New derivatives of a natural nordentatin

Abstract New derivatives were obtained from natural nordentatin (1) previously isolated from the methanol fraction of Clausena excavata by an acylation method. Herein, we report ten new pyranocoumarin derivatives 1a–1j. Their structures were elucidated based on UV-vis, FT-IR, NMR, and DART-MS data. The α-glucosidase inhibition and anticancer activities of nordentatin (1) and its derivatives were also evaluated. The α-glucosidase inhibition assay exhibited that the derivatives 1b, 1d, 1e, 1f, 1h, 1i, and 1j possess higher inhibitory activity for α-glucosidase with IC50 values of 1.54, 9.05, 4.87, 20.25, 12.34, 5.67, and 2.43 mM, whereas acarbose was used as the positive control, IC50 = 7.57 mM. All derivatives exhibited a weak cytotoxicity against a cervical cancer (HeLa) cell line with the IC50 between 0.25 and 1.25 mM. They also showed moderate to low growth inhibition of a breast cancer (T47D) cell line with IC50 values between 0.043 and 1.5 mM, but their activity was lower than that of the parent compound, nordentatin (1) (IC50 = 0.041 mM).


Introduction
Clausena excavata Burm f. belongs to the Rutaceae family and grows mainly in south and southeast Asia [1]. The plant is a wild shrub; its leaves, twigs, and root barks are used as traditional medicine for the treatment of cold, malaria, abdominal pain, snake-bite, viral infections (e.g., HIV), and dermatopathy. The plant is reported to contain bioactive constituents with antibacterial, antifungal, antimycobacterial, antinociceptive, in vivo immunomodulating, and insecticidal properties. Phytochemical studies have revealed that C. excavata is a rich source of coumarins [2,3], carbazole alkaloids [4], and limonoid [5]. Coumarins are plant-derived compounds with a benzopyrone moiety. They possess a wide variety of biological activities. Coumarins and their derivatives are being extensively studied for their biological activities, low toxicity, and low drug resistance properties [6,7].
It has been reported that nordentatin exhibits hypoglycemic activity [8]. The conversion reaction of this compound into its ester derivative is desired, as the derivative is expected to possess more potent biological activities, especially as an antidiabetic medicine in the future. Diabetes mellitus is a chronic disease caused by the dysfunction of carbohydrate metabolism. It has been recognized as one of the most serious public health problems globally. The International Diabetes Federation has estimated that 425 million people worldwide have diabetes. The number will increase to 642 million by 2040 [9][10][11]. Recently, there has been immense interest in the study of enzymes as drug targets. Especially, the inhibition of α-glucosidase can help in controlling postprandial glucose levels in diabetic patients. α-Glucosidases (α-Dglucoside glucohydrolase EC. 3.2.1.20) are membranebound enzymes, located in the epithelium of the small intestine, and the key enzymes of carbohydrate digestion. The inhibitors of α-glucosidase can delay the digestion of starch and other dietary sugars, and thus they prevent the onset of hyperglycemia and maintain the normal blood sugar level [9][10][11]. Clinical trials have already shown that α-glucosidase inhibitors can improve long-term glycemic control and decrease hemoglobin A1c (HbA1c) levels in patients with type II diabetes. They can also delay the development of type II diabetes in patients with impaired glucose tolerance [12]. Inhibitors of α-glucosidase such as voglibose, miglitol, and acarbose are currently used clinically, but their use is limited due to their adverse effects, such as diarrhea, abdominal cramping, flatulence, and vomiting. Therefore, much effort has been focused to develop effective α-glucosidase inhibitors from natural sources [13][14][15].
Cancer, in its various forms, is one of the major causes of death of the human population. Among them, cervical and breast cancers are the most common causes of cancer-related deaths in women globally [15,17]. Several natural products of plant origin could potentially be used as cancer chemotherapeutic agents. Some of the currently used plant-derived anticancer agents include podophyllotoxin, taxol, vincristine, and camptothecin [17]. The conversion reaction of marchantin to its ester derivatives increases the anticancer activity [18]. In the present work, we semi-synthesized pyranocoumarin benzoate derivatives from nordentatin (1) by using different benzoyl chlorides. Subsequently, the inhibitory effects of the resulting derivatives and of the parent compound against α-glucosidase enzymes and two cancer cell lines (HeLa and T47D) were evaluated and compared. Infrared spectra were recorded on an IR Tracer-100, ν in cm −1 . The UV-vis spectra were obtained on a Shimadzu (UV-1800) UV-vis spectrometer. The target compounds (1a-1j) were semi-synthesized by an acylation method with slight modification [19,20]. The starting material, nordentatin (1), was collected from our previous research [2]. Reagents such as 4-bromobenzoyl chloride, 3-chlorobenzoyl chloride, 2-chloro-4-nitrobenzoyl chloride, 3-chloro-4-fluoro benzoyl chloride, 3-bromo-benzoyl chloride, 4-butylbenzoyl chloride, 2,4,6-trichlorobenzoyl chloride, 3-trifluoromethylbenzoyl chloride, 3,5-bis (trifluoromethyl)benzoyl chloride, and 4-iodobenzoyl chloride (Wako, Japan) were used for the modification of compound 1 (nordentatin). Pyridine and 4-dimethylamino pyridine were used as catalysts. The reaction products were isolated by column chromatography on silica gel 60 (0.063-0.200 mm; Merck, Germany), by eluting with n-hexane and dichloromethane (20-100%). The reaction progress and the purity of the obtained compounds were monitored by p-TLC (preparative thin layer chromatography) on UV-254 plates (n-hex:CH 2 Cl 2 , 9:1; detection under UV light and by spraying anis aldehyde, followed by heating). The solvents (CH 2 Cl 2 , n-hex, and EtOAc) were purified by standard methods and distilled just before use.

General procedure for the modification of nordentatin (1) with benzoyl chloride derivatives by an acylation method
Esterification of nordentatin (1) was carried out by an acylation method [6,15] (Figure 1). First, around 100 mg of sample (1) was put in each flask. It was dissolved by adding 2 mL of pyridine and 10 mg of 4-(dimethylamino)-pyridine (DMAP) to the mixture. Subsequently, the mixture was stirred on a magnetic stirrer, and different kinds of benzoyl chlorides were added slowly. Then, the reaction was allowed to proceed at room temperature for 1 h. The mixture was checked with TLC at 30 min intervals until the reaction was completed. A volume of 30 mL of EtOAc and 30 mL of distilled water were added to the product mixture. Pyridine was neutralized with 1 M concentrated HCl. The mixture was shaken vigorously three times by using a separating funnel and the organic layer was collected. The aqueous layer was exacted three times with 30 mL of EtOAc. The EtOAc portion was combined and dehydrated with brine water and anhydrous magnesium sulfate. The solvent was removed using a rotary evaporator. Finally, the product mixtures were chromatographed and the pure compounds 1a to 1j were obtained.

α-Glucosidase inhibition assay
The inhibition activity of all isolated compounds against yeast was determined by the method disclosed by Ramadhan et al. [21] with slight modifications. A sample (10 µL) was mixed with yeast (0.4 U/mL) in 1 mM phosphate buffer (pH 6.9), followed by shaking with a microplate shaker for 2 min and pre-incubation at 37°C for 10 min. The reaction mixture was added to 50 µL of p-nitrophenyl-α-Dglucopyranoside (p-NPG). Subsequently, the mixture was placed in an incubator at 37°C for 20 min. After the incubation, the reaction was quenched by adding Na 2 CO 3 (100 µL). The release of p-nitrophenoxide from p-NPG was detected with a microplate reader at 415 nm (i-Mark ELISA [enzyme-linked immunosorbent assay] reader). Inhibition of the reaction was calculated using where A 0 is the absorbance without the sample and A 1 is the absorbance with the sample. The IC 50 value was determined from a plot of percentage inhibition versus sample concentration. Acarbose was used as the standard control, and the experiment was performed in triplicate.

MTT assay
The cytotoxicity of parent compound (1) and modified compounds (1a-1j) was measured by using the MTT assay method following the protocol of Suwito et al [22].
The cancer cells were seeded in a 96-well plate at a density of 1 × 10 4 cells/well with a phenol red-free RPMI (Roswell Park Memorial Institute) 1640 medium (containing 10% FBS) and kept for 24 h. Subsequently, the tested compound (various concentrations) was applied for 24 h. After addition of 0.5% MTT solution, the incubation was continued for a further 4 h at 37°C/5% CO 2 . The stop solution (0.04 N HCl in isopropanol) was added to the culture medium in each well. The spectroscopic measurements were carried out at 570 nm (peak) and 630 nm (bottom) using an ELISA reader. The experiment was conducted in triplicate. Doxorubicin was used as a positive control.
Ethical approval: The conducted research is not related to either human or animal use.

Semi-synthesis
In this study, derivatization of nordentatin (1) was carried out by using various benzoyl chlorides ( Figure  1). The reaction was simple, quick, and has resulted in ten new pyranocoumarin benzoates (1a-1j) ( Figure 2). Their structures were not only elucidated based on spectroscopic data but also compared with the structure of nordentatin (1) (Figures S1-S46). The FT-IR spectral data supported the presence of the -OH stretching group broad band in compound 1, and the absence of -OH stretching in compounds 1a-1j. In addition, the 13 C-NMR spectral data revealed the presence of one more carbonyl carbon from the benzoyl group at δ c ∼ 161 ppm. The presence of fluoro-containing compounds was inferred from splitting patterns and coupling constants in 13 C-NMR.    [23]. The highest inhibition activity was shown when chloride was at the meta-position as in 1b, followed by para-substituted iodide (1j) and the metaposition of bromide (1e). They are also comparable with the standard control acarbose. In addition, compounds containing bistrifluoromethane (1i), trifluoromethane (1h), 3-chloro, 5-fluoro (1d), and para-butyl groups (1f) showed higher activity than nordentatin (1) with an IC 50 value of 36.7 mM. Based on this study, we can conclude that the activity of some compounds increased after modification, while that of some compounds decreased, e.g., 1a, 1c, and 1g showed no inhibition. The cytotoxicity of all modified compounds was tested against a HeLa cell line, and their IC 50 values were compared with those of nordentatin (1) and the standard control doxorubicin ( Table 2). All tested compounds showed low activity with IC 50 values ranging from 0.25 to 1.25 mM against HeLa cells. However, the results indicated that the modified compounds that contain fluorobenzoyl groups (1i, 1d, 1h, and 1b) have more cytotoxic activity than others and nordentatin (1). On the other hand, the activity of compounds 1j, 1c, 1a, 1g, 1e, and 1f was lower ( Table 2). The investigation of cell proliferation of modified compounds was also performed on a T47D cell line ( Table 2). The results revealed that the modified compounds have moderate to low inhibition against the cancer cell line with the IC 50 values ranging from 0.043 to 1.57 mM, but their activity was lower than the activity of the parent compound, nordentatin (1) with an IC 50 value of 0.041 mM ( Table 2). The structure-activity relationship study showed that the modified compounds with chloro and bromo compounds at meta and para positions had stronger inhibition than others.

Conclusions
In the current work, we continue our previous research concerning bioactive secondary metabolites from natural medicinal plants and their derivatives. As a result, ten new pyranocoumarin benzoate derivatives were semisynthesized in good to excellent yields from natural nordentatin (1) by an acylation method. Both parent and modified compounds were tested for their antidiabetic activity by an α-glucosidase assay and anticancer activity (HeLa and T47D cell lines) by an MTT assay. Among the tested compounds, modified compound 1b showed the highest inhibition activity against yeast α-glucosidase enzymes, in particular 5 times higher than that of acarbose and 20 times higher than that of the parent compound, nordentatin (1). Among the tested compounds, modified compound 1i showed the highest cytotoxic activity against the HeLa cell line. Compound 1b showed a moderate cytotoxic activity against T47D but it was less effective than the parent compound, nordentatin (1). As a result of this study, we can conclude that some modified compounds are promising, since their activity is comparable with that of standard drugs. Especially, active compound 1b, 2,2-dimethyl-10-(2-methylbut-3-en-2-yl)-8-oxo2H,8H-pyrano[3,2-g] chromen-5-yl-3-chlorobenzoate, should be studied as a potential alternative α-glucosidase inhibitor.