room temperature for 12 h. After the reaction was completed, the mixture was extracted with 20 mL of distilled water and partitioned with ethyl acetate (10 mL), and the organic layers were dried over Na₂SO₄ and evaporated. The target compounds were purified on a flash column with n-hexane/EtOAc (8:2, v/v) to yield 45 mg of the compound (22.5%). δ_H (600 MHz, CDCl₃) ¹H-NMR δ 7.46 (d, J = 6.4 Hz, 1H), 7.33 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 4.71 (s, 1H), 4.60 (s, 1H), 4.51 (d, J = 12.2 Hz, 1H), 4.13 (d, J = 11.2 Hz, 1H), 3.20 (dd, J = 11.4, 4.7 Hz, 1H), 2.50 (m, 1H), 2.46 (s, 3H), 1.70 (s, 3H), 1.07 (s, 3H), 0.99 (s, 3H), 0.97 (s, 3H), 0.83 (s, 3H), 0.76 (s, 3H), 0.68 (d, 1H, J = 9 Hz). UV (CH₂Cl₂) peaks 229 and 280 nm, IR (KBr) 3430, 2924, 1720, 1651, 1458, 1404, 1265, 1095, 1033, 802 cm⁻¹. δ_C (150 MHz, CDCl₃) 166.8, 149.1, 138.6, 133.5, 131.9, 126.8, 125.8, 121.9, 108.0, 77.9, 63.1, 54.2, 51.5, 49.3, 47.9, 46.7, 45.5, 41.7, 39.8, 37.8, 37.7, 36.7, 36.1, 33.6, 33.1, 28.8, 28.6, 26.9, 26.3, 26.0, 24.2, 22.7, 19.7, 18.1, 17.2, 15.0, 14.3, 13.7 ppm; Anal. found C 71.1%, H 8.6%, Br 12.2%, O 7.4% Calc. for C₃₈H₅₅BrO₃: C 71.3%, H 8.6%, Br 12.4%, O 7.5%. UV (CH₂Cl₂) peaks 229, 280, 324 and 358 nm, IR (KBr) 3480, 2924, 1720, 1651, 1458, 1265, 1095, 802 cm⁻¹.

3.1.2. Synthesis of ((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-Acetoxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)methyl 2-bromo-3-methylbenzoate (3)

To a stirred solution of 2 (45 mg, 0.07 mmol), TEA (29.2 uL, 0.21 mmol) and DMAP (34.2 mg, 0.28 mmol) in dry DCM (10 mL), acetic anhydride (57 µL, 0.60 mmol) was added dropwise at 0 °C. Then, the reaction mixture was stirred at room temperature overnight. The mixture was slowly diluted with H₂O (100 mL) and extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with H₂O (2 × 150 mL) and brine (150 mL), dried over Na₂SO₄, and concentrated. The residue was purified by flash chromatography using 5% EtOAc in petroleum ether to yield 21 mg (50%) of the product as a white pure solid. δ_H (600 MHz, CDCl₃) δ 7.45 (d, J = 7.3 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 4.71 (s, 1H), 4.61 (s, 1H), 4.52 (d, J = 10.9 Hz, 1H), 4.46 (m, 1H), 4.12 (d, J = 11.0 Hz, 1H), 2.51 (m, 1H), 2.46 (s, 3H), 2.04 (s, 3H), 1.70 (s, 3H), 1.07 (s, 3H), 0.99 (s, 3H), 0.86 (s, 3H), 0.84 (s, 6H), 0.78 (d, 1H, J = 9 Hz); δ_C (150 MHz, CDCl₃) 170.0, 166.8, 149.0, 138.7, 133.5, 131.9, 126.8, 125.8, 122.0, 109.0, 79.9, 63.1, 54.2, 51.5, 49.3, 47.9, 46.7, 45.5., 41.7, 39.9, 37.3, 36.7, 36.6, 36.1, 33.6, 33.1, 28.8, 28,6, 26.9, 26.0, 24.2, 22.7, 20.3, 19.8, 18.1, 17.1, 15.4 (2CH₃), 15.0 13.7. Anal. found C 70.2%, H 8.4%, Br 11.5%, O 9.1% Calc. for C₄₀H₅₇BrO₄: C 70.4, H 8.4%, Br 11.7%, O 9.3%. UV (CH₂Cl₂) peaks 211, 229, 274 and 360 nm, IR (KBr) 3110, 2962, 1735, 1257, 1095, 802 cm⁻¹.

4. Conclusions

The synthesis of a betulin analog with a hybrid structure and potential for use in anticancer applications was presented. The chemical structure of the synthesized compound was confirmed using NMR, IR, and UV spectroscopies.