28-O-Acetyl-3-O’-(Phenylpropynoyl)Betulin

Abstract

New derivative of 28-acetylbetulin containing a phenylpropynoyl moiety at the C-3 position was obtained by Steglich method. The chemical structure of this compound has been determined through ¹H NMR, ¹³C NMR, IR, EI MS and HRMS. The antiproliferative effects of 28-O-acetyl-3-O’-(phenylpropynoyl)betulin were evaluated against three human cancer cell lines: T47D (breast cancer), CCRF/CEM (leukemia), SW707 (colorectal adenocarcinoma) and murine cell line P388 (leukemia). The synthesized compound exhibited moderate antiproliferative activity against P388 cells (IC₅₀ = 35.51 µM). The in silico analysis showed that the title compound meets the criteria of Veber’s rule.

1. Introduction

Triterpenes are one of the important classes of natural products with a wide range of biological activity. Due to their chemical structure and the type of substituents present in their structure, they can be classified into compounds with an oleanane, ursane or lupane skeleton. Pentacyclic triterpenes have not yet found clinical application, although they are the subject of extensive biological research. One of the possibilities for improving the pharmacological properties and increasing the biological effects of triterpenes is the chemical transformation of their structure leading to new derivatives [1,2,3,4].

Betulin (lup-20(29)-ene-3β,28-diol), a pentacyclic triterpene of the lupane type, is a substance of plant origin found in significant amounts in birch bark. Two hydroxyl groups at the C-28 and C-3 positions and an alkenyl group at the C-20 carbon present in the betulin structure are the locations most frequently subjected to chemical modifications [2].

Due to its high reactivity and the possibility of further functionalization, the alkynyl moiety is often used as a functional group in the synthesis of drug candidates [5]. The introduction of an alkynyl moiety into the structure of betulin, betulonic acid and 3-oxo-oleanolic acid makes it possible to obtain new bioactive agents with anticancer, anti-inflammatory, hepatoprotective, antiviral and antibacterial properties (Figure 1) [6,7,8,9].

Among the derivatives of pentacyclic triterpenes with carbon–carbon triple bonds, a large group constitutes substances showing anticancer activity. The alkyne-modified triterpenes have cytotoxic potential against various human cancer cell lines, namely lung cancer (NCI-H460, A549), colon cancer (HT-29, SW-480, DLD-1, SW707, HCT-8, HCT-116), melanoma (MALME-3M, 518A2), breast cancer (T47D, MCF-7), leukemia (K-562, CCRF/CEM, HL-60) and ovarian cancer (A2780) [10,11,12].

This study presents the synthesis and spectroscopic characterization of the new 28-acetylbetulin derivative containing an alkynyl group at the C-3 position. Additionally, the cytotoxic effects of the 28-O-acetyl-3-O’-(phenylpropynoyl)betulin 3 have been tested against four different cancer cell lines (T47D, CCRF/CEM, SW707 and P388).

2. Results and Discussion

The 28-acetyl derivative 2 was obtained in the reaction of betulin 1 with acetic anhydride (Ac₂O) in the presence of imidazole in anhydrous chloroform based on the procedure described by Tietze et al. [13]. The introduction of the acetyl group at the C-28 position made it possible to carry out the esterification reaction of the secondary hydroxyl group at the C-3 carbon in the triterpene scaffolds. Application of phenylpropynoic acid and reagents typical of the Steglich method DCC (N,N′-dicyclohexylcarbodiimide) and DMAP (4-aminopyridine) in dichloromethane (CH₂Cl₂) gave the alkynyl derivative 3 with good yield (74%). The synthetic steps involved in the preparation of Compound 3 are shown in Scheme 1.

The structure of 28-O-acetyl-3-O’-(phenylpropynoyl)betulin 3 was elucidated through ¹H NMR, ¹³C NMR, IR, EI MS and HRMS. The ¹H NMR data of Compound 3 show characteristic signals for 28-acetylbetulin protons, namely six singlets of the methyl groups at δ_H 0.89, 0.94, 0.98, 0.99, 1.06 and 1.71 and protons of the acetyl group at δ_H 2.01 (s, 3H, CH₃C=O). The chemical shifts of the H-3, H-19, H-28 and H-29 protons in the ¹H NMR spectrum of Compound 3 correspond to the values for 3,28-disubstituted betulin derivatives [14]. In the ¹³C NMR spectra of Compound 3, the signals of the carbon–carbon triple bond of the phenylpropynoyl moiety are at δ_C 83.2 and 85.7.

The IR spectrum of derivative 3 shows the typical bands for the lupane skeleton of pentacyclic triterpenes [15]. The signals at 1375 cm⁻¹, 1032 cm⁻¹ and 977 cm⁻¹ correspond to the vibration of C-O bonds. The vibration band of the C=C bond (isopropenyl group at the C-19) is observed at 1640 cm⁻¹ and 883 cm⁻¹. Additionally, the IR spectrum confirmed the presence of a carbon–carbon triple bond (2224 cm⁻¹) and C=O carbonyl bonds (1705 cm⁻¹ and 1740 cm⁻¹) of ester groups in the structure of Compound 3. The EIMS spectrum gave a molecular ion peak at m/z 612 consistent with the molecular formula C₄₁H₅₅O₄ for Compound 3. Additionally, the EIMS spectrum contains fragment ions with m/z 466 (78%), 203 (43%), 189 (100%) and 135 (39%). The proposed structures of fragment ions are presented in the Supplementary Materials. In the HRMS spectrum, the main peak (100%) with m/z of 644.3954 observed for Compound 3 is most likely an adduct of the tested compound with the solvent used.

For Compound 3, the physicochemical properties and lipophilicity related to the Lipinski and Veber rules were determined using an in silico method (

Table 1. Drug-likeness of Compound 3 based on Lipinski and Veber analysis.

Estimated Properties of Compound 3	Lipinski’s Rule	Veber’s Rule
Molecular weight (MW) 612.88 g/mol	No 2 violations: MW > 500, MLOGP > 4.15	Yes
Log Po/w (MLOGP) 7.32
Num. H-bond acceptors (HBA) 4
Num. H-bond donors (HBD) 0
Num. rotatable bonds (nRB) 6
Topological polar surface area (TPSA) 52.60 Å2

Lipinski guidelines—MW ≤ 500, MLOGP ≤ 4.15, HBA ≤ 10, HBD ≤ 5. Veber guidelines—nRB ≤ 10, TPSA ≤ 140 Ų [16].

). The Lipinski drug-likeness guidelines were violated twice by 28-O-acetyl-3-O’-(phenylpropynoyl)betulin 3 due to the value of molecular weight and lipophilicity (MW > 500, MLOGP > 4.15).

Derivative 3 only meets the criteria of Veber’s rule with a TPSA (topological polar surface area) value of 52.60 Ų and an nRB (rotatable bonds) number, indicating oral availability.

New 3-alkynyl substituted derivative of 28-acetylbetulin 3 was tested for antiproliferative activity in vitro against three human cancer cell lines like T47D (breast cancer), CCRF/CEM (leukemia), SW707 (colorectal adenocarcinoma) and murine cell line P388 (leukemia). Normal BALB/3T3 fibroblast cells were also used in the study. In vitro antiproliferative activity was expressed as IC₅₀ values (half maximum inhibitory concentration) in µM. Cisplatin was applied as a reference drug. The results of the proliferation inhibition of tested compounds are shown in

Table 2. Antiproliferative activity of Compound 3 and cisplatin.

Compound	Antiproliferative Activity IC50 ± SD [µM]
	Human			Murine
	T47D	CCRF/CEM	SW707	P388	BALB/3T3
3	Neg	Neg	Neg	35.51 ± 18.45	Neg
Cisplatin	10.4 ± 3.5	6.6 ± 1.8	7.4 ± 1.7	1.8 ± 0.9	8.9 ± 1.2

Neg—negative in the concentration used.

.

Compound 3 shows selective antiproliferative activity towards P338 cells with an IC₅₀ value of 35.51 µM. Comparison of this effect of derivative 3 with 28-acetylbetulin 2 on P338 cells shows that the introduction of a phenylpropynoyl group at the C-3 position causes an almost 3-fold decrease in activity. The introduction of a phenylpropynoyl moiety at C-3 gave Compound 3, which does not cause a cytotoxic effect on normal BALB/3T3 fibroblast cells, unlike the 28-acetyl derivative 2 (IC₅₀ = 29.7 µM) [17].

3. Materials and Methods

3.1. General Information

All reagents and solvents applied in the synthesis of Compounds 2 and 3 were purchased from Sigma–Aldrich (Sigma–Aldrich, Saint Louis, MO, USA). Merck silica gel 60 254F plates (Merck, Darmstadt, Germany) were used for TLC. The spots of the chromatograms were visualized by spraying with H₂SO₄-C₂H₅OH and heating to 100 °C for 2 min. Merck silica gel 60 (0.063–0.200 mm) and solvent system CH₂Cl₂: C₂H₅OH (60:1, v/v) were used for column chromatography. Melting point (mp) values were determined on an Electrothermal IA 9300 apparatus (Bibby Scientific Limited, Stone, Southampton, UK). IR spectra were recorded in KBr pellets on an IRAffinity-1 Shimadzu spectrometer (Shimadzu Corporation, Kyoto, Japan). NMR spectra (¹H and ¹³C-NMR at 600 MHz and 150 MHz, respectively) were measured in CDCl₃ solution on a Bruker Avance III 600 spectrometer (Bruker, Billerica, MA, USA). EI MS spectra were made on a Finnigan MAT 95 instrument. High resolution mass spectra (HRMS) were obtained using a Bruker Impact II instrument mass spectrometer (Bruker).

3.2. General Procedure for the Synthesis of 28-O-acetyl-3-O’-(Phenylpropynoyl)Betulin 3

A mixture of Compound 2 (0.5 mmol) and phenylpropynoic acid (0.6 mmol) in dry dichloromethane (10 mL) was stirred at 0 °C for 30 min. DCC (0.73 mmol) and DMAP (0.12 mmol) were dissolved in dichloromethane (0.7 mL). Then, to the reaction mixture a solution of Steglich reagents (DCC and DMAP) was added dropwise. Stirring was continued first at 0 °C and then at room temperature for 24 h. The resulting white precipitate was filtered off, and the filtrate was concentrated to dryness using a vacuum evaporator. The product was purified by column chromatography (SiO₂, dichloromethane: ethanol 60:1, v/v).

28-O-Acetyl-3-O’-(phenylpropynoyl)betulin 3. Yield 74%; mp 105–107 °C; R_f 0.71 (di-chloromethane/ethanol, 60:1, v/v); ¹H NMR (600 MHz, CDCl3) δ: 0.89 (s, 3H, CH₃), 0.94 (s, 3H, CH₃), 0.98 (s, 3H, CH₃), 0.99 (s, 3H, CH₃), 1.06 (s, 3H, CH₃), 1.71 (s, 3H, CH₃), 2.10 (s, 3H, CH₃C=O), 2.47 (m, 1H, H-19), 3.87 (d, J = 10.8 Hz, 1H, H-28), 4.26 (d, J = 10.8 Hz, 1H, H-28), 4.62 (s, 1H, H-29), 4.67 (m, 1H, H-3), 4.71 (s, 1H, H-29), 7.38–7.63 (m, 5H, Ar-H) (Figure S1-Supplementary Materials); ¹³C NMR (150 MHz, CDCl3) δ: 14.7, 16.0, 16.2, 16.6, 18.2, 19.1, 20.8, 21.1, 23.6, 25.1, 27.0, 27.9, 29.6, 29.7, 34.1, 34.6, 37.1, 37.6, 38.0, 38.4, 40.9, 42.7, 46.3, 47.7, 48.8, 50.2, 55.4, 62.8, 81.1, 83.2, 85.8, 109.9, 128.5, 130.5, 132.9, 150.2, 154.2, 171.6 (Figure S2-Supplementary Materials); IR (ν max cm⁻¹, KBr): 883, 997, 1032, 1375, 1457, 1640, 1705, 1740, 2224, 2945 (Figure S3-Supplementary Materials); EI MS (70 eV) m/z (rel. intensity): 612 (M+, 5), 466 (78), 203 (42), 189 (100), 135 (39) (Figure S4-Supplementary Materials); HRMS (APCI) m/z (neg): 644.3954 (100%) (Figure S5-Supplementary Materials).

3.3. Antiproliferative Activity

The cell lines—T47D (human breast cancer), CCRF/CEM (human leukemia), SW707 (human colorectal adenocarcinoma), P388 (mouse leukemia) and BALB/3T3 (normal mouse fibroblasts)—were obtained from the American Type Culture Collection (Rockville, MD, USA). First, the cells were plated in 96-well plates (Sarstedt, Numbrecht, Germany) at a density of 10⁴ cells per well in 100 μL of culture medium. The tested cells were cultured in the opti-MEM medium supplemented with 2 mM glutamine (Gibco, Grand Island, NY, USA), streptomycin (50 μg/mL), penicillin (50 U/mL) and 5% fetal calf serum (Gibco).

The cell cultures were maintained at 37 °C in humid atmosphere saturated with 5% CO₂. After 24 h, the tested compounds were added to cells in concentrations ranging from 1 to 100 mg/mL. Compounds in given concentration were tested in triplicates in each experiment, which was repeated 3 times. The antiproliferative effects of derivative 3 and cisplatin were tested using the SRB assay for T47D, SW707, BALB/3T3 cells and the MTT assay for leukemic cells, as previously described in the literature [18].

3.4. In Silico Analysis

The physicochemical properties and lipophilicity were predicted using the pharmacokinetic webserver SwissADME (http://www.swissadme.ch/, accessed on 22 September 2023) [19].