28-O-Acetyl-3-O′-(prop-2-enoyl)betulin

Abstract

28-Acetylbetulin is a good starting compound for the synthesis of 3- or 3,28-substituted betulin derivatives with biological activity. The final product of the reaction of 28-acetylbetulin and acrylic acid under Steglich esterification conditions produced a new 3-alkenyl betulin derivative. The structure of the obtained compound was confirmed based on the analysis of NMR, IR, EI MS, and HRMS spectra. Selected pharmacokinetic parameters related to the absorption and distribution were calculated for the new betulin derivative using in silico methods.

1. Introduction

Plant-based natural compounds (secondary metabolites) do not affect the phases of plant growth and development, and their presence is limited to protection against pathogens. Secondary metabolites are formed from the biosynthetic intermediates of cellular processes such as photosynthesis, glycolysis, and the Krebs cycle. Natural compounds of plant origin are characterized by a wide structural diversity. The complexity of the chemical structure of these compounds determines a wide range of biological activity [1].

Betulin, a plant metabolite from the group of lupane-type pentacyclic triterpenes, can be found in large amounts in the outer bark of the birch tree (Betula alba, Betula pubescens, Betula platyphylla, and Betula pendula). Betulin is most often obtained by extracting birch bark with organic solvents (ethanol, chloroform, acetone) [2]. In addition to betulin, its derivatives are also obtained from plant material. Examples of such compounds include 28-acyl substituted betulin derivatives, which are isolated from the bark of Quercus suber L. and the leaves of Nerium oleander L. [3,4].

Chemical synthesis is a method of obtaining new semi-synthetic betulin derivatives. The reaction of betulin with acetic anhydride leads to two products, i.e., 28-acetylbetulin 1 and 3,28-diacetylbetulin 2 (Figure 1). The yield of substances 1 and 2 depends on the conditions of this synthesis [5,6].

Although both compounds find practical use in the preparation of new derivatives, 28-acetylbetulin 1 shows greater potential for biological activity. The activity of 28-acetylbetulin 1 was observed toward different tumor cell lines such as anaplastic thyroid (8505C, SW1736), ovarian (A2780), colon (SW480, HCT-8, HCT-116, DLD-1, HT-29), lung (A549), melanoma (518A2), head and neck (A253, FaDu), cervica (A431), breast (MCF-7) and liposarcoma with values of IC₅₀ in the range 10.71–15.84 µM [7]. Antiviral activity studies have shown that 28-acetylbetulin 1 is a potent inhibitor of the Semliki Forest Virus (SFV) (IC₅₀ value 12.1 µM) compared to ribavirin (IC₅₀ value 95.1 µM) [8]. The performed antibacterial activity studies confirmed that compound 1 had a moderate inhibitory activity (22%) on the growth of Chlamydia pneumoniae at a concentration of 1 µM [9].

This work presents the synthesis, structural characterization, and selected ADME parameters of the new 3-alkenyl 28-diacetylbetulin derivative.

2. Results and Discussion

28-Acetylbetulin 1 was prepared by the treatment of betulin with acetic anhydride (Ac₂O) in the presence of imidazole in dry chloroform (CHCl₃), as described in the literature [6]. Selective acetylation of the primary hydroxyl group of betulin at position C-28 (compound 1) allowed the reaction with acrylic acid in anhydrous dichloromethane in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-aminopyridine (DMAP). The 3-alkenyl derivative of 28-acetylbetulin 3 was obtained as a product of the Steglich reaction at a 68% yield. The conditions for the synthesis of compounds 1 and 3 are shown in Scheme 1.

The chemical structure of 28-O-acetyl-3-O′-(prop-2-enoyl)betulin 3 was established with NMR, IR spectroscopy, and mass spectroscopy (EI MS, HRMS).

Compound 3 contained a prop-2-enoyl group at the C3 position, as evidenced by the shifts at 5.73 ppm (d, 1H, J = 10.8 Hz, CH=CH₂), 6.02–6.07 ppm (dd, 1H, J = 18.0, 12.0 Hz, CH=CH₂) and 6.29 ppm (d, 1H, J = 17.4 Hz, CH=CH₂). The position of the H3 proton (δ = 4.47–4.49 ppm) was characteristic of the 3,28-disubstituted betulin derivatives [10]. The analysis of the ¹³C NMR spectrum showed that the signal of the ester groups corresponded to the shift values 165.1 and 170.6 ppm. The EI MS spectrum showed a molecular ion peak (M⁺) with an intensity of 8%, which could indicate that derivative 3 easily broke down. The HR-MS analysis was performed using the APCI method and did not show the presence of a molecular ion at all. According to the literature, in the case of the APCI method, triterpene derivatives are fragmented under measurement conditions [11]. A major peak (100%) with m/z of 336.3251 was observed for compound 3. The proposed structure of this fragmentation ion is shown in Figure S6 in the Supplementary Materials.

The determination of the ADME profile for new compounds considered potential therapeutic substances enabled a quick assessment of the parameters in relation to absorption, distribution, metabolism, excretion, and toxicity [12]. For compound 3, the parameters determining the absorption properties were designated, such as Caco-2 permeability (logPapp), human intestinal absorption (HIA), and skin permeability (logKp) (

Table 1. Selected ADME parameters of compound 3 and the anticancer drug doxorubicin.

Parameters	Compound 3	Doxorubicin
Absorption
Caco-2 permeability (logPapp) a	1.27	0.48
Skin permeability (logKp) b	−2.56	−2.73
Human intestinal absorption (HIA) c	100%	55%
Distribution
BBB permeability (logBB)	−0.64	−1.27
CNS permeability (logPS)	−1.09	−4.29

^a 10⁻⁶ cm/s, ^b cm/h, ^c %.

). The predicted values of these parameters obtained for compound 3 were related to the values that were calculated for doxorubicin: a drug used in the treatment of various cancer diseases [13].

Studies using the Caco-2 cell monolayer are being used to predict the absorption of orally administered drugs through the human intestinal mucosa. It has been assumed that the value of logPapp determined by the pkCSM web server > 0.9 indicated the high Caco-2 permeability of the compound [12,14]. Derivative 3 was characterized by the high permeability of Caco-2 (logPapp = 1.27), which was almost three times higher than the value predicted for doxorubicin (logPapp = 0.48).

Skin permeability (logKp) describes the ability of molecules of a given compound to proceed through the transdermal route. LogKp values of less than −2.50 indicated that the test compound could be used as a drug for skin diseases [12]. The in silico logKp values of derivative 3 (−2.56) and doxorubicin (−2.73) indicated good skin permeability in both compounds.

In the case of orally administered drugs, an important parameter to determine their bioavailability is their absorption in the small intestine. The high value of the intestinal absorption coefficient of HIA (HIA = 100%) indicated that derivative 3 could be better absorbed from the gastrointestinal tract after oral administration compared to doxorubicin (predicted HIA = 55%).

The blood–brain barrier (BBB) protects the brain against absorption from the outside and the distribution of various substances in the brain cells. The log BB value (the ratio of drug concentration in the brain to the concentration of the drug in plasma) for compounds that easily crossed BBB was >3 [12]. The LogBB values of −0.64 and −1.27 for derivative 3 and doxorubicin, respectively, indicated that these compounds were poorly absorbed and distributed in the brain cells.

Permeability to the central nervous system (CNS) plays an important role in the distribution of oral drug delivery for brain-related disorders. Their permeability to the CNS could be related to the logPS (the blood–brain permeability–surface area). Compounds with a logPS value < −3 showed lower permeability to the CNS [12]. The logPS value = −1.09 for betulin derivative 3 indicated good CNS penetration. It could, therefore, be assumed that compound 3 can be used in the treatment of diseases in relation to the nervous system.

3. Materials and Methods

3.1. General Information

The acetic anhydride, imidazole, acrylic acid, DCC, DMAP, and solvents were purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA). The melting points were detected on an Electrothermal IA 9300 apparatus (Bibby Scientific Limited, Stone, Southampton, UK) and were uncorrected. IR spectra were obtained using an IRAffinity-1 Shimadzu spectrometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra (¹H and ¹³C-NMR at 600 MHz and 150 MHz, respectively) were recorded in CDCl₃ on a Bruker Avance III 600 spectrometer (Bruker, Billerica, MA, USA). The EI MS spectra were recorded on a Finnigan MAT 95 instrument. The HR-MS spectra were obtained using a Bruker Impact II instrument mass spectrometer (Bruker). Silica gel 60 254F plates (Merck, Darmstadt, Germany) were used for TLC chromatography. The spots of the chromatograms were visualized by spraying with an ethanolic solution of sulfuric acid and heating at 100 °C. The new derivative 3 was purified by column chromatography (silica gel 60 (0.063–0.200 mm)) in the stationary phase; dichloromethane:ethanol was used at a ratio of 60:1, (v/v—the mobile phase).

3.2. General Procedure for the Synthesis of 28-O-Acetyl-3-O′-(prop-2-enoyl)betulin 3

28-acetylbetulin 1 (0.5 mmol) was dissolved in 10 mL dry dichloromethane. Acrylic acid (0.6 mmol) was added to this mixture and stirred at 0 °C for 30 min. Then, a solution of DCC (0.73 mmol) and DMAP (0.12 mmol) was added dropwise in dichloromethane (0.7 mL). After 5 h of stirring at 0 °C, the cooling bath was removed. Stirring continued at room temperature for 24 h. The resulting precipitate was filtered off. Dichloromethane was removed under reduced pressure. The column chromatography (SiO₂, dichloromethane:ethanol 60:1, v/v) of the residue gave targeted compound 3.

28-O-Acetyl-3-O′-(prop-2-enoyl)betulin 3. Yield 68%; mp 202–204 °C; R_f 0.64 (dichloromethane/ethanol, 60:1, v/v); ¹H NMR (600 MHz, CDCl₃) δ: 0.73 (s, 3H, CH₃), 0.75 (s, 3H, CH₃), 0.77 (s, 3H, CH₃), 0.93 (s, 3H, CH₃), 0.98 (s, 3H, CH₃), 1.62 (s, 3H, CH₃), 2.00 (s, 3H, CH₃C=O), 2.35–2.41 (m, 1H, H-19), 3.78 (d, J = 12.0 Hz, 1H, H-28), 4.18 (d, J = 12.0 Hz, 1H, H-28), 4.47–4.49 (m, 1H, H-3), 4.52 (s, 1H, H-29), 4.62 (s, 1H, H-29), 5.73 (d, 1H, J = 10.8 Hz, CH=CH₂), 6.02–6.07 (dd, 1H, J = 18.0, 12.0 Hz, CH=CH₂), 6.29 (d, 1H, J = 17.4 Hz, CH=CH₂) (Figure S1—Supplementary Materials); ¹³C NMR (150 MHz, CDCl₃) δ: 13.7, 15.0, 15.1, 15.5, 17.2, 18.1, 19.8, 20.0, 22.7, 24.1, 26.0, 26.9, 28.6, 28.7, 33.1, 33.5, 36.1, 36.6, 37.0, 37.4, 39.9, 41.7, 45.3, 46.7, 47.8, 49.3, 54.4, 61.8, 80.1, 108.9, 128.1, 129.0, 149.1, 165.1, 170.6 (Figure S2—Supplementary Materials); IR (ν _max cm⁻¹, KBr): 1240, 1263, 1269, 1372, 1466, 1638, 1732, 1741, 2941 (Figure S3—Supplementary Materials); EI MS (70 eV) m/z (rel. intensity): 538 (M⁺, 8), 466 (85), 203 (45), 189 (100), 135 (39) (Figure S4—Supplementary Materials).; HR-MS (APCI) m/z (neg): 336.3251; C₂₅H₃₆ (Calculated. 336.2817) (Figure S5—Supplementary Materials).

3.3. In Silico Analysis

The ADME parameters (Caco-2 permeability, skin permeability, human intestinal absorption, BBB permeability, and CNS permeability) were predicted using the pharmacokinetic webserver: pkCSM (https://biosig.lab.uq.edu.au/pkcsm/, accessed on 3 May 2023) [14].