New Derivatives of Lupeol and Their Biological Activity

: The natural product lupeol ( 1 ) was isolated from Bombax ceiba leaves, which were used as starting material in the semisynthetic approach. Three new derivatives ( 2a , 2b , and 3 ) were synthesized using oxidation and aldolization. Their chemical structures were elucidated by spectroscopic analyses (HRESIMS and NMR). Compounds 3 showed signiﬁcant α -glucosidase inhibition with an IC 50 value of 202 µ M, whereas 2a and 2b were inactive.


Introduction
Diabetes mellitus (DM) causes high blood glucose after the consumption of a carbohydrate-enriched diet, leading to hyperglycemia. Uncontrolled diabetes is manifested by a very high rise in triglycerides and fatty acid levels [1]. Diverse antidiabetic drugs derived from synthetic compounds are of interest to chemists. However, these synthetic drugs come with several serious complications [1]. Due to the limitations associated with the use of existing synthetic antidiabetic drugs, the search for newer antidiabetic agents from natural sources continues. Lupeol is a pharmacologically active pentacyclic triterpenoid found in several medicinal plants worldwide [2]. It has several potential medicinal properties and is found in a variety of botanical sources [3]. Notably, lupeol has been reported to selectively target diseased and unhealthy human cells, while sparing normal and healthy cells [4]. Dozens of novel lupeol derivatives were synthesized and screened for their in vivo antihyperglycemic activity [5,6]. Most derivatives lowered the blood glucose levels, in a sucrose-challenged streptozotocin-induced diabetic rat (STZ-S) model [5]. To continue our ongoing search for highly efficient antidiabetic agents from derivatized lupeol [6,7], we herein describe the synthesis of lupeol derivatives 2, 2a, 2b, and 3 ( Figure 1). The structures of all the obtained compounds were characterized by 1 H, 13 C NMR, and HRESIMS. All derivatives were evaluated for α-glucosidase inhibition.

Synthesis
Lupeol was isolated from the Vietnamese plant Bombax ceiba, following our previously reported procedure [8]. Lupeol was transformed to products 2, 2a, and 2b using oxidation with Oxone ® , a potassium triple-salt (KHSO5·1/2KHSO4·1/2K2SO4) [6,9]. The conditions followed our previously reported method [6], with slight modifications. Both 2a and 2b had the same molecular formula as C32H52O4. Comparison of NMR data of 2a/2b and 1 indicated that oxidation occurred. The 1 H NMR spectrum of 2a/2b showed differences with 1: the downfield methine at δH 8.11, two oxymethines at δH 5.26 and 4.48, and a doublet methyl at δH 1.22. These signals indicated that the isopropenyl group of 1 was transformed to a 2-formylethyl group at C-19. Moreover, the downfield signal of H-3 (δH 4.48) indicated that 3-OH was esterified by acetic acid. The 13 C NMR spectrum of 2a/2b showed one carbonyl ester at δC 171.1, one formyl group at δC 163.7 and two oxygenated carbons at δC 81.1 and 72.7, supporting the previous findings. Interestingly, 2a and 2b are C-20 epimers. Corbett and co-workers [10,11] indicated the method to define the absolute configuration of C-20 of lupane-type triterpenes. Particularly, the (20S) and (20R) isomers exhibited differences in the chemical shifts of C-19, C-20, C-29, and C-30, especially C-30. According to Corbett et al., 2a, having C-30 at δC 20.1, would have a 20R configuration. On the other hand, 2b would have the 20S configuration due to the lower chemical shift of C-30 at δC 14.2.
Compound 2 was further aldolized with 4-bromobenzaldehyde to afford compound 3. Compound 3 had the same molecular formula as C36H51BrO2, determined by a protonated ion peak at m/z 595.3188 in HRESIMS. Comparison of 1D NMR data of 2 and 3 indicated obvious differences. The first difference is the presence of a 1,4-disubstituted benzenoid characterized by two ortho-coupled protons at δH 7.51 and 7.42, and a trans double bond at δH 6.75 and 7.46. This was confirmed by the disappearance of a methyl ketone group at δH 2.15 (CH3-29). This finding indicated that the aldolization occurred exclusively at C-29. The second difference was in the 13 C NMR spectrum. This spectrum showed the presence of seven aromatic carbons at δC 141.0 (C-1), 133.9 (C-5′), 132.3 (C-2′), 129.8 (C-3′,7′), and 126.9 (C-4′, 6′), supporting the reaction at C-29.

Synthesis
Lupeol was isolated from the Vietnamese plant Bombax ceiba, following our previously reported procedure [8]. Lupeol was transformed to products 2, 2a, and 2b using oxidation with Oxone ® , a potassium triple-salt (KHSO 5 ·1/2KHSO 4 ·1/2K 2 SO 4 ) [6,9]. The conditions followed our previously reported method [6], with slight modifications. Both 2a and 2b had the same molecular formula as C 32 H 52 O 4 . Comparison of NMR data of 2a/2b and 1 indicated that oxidation occurred. The 1 H NMR spectrum of 2a/2b showed differences with 1: the downfield methine at δ H 8.11, two oxymethines at δ H 5.26 and 4.48, and a doublet methyl at δ H 1.22. These signals indicated that the isopropenyl group of 1 was transformed to a 2-formylethyl group at C-19. Moreover, the downfield signal of H-3 (δ H 4.48) indicated that 3-OH was esterified by acetic acid. The 13 C NMR spectrum of 2a/2b showed one carbonyl ester at δ C 171.1, one formyl group at δ C 163.7 and two oxygenated carbons at δ C 81.1 and 72.7, supporting the previous findings. Interestingly, 2a and 2b are C-20 epimers. Corbett and co-workers [10,11] indicated the method to define the absolute configuration of C-20 of lupane-type triterpenes. Particularly, the (20S) and (20R) isomers exhibited differences in the chemical shifts of C-19, C-20, C-29, and C-30, especially C-30. According to Corbett et al., 2a, having C-30 at δ C 20.1, would have a 20R configuration. On the other hand, 2b would have the 20S configuration due to the lower chemical shift of C-30 at δ C 14.2.
Compound 2 was further aldolized with 4-bromobenzaldehyde to afford compound 3. Compound 3 had the same molecular formula as C 36 H 51 BrO 2 , determined by a protonated ion peak at m/z 595.3188 in HRESIMS. Comparison of 1D NMR data of 2 and 3 indicated obvious differences. The first difference is the presence of a 1,4-disubstituted benzenoid characterized by two ortho-coupled protons at δ H 7.51 and 7.42, and a trans double bond at δ H 6.75 and 7.46. This was confirmed by the disappearance of a methyl ketone group at δ H 2.15 (CH 3 -29). This finding indicated that the aldolization occurred exclusively at C-29. The second difference was in the 13 C NMR spectrum. This spectrum showed the presence of seven aromatic carbons at δ C 141.0 (C-1), 133.9 (C-5 ), 132.3 (C-2 ), 129.8 (C-3 ,7 ), and 126.9 (C-4 , 6 ), supporting the reaction at C-29. Inhibition of 2a, 2b, and 3 Compounds 2a, 2b, and 3 were evaluated for α-glucosidase inhibition. Only compound 3 exhibited moderate α-glucosidase inhibition with an IC 50 value of 202 µM, compared with an acarbose-positive control (IC 50 360 µM). Other compounds were inactive.

Materials
Reagents and solvents were obtained from commercial suppliers and were used without further purification. Column chromatography was carried out using Merck Kieselgel 60 silica gel (particle size: 32-63 Å). Analytical TLC was performed using Merck precoated silica gel 60 F-254 sheets.
NMR spectroscopic data were acquired on Bruker Avance III apparatus at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. HRESIMS spectra were recorded on a Bruker MICROTOF-Q 10187.

Synthesis Procedure
Synthesis of 2, 2a, and 2b: Lupeol (1, 200 mg, 0.469 mmol) was oxidized with Oxone ® (951 mg, 1.548 mmol) in acetic acid (40 mL) at 100 • C for 3 h. The mixture was stirred and continuously monitored by TLC. The mixture was extracted with EtOAc-water (1:1) to gain the organic layer. This solution was evaporated to afford a residue. Then, the residue was purified by silica gel CC to give compounds 2, 2a, and 2b.

α-Glucosidase Inhibitory Assay
The α-glucosidase (0.2 U/mL) and substrate (5.0 mM p-nitrophenyl-α-D-glucopyranoside) were dissolved in 100 mM pH 6.9 sodium phosphate buffer [12]. The inhibitor (50 µL) was preincubated with α-glucosidase; then, the substrate (40 µL) was added to the reaction mixture. The enzymatic reaction was carried out at 37 • C for 20 min and stopped by the addition of 0.2 M Na 2 CO 3 (130 µL). Enzymatic activity was quantified by measuring absorbance at 405 nm. All samples were analyzed in triplicate at five different concentrations around the IC 50 values, and the mean values were retained. The inhibition percentage (%) was calculated as follows: Inhibition (%) = [1 − (A sample /A control )] × 100.

Conclusions
Three new derivatives, 2a, 2b, and 3, from the natural product lupeol have been synthesized via oxidation and aldolization routes and evaluated for their α-glucosidase inhibition. Synthetic compound 3 showed much stronger α-glucosidase inhibitory activity (IC 50 202 µM) than acarbose (IC 50 360 µM). Synthetic products 2a and 2b, which lacked the 3-OH group, exhibited lower activity than 3 toward α-glucosidase. This result confirmed that this substituted group might be involved in α-glucosidase inhibition.