**3-Hydroxy-8,14-secogammacera-7,14-dien-21-one: A New Onoceranoid Triterpenes from** *Lansium domesticum* **Corr. cv** *kokossan*

**Zulfikar 1, Nurul Kamila Putri 1, Sofa Fajriah 2, Muhammad Yusuf 1, Rani Maharani 1, Jamaludin Al Anshori 1, Unang Supratman 1,3 and Tri Mayanti 1,\***


Received: 24 August 2020; Accepted: 10 September 2020; Published: 30 September 2020

**Abstract:** A new onoceranoid triterpenes, namely 3-hydroxy-8,14-secogammacera-7,14-dien-21-one (**1**), has been isolated from the fruit peels of *Lansium domesticum* Corr. cv *kokossan*. The structure of **1** was determined on the basis of spectroscopic data including infrared, 1D and 2D-NMR, as well as high resolution mass spectroscopy analysis. Compound **1** showed a weak activity against MCF-7 breast cancer cell lines.

**Keywords:** *Lansium domesticum*; Meliaceae; MCF-7; triterpene onoceranoid

#### **1. Introduction**

*Lansium domesticum* Corr. (Meliaceae) is a popular plant that widely grows in southeastern Asia [1,2]. *L. domesticum* Corr. cv *kokossan* (Meliaceae) is a higher tree growing up to 30 m in height, commonly called "kokosan" in Indonesia [3]. Several bioactive triterpenoids have been isolated from *L. domesticum* [4–9], of which some have shown potential anticancer [10], antibacterial [11], insecticides [12], antimalarial [13], antimutagenic [14,15], and antifeedant activities [16]. Previously, we isolated six triterpenes from the seeds and bark of *L. domesticum* Corr. cv *kokossan* [2,16]. In this paper, we report the isolation and structural elucidation of the new onoceranoid triterpenes **1** (Figure 1), along with its cytotoxic activity against MCF-7 breast cancer lines.

**Figure 1.** Chemical Structure of compound **1**.

#### **2. Results**

#### *Extraction and Isolation*

The dried fruit peel (1.7 kg) was extracted with *n*-hexane, ethyl acetate, and methanol three times, respectively, for 24 h, at room temperature. After solvent removal under reduced pressure, we obtained the *n*-hexane (86 g), ethyl acetate (110 g), and methanol (75 g) crude extracts. A portion of *n*-hexane (60 g) was subjected to vacuum liquid chromatography over silica gel using a 10% gradient of *n*-hexane/EtOAc (10:0–0:10) to afford eight fractions (A–H). Fraction D (6.7 g) was subjected to vacuum liquid chromatography, eluted by a 1% gradient of *n*-hexane/EtOAc (100:0–85:15) to afford five combined fractions (D1–D5). Fraction D4 was then subjected to silica gel column chromatography, eluted with *n*-hexane:acetone:methanol (7:2.5:0.5), resulting in four fractions (D4A–D4D). Compound **1** was obtained from fraction D4B as a white crystal (0.27 g).

3-Hydroxy-8,14-secogammacera-7,14-dien-21-one (**1**), white crystal, m.p. 153–155 ◦C, [α] 20D <sup>−</sup> 3.0◦ (c 0.1, MeOH), HR-TOF-MS *m*/*z* 463.3569 [M + Na]<sup>+</sup> (calcd. for C30H48O2Na, *m*/*z* 463.3552); IR (KBr) νmax (cm<sup>−</sup>1): 3533, 2932, 1700, 1456; 1H-NMR and 13C-NMR showed in the Table 1. Compound **1** was evaluated for its cytotoxic acitivity against MCF-7 breast cancer cell line, and compared to doxorubicin (35.7 μM) as a positive control. Compound **1** exhibited weak activity against MCF-7 with an IC50 value of 717.5 μM.


**Table 1.** 13C and 1H NMR Spectral Data of Compounds **1** in CDCl3.

#### **3. Discussion**

The molecular composition of **1** was proposed as C30H48O2 with seven degrees of unsaturation, based on HR-TOF-MS and NMR spectral data. Mass spectra showed molecular ion peak at *m*/*z* 463.3569 [M + Na]+, with calculated *m*/*z* 463.3552 for C30H48O2Na. The IR spectrum exhibited bands at νmax (cm<sup>−</sup>1) 3533 (hydroxy), 2932 (C-H stretching of aliphatic), 1700 (ketone), and 1456 (*gem* dimethyl).

The 13C NMR data (Table 1) with DEPT and HSQC experiments (Figures S2–S4) revealed the presence of total of 30 carbon signals, which were classified as eight methyls, eight methylenes, seven methines (two olefinic and one oxygenated), and seven quaternary carbons (two olefinic and one carbonyl). The two trisubstituted double bonds and the carbonyl in a system with seven degrees of unsaturation suggested that **1** possess a tetracyclic structure. Previous studies supported that evidence, indicating that compound **1** has an onoceranoid-type triterpenoid [3,14].

The 1H NMR spectrum of **1** (Figure S1) displayed the presence of eight methyl groups at δ<sup>H</sup> (ppm) 1.00 (3H, Me-23), 0.87 (3H, Me-24), 0.99 (3H, Me-25), 1.75 (3H, Me-26), 1.72 (3H, Me-27), 0.76 (3H, Me-28), 1.12 (3H, Me-29), and 1.08 (3H, Me-30). Two olefinic protons (H-7 and H-15) were observed at δ<sup>H</sup> (ppm) 5.45 and 5.42 (each 1H, brs), together with two vinyl methyls proton resonating at δ<sup>H</sup> (ppm) 1.75 (3H, s, H-26) and δ<sup>H</sup> 1.72 (3H, s, H-27), indicating two trisubstituted double bonds of **1**. One oxygenated methine signal was also observed at δ<sup>H</sup> 3.29 ppm (1H, dd, H-3).

The structure of **1** was further defined by 1H-1H COSY (correlated spectroscopy) and HMBC (heteronuclear multiple bond connectivity) spectra (Figure 2, Figures S5 and S6). The 1H-1H COSY spectra showed couplings between H-1 (δ<sup>H</sup> 1.14)/H-2 (δ<sup>H</sup> 1.65), H-2 (δ<sup>H</sup> 1.65)/H-3 (δ<sup>H</sup> 3.29), H-9 (δ<sup>H</sup> 1.66)/H-11 (δ<sup>H</sup> 2.10), H-11 (δ<sup>H</sup> 2.10)/H-12 (δ<sup>H</sup> 1.98), H-12 (δ<sup>H</sup> 1.98)/H-13 (δ<sup>H</sup> 1.59), and H-19 (δ<sup>H</sup> 1.51)/H-20 (δ<sup>H</sup> 2.77), supporting the presence of a onoceranoid-type triterpenoid structure. The oxygenated methine bearing a hydroxy group was located at C-3 by the HMBC correlations of H2-1 (δ<sup>H</sup> 1.86), H3-23 (δ<sup>H</sup> 1.00) and H3-24 (δ<sup>H</sup> 0.87) to C-3 (δ<sup>C</sup> 79.1). The Δ7,8 and Δ14,15 double bonds were assigned by the HMBC correlations from H3-26 (δ<sup>H</sup> 1.75) to C-7 (δ<sup>C</sup> 121.7), C-8 (δ<sup>C</sup> 135.4), C-9 (δ<sup>C</sup> 55.3); and H3-27(δ<sup>H</sup> 1.72) to C-13 (δ<sup>C</sup> 56.0), C-14 (δ<sup>C</sup> 134.9), C-15 (δ<sup>C</sup> 122.3). The ketone group was attached to C-21 by the HMBC correlations of H2-20 (δ<sup>H</sup> 2.77) and H3-29 (δ<sup>H</sup> 1.12) to C-21 (δ<sup>C</sup> 217.0). This analysis indicated that compound **1** was similar to 3β-hydroxyonocera-8(26),14-dien-21-one [5], uniquely differing on the double bond position in the B ring. Based on the coupling constants of H-3 (dd, *J* = 11.4; 4.3 Hz), the configuration of the hydroxyl group at C-3 was indicated in the β-equatorial position [5,17]. From the analyses, compound **1** was determined as 3-hydroxy-8,14-secogammacera-7,14-dien-21-one.

**Figure 2.** 1H-1H COSY and HMBC correlations of compound **1**.

#### **4. Materials and Methods**

#### *4.1. General Experimental Procedures*

Mass spectrum was recorded on a waters Xevo QTOFMS (Waters, Milford, MA, USA). IR spectrum was measured on a One Perkin Elmer infrared-100 (Waltham, MA, USA) in KBr. NMR data were recorded on a Brucker spectrometer (Billerica, MA, USA) at 400 MHz for 1H and 120 MHz for 13C using TMS as an internal standard. Chromatographic separations were carried out on silica gel G60 (Merck, Darmstadt, Germany) and RP18 (Merck). TLC plates were precoated with silica gel GF254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% (*v*/*v*) H2SO4 in ethanol, followed by heating.

#### *4.2. Plant Material*

The fruit peels of *L. domesticum* Corr. cv *kokossan* (Meliaceae) was collected from Cililin, West Java, Indonesia in April 2018. The plant was identified and deposited in The Herbarium of Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia (Identification Number: 195/HB/08/2018).

#### *4.3. Cytotoxic Bioassay*

Cytotoxicity of the compound against MCF-7 human breast cancer cells was measured using MTT (*Methyl Thiazoldiphenyl-Tetrazoliumbromide*) assay. Stock culture was grown in flasks, containing Roswell Park Memorial Institute (RPMI) medium, respectively supplemented with 10% (*v*/*v*) feta bovine serum (FBS) and 1% (*v*/*v*) penicillin-streptomicin as an antibiotic. The culture was incubated at 37 ◦C for 24 h. The medium was changed, and tumor cells were detached and seeded in 96-well microliter plates. After, 24 h, compounds were added to the wells. After 48 h, cell viability was determined by measuring the metabolic conversion of yellow salt or 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color product resulting from reduction in viable cells. Insoluble formazan was diluted with DMSO. The MTT assay results were read using spectrophotometer UV at 450 nm. Compound **1** was evaluated at eight concentrations (15.9; 34.0; 70.3; 140.7; 283.6; 567.3; 1134.6; 2269.1 μM) in 100% DMSO with the final concentration of DMSO of 2.5% (*v*/*v*) in each well. Doxorubicin was used as a positive control. IC50 values were calculated by the linear regression method using Microsoft Excel software.

#### **5. Conclusions**

A new onoceranoid triterpene, namely 3-hydroxy-8,14-secogammacera-7,14-dien-21-one (**1**), was isolated from fruit peels of *L. domesticum* Corr. cv *kokossan* (Meliaceae). This compound exhibited weak cytotoxic activity against MCF-7 human breast cancer cell lines with IC50 value of 717.5 μM.

**Supplementary Materials:** The following are available online, Figure S1. 1H-NMR spectrum of **1** (500 MHz in CDCl3), Figure S2. 13C-NMR spectrum of **1** (125 MHz in CDCl3), Figure S3. DEPT-135◦\_spectrum of **1** (125 MHz in CDCl3), Figure S4. HSQC Spectrum of **1**, Figure S5. 1H-1H COSY spectrum of **1**, Figure S6. HMBC spectrum of **1**, Figure S7. Infrared Spectrum of **1** (in KBr), Figure S8. HR-TOF-MS Spectrum of **1**, Figure S9. TLC Profile of **1**.

**Author Contributions:** Conceptualization, T.M. and U.S.; methodology, N.K.P. and Z.; software validation, S.F.; formal analysis, S.F.; investigation, R.M. and J.A.A.; resources, T.M. and M.Y.; data curation, N.K.P. and Z.; writing—original draft preparation, Z.; writing—review and editing, J.A.A. and M.Y.; visualization, R.M.; supervision, T.M., M.Y., and U.S.; project administration, N.K.P.; funding acquisition T.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PTM, Ministry of Research, Technology and Higher Education, Indonesia, grant number: 1827/UN6.3.1/LT/2020 (T.M.).

**Acknowledgments:** We thank to Joko Kusmoro, M.P. at Jatinangor Herbarium for identification of the plant material, Ahmad Darmawan at the Research Center for Chemistry, Indonesian Science Institute, for performing the NMR measurements, Kansi at the Center Laboratory of Universitas Padjadjaran for performing the HR-TOF-MS measurements, Tenny Putri Wikayani and Nurul Qomarilla at Cells and Tissues Culture Laboratory, Faculty of Medicine, Universitas Padjadjaran for the cytotoxic assay.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples of the compounds **1** are available from the authors.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Short Note* **2-Propyl-***N*- **-[1,7,7-trimethylbicyclo[2.2.1]hept-2 ylidene]pentanehydrazide**

#### **Mariia Nesterkina 1,\*, Dmytro Barbalat 2, Ildar Rakipov 1,3 and Iryna Kravchenko <sup>1</sup>**


Received: 28 September 2020; Accepted: 19 October 2020; Published: 26 October 2020

**Abstract:** 2-Propyl-*N*- -[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide was obtained in 80% yield via the Einhorn variation of the Schotten–Baumann method by (+)-camphor hydrazide condensation with valproic acid (VPA) chloride. The structure of the titled compound was verified by Raman, FTIR, 1H-NMR, and 13C-NMR spectral analysis along with FAB-mass spectrometry. Thermal properties of synthesized derivative were elucidated by DSC and its purity by HPLC. The compound was successfully tested as a potential anticonvulsant agent based on models of chemically- and electrically-induced seizures.

**Keywords:** hydrazone; (+)-camphor; valproic acid; technology; terpenoid; anticonvulsant activity

#### **1. Introduction**

Valproic acid (VPA) refers to extensively used antiepileptic drugs (AEDs) that have been found to be effective against all types of seizures [1]. However, clinical use of VPA is associated with a significant adverse effect such as hepatotoxicity [2], causing the need for chemical modification of the VPA molecule aimed at dose reducing VPA-containing derivatives. In this context, particular interest is focused on terpenoids, which have proven to be anticonvulsant agents and serve as versatile scaffolds for enhancing the permeability of drugs [3,4]. Recently, this strategy has been applied to facilitate penetration of gamma-amino butyric acid (GABA) through the blood-brain barrier (BBB) by its conjugation with *l*-menthol, resulting in an increase in anticonvulsant potency [5]. Notably, the binding of biologically active molecules to terpenoids is expedient by the formation of enzymatically degradable bonds such as -NH-N=C-, -CO-OR-, -CO-NH-, etc.

Bearing the aforementioned in mind, bicyclic terpenoid (+)-camphor possessing its own antiseizure action [6] has been used as a base for the synthesis of conjugates containing the VPA moiety. Thus, we report herein on the synthesis and detailed structural determination of 2-propyl-*N*- -[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide comprising both (+)-camphor and VPA residues. The obtained compound was then investigated as a potential anticonvulsant agent on the models of maximal electroshock seizure (MES) and pentylenetetrazole (PTZ)-induced convulsions.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

2-Propyl-*N*- -[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide **3** was synthesized via the Einhorn variation of the Schotten-Baumann method, as shown in Scheme 1. For this purpose, camphor hydrazine **1** was acylated with valproic acid chloride **2** in the presence of triethylamine (TEA) both as an acid scavenger and nucleophilic acylation catalyst. Initial hydrazine **1** was obtained by camphor treatment with hydrazine hydrate and acid chloride **2** through VPA reflux with SOCl2 [7,8]. Target compound was isolated in an 80% yield as white crystals well soluble in organic solvents such as chloroform, hexane, acetonitrile, and benzene. The structure of hydrazone **3** was verified by 13C-NMR, 1H-NMR, FT-IR, Raman spectroscopy, and FAB-mass spectrometry. Thermal transition of compound **3** was carried out by differential scanning calorimetry (DSC) along with the determination of melting enthalpy (Δ*H*m) as 120.2 J/g. The purity was assessed by HPLC analysis using the reversed-phase method with isocratic elution by a system composed of acetonitrile: 0.01% formic acid aqueous solution (70:30). According to the HPLC assay, hydrazone purity was established by internal normalization with detection at ultraviolet wavelengths (230 nm and 260 nm) as 100% and 99%, respectively.

**Scheme 1.** Synthesis of 2-propyl-*N*- -[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide.

Fast-atom bombardment (FAB) has been applied as an ionization method in mass spectrometry investigation; the FAB-MS spectrum of derivative **3** displays the protonated molecular ion peak [M + H]<sup>+</sup> at *m*/*z* 293. The FT-IR spectrum exhibits absorption bands of N–H bonds (3179 cm−1), C=O groups (1697 cm<sup>−</sup>1), and alkyl C–H (2970-2834 cm−1). Taking into account the overlapping of C=N and C=O vibrations in the FT-IR spectrum, the formation of the imine (C=N) group was confirmed by Raman spectroscopy as an intense peak at 1646 cm−1. The 1H-NMR spectrum corroborates the proposed structure of compound **3** by chemical shift, integration, and multiplicity of resonance signals. The characteristic proton signal of the -NH group was observed as a singlet at 9.51 ppm. Likewise, the 13C-NMR spectrum detected the presence of all carbon atoms in the molecule.

#### *2.2. Anticonvulsant Activity*

Anticonvulsant activity of the title compound was determined by two pharmacological seizure models including intravenous pentylenetetrazole (i.v. PTZ) and maximal electroshock seizure (MES) tests. In the PTZ-induced model, the anticonvulsant effect was established by the assessment of PTZ minimum effective doses (MED) that provoke clonic-tonic convulsions (DCTC) and tonic extension (DTE). As illustrated in Figure 1, camphor, hydrazone **3**, along with VPA, demonstrated protection against PTZ-induced seizures at 3 h after administration, as validated by the increase of DCTC and DTE values to 165–187% and 170–197%, respectively, compared with the control (100%). However, at this time point, there were no statistically significant differences between the afore-mentioned experimental groups, indicating comparable activity of the investigated compounds. In marked

contrast, synthesized derivative **3** was found to possess significant antiseizure action over a long time period (24 h after administration) with the average values of 277% for DCTC and 238% for DTE. As seen, hydrazone **3** exhibited higher potency versus initial camphor and VPA (*p* < 0.01), which highlighted its prolonged effect.

**Figure 1.** At 3 h and 24 h after oral administration. Values are given as mean ± SEM, *n* = 5 mice; for all groups *p* < 0.01 compared with the control; \*\* *p* < 0.01 compared with VPA.

In the MES test, camphor hydrazone **3** substantially prevented the animals' mortality at 3 h after administration, manifesting 80% protection that is equivalent to the VPA effect (80%), whereas moderate antiseizure action was observed for the initial camphor (60%) (Table 1). Conversely, the activity of the titled compound was maintained at a long time period (24 h) with 100% of mortality protection, which confirms the idea toward enzymatic cleavage of labile bonds (C=N, N-NH, or CO-NH) in hydrazone molecules, followed by gradual release of pure terpenoid and VPA.


**Table 1.** Against maximal electroshock (MES)-induced seizures.

Thus, camphor derivative **3** protects against seizures induced by chemical and electrical stimuli both in short (3 h) and long (24 h) time periods.

#### **3. Materials and Methods**

#### *3.1. General Information*

(+)-Camphor and valproic acid (VPA) were obtained from commercial sources. Hydrazine **1** and acid chloride **2** were synthesized according to the standard procedure [7,8]. The progress of reaction was monitored by TLC on Merck-made (TLC Silica gel 60 F254) plates (Darmstadt, Germany) visualized by UV light using ethyl acetate-benzene (1:1) as the eluent system. Structure of the obtained compound was established by 1H-NMR spectroscopy on a Varian VXR-300 (300 MHz) instrument (Varian Inc., Palo Alto, CA, USA) and by 13C-NMR spectroscopy on a Varian-Mercury 400 spectrometer

(Varian Inc., Palo Alto, CA, USA) using DMSO-*d6* as a solvent and TMS as an internal standard. FAB mass spectra were obtained on a VG 70-70EQ mass spectrometer (VG Analytical Ltd., Manchester, UK) equipped with a Xe ion gun (8 kV); the sample was mixed with the *m*-nitrobenzyl-alcohol matrix. The purity of the compound was checked by high-performance liquid chromatography on an Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA). IR spectra were measured with a Frontier FT-IR spectrometer (Perkin-Elmer, Hopkinton, MA, USA) using KBr pellets. Raman spectra were undertaken with a DXR Raman Microscope (Thermo Fisher Scientific, Madison, WI, USA). DSC curves were recorded in a Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE, USA) using aluminum crucibles containing approximately 2 mg of samples, under a dynamic nitrogen atmosphere and a heating rate of 5 ◦C min−<sup>1</sup> in the temperature range of 20 to 200 ◦C.

#### *3.2. Synthesis of 2-Propyl-N*- *-[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide* (**3**)

To a stirred solution of (+)-camphor hydrazine **1** (0.8 g, 5.26 mmol) in CH2Cl2 (25 mL) at room temperature, valproic acid chloride (0.897 g, 5.52 mmol) was added. The reaction mixture was cooled to 0 ◦C, stirred for 10 min, and triethylamine (TEA) was added dropwise (0.77 mL, 5.52 mmol). Stirring was continued for 30 min, then the flask was gradually warmed to room temperature and the stirring continued for an additional 4 h. The reaction completion was monitored by TLC. The reaction mixture was filtered, the filtrate was diluted with CH2Cl2 to 100 mL, and washed with 1 M aqueous HCl, 10% aqueous NaHCO3, and water. The combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by recrystallization from methanol.

White crystals (80%). 1H-NMR (300MHz, DMSO-*d6*) δ: 0.65 (s, 3H, CH3), 0.82 (s, 6H, 2CH3), 0.86 (s, 3H, CH3), 0.90 (s, 3H, CH3), 1.21 (m, 8H, 2CH2-CH2), 1.46-1.51 (m, 2H, CH2), 1.65 (t, 1H, CH), 1.77 (m, 1H, CH), 1.89 (m, 2H, CH2), 2.30 (m, 1H, CH), 9.51 (s, 1H, NH). 13C-NMR (100 MHz, DMSO-*d6*) δ: 177.4 (C=O), 171.3 (C-2), 52.3 (C-1), 47.8 (C-7), 47.6 (C-4), 43.9 (CH), 35.1 (C-3), 34.5 (CH2), 32.7 (C-6), 27.3 (C-5), 20.5 (CH2), 18.8 (C-8,9), 14.2 (C-10), 11.5 (CH3). FT-IR (νmax, CM<sup>−</sup>1): 3179 (N-H), 2970–2834 (C-H), 1697 (C=O). Raman (νmax, CM<sup>−</sup>1): 2943-2872 (C-H), 1646 (C=N). MS (FAB) *m*/*z*: 293 [M + H]+. HPLC: tr = 23.43 min. M.p. (DSC) onset: 154.26 ◦C, peak max: 155.46 ◦C (Supplementary Materials).

#### *3.3. Anticonvulsant Screening*

Anticonvulsant activity was studied using outbreed male white mice (18–22 g) as experimental animals. Animals were maintained under a 12 h light regime and in a standard animal facility with free access to water and food. All the animals were purchased from Odessa National Medical University, Ukraine. The Animal Ethics Committee (agreement No. 03/2020) of Odessa National Polytechnic University (Ukraine) approved the study. (+)-Camphor, VPA, and compound **3** were administered orally (preliminarily dissolved in in Tween 80/water emulsion): camphor at a dose of 50 mg/kg; VPA and hydrazone **3** in equimolar amounts. Antiseizure evaluation was carried out at 3 h and 24 h after administration both on MES and PTZ-induced convulsions according to the earlier reported procedures [9,10].

#### **4. Conclusions**

Einhorn variation of the Schotten–Baumann method was successfully applied for the synthesis of 2-propyl-*N*- -[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]pentanehydrazide via (+)-camphor hydrazide condensation with valproic acid (VPA) chloride, followed by structure confirmation using Raman, FT-IR, 1H-NMR, and 13C-NMR spectral analysis along with FAB-mass spectrometry. The title compound was found to demonstrate prolonged anticonvulsant activity both on PTZ- and MES-induced seizures.

**Supplementary Materials:** Copies of the 1H-NMR 13C-NMR, FT-IR, Raman, FAB mass spectra, DSC thermograms, and HPLC chromatograms are available online.

**Author Contributions:** I.K. conceived and designed the experiments; M.N. performed the synthesis and analyzed the NMR spectral data; D.B. performed the analysis of FT-IR, Raman, DSC, and HPLC experiments; I.R. carried out FAB characterization of the compound. All authors contributed in manuscript writing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Short Note N***-[7-Chloro-4-[4-(phenoxymethyl)-1***H***-1,2,3-triazol-1 yl]quinoline]-acetamide**

**Paolo Coghi 1,2,\*, Jerome P. L. Ng 2,3, Ali Adnan Nasim 2,3 and Vincent Kam Wai Wong 2,3,\***


**Abstract:** The 1,2,3-triazole is a well-known biologically active pharmacophore constructed by the copper-catalyzed azide–alkyne cycloaddition. We herein reported the synthesis of 4-amino-7-chloro-based [1,2,3]-triazole hybrids via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of 4-azido-7-chloroquinoline with an alkyne derivative of acetaminophen. The compound was fully characterized by Fourier-transform infrared (FTIR), proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), heteronuclear single quantum coherence (HSQC), ultraviolet (UV) and high-resolution mass spectroscopies (HRMS). This compound was screened in vitro with different normal and cancer cell lines. The drug likeness of the compound was also investigated by predicting its pharmacokinetic properties.

**Keywords:** 1,2,3-triazole; anticancer; aminoquinoline; hybrid compound

#### **1. Introduction**

The 7-chloroquinoline moiety, a pharmacophore of several established antimalarial drugs such as chloroquine (Figure 1a) [1], is recently being focused on as a potential anticancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs [2].

With increasing drug resistance to available agents, intensive drug discovery efforts on developing new antimalarial/anticancer drugs or modifying existing agents are ongoing [3]. Molecular hybridization as a drug discovery strategy involves the rational design of new chemical entities by the fusion (usually via a covalent linker) of two drugs, of which both active compounds and/or pharmacophoric units are recognized and derived from known bioactive molecules [3].

In order to broaden the structural diversity of the compounds and intensify their biological activities, covalently linked hybrids were created with a pharmacologically significant class of compounds known as [1,2,3]-triazole (Figure 1b–d). These [1,2,3]-triazoles have exhibited a myriad of biological activities including antifungal, [4] antibacterial [5] and antitubercular activities [6]. Some structures of quinolone- and triazole-based hybrids reported in literature [7–10] (Figure 1e–h).

We have previously reported the conversion of the commercially available 4,7 dichloroquinoline **1** to a series of 4-amino-7-chloroquinolone derivatives [11]. Herein, we reported the synthesis of a novel 4-amino-7-chloroquinoline-based 1,2,3-triazole hybrid **5** by Cu(I)-catalyzed azide–alkyne cycloaddition. The structure of compound **5** was characterized by NMR, MS, FT-IR and UV spectra. The cytotoxicity of **5** was also evaluated against different cell lines.

**Citation:** Coghi, P.; Ng, J.P.L.; Nasim, A.A.; Wong, V.K.W. *N*-[7-Chloro-4-[4- (phenoxymethyl)-1*H*-1,2,3-triazol-1 yl]quinoline]-acetamide. *Molbank* **2021**, *2021*, M1213. https://doi.org/ 10.3390/M1213

Academic Editor: Bartolo Gabriele

Received: 20 April 2021 Accepted: 13 May 2021 Published: 20 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Clinically approved quinoline (red) and 1,2,3-triazole (blue) containing drugs. (**a**) Chloroquine; (**b**) tazobactam; (**c**) carbonic anhydrase inhibitors (CAI); (**d**) cefatrizine; (**e**–**h**) some structures of quinolone- and triazole-based hybrids reported in literature and compound **5** reported in this paper.

#### **2. Results and Discussion**

The synthesis of the triazole hybrid compound, *N*-[7-chloro-4-[4-(phenoxymethyl)-1*H*-1,2,3-triazol-1-yl] quinoline]-acetamide **5**, involved the initial synthesis of the precursors 4-azido-7-chloroquinoline **2** and *O*-acetylenic acetaminophen **4**.

Quinoline **2** was furnished by applying the modified method reported by de Souza et al. [12], whereby 4,7-dichloroquinoline **1** reacted with two equivalents of NaN3 in anhydrous DMF at 65 ◦C for 6 h (Scheme 1a). The recrystallization of the crude product from CH2Cl2/hexane afforded 2 in an 86% yield.

**Scheme 1.** (**a**) Synthesis of 4-azido-7-chloro-quinoline. (i) NaN3, DMF; (**b**) Synthesis of *N*-[7-chloro-4- [4-(phenoxymethyl)-1*H*-1,2,3-triazol-1-yl]quinoline]-acetamide. (ii) propargyl bromide, DMF, K2CO3; (iii) **2**, CuSO4, ascorbic acid, *t*BuOH/water (1:1).

The *O*-alkylation reaction of acetaminophen **3** with 1.5 equivalents of propargyl bromide and anhydrous K2CO3 in anhydrous DMF yielded the acetylenic intermediate **4** in a good yield after the recrystallization from CH2Cl2/hexane (82%, Scheme 1b).

The hybrid compound **5** was finally obtained by using a modified cycloaddition procedure reported by Fokin et al. [13].

Equimolars of quinoline **2** and acetylene **4** were dissolved in *t*BuOH/water (1:1) and were treated with sodium ascorbate (0.4 equiv.) and CuSO4 (20 mol%) sequentially

(Scheme 1b). The reaction mixture was then stirred vigorously at 65 ◦C for 24 h. Compound 5 was isolated by column chromatography in a high yield (72%).

The structure of **5** was verified by 1H and 13C NMR spectra (Supplementary Materials, Figures S3 and S4). The 1H-NMR spectrum showed a singlet at 5.33 ppm associated with the C13-methylene group and a singlet at 2.07 ppm associated with the α-position of the C18-carbonyl group.

Regarding the 13C NMR signals, the disappearance of characteristic peaks of acetylenic group at 78.5 and 75.5 ppm, and the appearance of a C-13 methylene signal at 61.5 ppm and C-11 vinylic signal at 127.3 ppm were probably the most relevant features to verify the incorporation of a triazole moiety.

Heteronuclear single quantum coherence spectroscopy (HSQC) was used to assign 13C signals of compound **5** as shown in Table S1 (see Supplementary Materials for 2D spectra, Figures S5 and S6).

The IR spectrum of compound **5** showed characteristic N–H stretching at 3448 cm<sup>−</sup>1, C=O stretching at 1674 cm−<sup>1</sup> and C=C stretching at 1612 cm−<sup>1</sup> (Supplementary Materials, Figure S7). Other vibrational peaks at 1550, 1512 and 1411 cm−<sup>1</sup> were corresponding to the N–H bending in the amide. Moreover, the absence of the azide stretching peak of **2** at 2123 cm−<sup>1</sup> in the IR spectrum of **5** confirmed the conversion of azide.

The UV and HRMS of **5** was also recorded for further characterization (Supplementary Materials, Figures S8 and S9).

The target compound **5** showed a low cytotoxicity by evaluating its in vitro cytotoxic activities against normal (LO2 and BEAS-2B), immortalized (HEK293) and cancer (HepG2 and A549) cell lines (IC50 values > 100 μM, Supplementary Materials Figure S10).

In addition, the promising compound **5** was assessed by predicting its physicochemical properties and oral bioavailability. From the calculated physicochemical properties (Supplementary Materials Table S2), compound **5** did not violate any of Lipinski's rules [14], indicating its drug-like character and a good chance for oral administration.

This finding corroborates the results of the gastrointestinal absorption from SwissADME, in which compound 5 was predicted with high absorption according to BOILED-Egg model [15] (Figure S11) and data from admetSAR 2 [16], in which the compound was predicted to be orally bioavailable and absorbed in human intestine.

#### **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 E. Merck precoated silica gel 60 F254 plates and visualized with phosphomolybdic acid, iodine, or a UV-visible lamp.

All chemicals were purchased from Bide Pharmatech., Ltd. (Shanghai, China) and J & K scientific (Hong Kong, China). 1H-NMR and 13C-NMR spectra were collected in CDCl3 at 25 ◦C on a Bruker Ascend®-600 NMR spectrometer (600 MHz for 1H and 150 MHz for 13C) (Bruker, Billerica, MA, USA). All chemical shifts were reported in the standard δ notation of parts per million using the peak of residual proton signals of CDCl3 or DMSOd6 as an internal reference (CDCl3, δ<sup>C</sup> 77.2 ppm, δ<sup>H</sup> 7.26 ppm; DMSO-d6, δ<sup>C</sup> 39.5 ppm, δ<sup>H</sup> 2.50 ppm). High-resolution mass spectra (HRMS) were measured using electrospray ionization (ESI). The measurements were done in a positive ion mode (interface capillary voltage 4500 V); the mass ratio was from *m*/*z* 50 to 3000 Da; external/internal calibration was done with electrospray calibration solution.

HRMS analyses were performed by an Agilent 6230 electrospray ionization (ESI) timeof-flight (TOF) mass spectrometer with Agilent C18 column (4.6 mm × 150 mm, 3.5 μm). The mobile phase was isocratic (water +0.01% TFA; CH3CN) at a flow rate of 0.5 mL/min. The peaks were determined at 254 nm under UV.

UV analysis was performed by a Shimadzu UV—2600 (Osaka, Japan) with 1 cm quartz cell and a slit width of 2.0 nm. The analysis was carried out using a wavelength in the range of 200–400 nm.

IR analysis (KBr) was performed by a Shimadzu IRAffinity-1S (Osaka, Japan) with a frequency range of 4000–500 cm<sup>−</sup>1.

#### 3.1.1. Synthesis of 4-Azido-7-chloro-quinoline (**2**)

The 4,7-Dichloroquinoline (2.0 g, 10 mmol) was dissolved in 5 mL anhydrous DMF. NaN3 (1.3 g, 20 mmol) was then added in one portion, and the resulting mixture was stirred at 65 ◦C for 6 h, whereupon TLC indicated reaction completion. The reaction mixture was then allowed to cool to ambient temperature, after which it was diluted with 100 mL CH2Cl2, washed with water (3 × 30 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The resulting product residue was recrystallized from a CH2Cl2/hexane 1:1 mixture to yield the final pure product **2** as colorless, needle-like crystals in 86% yield.

*δ*<sup>H</sup> (600 MHz, CDCl3) 8.82 (1H, d, *J =* 4.9 Hz, H-2), 8.09 (1H, d, *J* = 2.4 Hz, H-8), 8.01 (1H, d, *J* 9.3, H-5), 7.49 (1H, dd, *J* 2.4 and 9.3, H-6) 7.12 (1H, d, *J* 4.9, H-3) ppm; *δ*<sup>C</sup> (150 MHz, CDCl3) 150.9, 149.1, 146.8, 136.9, 127.9, 123.8, 119.9, 108.7 ppm. The spectral characteristics are consistent with those of **2** in the literature [17].

#### 3.1.2. Synthesis of *N*-[4-(propargyloxy) Phenyl] acetamide (**4**)

Acetaminophen (*N*-acetyl-*para*-aminophenol) (13 mmol) was dissolved in 10 mL of anhydrous DMF. Anhydrous K2CO3 (2.7 g, 19.5 mmol) was then added to the solution, and the mixture was stirred at 30 ◦C for 30 min. Propargyl bromide (3-bromopropyne, 2.2 mL, 19.5 mmol) was then added slowly to the reaction mixture, and subsequently stirred at 30 ◦C for 6 h upon which TLC indicated completion of the reaction. The reaction mixture was then diluted with 50 mL water and extracted with ethyl acetate (3 × 50 mL). These extracts were then combined, washed with water (2 × 50 mL), dried over anhydrous Na2SO4 and evaporated in vacuo to yield the product residue that was then recrystallized from the CH2Cl2/hexane 1:1 mixture to yield the compound 4 in 82% yield. *δ*<sup>H</sup> (600 MHz, CDCl3) 2.15 (3H, s, Me), 2.51 (1H, s), 4.67 (2H, s, CH2), 6.94 (2H, d, *J* = 8.99 Hz, HAr), 7.22 (1H, br, NH), 7.40 (2H, d, *J* = 8.99 Hz, HAr) ppm; *δ*<sup>C</sup> (150MHz, CDCl3) 24.2, 56.3, 75.5, 78.5, 115.5, 121.8, 131.9, 154.6, 168.3 ppm. The spectral characteristics are consistent with those of **4** in the literature [18].

3.1.3. Synthesis of N-[7-chloro-4-[4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl] quinoline]-acetamide (**5**)

The derivative of acetaminophen 4 (1mmol) and the appropriate azide **2** were dissolved in 5 mL *t*BuOH/water (1:1) and, while stirring at 65 ◦C, 1 M sodium ascorbate (0.4 mL, 0.4 mmol) and 1 M CuSO4 (0.2 mL, 20 mol%) were added sequentially, in that order. The reaction mixture was then stirred at 65 ◦C for 24 h. The crude product was then precipitated out by slowly adding cold water to the reaction mixture, after which it was filtered, washed with water, air dried and purified by silica column chromatography (eluents ranging in polarity from EtOAc/hex 3:7 to 5% MeOH in EtOAc). Yield 72%, *δ*<sup>H</sup> (600 MHz, CDCl3) 2.07 (3H, s, Me), 5.33 (2H, s, CH2-O), 7.09 (2H, d, *J* = 9 Hz, H-15), 7.57 (2H, d, *J* = 9 Hz, H-16), 7.86 (1H, dd, *J* = 9.1 and 2.2 Hz, H-6), 7.93 (1H, d, *J* = 4.65 Hz, H-3), 8.03 (1H, d, *J* = 9.1 Hz, H-5), 8.36 (1H, d, *J* = 2.3 H-8), 9.01 (1H, s, H-11), 9.23 (1H, d, *J* = 4.65 Hz, H-2), 9.89 (1H, s, NH) ppm; *δ*<sup>C</sup> (150MHz, CDCl3) 24.2 (C-19), 61.7 (C-13), 115.3 (C-15), 117.6 (C-3), 120.8 (C-16), 121 (C-10), 125.8 (C-5), 127.3 (C-11), 128.6 (C-8), 129.5 (C-6), 133.5 (C-17), 135.9 (C-4), 140.8 (C-7), 144.2 (C-12), 149.8 (C-9), 152.8 (C-2), 154.1 (C-14), 168.2 (CO) ppm; HRMS-ESI *m*/*z* 394.1083 [M + H]+ (calculated for C20H17ClN2O2, *m*/*z* 394.1065); UV (CH2Cl2) peaks 211, 234 and 287 nm; IR (KBr) (νmax/cm<sup>−</sup>1) 3478 (NH), 2924 (CH), 2368, 1674 (NH amide), 1612, 1550, 1512, 1458, 1411, 1373, 1319, 1242, 1049, 825 cm<sup>−</sup>1.

#### *3.2. Biological Studies*

Compound **5** was dissolved in DMSO at a final concentration of 50 mM and stored at −20 ◦C before use. Cytotoxicity was assessed by using the 3-(4,5-dimethylthiazole-2yl)- 2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) assay as previously described [19]. Briefly, 4 × 103 cells per well were seeded in 96-well plates before drug treatments. After

overnight cell culture, the cells were then exposed to different concentrations of selected compounds (0.19–100 μM) for 72 h. Cells without drug treatment were used as controls. Subsequently, 10 μL of 5 mg/mL MTT solution was added to each well and incubated at 37 ◦C for 4 h followed by addition of 100 μL solubilization buffer (10 mM HCl in solution of 10% of SDS) and overnight incubation. Then A570 nm was determined in each well on the next day. The percentage of cell viability was calculated using the following formula: cell viability (%) = Atreated/Acontrol × 100. A representative graph of at least three independent experiments was shown in Supplementary Materials Figure S8.

#### **4. Conclusions**

The synthesis of a potential triazole-based quinoline was presented. The chemical structure of the synthesized compound was verified by using NMR, mass, IR and UV spectrometries. The cytotoxicity and drug likeness of the compound were also determined by MTT assay and computations, respectively.

**Supplementary Materials:** The following are available online, Figure S1: 1H NMR compound **4**, Figure S2: 13C NMR compound **4**, Figure S3: 1H NMR compound **5**, Figure S4: 13C NMR compound **5**, Figures S5 and S6: HSQC compound **5**, Figure S7: IR spectrum compound **5**, Figure S8: HRMS of **5**, Figure S9: UV spectrum, Figure S10: cytotoxicity results, Table S1: 1H and 13C-nuclear magnetic spectroscopy (NMR) chemical shifts, Table S2: physicochemical properties of **5** calculated by SwissADME, Figure S11: BOILED-Egg graph.

**Author Contributions:** Conceptualization, P.C.; methodology, P.C. and J.P.L.N.; investigation, A.A.N.; data curation, J.P.L.N.; writing—original draft preparation, P.C.; writing—review and editing, J.P.L.N. and V.K.W.W.; supervision, P.C. and V.K.W.W.; project administration, P.C.; funding acquisition, P.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by FDCT grants from Macao Science and Technology Development Fund to PC (project code: 0096/2020/A, 0087/2020/A).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Giovanni Ribaudo for his kind and grateful support and Yuhan Xie for support during analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Short Note* **Kokosanolide D: A New Tetranortriterpenoid from Fruit Peels of** *Lansium domesticum* **Corr. cv Kokossan**

**Fawwaz M. Fauzi, Sylvia R. Meilanie, Zulfikar, Kindi Farabi, Tati Herlina, Jamaludin Al Anshori and Tri Mayanti \***

Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor 45363, Indonesia; fawwaz18002@mail.unpad.ac.id (F.M.F.); sylvia14001@mail.unpad.ac.id (S.R.M.); zulfikar15002@mail.unpad.ac.id (Z.); kindi.farabi@unpad.ac.id (K.F.); tati.herlina@unpad.ac.id (T.H.); jamaludin.al.anshori@unpad.ac.id (J.A.A.)

**\*** Correspondence: t.mayanti@unpad.ac.id; Tel.: +62-8132-010-2633

**Abstract:** A novel tetranortriterpenoid named kokosanolide D has been isolated from fruit peels of *Lansium domesticum*. The structure of kokosanolide D was elucidated primarily on the basis of spectroscopic data including infrared, 1D and 2D-NMR, as well as high resolution mass spectroscopy analysis and comparison with related compounds previously reported.

**Keywords:** kokosanolide; tetranortriterpenoid; *Lansium domesticum*; Meliaceae

#### **1. Introduction**

*Lansium domesticum* Corr. (Meliaceae) is a tree that grows widely in Indonesia and various countries in Southeast Asia [1,2]. In addition, this tree is also found in Australia, Suriname and Puerto Rico [3]. *Lansium domesticum* Corr. cv *Kokossan*, which is commonly called "kokosan" in Indonesia, thrives and bears fruit during the rainy season [4]. Several types of triterpenoid compounds have been reported from *L. domesticum* Corr., such as tetranortriterpenoid [5], triterpenoid glycosides [6,7], onoceranoids [4,8–10] and cycloartane triterpenoids [11], which showed various biological activities, such as being antifeedant [12], anticancer [13], antibacterial [14], antimutagenic [15,16], and antimalarial [17].

During our previous research on triterpenoid compounds from Indonesian Meliaceae plants, we have isolated two tetranortriterpenoid compounds (kokosanolide A and C) from the fruit seeds and one onoceranoid type triterpenoid compound (kokosanolide B) from the bark of *L. domesticum* cv *Kokossan* [12]. In the present study, we isolated a new tetranortriterpenoid compound from the methanol extract of fruit peels of *L. domesticum* Corr. cv *Kokossan* which is named kokosanolide D (**1**) (Figure 1). The chemical structure of compound **1** is determined by spectroscopic data, including infrared, 1D and 2D-NMR and HRMS.

**Figure 1.** Chemical Structure of Compound **1**.

**Citation:** Fauzi, F.M.; Meilanie, S.R.; Zulfikar; Farabi, K.; Herlina, T.; Al Anshori, J.; Mayanti, T. Kokosanolide D: A New Tetranortriterpenoid from Fruit Peels of *Lansium domesticum* Corr. cv Kokossan. *Molbank* **2021**, *2021*, M1232. https://doi.org/ 10.3390/M1232

Academic Editor: Giovanni Ribaudo

Received: 9 May 2021 Accepted: 5 June 2021 Published: 10 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Results**

#### *Extraction and Isolation*

The dried fruit peels of *L. domesticum* (1.7 Kg) was macerated at room temperature with *n-*hexane (5 × 2 L), ethyl acetate (5 × 2 L), and methanol (5 × 2 L). The solvents were removed by evaporation to give a crude *n-*hexane extract (86 g), ethyl acetate (110 g), and methanol (75 g). The methanol extract (75 g) was partitioned using butanol:H2O (1:1) to give the butanol fraction (24 g). The butanol fraction (24 g) was fractionated by vacuum liquid chromatography on silica gel using a 10% gradient of *n-*hexane-ethyl acetatemethanol to give seventeen fractions (A–Q). Fraction G–H (2.9 g) was separated by silica gel open column chromatography using a 5% gradient of dichloromethane-ethyl acetate to give thirty-four fractions (GH1–GH34). Fraction GH13–15 (231.0 mg) was separated with silica gel open column chromatography using a 0.5–1% gradient of chloroform-acetone to give compound **1** (14.2 mg).

Kokosanolid D, colorless amorphous powder; HR-TOFMS *m*/*z* 517.2075 [M + H]<sup>+</sup> (calcd. for C27H33O10, *m/z* 517,2074); IR (KBr) vmax (cm<sup>−</sup>1): 3441, 1760, 1726, 1698, 1440, 1392; 1H-NMR (CDCl3, 500 MHz) and 13C-NMR (CDCl3, 125 MHz) shown in Table 1.

**Table 1.** 13C-NMR, 1H NMR and HMBC Spectral Data of Compound **1** in CDCl3.


#### **3. Discussion**

Kokosanolide D (**1**) is obtained as colorless and amorphous from chloroform acetone. The molecular formula is determined to be C27H32O10 by LC-ESI-MS data (*m/z* 517.2076, [M + H]+), which is combined with 1H and 13C-NMR spectral data (Table 1) with twelve degrees of unsaturation. The IR (KBr) spectrum shows bands which can be assumed to be derived from the hydroxyl groups (*v*max: 3441 cm<sup>−</sup>1), ketones (*v*max: 1760 cm<sup>−</sup>1), unsaturated ketones (*v*max: 1726 cm<sup>−</sup>1) isolated double bonds (*v*max: 1698 cm<sup>−</sup>1) and *gem*-dimethyl (*v*max: 1440 cm−<sup>1</sup> and 1392 cm<sup>−</sup>1). 1H-NMR (Figure S1) showed three singlet signals (<sup>δ</sup> 0.98, 1.26, and 1.37) from the

tertiary methyl group and one doublet from the secondary methyl group at δ 1.16 which correlated with H-10 (δ 3.29, 1H, *d*, 7). The singlet signal also appears in the lower field area (δ 3.67), which is thought to be the C-27 methoxy proton signal. A more refined analysis of the 1H-NMR spectrum reveals the signal characteristics of the tetranortriterpenoid skeleton in the presence of *β*-substituted furan signals (δ 7.48, 7.39 and 6.43) and olefinic signals of *α, β*-unsaturated ketone (δ 6.46, 1H, *s*).

13C-NMR, DEPT and HMQC spectrum (Figures S2–S4) shows 27 carbon signals referring to the signal characteristics of the furan ring (δ 142.8, 141.5, 119.7, and 110.2), ketone (δ 209.7), two ester groups (δ 172.0 and 165.7), one carbon oxygenated by two oxygens (δ 107.0) and *α, β-*unsaturated ketone (δ 167.5 and 117.4), thus showing that compound **1** has a hexacyclic structure with the presence of furan groups. 1H-1H COSY (Figure S5) shows the proton correlation of H6/H5, H9/H11, H10/H19, H11/H12, H21/H20, H25/H24 and H26/H24. These correlations indicate the presence of a tetranortriterpenoid skeleton with a furan ring [17]. The correlation of the partial structure was further explained by the HMBC correlation spectral data (Figures 2 and S6).

**Figure 2.** (**a**) 1H-1H COSY and HMBC and (**b**) NOESY correlations for compound **1**.

The correlation of oxygenated H-17 (δ 5.17) to C-23 (δ 119.7), C-24 (δ 110.2) and C-26 (δ 141.5) shows that C-17 binds to the furan ring. The pyran ring position is confirmed by the correlations of H-22 (δ 2.40 and 3.02) with C-1 (δ 107.0), C-2 (δ 77.1), C-8 (δ 67.8), C-9 (δ 73.4) and C-14 (δ 167.5). An *α,β*-unsaturated *δ*-lactone ring system is determined by the correlation of H-15 (δ 6.46) which connected with C-8 (δ 67.8), C-13 (δ 38.4) and C16 (δ 165.7). The correlations arising from two methyl (δ 0.98 and 1.37) to C-3 (δ 209.7), C-4 (δ 47.8) and C-5 (δ 55.9) indicate that *gem*-dimethyl is bound to C-4. Another correlation between the carbomethoxyl (δ 3.67) and H-6 (δ 4.82) signals to the carbonyl ester (δ 172.0) determines the position of the ester group attached to C-6. Basically, the NMR data of compound **1** were similar to those of kokosanolide A [12]. The only difference lies in C-8. Kokosanolide A has C-8 (δ 34.6), which is a metine, while in compound **1**, C-8 (δ 67.8) is a quaternary carbon that binds the hydroxyl group. The absence of a C-8 correlation with any protons in the HMQC data supports this suggestion. A NOESY spectrum shows H-5/ H-6/H20 correlation. This NOESY correlation is also found in kokosanolide A [12]. It can be assumed that kokosanolide A and compound **1** have the same stereochemical configuration and indicated that hydroxyl group C-8 should have *α-*orientation. Thus, the structure of compound **1** is determined to be a new tetranortriterpenoid from kokosanolide group named kokosanolide D.

#### **4. Materials and Methods**

#### *4.1. General Experimental Procedures*

Mass spectra were measured with a waters Xevo QTOFMS (Waters, Milford, MA, USA). IR spectra were measure on a One Perkin Elmer infrared-100. NMR data were recorded on a JNM-ECZ500R/S1 spectrometer at 500 MHz for 1H and 125 MHz for 13C using TMS as an internal standard. Chromatographic separations were carried out on silica gel G60 (Merck, Darmstadt, Germany) and RP18 (Merck). TLC plates were pre-coated with silica gel GF254 (Merck, 0.25 mm), and detection was achieved by spraying with 10% H2SO4 in ethanol, followed by heating.

#### *4.2. Plant Material*

The fruit peels of *L. domesticum* were collected from Cililin, West Java, Indonesia in April 2018. The plant was identified and deposited in the Herbarium Laboratory of the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia (Identification Number: 195/HB/08/2018).

#### **5. Conclusions**

A new tetranortriterpenoid was isolated from the methanol extract of fruit peels of *L. domesticum* Corr. cv *kokossan,* which is named kokosanolide D.

**Supplementary Materials:** The following are available online, Figure S1. 1H-NMR spectrum of 1 (500 MHz in CDCl3), Figure S2. 13C-NMR spectrum of 1 (125 MHz in CDCl3), Figure S3. DEPT\_135◦ spectrum of 1 (125 MHz in CDCl3), Figure S4. HMQC spectrum of 1, Figure S5. 1H-1H COSY spectrum of 1, Figure S6. HMBC spectrum of 1, Figure S7. NOESY spectrum of 1, Figure S8. Infrared spectrum of 1 (in KBr), Figure S9. HR-TOF-MS spectrum of 1, Figure S10. TLC profile of 1.

**Author Contributions:** The following statements should be used Conceptualization, F.M.F. and T.M.; methodology, F.M.F., T.H.; software, F.M.F. and Z.; validation, K.F., T.M. and T.H.; formal analysis, F.M.F. and S.R.M.; investigation, T.M. and J.A.A.; resources, T.M.; data curation, K.F. and J.A.A.; writing—original draft preparation, F.M.F.; writing—review and editing, T.M. and J.A.A.; visualization, S.R.M. and T.H.; supervision, T.M. and T.H.; project administration, S.R.M.; funding acquisition, T.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PD, Ministry of Research, Technology and Higher Education, Indonesia, grant number 1827/UN6.3.1/LT/2020 (T.M).

**Acknowledgments:** We thank Joko Kusmoro, M.P. at Jatinangor Herbarium for identification of the plant material, Ahmad Darmawan and Sofa Fajriah at the Research Center for Chemistry, Indonesian Science Institute, for performing the NMR measurements, Kansy Haikal at the Center Laboratory of Universitas Padjadjaran for performing the HR-TOF-MS measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


#### *Communication*

## **Bioactive Antidiabetic Flavonoids from the Stem Bark of** *Cordia dichotoma* **Forst.: Identification, Docking and ADMET Studies**

**Nazim Hussain 1, Bibhuti Bhushan Kakoti 2, Mithun Rudrapal 3,\*, Khomendra Kumar Sarwa 4, Ismail Celik 5, Emmanuel Ifeanyi Attah 6, Shubham Jagadish Khairnar 7, Soumya Bhattacharya 8, Ranjan Kumar Sahoo <sup>9</sup> and Sanjay G. Walode <sup>3</sup>**


**Abstract:** *Cordia dichotoma* Forst. (F. Boraginaceae) has been traditionally used for the management of a variety of human ailments. In our earlier work, the antidiabetic activity of methanolic bark extract of *C. dichotoma* (MECD) has been reported. In this paper, two flavonoid molecules were isolated (by column chromatography) and identified (by IR, NMR and mass spectroscopy/spectrometry) from the MECD with an aim to investigate their antidiabetic effectiveness. Molecular docking and ADMET studies were carried out using AutoDock Vina software and Swiss ADME online tool, respectively. The isolated flavonoids were identified as 3,5,7,3- ,4- -tetrahydroxy-4-methoxyflavone-3-*O*-L-rhamnopyranoside and 5,7,3- -trihydroxy-4-methoxyflavone-7-*O*-L-rhamnopyranoside (quercitrin). Docking and ADMET studies revealed the promising binding affinity of flavonoid molecules for human lysosomal α-glucosidase and human pancreatic α-amylase with acceptable ADMET properties. Based on computational studies, our study reports the antidiabetic potential of the isolated flavonoids with predictive pharmacokinetics profile.

**Keywords:** *C. dichotoma*; flavonoids; antidiabetic; α-glucosidase; α-amylase; docking; ADMET

#### **1. Introduction**

*Cordia dichotoma* Forst. (also known as Indian cherry, F. Boraginaceae) is a traditionally important deciduous medicinal plant widely grown in India, Sri Lanka and other tropical countries of the world [1]. This plant has been traditionally (Ayurveda, Unani and Siddha medicines) used for the management of a variety of human ailments/disorders [1]. Leaves and stem bark have been used traditionally in the treatment of fever, dyspepsia, diarrhea, leprosy, gonorrhea and wounds [2]. Leaves, seeds, bark and fruits have been reported to

**Citation:** Hussain, N.; Kakoti, B.B.; Rudrapal, M.; Sarwa, K.K.; Celik, I.; Attah, E.I.; Khairnar, S.J.; Bhattacharya, S.; Sahoo, R.K.; Walode, S.G. Bioactive Antidiabetic Flavonoids from the Stem Bark of *Cordia dichotoma* Forst.: Identification, Docking and ADMET Studies. *Molbank* **2021**, *2021*, M1234. https://doi.org/10.3390/M1234

Academic Editor: Giovanni Ribaudo

Received: 7 May 2021 Accepted: 9 June 2021 Published: 11 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

exhibit anti-inflammatory, anthelmintic, antibacterial, antileprotic, antiviral, diuretic, astringent, demulcent, laxative/purgative, expectorant/antitussive, tonic, immunomodulatory, hepatoprotective and gastroprotective/antiulcer activities [3,4].

Recent literature have also reported the anti-inflammatory [3], antidiabetic [4], anticancer [2] and antioxidant [3] activities for the bark extract of *C. dichotoma*. Phytoconstituents like3- ,5-dihydroxy-4- -methoxyflavanone-7-*O*-α-L-rhamnopyranoside, β-sitosteryl-3β-glucopyranoside-6- -*O*-palmitate, quercitrin and β-sitosterol have already been isolated from the leaves of this plant [5]. Betulin, lupeol-3-rhamnoside, β-sitosterol, taxifolin-3-5 dirhamnoside, hesperitin-7-rhamnoside, rutin, chlorogenic acid and caffeic acid have been reported from seeds of *C. dichotoma* [2–5]. Flavonoids, the most important class of plant polyphenolics possessing diverse range of biological/pharmacological potential [6–8], are attributed to be the most dominant phytochemical components in various plant parts of *C. dichotoma*. In this work, the isolation and identification of bioactive flavonoids from the methanolic bark extract of *C. dichotoma* Forst was carried out. The isolated flavonoid molecules were further investigated for their antidiabetic potential and pharmacokinetic properties by molecular docking and ADMET studies.

#### **2. Results and Discussion**

The phytochemical analysis revealed the presence of flavonoids, alkaloids, glycosides, saponins, steroids, carbohydrates and proteins in the methanolic bark extract of *C. dichotoma* Forst (MECD) [2–4].

#### *2.1. Identification of Isolated Phytocompounds*

#### 2.1.1. Compound 1 (MECD-1)

Subfraction 20–78 was purified by column chromatography on silica using methanol: ethyl acetoacetate to yield the pure compound 1 (120 mg). The isolated compound 1 was obtained as pale yellow amorphous powder with a melting range of 163–165 ◦C and a [α] 25D value of +0.34 (conc. 0.25 mg/mL, MeOH). The structure of the isolated compound 1, represented in Figure 1, was elucidated by UV, IR, 1H-NMR, 13C-NMR and mass spectroscopic/spectrometric analyses (Figure S1A–D).

**Figure 1.** Structure of compound 1 (MECD-1).

UV (λmax, nm, MeOH): 307, 334; FT-IR (υmax, cm<sup>−</sup>1, KBR): 3265, 2960, 2895, 1668, 1616, 1429, 1367; 1H-NMR (δ, 400 MHz, DMSO-d6): 0.79 (J = 6.0 Hz), 3.98 (J = 15.0, 3.5 Hz), 2.96 (J = 15.0, 11.0 Hz), 3.02 (J = 11.0, 3.5 Hz), 4.41, 4.43, 5.25, 6.21, 6.42 (J = 2.0 Hz), 7.26 (J = 6.5, 2.0 Hz); 13C-NMR (δ, 100 MHz, DMSO-d6): 193.56, 102.25, 55.78, 52.55.

The IR bands at 3265 cm−<sup>1</sup> (O-H stretching) revealed the presence of the hydroxyl group in the structure of compound 1. Other prominent absorption bands at 2960 and 2895 cm−<sup>1</sup> (aliphatic C-H stretching), 1668 cm−<sup>1</sup> (C=O stretching) and 1616 cm−<sup>1</sup> (aromatic C=H stretching) indicated the presence of methyl group (CH3), α,β unsaturated carbonyl group and aromatic rings. In 1H-NMR spectrum, three one-proton double doublets at δ 3.98 (J = 15.0, 3.5 Hz), 3.02 (J = 11.0, 3.5 Hz) and 2.96 (J = 15.0, 11.0 Hz) were

ascribed to H-2, H-3α and H-3β protons, respectively of ring C of a flavone moiety. Two one-proton doublets at δ 6.21 and 6.42 (J = 2.0 Hz, each) were assigned to H-6 and H-8 aromatic protons. Two doublets at δ 7.26 (J = 6.5, 2.0 Hz), each integrating for one proton, were ascribed correspondingly to H-2, H-5 and H-6 of aromatic protons. A three-proton singlet at δ 3.61 was attributed to methoxy (OCH3) protons. A broad singlet at δ 5.25 was accounted to H-1- anomeric proton, while a three proton doublet at δ 0.79 (J = 6.0 Hz) was appeared due to secondary methyl proton H-6- of rhamnose unit. The remaining protons of sugar unit appeared between δ 4.43 and 4.41. The 13C-NMR spectra showed twenty two distinct signals suggesting that the compound contains twenty two carbon atoms. The important signals appeared at δ 193.56 (C-4, carbonyl carbon), 102.25 (C-1 anomeric carbon), 55.78 (methoxy carbon OCH3) and 17.95 (C-6- methyl carbon). The presence of an aromatic methoxy group was confirmed by position of the methyl signal at δ 52.55. The molecular ion [M]+ peak was obtained at *m/z* 446.0, which concord the molecular formula of the compound 1 as C22H22O11. The NMR spectral data also supported the structure of the compound. A thorough spectral interpretation suggests that the compound 1 (MECD-1) is **5,7,3,-trihydroxy-4-methoxyflavone-7-***O***-L-rhamnopyranoside**.

#### 2.1.2. Compound 2 (MECD-2)

Subfraction 120–184 was purified by column chromatography on silica gel using methanol:ethyl acetoacetate to obtain the pure compound 1 (160 mg). The isolated compound 1 was obtained as pale yellow crystalline powder with a melting range of 176–178 ◦C and a [α] 25D value of +0.36 (conc. 0.25 mg/mL, MeOH). The structure of the isolated compound 1, represented in Figure 2 was elucidated by UV, IR, 1H-NMR, 13C-NMR and mass spectroscopic/spectrometric analyses (Figure S2A–D).

**Figure 2.** Structure of compound 2.

UV (λmax, nm, MeOH): 312, 346; FT-IR (υmax, cm<sup>−</sup>1, KBR): 3265, 2950, 2880, 1654, 1502, 1454, 1354, 810. 1H-NMR (δ, 400 MHz, DMSO-d*6*): 0.73 (J = 7.1 Hz), 3.96–4.81, 6.11, 6.29 (J = 2.1 Hz), 6.78 (J = 9.6 Hz), 7.14 (J = 9.6, 2.2 Hz), 7.20 (J = 2.2 Hz), 12.55; 13C-NMR (δ, 100 MHz, DMSO-d*6*): 156.90 (C-2), 134.69 (C-3), 178.20 (C-4), 104.56 (C-4), 161.75 (C-5), 99.15(C-6), 164.63 (C-7), 64.09 (C-8), 157.76(C-8), 121.22 (C-1- ), 116.13 (C-2- ),145.65 (C-3- ), 148.88 (C-4- ), 2115.93 (C-5- ), 121.58 (C-6- ), 102.29 (C-1--), 71.03 (C-2--), 70.83(C-3--), 71.66 (C-4--), 70.51 (C-5--), 17.95 (C-6--).

The IR bands at 3265 cm−<sup>1</sup> (O-H stretching) revealed the presence of hydroxyl group in the structure of compound 2. Other prominent absorption bands at 2950 and 2880 cm−<sup>1</sup> (aliphatic C-H stretching), 1654 cm−<sup>1</sup> (C=O stretching) and 1502 cm−<sup>1</sup> (aromatic C=H stretching) indicated the presence of methyl group (CH3), α,β unsaturated carbonyl group and aromatic rings. The 1H-NMR spectrum exhibited a set of two coupled doublets at δ 6.11 and 6.29 (J = 2.1 Hz, each), which was ascribed to H-6 and H-8 aromatic protons. Another set of coupled signals consisting of two doublets at δ 7.20 (J = 2.2 Hz), 6.78 (J = 9.6 Hz) and

a double-doublet at δ 7.14 (J = 9.6, 2.2 Hz) were ascribed to H-2- , H-5 and H-6 aromatic protons of ring B. A doublet at δ 5.15 (J = 8.1 Hz) was assigned to H-1- anomeric proton, while as another doublet at δ 0.73 (J = 7.1 Hz) was attributed to methyl protons (H-6--) of rhamnose unit. The remaining protons of rhamnose resonated from δ 4.81 to 3.96. A single proton singlet at δ 12.55 was attributed to hydroxyl proton. The 13C-NMR spectrum displayed signals for twenty-one carbons. Important signals appeared for carbonyl carbon (δ 178.20, C-4), anomeric carbon (δ 102.29, C-1--) and methyl carbon (δ 17.94, C-6--). The molecular ion [M]+ peak was obtained at *m/z* 448.0, which concord the molecular formula of the compound as C21H20O11. The 1H and 13C NMR data was compared with other reported flavonoids and was found to be **3,5,7,3,4,-tetrahydroxy-4-methoxyflavone-3-***O***-Lrhamnopyranoside** (Quercitrin (Quercetin-3-*O*-L-rhamnoside).

#### *2.2. Molecular Docking*

Molecular docking is used to understand the drug–receptor interaction, binding affinity and binding orientation of bioactive molecules into the target protein molecule. The objective behind docking study is to predict a particular biological activity based on the binding orientation/affinity of small molecule ligands to the appropriate target active site [9]. In the docking study, the binding affinity was predicted in terms of the interaction energy (kcal/mole). Results of docking (binding) energies are given in Table 1. Both the compounds exhibited very good binding affinity against both α-glucosidase and α-amylase enzyme. Compound 1 (MECD-1) exhibited more binding affinity against alpha-amylase compared to the alpha-glucosidase. On the hand, the compound 2 (MECD-2) showed better affinity against alpha-glucosidase than alpha-amylase. Not much variation in binding energies between these two enzymes were observed. Docking scores of isolated compounds were compared with that of the standard drug, acarbose. Against α-glucosidase, the binding affinity of compounds 1 and 2 was comparatively more affinity than the standard drug. On the other hand, the binding affinity of compounds 1 and 2 were less to some extent than that the standard drug against α-amylase. No significant difference in activities between isolated test compounds and the standard compound was observed. The test compounds were found to have α-glucosidase and α-amylase inhibitory potential to a similar degree as that of the standard drug, acarbose. Overall, both the isolated flavonoids exhibited significant inhibitory potential of human glucosidase and amylase enzymes.


**Table 1.** Docking data of compounds.

5NN8: Human lysosomal acid α-glucosidase; 1B2Y: Human pancreatic α-amylase; MECD: Metanolic bark extract of *C. dichotoma.*

Post-docking visualization of protein–ligand complexes revealed that the compounds interacted with active site residues of the protein molecules through the formation of predominantly hydrogen bonding interactions (Figures 3 and 4). From the observation of 2D interaction diagrams of compound 1-α-glucosidase complexes, it is clear that compound 1 formed H-bonds with Trp59, Gln63, Asp197, Asp300 and Asp356 residues, whereas the compound 2 interacted with Tyr62, His101, His201 and Gly306 residues through H-bonds (Figure 3a,b). The 3D diagrams revealed the binding conformation and binding poses of the compounds at the catalytic site of α-glucosidase (Figure 3c,d) were observed.

**Figure 3.** (**a**) Two-dimensional interactions between compound 1 and α-glucosidase, (**b**) 2D interactions between compound 2 and α-glucosidase showing hydrogen bonding and other non-covalent interactions with amino acid residues at the active site, (**c**) 3D representation of protein-ligand interactions showing binding conformation and (**d**) binding poses/binding modes of both the compounds at the catalytic site of α-glucosidase.

From the observation of 2D interaction diagrams of compound 2-α-amylase complexes, it is clear that compound 1 formed prominent H-bonds with Asp404, Ser523 and Ser524 residues, whereas the compound 2 interacted with Asp616, His674 and Leu678 residues through H-bonds (Figures 3b and 4a). The 3D diagrams revealed the binding conformation and binding poses of the compounds at the catalytic site of alpha-amylase (Figures 3d and 4c) were observed.

Upon critical analysis of protein–ligand interactions, favorable binding orientations and/or binding modes of flavonoid molecules for both the α-glucosidase and α-amylase enzyme were evident. Both the compounds structurally represent glycosides of flavones, which are abundantly found in plant kingdom. The glycone part (sugar), i.e., the rhamnose moiety is similar in both the compounds, while the aglycone part (non-sugar bioactive principle, flavanone moiety) is dissimilar. In compound 1, it is 5,7,3- -trihydroxy-4 methoxyflavone, whereas, it is 3,5,7,3- ,4- -tetrahydroxy-4-methoxyflavone, i.e., quercetin in compound 2. The aglycone, i.e., the flavonoid moiety is a polyhydroxylated C6-C3-C6 tricyclic heteroaromatic system (phenylchromone) [10–12] with distinct structural features, particularly in terms of nature and pattern of ring substitutions. There is a close structural resemblance between these two isolated flavonoid glycosides. The basic flavone skeleton along with hydroxylated/methoxylated aromatic ring interacted predominantly with the active site residues of target protein molecules. Polar groups such as phenolic hydroxy groups and carbonyl moiety contributed significantly in protein-ligand interactions with

the formation of hydrogen bonds. Apart from hydrogen bonding, other non-bonding interactions such as hydrophobic interactions also exist, but to a lesser extent. Aromatic bulky moieties chromone system and phenyl ring were mainly involved in non-polar hydrophobic interactions.

**Figure 4.** (**a**) Two-dimensional interactions between compound 1 and α-amylase, (**b**) 2D interactions between compound 2 and α-glucosidase showing hydrogen bonding and other non-covalent interactions with amino acid residues at the active site, (**c**) 3D representation of protein–ligand interactions showing binding conformation and (**d**) binding poses/binding modes of both the compounds at the catalytic site of α-amylase.

The human lysosomal α-glucosidase and pancreatic α-amylase enzymes play an important role in the digestion of dietary long-chain complex carbohydrates (breakdown of starch and disaccharides to glucose) and hence, their inhibition is believed to facilitate the reduction of post-prandial (post meal) blood glucose level in type 2 diabetes [13]. The traditional usefulness about the antidiabetic potential of *C. dichotoma* is mentioned in literature [2–4]. In our earlier studies, the antidiabetic activity of the methanolic bark extract of *C. dichotoma* has already been reported [4]. Moreover, the literature suggest that the flavonoids content demonstrates antidiabetic efficacy of many plants [14–18]. Our docking study validates the antidiabetic claim about *C. dichotoma* reported in traditional medicines and in recent literature. Although the isolated phytocompounds are already established bioactive flavonoids with many scientific reports from past literature, their antidiabetic potential determined by in silico (molecular docking) methods with α-glucosidase and α-amylase inhibitory activities has been reported for the first time. Our study may thus provide an avenue for further investigation with these bioflavonoids for their development

as potent antidiabetic molecules with alpha-glucosidase and alpha-amylase inhibitory agents for the treatment of type 2 diabetes mellitus.

#### *2.3. ADMET*

Results of predicted ADMET (absorption, distribution, metabolism, excretion and toxicity) data showed that both the isolated compounds possess good solubility profile, which is in favor of their oral bioavailability. There is a prediction of poor intestinal absorption, while the compounds were predicted to be non-inhibitors of the cytochromes (CYP450) [19]. Poor intestinal absorption might be due to their limited oil/water partition coefficient (logP) values (−1.64 and −1.84). CYP450 enzymes are largely involved in drug metabolism. Non-inhibition of CYP450 enzymes suggests that compounds do not suppress the metabolic function of the enzymes. Inhibition can lead to increased bioavailability of compounds that normally undergo extensive first-pass elimination or to decreased elimination of compounds dependent on metabolism for systemic clearance. Compounds did not exhibit the property of blood brain barrier (BBB) penetration. It substantiates that the compounds are devoid of producing CNS toxicities. Furthermore, quercitrin (compound 2) was predicted to be a substrate to permeability of glycoprotein (p-gp), whereas the other flavonoid molecule (compound 1) did not show such property. Glycoprotein is responsible for the efflux of drug molecules out of the target cells [20]. A good drug candidate should not only have sufficient efficacy against the therapeutic target, but also show appropriate ADMET properties at a therapeutic dose. It is therefore inevitable to evaluate the ADMET profile of drug-like molecules to avoid the failure of candidate drugs at the clinical stage of drug development [21].

#### **3. Materials and Methods**

#### *3.1. Collection of Plant*

The barks of *Cordia dichotoma* Forst. were collected from the Duhai forest of Ghaziabad, Uttar Pradesh, India. The plant species was identified from National Institute of Science Communication and Information Resources, New Delhi, India. The voucher specimen (NISCAIR/RHMD/Consult/2012-13/2025/33) of the bark of *Cordia dichotoma* Forst. was submitted at the herbarium of the department for future reference.

#### *3.2. Preparation of Methanolic Bark Extract*

The shade-dried barks of *C. dichotoma* were pulverized to a coarse powder and defatted using petroleum ether by the cold maceration method [2] to remove fat, latex and non-polar compounds of high molecular weights. The defatted plant residues were then macerated successively with methanol to obtain the desired extract [3,4]. The collected extract was filtered through Whatman No. 1 filter paper. Rotary evaporator was used to concentrate the filtrate. The concentrated extract was preserved in refrigerator at 4 ◦C for further use. The percentage yield of the methanolic bark extract of *C. dichotoma* (MECD) was 7.11%.

#### *3.3. Phytochemical Analysis*

Chemical tests for the screening and detection of phytochemical constituents of the MECD were carried out using the standard procedures [22,23].

#### *3.4. Isolation of Phytocompounds*

The MECD was subjected to column chromatographic separation using silica gel (packed column, 100–200 mesh) and a glass column (6.0 × 3 inch dimension) [6] for the isolation of bioactive phytoconstituents in pure form. The elution was carried out by gradient separation technique using the solvent system of n-hexane/ethyl acetate. The column was eluted successively with n-hexane:ethyl acetate in increasing order of polarity (98:2, 95:5, 90:10, 80:20, 60:40, 50:50, 35:65, 30:70, 25:75, 20:80 and 100%). The fractions collected were subjected to thin-layer chromatography (TLC) to check their homogeneity. Chromatographically identical fractions (having the same Rf values) were combined together and concentrated. The concentrated fractions were purified by crystallization with methanol/benzene and confirmed by their sharp melting points.

#### *3.5. Identification of Isolated Compounds*

Ultraviolet (UV)–visible spectra were recorded on Shimadzu UV-1700 UV–visible spectrophotometer (Shimadzu, Kyoto, Japan) and the wave lengths of maximum absorption (λmax, nm) were reported. Infrared (IR) spectra were obtained on a Bruker alpha Fourier transform (FT-IR) spectrometer (Bruker, MA, USA) using the KBR disc and reported in terms of frequency of absorption (υmax, cm<sup>−</sup>1). 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance II 400 FT-NMR spectrometer (Bruker, MA, USA) at 400 and 100 MHz, respectively using tetramethylsilane (TMS) as an internal standard (δ 0.00 ppm) and CDCl3 as a solvent. Mass spectra were obtained on a LC–MS Water 4000 ZQ instrument (Waters, Massachusetts, USA) using electrospray ionization (ES+). The *m/z* values were recorded in the range of *m/z* between 100 and 500 and the *m*/*z* values of the most intense molecular ion [M]+ peak were considered. Melting points were determined on an electric melting point apparatus (JSGW, Model 3046). (Jain Scientific Glass Works, Ambala, India)

#### *3.6. Molecular Docking*

The X-ray crystal structure of proteins, viz., human lysosomal acid α-glucosidase (PDB ID: 5NN8) and human pancreatic α-amylase (PDB ID: 1B2Y) were reposited by Roig-Zamboni et al. [24] and Nahoun et al. [25] having resolution of 2.45 Å and 3.20 Å, respectively were retrieved from the RCSB protein data bank (http://www.rcsb.org/ (accessed on 13 March 2021)).

Prior to docking, The docking was performed in the AutoDock Vina software(The Scripps Research Institute, La Jolla, CA, USA) [26] in accordance with the standard procedure. The protein crystal structure was prepared prior to the docking process. Hydrogen atoms were added to the protein structure, and all ionizable residues were set at their default protonation at pH 7.4. The active site coordinates were determined with the dimensions of *x* = −15.941, *y* = −37.643, *z* = 92.912 for 5NN8 and *x* = 22.116, *y* = 4.749, *z* = 45.878 for 1B2Y, and, a grid box with radius of 25 × 25 × 25 A3 was generated for both the proteins. Similarly, the ligands were prepared and energy minimized using Chem3D 17.0 software. During the docking process, the receptor was rigidly held, while the ligands were allowed to flex during the refinement. Binding energies of docking were recorded and analyzed. The best docked poses and binding modes of protein–ligand interactions were obtained using the Discovery Studio visualizer.

#### *3.7. ADMET Prediction*

Predictive ADMET (pharmacokinetics) parameters were studied using web-based Swiss ADME tool developed by Daina et al. [27]. Solubility, intestinal absorption, oil/water partition coefficient (logP), CYP450 inhibition, blood brain barrier penetration and p-gp substrate were predicted [28].

#### **4. Conclusions**

This study reports two bioactive flavonoids, viz., 3,5,7,3- ,4- -tetrahydroxy-4-methoxyflavone-3-*O*-L-rhamnopyranoside and 5,7,3- -trihydroxy-4-methoxyflavone-7-*O*-L-rhamnopyranoside (Quercitrin) isolated and identified from the methanolic bark extract *Cordia dichotoma* Forst. The molecular docking study investigated the antidiabetic potential of the isolated flavonoids against human lysosomal acid α-glucosidase and human pancreatic α-amylase enzymes. The predictive ADMET study demonstrated acceptable pharmacokinetics of the isolated compounds. The in silico study needs to be further validated by in vitro and in vivo experimental assays in order to confirm the antidiabetic effectiveness for the flavonoids reported herein. Our study may thus provide an avenue for further investigation with these bioflavonoids for their development as potent antidiabetic molecules with α-glucosidase and α-amylase inhibitory agents for the treatment of type 2 diabetes mellitus.

**Supplementary Materials:** The following are available online, Figure S1A: FT-IR spectrum of compound 1, Figure S1B: 1H-NMR spectrum of compound 1, Figure S1C: 13C-NMR spectrum of compound 1, Figure S1D: Mass spectrum of compound 1, Figure S2A: FT-IR spectrum of compound 2, Figure S2B: 1H-NMR spectrum of compound 2, Figure S2C(1) and Figure S2C(2): 13H-NMR spectrum of compound 2, Figure S2D: Mass spectrum of compound 2.

**Author Contributions:** Conceptualization, N.H. and B.B.K.; methodology, N.H.; software, I.C.; validation, I.C. and M.R.; formal analysis, M.R. and E.I.A.; investigation, N.H. and I.C.; resources, B.B.K. and I.C.; data curation, M.R., I.C. and S.J.K.; writing—original draft preparation, E.I.A. and S.J.K.; writing—review and editing, M.R. and E.I.A.; visualization, E.I.A.; supervision, B.B.K.; project administration, B.B.K.; funding acquisition, S.J.K., K.K.S., S.B., R.K.S. and S.G.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Additional data will be made available on request.

**Acknowledgments:** Authors gratefully acknowledge the TÜB˙ ITAK (The Scientific and Technological Research Council of Turkey), ULAKB˙ IM (Turkish Academic Network and Information Centre), High Performance and Grid, Computing Centre (TRUBA resources), Turkey for providing necessary facilities for performing the computational studies. Authors extend their sincere thanks to Sagarika Chandra for her needful help towards editing the images incorporated in the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **2-Chloro-4,6-***bis***{(***E***)-3-methoxy-4-[(4-methoxybenzyl)oxy]styryl} pyrimidine: Synthesis, Spectroscopic and Computational Evaluation**

**Otávio Augusto Chaves 1, Vitor Sueth-Santiago 2,3,†, Douglas Chaves de Alcântara Pinto 2, José Carlos Netto-Ferreira 2,4, Debora Decote-Ricardo <sup>5</sup> and Marco Edilson Freire de Lima 2,\***


**Abstract:** A novel curcumin analog namely 2-chloro-4,6-*bis*{(*E*)-3-methoxy-4-[(4-methoxybenzyl)oxy] styryl}pyrimidine (compound **7**) was synthesized by three-step reaction. The condensation reaction of protected vanillin with 2-chloro-4,6-dimethylpyrimidine (**6**) was the most efficient step, resulting in a total yield of 72%. The characterization of compound **7** was performed by 1H and 13C nuclear magnetic resonance (NMR), as well as high-resolution mass spectrometry. The experimental spectrometric data were compared with the theoretical spectra obtained by the density functional theory (DFT) method, showing a perfect match between them. UV-visible spectroscopy and steady-state fluorescence emission studies were performed for compound **7** in solvents of different polarities and the results were correlated with DFT calculations. Compound **7** showed a solvatochromism effect presenting higher molar extinction coefficient (log ε = 4.57) and fluorescence quantum yield (φ = 0.38) in toluene than in acetonitrile or methanol. The simulation of both frontier molecular orbitals (FMOs) and molecular electrostatic potential (MEP) suggested that the experimental spectra profile in toluene was not interfered by a possible charge transfer. These results are an indication of a low probability of compound **7** in reacting with unsaturated phospholipids in future applications as a fluorescent dye in biological systems.

**Keywords:** curcumin analog; organic synthesis; photophysical properties; steady-state fluorescence; DFT calculation

#### **1. Introduction**

Turmeric, obtained from the dried rhizomes of *Curcuma longa* (Zingiberaceae), is a golden colored material, commonly used around the world for seasoning and food coloring. Since antiquity, turmeric has been widely used in the treatment of several diseases in traditional Chinese and Indian medicine (traditionally known as Ayurveda, e.g., for the treatment of inflammatory diseases) [1–3]. Curcumin ((1*E*,6*E*)-1,7-*bis*(4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione) also known as diferuloylmethane, is the main chemical component of turmeric (accounting for up to 70%), belonging to the class of diarylheptanoid

Sueth-Santiago, V.; Pinto, D.C.d.A.; Netto-Ferreira, J.C.; Decote-Ricardo, D.; Lima, M.E.F.d. 2-Chloro-4,6-*bis*{(*E*)-3-methoxy-4-[(4 methoxybenzyl)oxy]styryl}pyrimidine: Synthesis, Spectroscopic and Computational Evaluation. *Molbank* **2021**, *2021*, M1276. https://doi.org/ 10.3390/M1276

**Citation:** Chaves, O.A.;

Academic Editor: Giovanni Ribaudo

Received: 31 July 2021 Accepted: 29 August 2021 Published: 7 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

metabolites. This compound is mainly responsible for both biological, metal chelator, flavoring, reactivity and pigment properties of turmeric (Figure 1A) [1,4]. Furthermore, curcumin stems present potent antioxidant, anti-inflammatory and anticancer activity due to their capacity to suppress the proliferation of a wide variety of tumor cells and regulate the expression of different enzymes [5]. Some structural modifications in the curcumin moiety based on structure-activity relationship have improved its drug profile (Figure 1). As an example, dimethoxycurcumin showed potential trypanocidal properties with a half-maximal inhibitory concentration (IC50) value of 11.07 μM [6], while 1-methyl-3,5-*bis*[(*E*)-4-pyridyl) methylidene]-4-piperidone and 1-isopropyl-3,5-*bis*[(pyridine-3-yl) methylene] piperidin-4-one showed potential anticancer properties with IC50 values in the 0.7–1.0 μM range for H3122 cells lines (lung cancer cell lines) [7]. In addition, a series of asymmetric dihydrothiopyran curcumin analogs demonstrated high inhibition at a submicromolar level against acute promyelocytic leukemia cells [8], and integrating a tetrahydro-4-pyrone linker into curcumin structure led to cell growth inhibition, promoted apoptosis and enhanced irinotecan sensitivity against gastric cancer cells [9]. Finally, theoretical evaluation for some curcumin derivatives (replacing methoxyl groups in the aromatic moieties for bromo, chloro or hydroxyl groups) indicated these novel derivatives as potential inhibitors of amyloid-β peptides aggregation in the human brain (Aβs—aggregation process involved in the onset of Alzheimer's disease) [10].

**Figure 1.** (**A**) Chemical structure of curcumin and its simplified structural-activity relationship. (**B**) Tautomerism form for curcumin. (**C**) Chemical structure for the curcumin analog under study compound **7**.

From a structural point of view, the *ortho*-methoxy phenolic groups are symmetrically connected to a seven-carbon chain through an α,β-unsaturated β-diketone group. All these connections will result in a highly conjugated structure that shows UV-visible absorption in the 200–500 nm range, depending on the solvent polarity; however, the spectral properties of curcumin are related to the tautomeric forms (*bis*-keto or enol—Figure 1B) [11]. For this reason, curcumin has been widely explored in terms of spectroscopic properties, showing maximum absorption in the 408–430 nm range in different organic solvents; however, its maximum steady-state fluorescence emission (460–560 nm) is more sensitive than absorption to the solvent polarity, presenting Stokes' shift varying from 2000 to 6000 cm<sup>−</sup>1.

Furthermore, curcumin shows a low fluorescence quantum yield in most of the solvents, which is significantly reduced in the presence of water [12]. In this sense, novel organic and inorganic compounds based on curcumin moiety have been proposed to improve the photophysical behavior, e.g., curcumin boron complexes can act as a near-infrared imaging fluorescent probe [13], chemical sensor [14] and larger second-order nonlinear property [15].

Based on the background described above on the biological and photophysical importance of curcumin and both its derivatives and analogs, the main goal of the present work is the synthesis of a novel symmetric curcumin analog, namely 2-chloro-4,6-*bis*{(*E*)-3 methoxy-4-[(4-methoxybenzyl)oxy]-styryl}pyrimidine (compound **7**—number according to the synthetic steps shown in Scheme 1) (Figure 1C). Its characterization via high-resolution mass spectrometry (HRMS) and experimental or computational Nuclear Magnetic Resonance spectra (NMR—1H and 13C) will follow the synthetic procedure. The preliminary spectroscopic characterization of compound **7** was also described by UV-visible and steadystate fluorescence techniques for three different solvents (methanol, acetonitrile or toluene), combined with computational results based on Density Functional Theory (DFT) calculations.

**Scheme 1.** Synthetic procedure to obtain the curcumin analog **7**.

#### **2. Results and Discussion**

#### *2.1. Organic Synthesis and Structure Determination*

The organic synthesis of compound **7** (curcumin analog) was performed through three main steps, as shown in Scheme 1. Basically, the first step was aimed at preparing para-methoxybenzyl chloride (**3**), which was employed in the derivatization of vanillin (**4**). Protected vanillin, namely 3-methoxy-4-[(4-methoxybenzyl)oxy]benzaldehyde (**5**), was then condensed with 2-chloro-4,6-dimethylpyrimidine (**6**) based on a protocol from Lee and coworkers [16], resulting in the formation of the target compound **7**.

Compound **7** was characterized by experimental and computational 1H- and 13C-NMR (Figures S1, S2 and Table S1 in the Supplementary Material), as well as by high resolution mass spectrometry (HRMS). As can be seen in Figure S1 and Table S1, the experimental 1H-NMR signals (δ) at 2.50 and 3.30 correspond to DMSO-d6 (solvent) and remnants hydration molecules, respectively, while δ at 3.77 and 3.85 correspond to the hydrogens for the methoxy groups connected to C5 and C7, respectively. In addition, the signals (δ) in the 6.96–7.89 range were assigned to the hydrogens of the aromatic moiety of compound **7** (details in Figure S1). On the other hand, the experimental 13C-NMR δ (via Distorsionless Enhancement by Polarization Transfer Including the Detection of Quaternary Nuclei—DEPTQ—Figure S2 and Table S1), revealed absorptions compatible with the proposed structure, e.g., δ at 55.58 and 56.09 corresponding to the carbon from the methoxyl groups connected to C5 and C7, respectively. In addition, δ in the 111.00–166.28 range can be assigned to the carbons present in the aromatic moiety (details in Figure S2) of

compound **7**. Overall, the proposed structure for **7** is in agreement with the experimental NMR data described in the literature for a similar compound: (E,E)-4,6-bis-(40-hydroxy-30 methoxystyryl)pyrimidine [16]. To further confirm the structure for compound **7**, HRMS experiments revealed the value of *m/z* 650.2184 [M]<sup>+</sup> which is in full agreement with the chemical formula C38H35ClN2O6 (calculated monoisotopic mass: 650.2184 g/mol).

Through the rise of reliable quantum chemical computational methods, such as Density Functional Theory (DFT), the DFT-NMR spectra of different organic compounds have been extensively reported. A comparison between the experimental and theoretical spectra in most cases reveals that they show the same profile [17–19]. Figures S1B and S2B show the theoretical DFT spectra (1H- and 13C-NMR) for the compound **7** and Table S1 compares the δexp and δcalc values (experimental and theoretical, respectively). An inspection of the experimental and theoretical δ values revealed that they are practically the same in both cases. As an example, the methyl protons from methoxyl group in C5--OCH3 presented δcalc 3.77/δexp 3.67 and δcalc 55.58/δexp 54.00 for 1H- and 13C-NMR, respectively. These results are clear indication that the chemical structure of the synthesized compound **7** is in accordance with the proposed structure. In addition, it was also shown that DFT is a good method to simulate NMR spectra for this curcumin analog.

#### *2.2. Spectroscopic Study: UV-Vis Absorption and Steady-State Fluorescence Emission*

In general, organic compounds based on curcumin are dyes whose structure allows them to be classified as donor-acceptor-donor species (D-A-D) due to the presence of electron donor groups at both ends of the conjugated π system and an electron acceptor group at their central portion [20]. The synthetic compound under study, i.e., compound **7**, also shows a D-A-D structure (Figure 2A), with a large absorption band in about 380, 390 and 395 nm in methanol, acetonitrile and toluene, respectively (Figure 2B and Table 1). These bands are probably due to a n–π\* electronic transition, with an additional absorption at low wavelength (<325 nm), corresponding to a π-π\* electronic transition in both methanol and acetonitrile [21]. It is important to note that toluene has a cut-off band of about 285 nm. Therefore, for toluene, only maximum absorption bands at 300 and 395 nm were considered. The absorption spectrum in each solvent (Figure 2B) shows a broad band in the high absorption wavelength region (325–450 nm range). The absence of shoulders in the absorption band in this region probably indicates that compound **7** does not present isomeric forms in the ground-state, which is in accordance with the proposed structure (Figure 1C). These results are in opposition to those described for curcumin, which show keto-enol tautomerism (Figure 1B) [22]. A considerable change in the energy of electronic transition in the 325–450 nm range can also be observed (Figure 2B) due to a solvatochromism effect, starting from a polar protic solvent (methanol) to a nonpolar solvent (toluene). For methanol, the presence of a blue shift in the maximum absorption band at 380 nm can be clearly observed, which is probably due to interactions by hydrogen bonding between the solvent and the electron acceptor groups (alkoxyl and the nitrogen atoms of the pyrimidine ring) present in compound **7** [12,22]. In addition, compound **7** showed a higher extinction coefficient (log ε at λmax = 380, 390 and 395 nm for methanol, acetonitrile and toluene, respectively—Figure 2C and Table 1) in nonpolar and polar aprotic solvents (e.g., log ε = 4.57 in toluene), a spectroscopic behavior that is very suitable for photosensitizer and fluorescent dyes [12]. These results are similar to those reported in the literature for photosensitizers and fluorescent synthetic diacetoxyboron complexes, also based on curcumin structure [20].

**Table 1.** Photophysical results for the compound **7** in three different solvent polarities.


<sup>a</sup> UV-cutoff for methanol, acetonitrile and toluene: 210, 190 and 285 nm, respectively [23]. <sup>b</sup> Corresponding to the highest wavelength UV absorption (λmax = 380, 390 and 395 nm for methanol, acetonitrile and toluene, respectively).

**Figure 2.** (**A**) Chemical structure of the compound **7** highlighting the D-A-D moieties. (**B**) UV-visible spectra for the compound **7** in methanol, acetonitrile and toluene. [**7**] = 2.32 × 10−<sup>6</sup> mol/L (**C**) Beer-Lambert plots for the determination of ε value for **7** in three different solvents (at λmax = 380, 390 and 395 for methanol, acetonitrile and toluene, respectively). [**7**] = 0.17–2.44 × 10−<sup>5</sup> mol/L.

Steady-state fluorescence measurement is a comprehensive approach used to assess the excited state behavior of organic dyes in the presence of metallic species or solvents [20]. As can be seen in Figure 3A and Table 1, compound **7** shows maximum fluorescence emission in methanol, acetonitrile and toluene at 507, 504 and 443 nm, respectively (λexc = 390 nm, a wavelength in which none of these solvents contribute for the absorption phenomenon). By inspecting the spectra shown in Figure 3A, a significant red shift in the maximum fluorescence emission of compound **7** can be observed when the polarity of the solvent is varied. Thus, for toluene, a non-polar solvent, the maximum fluorescence emission for compound **7** was recorded at 443 nm. In contrast, for the polar solvents methanol or acetonitrile, maximum values were observed at 507 and 504 nm, respectively. The maximum fluorescence emission did not change significantly when using polar protic (methanol) or non-protic solvent (acetonitrile). Thus, it can be concluded that the process of solvation of the lowest energy unoccupied molecular orbital (LUMO) does not involve hydrogen bonding. Further confirmation of these results was made using computational calculations, which showed similar values, for both LUMO (−2.41 and −2.31 eV) and |ΔE| (3.29 and 3.27 eV) in methanol and acetonitrile, respectively (Table 2). These values justify the similarity between the spectroscopic data obtained in these two solvents. Surprisingly, compound **7** showed LUMO and |ΔE| values (−2.25 and 3.34 eV, respectively—Table 2) in toluene comparable in magnitude to those calculated for methanol and acetonitrile. However, the maximum fluorescence emission in the former solvent showed a significant blue-shift compared to that observed in the two later solvents (Figure 3).

**Table 2.** Comparison between experimental and theoretical (calculated—DFT) signals (δ) for 1H- and 13C-NMR to the compound **7**.


**Figure 3.** (**A**) Normalized steady-state fluorescence emission spectra for the compound **7** in three different solvents (λexc = 390 nm and [**7**] = 2.32 × 10−<sup>6</sup> mol/L). (**B**–**D**) Normalized steady-state and excitation spectra for the compound **7** in methanol, acetonitrile and toluene, respectively.

The fluorescence quantum yield (ΦF) for compound **7** was recorded in methanol, acetonitrile and toluene. All Φ<sup>F</sup> determinations were performed using λexc = 390 nm for air saturated samples, with its value varying according to the polarity of the solvent. Compound **7** in methanol presented an extremely low fluorescence quantum yield (Φ<sup>F</sup> = 0.09) when compared to acetonitrile (Φ<sup>F</sup> = 0.23) and toluene (Φ<sup>F</sup> = 0.38) (Table 1), suggesting that its singlet excited state in methanol must be deactivated mainly by a non-radiative process. The excitation spectrum for compound **7** (Figure 3B–D) showed a broadband in the 325–450 nm range, which can be attributed to the S0–S1 transition due to its fully superimposed to the corresponding UV–vis absorption spectrum (Figure 2) [22]. In addition, the excitation spectrum in all three solvents is the specular image of the corresponding fluorescence emission spectrum. The Stokes shift reflected the difference between the spectral position at the maximum for the excitation spectrum and the fluorescence emission, dependent on the fluorophore and the solvation environment. The maximum fluorescence emission for compound **7** in methanol, acetonitrile and toluene showed Stokes's shift of 115, 109 and 47 nm, respectively. These results agree with the computational ones, which indicated that more polar solvents typically lead to larger Stokes shifts [24].

In general, dye molecules with a large change in their permanent dipolar moment (μ) exhibit a strong solvatochromism after excitation [22,25]. As shown above, spectroscopic studies for compound **7**, regarding ground-state absorption and steady-state fluorescence emission indicated a different behavior depending on the solvent polarity. Thus, while the absorption spectrum for compound **7** showed a blue shift when the solvent polarity changed from non-polar to polar, a red shift was observed in the case of fluorescence emission spectrum (inverted solvatochromism). These results indicated that there must be a significant change in the dipole moment value (μ) for compound **7**, according to the polarity of the solvent [25]. To gain further insights into the behavior of the dipole moment for compound **7** in the presence of solvents of different polarities, the theoretical values for μ were calculated using the DFT method (Table 2). From these calculations, a significant change in the μ value was observed from methanol (6.26 D) to toluene (5.46 D). On the

other hand, the μ value was quite similar in methanol and acetonitrile (6.26 to 6.21 D, respectively), also justifying the position of the maximum fluorescence in these two solvents, which was red-shifted when compared to toluene. The same solvatochromic profile for curcumin has already been reported [22]; however, these effects are mainly related to the keto-enol equilibrium, which is not the case for compound **7**, whose structure does not allow the existence of such equilibrium.

It is known that curcumin at a relatively high concentration can form dimers through a cycloaddition reaction due to its enolic form, in which a C=C double bond is present. This phenomenon directly affects the biological, photophysical and spectral profile of curcumin [26,27]. Even though the structure of compound **7** cannot lead to the formation of dimers due to the impossibility of the presence of an enolic form, it is important to theoretically assess its possible reactivity concerning the occurrence of a charge transfer process between compound **7** and model compounds that have an aromatic ring. The knowledge of this reactivity can help to understand the spectral profile discussed above when using toluene as a non-polar solvent.

The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) are the most important frontier molecular orbitals (FMOs), playing a crucial role in understanding the stability and chemical reactivity of different compounds. In general, FMOs are responsible for predicting interactions between molecules (e.g., the interaction between small compounds and phospholipids in biological systems), electronic spectra and chemical reactions, such as the dimers' formation [28]. Theoretical studies on the curcumin reaction using FMOs indicated the contribution of HOMO and LUMO from the o-methoxyphenol and unsaturated β-diketone groups, respectively [26]. Figure 4A shows the theoretical FMOs representation (DFT method) for compound **7** in methanol, acetonitrile and toluene. The HOMO density for compound **7** is delocalized through the ortho-methoxyphenol portion and the α,β-unsaturated pyrimidine base. It was not found any evidence for the HOMO density delocalized over the oxygen connected to the aromatic group, which is probably due to the presence of the vanillin protected by the para-methoxybenzyl group. On the other hand, the LUMO density is located mainly in the α,β-unsaturated group linked to the pyrimidine ring [28]. Even though the LUMO density is in a possible reactive fraction of compound **7**, the fact that this region does not have reactive groups (as an example, a carbonyl group), in association with the presence of the protected vanillin, also affected the localized HOMO density. These results indicated that compound **7** has a low probability of reaction, including a charge transfer process in the presence of toluene.

An alternative that allows examining the reactive behavior of a molecule and can be used to understand the charge transfer process between a target compound and solvents, is the molecular electrostatic potential (MEP) map. The MEP is generated in space around a molecule by charge distribution, especially useful in understanding the sites for electrophilic attack and nucleophilic reactions in the study of biological recognition processes, hydrogen bonding interactions and dimer formation [28,29]. The prediction of the reactive molecular sites via MEP was obtained for compound **7** in the ground and excited singlet states (S0 and S1 states, respectively) by applying the DFT method in methanol, acetonitrile and toluene. Figure 4B shows the MEP maps with negative regions (assigned in red), corresponding to sites prone to electrophilic attack, and positive regions (assigned in blue) corresponding to nucleophilic reactivity. These computational results suggested that the possible sites for nucleophilic attack are found around the chlorine atom. However, this negative region is diluted in the α,β-unsaturated system both in the ground-state and excited singlet state, indicating a low possibility of reacting with other compounds, including charge transfer interaction, corroborating the FMOs' results. Overall, the computational results suggested low reactivity, which indicated that compound **7** probably would not react with unsaturated phospholipids in possible applications as a fluorescent dye in biological systems. Furthermore, these results also indicated a low probability of charge transfer between compound **7** and toluene, which cannot be responsible for the spectral

profile for this compound in toluene [26]. In addition, the comparison between the MEP map and the Mulliken charge (Table 3) for the optimized S0 and S1 states revealed small changes in the distribution of the associated electronic charge in the ground and excited single states (only for the p-methoxyphenyl moiety). A slight increase was observed in the positive regions of the p-methoxyphenyl moiety; however, it is less rich in electrons when compared to the chlorine atom. These results suggested the pyrimidine ring contributes to the photophysical characteristics of compound **7** and the protected vanillin groups at its extremity can be responsible for possible further effects [30]. The fact that Mulliken's charge in the ground and excited states for compound **7** are practically the same when toluene is used as a solvent reinforced the low probability of charge transfer between this compound and non-polar aromatic solvents.

**Figure 4.** (**A**) HOMO-LUMO density for the compound **7** in methanol, acetonitrile and toluene. (**B**) Molecular electrostatic potential (MEP) map for the compound **7** (in a.u.) in three different solvents.


**Table 3.** Mulliken charge values for the compound **7** in both ground and excited state.

<sup>a</sup> Carbon numeration according to denomination presented in Scheme 1.

#### **3. Materials and Methods**

All reagents and solvents were purchased from commercial sources (Tedia Ltda, Rio de Janeiro, Brazil and Sigma-Aldrich, Saint Louis, MO, USA) and used without further purification. The Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker Ultrashield Plus Spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 500 and 125 MHz for 1H and 13C (DEPTQ), respectively, with tetramethyl silane (TMS) as internal reference and deuterated dimethyl sulfoxide (DMSO-d6) as solvent (signals for DMSO-d6: δ 2.50 and 39.7 for 1H and 13C-NMR, respectively). Chemical shifts (δ) were reported in ppm and the coupling constants (*J*) in Hertz [Hz]. The High-Resolution Mass Spectrometry (HRMS) analyses were taken in the positive ion mode under electrospray ionization (ESI) method on a Bruker 9.4 T Apex-Qh (FT-ICR) (Bruker Daltonik GmbH Life Sciences, Bremen, Germany). Reactions were monitored by Thin Layer Chromatography (TLC) on Merck silica gel 60 F245 aluminum sheets and TLC spots were visualized by inspection of the plates under ultraviolet (UV) light (254 and 365 nm).

#### *3.1. Synthesis of 2-Chloro-4,6-bis{(E)-3-methoxy-4-[(4-methoxybenzyl)oxy]-styryl}pyrimidine (Compound 7)*

The organic synthesis of compound **7** was performed through three main steps: Firstly, synthesis of compound **3** was accomplished by reducing 1.80 mL of 4-methoxybenzaldehyde (**1**) (1.15 mmol) by sodium borohydride (NaBH4). Initially, 0.28 g (7.5 mmol) of NaBH4 was slowly added to a solution of (**1**) in 50 mL of methanol at 0 ◦C. The resulting solution (4-methoxybenzaldehyde + NaBH4 in methanol) was left under stirring at room temperature for 1 h. The reaction was stopped by the addition of 50 mL of distilled water. The product was extracted with ethyl ether (3 × 50 mL) and the combined organic phases were washed with saturated sodium chloride solution (2 × 50 mL) and dried over anhydrous sodium sulfate (Na2SO4). The solvent was removed under reduced pressure without heating. A white solid (para-methoxybenzyl alcohol, **2**) was formed, which was then dissolved in 30 mL of dry dichloromethane (CH2Cl2) and cooled in an ice bath. Then, 1.5 mL of thionyl chloride (SOCl2, 2.46 g, 20 mmol) was added dropwise to replace the hydroxyl group in **2** with a chlorine atom, a better leaving group for the second step of the reaction. The formed mixture was kept under a dry nitrogen atmosphere. Upon completion

of the reaction, the solvent and excess SOCl2 were removed by evaporation under reduced pressure. The crude product was isolated as a colorless oil with a quantitative yield and characterized by mass spectrometry (MS/MS: *m/z* 158 [M + 2]+; 156 [M]+; 121; 77; 51).

In the second synthetic step, in a bottom flask, compound **3** was solubilized in 30 mL of dry dimethylformamide (DMF), to which 2.20 g of vanillin (**4**, 15 mmol in DMF) and oven-dried potassium carbonate (K2CO3; 2.40 g, 18 mmol) were added. To remove the produced acid gas (HCl) without the entrance of atmospheric air, the flask containing the resulting suspension was coupled to a glass flowmeter and maintained at ambient pressure under stirring for 18 h. Then, the reaction mixture was poured into crushed ice, yielding a white crystalline solid corresponding to compound **5**, which was filtered and recrystallized from ethyl acetate. The yield (83%) was calculated from the initial amount of **1** used in the previous step, and compound **5** was characterized by mass spectrometry (MS/MS: *m/z* 272 [M]+; 121; 77).

Finally, in the last step, 4 mmol of the protected vanillin (**5**) were condensed with 2 mmol of 2-chloro-4,6-dimethylpyrimidine (**6**) under 20 mL of an aqueous sodium hydroxide solution (NaOH, 4 mol/L) containing 0.29 mmol of tetrabutylammonium bisulfate ([(Bu)4NH4] HSO4). This step was based on a protocol from Lee and coworkers [16]. The reaction mixture was refluxed until the formation of a yellow gum, which was then filtered and recrystallized from ethyl acetate, yielding a yellow solid (yield of 72% for the target compound **7**). 1H-NMR (500 MHz, DMSO-d6) δ (ppm): 7.87 (d, 2H, *J* = 16.0 Hz); 7.61 (s, 1H); 7.40 (m, 6H); 7.27 (d, 2H, *J* = 5.0 Hz); 7.20 (d, 2H, *J* = 16.0 Hz); 7.12 (d, 2H, *J* = 5.0 Hz); 6.96 (d, 4H, *J* = 7.0 Hz); 5.07 (s, 4H); 3.95 (s, 6H); and 3.77 (s, 6H). 13C-NMR DEPTQ (125 MHz, DMSO-d6) δ (ppm): 166.2 (C1- '); 160.8 (C2); 159.5 (C5- ); 150.1 (C8); 149.8 (C7); 138.7 (C4); 130.1 (C3- ); 129.1 (C5); 128.6 (C2- ); 123.1 (C3); 122.6 (C10); 114.9 (C6); 114.3 (C4- ), 113.7 (C9); 111.0 (C1); 70.1 (C1- ); 56.1 (C7-OCH3); 55.6 (C5- -OCH3). HRMS *m/z* 650.2184.

#### *3.2. Spectroscopic Measurements*

The UV-Vis spectra were measured in a Shimadzu model Mini 1240 (Shimadzu Scientific Instruments, Kyoto, Japan) at room temperature (ca 298 K). In a quartz cell (1.0 cm optical path), 3 mL of methanol, acetonitrile or toluene was used as solvent to measure the UV-spectra of compound **7** (concentration of 2.32 × 10−<sup>6</sup> mol/L) in the 200–500 nm range. The molar extinction coefficient (ε) for compound **7** in the three different solvents (methanol, acetonitrile or toluene) was calculated using Beer-Lambert law (Equation (1)):

$$A = \varepsilon l.c \tag{1}$$

where *l*, *A* and *c* are the cuvette cell pathlength, absorbance and molar concentration of the compound **7** (0.17–2.44 × 10−<sup>5</sup> mol/L), respectively.

Steady-state fluorescence spectra measurements were performed using an optical spectrometer Jasco J-815 (Jasco Easton, MD, USA). A thermostated cuvette holder Jasco PFD-425S15F (Jasco Easton, MD, USA) was employed to control the temperature in the quartz cell (1.0 cm optical path). All spectra were recorded as the average of three scans with appropriate background corrections. The steady-state fluorescence spectrum of the compound **7** (concentration of 2.32 × 10−<sup>6</sup> mol/L) in methanol, acetonitrile or toluene was obtained in the 406–650 nm range (λexc = 390 nm). The corresponding excitation spectrum was recorded at λem = 507, 504 and 443 nm in methanol, acetonitrile, and toluene, respectively. The evaluation of the aggregation process was carried out by recording the steady-state fluorescence spectrum upon successive additions of the compound **7** in the concentration range of 0.17–2.44 × 10−<sup>5</sup> mol/L in all three different solvents (data shown only for acetonitrile). The fluorescence quantum yield (φ) at 298 K was calculated using Equation (2) and anthracene as a reference (λexc = 390 nm) [31]:

$$\varphi = \varphi\_{ref} \cdot \frac{I\_{sample}}{I\_{ref}} \cdot \frac{A\_{ref}^{390 \text{nm}}}{A\_{sample}^{390 \text{nm}}} \cdot \frac{n^2}{n\_{ref}^2} \tag{2}$$

where *φref*, *I*, *A*390nm and *n*<sup>2</sup> are the quantum yield for the reference compound (anthracene) [32], integral of the steady-state fluorescence emission spectra, absorbance at 390 nm (0.070 a.u.) and refractive index of the solvent, respectively. The subscript "ref" denotes the respective parameters for the reference compound (anthracene).

#### **4. Conclusions**

The curcumin analog 2-chloro-4,6-*bis*{(*E*)-3-methoxy-4-[(4-methoxybenzyl)oxy]-styryl} pyrimidine (compound **7**) was synthesized by three-step reaction. Basically, the first step was aimed at preparing *para*-methoxybenzyl chloride, which was employed in the derivatization of vanillin. Protected vanillin, namely 3-methoxy-4-((4-methoxybenzyl) oxy) benzaldehyde, was then condensed with 2-chloro-4,6-dimethylpyrimidine resulting in the formation of the target compound **7** with a final yield of 72%. The experimental and theoretical (DFT) signals of compound **7** by 1H and 13C-NMR confirmed the proposed structure, reinforced by HRMS *m/z* 650.2184 [M]+ for C38H35ClN2O6. Compound **7** showed a large absorption band in about 380, 390 and 395 nm in methanol, acetonitrile and toluene, respectively, while steady-state fluorescence emission of compound **7** showed a solvatochromism effect with higher fluorescence quantum yield (φ = 0.38) in toluene than in acetonitrile or methanol. The FMOs and MEP results indicated the absence of a possible charge transfer when toluene was used as solvent. Overall, the results indicated a low probability of compound **7** reacting with unsaturated phospholipids for future applications as a fluorescent dye in biological systems.

**Supplementary Materials:** The following are available online, Figures S1 and S2: The experimental and theoretical (DFT) 1H- and 13C-NMR spectra for the compound **7**. Table S1: Comparison between experimental and theoretical (calculated-DFT) signals (δ) for 1H- and 13C-NMR to the compound **7**.

**Author Contributions:** Conceptualization, M.E.F.d.L. and O.A.C.; synthesis and molecule characterization, V.S.-S., D.C.d.A.P. and M.E.F.d.L.; spectroscopic analysis and computational calculations, O.A.C. and J.C.N.-F.; writing—original draft preparation, O.A.C., V.S.-S. and M.E.F.d.L.; writing—review and editing, J.C.N.-F., M.E.F.d.L. and D.D.-R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Brazilian agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge Professor Nanci Câmara de Lucas Garden from Institute of Chemistry at Universidade Federal do Rio de Janeiro (UFRJ, Brazil) for the spectroscopic facilities. O.A.C. also thanks Fundação para a Ciência e a Tecnologia (FCT—Portuguese Foundation for Science and Technology) for the PhD fellowship 2020.07504.BD.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Short Note* **2-Oxo-2***H***-chromen-7-yl 4-chlorobenzoate**

**Diana Becerra 1,\*, Jaime Portilla <sup>2</sup> and Juan-Carlos Castillo 1,2,\***


**Abstract:** We describe the synthesis of 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3** in 88% yield by the *O*-acylation reaction of 7-hydroxy-2*H*-chromen-2-one **1** with 4-chlorobenzoyl chloride **2** in dichloromethane using a slight excess of triethylamine at 20 ◦C for 1 h. The ester **3** was completely characterized by mass spectrometry, IR, UV–Vis, 1D, and 2D NMR spectroscopy.

**Keywords:** 7-hydroxy-2*H*-chromen-2-one; *O*-acylation reaction; coumarin

#### **1. Introduction**

The coumarin was first isolated from tonka beans by A. Vogel in 1820 [1], while W. H. Perkin described the first chemical synthesis in 1868 by heating acetic acid with the sodium salt of salicylaldehyde [2]. The coumarin is also known as 2*H*-chromen-2-one (1,2-benzopyrone or 2*H*-1-benzopyran-2-one) according to the IUPAC nomenclature. This oxa-heterocycle is a two-ring system, consisting of a benzene ring fused with a *α*-pyrone nucleus. It should be noted that coumarin-based fluorescent chemosensors have been widely employed in bioorganic chemistry, molecular recognition, and materials science [3]. Over the last decades, synthetic and naturally occurring coumarins have received considerable attention from organic and medicinal chemists due to their huge diversity of biological and pharmacological activities, including anti-inflammatory [4], antibacterial [5], antifungal [6], anticoagulant [7], antioxidant [8], antiviral [9], cholinesterase (ChE), and monoamine oxidase (MAO) inhibitory properties [10]. Besides, coumarins exhibited significant anticancer activity through diverse mechanisms of action, including inhibition of carbonic anhydrase, inhibition of microtubule polymerization, inhibition of tumor angiogenesis, regulating the reactive oxygen species, among others [11–14].

In particular, 7-hydroxycoumarin derivatives have been widely used as valuable building blocks for the preparation of novel coumarin-based anticancer agents [15–17]. For instance, umbelliferone analogs (**I**) and (**II**) had excellent activity against MCF-7 cells with IC50 values of 9.54 and 16.1 μM, respectively, as illustrated in Figure 1 [15]. Interestingly, the coumarin-containing ketone (**III**) showed potent activity against breast cancer MCF-7 cells, with an IC50 value of 0.47 μM [16]. In contrast, the coumarin-containing ester (**IV**) exhibited high selectivity towards tumor-associated hCA IX over the cytosolic hCA I isoform, with a value of 21.8 nM [17].

It should be noted that the post-functionalization of the 7-hydroxycoumarin skeleton has been scarcely studied in synthetic and medicinal chemistry [18]. Interestingly, the hydroxyl group at the 7-position of the coumarin skeleton can be exploited to perform alkylation and acylation reactions [19–22]. Herein, we describe the synthesis and complete characterization of 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3** through an *O*-acylation reaction of 7-hydroxy-2*H*-chromen-2-one **1** with 4-chlorobenzoyl chloride **2** in the presence of triethylamine under mild reaction conditions.

**Citation:** Becerra, D.; Portilla, J.; Castillo, J.-C. 2-Oxo-2*H*-chromen-7-yl 4-chlorobenzoate. *Molbank* **2021**, *2021*, M1279. https://doi.org/10.3390/ M1279

Academic Editor: Giovanni Ribaudo

Received: 29 August 2021 Accepted: 10 September 2021 Published: 14 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Biologically active 7-hydroxycoumarin derivatives.

#### **2. Results and Discussion**

In connection with the ongoing development of efficient and simple protocols for the acylation of heterocyclic compounds of biological interest [23,24], we describe an expeditious approach to synthesize 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3** through an *O*-acylation reaction between equimolar amounts of 7-hydroxy-2*H*-chromen-2-one **1** and 4-chlorobenzoyl chloride **2** in dichloromethane, using a slight excess of triethylamine with vigorous stirring at 20 ◦C for 1 h under normal atmospheric conditions (Scheme 1). After the specified reaction time, the solvent was removed under vacuum using a rotary evaporator. The resulting crude product was purified by flash chromatography on silica gel using dichloromethane as an eluent to furnish ester **3** in 88% yield. This procedure is distinguished by its short reaction times, high yield, clean reaction profile, and operational simplicity. Albeit the compound **3** was synthesized nine years ago [19], the structural and electronic information obtained from spectroscopic and spectrometry data has not been explained yet. For that reason, a complete spectroscopic and analytical characterization was performed in this work (see Section 3). Initially, the structure of **3** was determined by mass spectrometry, IR, UV–Vis, and 1D NMR spectroscopy (Figures S1–S7). Later, the analysis of 2D NMR spectra, including HSQC (Figure S8), HMBC (Figures S9 and S10), COSY (Figure S11), and NOESY (Figure S12), allowed the structural assignment without ambiguity.

**Scheme 1.** Time-efficient synthesis of 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3**.

The absorption bands at 1728 and 1589/1620 cm−<sup>1</sup> are assigned to the C=O and C=C stretching vibrations in the IR spectrum, respectively. The absorption bands at 1068/1092 and 1231/1261 cm−<sup>1</sup> are attributed to the C–O–C asymmetric stretching vibrations. It should be noted that the C–Cl stretching band is normally expected around 580–750 cm−<sup>1</sup> [25]; thus, a strong band at 744 cm−<sup>1</sup> is assigned to the C–Cl stretching vibration. The 1H-NMR spectrum of **3** recorded in DMSO-*d*<sup>6</sup> showed one doublet of doublets at 7.34 ppm and four doublets at 6.51, 7.48, 7.83, and 8.11 ppm for the coumarin ring, as well as two doublets at 7.70 and 8.15 ppm for the benzene ring (Table 1). The proton signal

of the hydroxyl group attached to the coumarin ring was not observed, indicating that the *O*-acylation process was successful. The 13C{1H} NMR and DEPT spectra of **3** showed 14 carbon signals, consisting of seven aromatic methines, five quaternary aromatic carbons, and two carbonyl carbons (Table 1 and Figure 2A). The complete assignment of the proton and carbon signals of **3** is described in Section 3, while the correlations 1H-1H and 1H-13C observed in COSY and HMBC experiments, respectively, are illustrated in Figure 2B. In the MS spectrum, two molecular peaks are observed at *m*/*z* 300 and 302 complying with the Cl-rule, along with two peaks at *m*/*z* 141 and 139 with 32% and 100% intensity, respectively, corresponding to the (4-chlorobenzylidyne)oxonium ion (C7H4ClO+). Additionally, the accurate mass (*m*/*z* 301.0261) of the pseudo-molecular ion ([M + H]+) and the elemental formula (C16H10ClO4 +) is confirmed by HRMS measurements, obtaining an error mass of 1.33 ppm.

**Figure 2.** (**A**) Structure of 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3**. (**B**) Connectivities of **3** based on COSY (bold red line) and HMBC (from H to C, blue arrow) data.


**Table 1.** 1H and 13C{1H} NMR assignments, and COSY, NOESY, and HMBC correlations of **3** a.

<sup>a</sup> Measured at 400 MHz (1H) and 101 MHz (13C) in DMSO-*d*<sup>6</sup> at 25 ◦C.

In summary, we described the expeditious and ambient-temperature synthesis of 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3** through an *O*-acylation reaction of 7-hydroxy-2*H*-chromen-2-one **1** with 4-chlorobenzoyl chloride **2** in dichloromethane, using a slight

excess of triethylamine. This protocol is distinguished by its short reaction times, high yield, clean reaction profile, and operational simplicity.

#### **3. Materials and Methods**

#### *3.1. General Information*

The 7-hydroxy-2*H*-chromen-2-one **1** (CAS 93-35-6) and 4-chlorobenzoyl chloride **2** (CAS 122-01-0) were purchased from Sigma–Aldrich (Saint Louis, MO, USA). The starting materials were weighed and handled in air at ambient temperature. The silica gel aluminum plates (Merck 60 F254, Darmstadt, Germany) were used for analytical TLC. The IR absorption spectrum was recorded at room temperature employing a Shimadzu FTIR 8400 spectrophotometer (Scientific Instruments Inc., Seattle, WA, USA) equipped with an attenuated reflectance accessory. 1H and 13C{1H} NMR spectra were recorded at 25 ◦C on a Bruker Avance 400 spectrophotometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 400 MHz and 101 MHz, respectively. The concentration of the sample was approximately 15 mg/0.5 mL of DMSO-*d*6. Chemical shifts of 1H and 13C{1H} NMR experiments were referenced by tetramethylsilane (δ = 0.0 ppm). Chemical shifts (δ) are given in ppm and coupling constants (*J*) are given in Hz. The 2D HSQC, HMBC, COSY, and NOESY experiments were performed using the standard Bruker pulse sequence. NMR data were analyzed using the MestReNova 12.0.0 (2017) software (Mestrelab, Escondido, CA, USA). The mass spectrum was recorded on a SHIMADZU-GCMS 2010-DI-2010 spectrometer (Scientific Instruments Inc., Columbia, WA, USA) equipped with a direct inlet probe operating at 70 eV. The high resolution mass spectrum (HRMS) was recorded using a Q-TOF spectrometer via electrospray ionization (ESI, 4000 V). The UV–Vis spectrum was obtained from an acetone solution (5.0 × 10−<sup>4</sup> M) in an Evolution 201 UV–Vis spectrophotometer (Thermo Fischer Scientific Inc., Madison, WI, USA).

#### *3.2. Synthesis of 2-Oxo-2H-Chromen-7-yl 4-Chlorobenzoate 3*

A mixture of 7-hydroxy-2*H*-chromen-2-one **1** (162 mg, 1.0 mmol), 4-chlorobenzoyl chloride **2** (128 μL, 1.0 mmol), and triethylamine (167 μL, 1.2 mmol) in dichloromethane (5.0 mL) was stirred at 20 ◦C for 1 h (Scheme 1). After a complete disappearance of the starting materials, as monitored by thin-layer chromatography (TLC), the solvent was removed using a rotary evaporator under vacuum. The resulting crude product was purified by flash chromatography on silica gel using dichloromethane as an eluent to afford 2-oxo-2*H*-chromen-7-yl 4-chlorobenzoate **3** as colorless, needle-like crystals (265 mg, 88% yield): Rf (DCM) = 0.38. M.p 228–229 ◦C. FTIR-ATR: ν = 3086, 1728 (ν C=O), (1620 and 1589 for ν C=C), 1497, 1396, (1261 and 1231 for *v*<sup>a</sup> C–O–C), (1092 and 1068 for *v*<sup>a</sup> C–O–C), 984, (880 and 837 for *v*<sup>s</sup> C–O–C), 744 (*v* C–Cl), 613, 540, 521 cm<sup>−</sup>1. UV–Vis (acetone) λmax (ε, L·mol−1·cm<sup>−</sup>1): 316 (469), 330 (3898) nm. 1H-NMR (400 MHz, DMSO-*d*6): δ = 6.51 (d, *J* = 9.6 Hz, 1H, H-3), 7.34 (dd, *J* = 8.4, 2.0 Hz, 1H, H-6), 7.48 (d, *J* = 2.0 Hz, 1H, H-8), 7.70 (d, *J* = 8.4 Hz, 2H, H-3- ), 7.83 (d, *J* = 8.4 Hz, 1H, H-5), 8.11 (d, *J* = 9.6 Hz, 1H, H-4), 8.15 (d, *J* = 8.4 Hz, 2H, H-2- ) ppm. 13C{1H}-NMR (101 MHz, DMSO-*d*6): δ = 110.4 (CH, C-8), 115.8 (CH, C-3), 117.0 (Cq, C-4a), 118.8 (CH, C-6), 127.4 (Cq, C-1- ), 129.2 (2CH, C-3- ), 129.5 (CH, C-5), 131.8 (2CH, C-2- ), 139.3 (Cq, C-4- ), 143.9 (CH, C-4), 152.9 (Cq, C-7), 154.1 (Cq, C-8a), 159.7 (Cq, C-2), 163.4 (Cq, C=O) ppm. MS (EI, 70 eV) *m*/*z* (%): 302/300 (3/8) [M+•], 141/139 (32/100), 113/111 (28/85), 105 (14), 75 (29), 51 (16). HRMS (ESI+): calcd for C16H10ClO4 +, 301.0257 [M + H]+; found, 301.0261.

**Supplementary Materials:** The following are available online. Figure S1: HRMS spectrum for compound **3**; Figure S2: EIMS spectrum of the compound **3**; Figure S3: IR spectrum for compound **3**; Figure S4: UV–Vis spectrum for compound **3**; Figure S5: 1H-NMR spectrum for compound **3**; Figure S6: 13C{1H} NMR and DEPT-135 spectra for compound **3**; Figure S7: Expansion 13C{1H} NMR and DEPT-135 spectra for compound 3; Figure S8: HSQC 2D C–H correlation spectrum for compound **3**; Figure S9: HMBC 2D C–H correlation spectrum for compound **3**; Figure S10: Expansion HMBC 2D C–H correlation spectrum for compound **3**; Figure S11: COSY 2D H–H correlation spectrum for compound **3**; Figure S12: NOESY 2D H–H correlation spectrum for compound **3**.

**Author Contributions:** Investigation, data curation, writing—original draft preparation, D.B.; writing—review and editing, resources, J.P.; conceptualization, data curation, writing—original draft preparation, J.-C.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The APC was sponsored by MDPI.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Acknowledgments:** The authors thank Universidad Pedagógica y Tecnológica de Colombia and Universidad de los Andes. D.B. and J.-C.C. acknowledge to the Dirección de Investigaciones at the Universidad Pedagógica y Tecnológica de Colombia (Project SGI-3073). J.P. thanks support from the Facultad de Ciencias at the Universidad de los Andes (Project INV-2019-84-1800).

**Conflicts of Interest:** The authors declare not conflict of interest.

#### **References**


### *Communication* **New Derivatives of Lupeol and Their Biological Activity**

**Hoang-Thuy-Tien Le 1, Quoc-Cuong Chau 1, Thuc-Huy Duong 1,\*, Quyen-Thien-Phuc Tran 1, Nguyen-Kim-Tuyen Pham 2, Thi-Hoai-Thu Nguyen 3, Ngoc-Hong Nguyen <sup>4</sup> and Jirapast Sichaem 5,\***


**Abstract:** 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 IC50 value of 202 μM, whereas **2a** and **2b** were inactive.

**Keywords:** lupeol derivative; benzylidene derivative; *α*-glucosidase inhibition; Oxone®

#### **1. 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 1H, 13C NMR, and HRESIMS. All derivatives were evaluated for *α*-glucosidase inhibition.

**Citation:** Le, H.-T.-T.; Chau, Q.-C.; Duong, T.-H.; Tran, Q.-T.-P.; Pham, N.-K.-T.; Nguyen, T.-H.-T.; Nguyen, N.-H.; Sichaem, J. New Derivatives of Lupeol and Their Biological Activity. *Molbank* **2021**, *2021*, M1306. https:// doi.org/10.3390/M1306

Academic Editors: Giovanni Ribaudo and Rodrigo Abonia

Received: 16 November 2021 Accepted: 6 December 2021 Published: 10 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

67

**Figure 1.** Synthesis of **2**, **2a, 2b**, and **3** from lupeol (**1**).

#### **2. Results and Discussion**

#### *2.1. 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 1H NMR spectrum of **2a**/**2b** showed differences with **1**: the downfield methine at *δ*<sup>H</sup> 8.11, two oxymethines at *δ*<sup>H</sup> 5.26 and 4.48, and a doublet methyl at *δ*<sup>H</sup> 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*<sup>H</sup> 4.48) indicated that 3-OH was esterified by acetic acid. The 13C NMR spectrum of **2a**/**2b** showed one carbonyl ester at *δ*<sup>C</sup> 171.1, one formyl group at *δ*<sup>C</sup> 163.7 and two oxygenated carbons at *δ*<sup>C</sup> 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 (20*S*) and (20*R*) 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*<sup>C</sup> 20.1, would have a 20*R* configuration. On the other hand, **2b** would have the 20*S* configuration due to the lower chemical shift of C-30 at *δ*<sup>C</sup> 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 *δ*<sup>H</sup> 7.51 and 7.42, and a *trans* double bond at *δ*<sup>H</sup> 6.75 and 7.46. This was confirmed by the disappearance of a methyl ketone group at *δ*<sup>H</sup> 2.15 (CH3-29). This finding indicated that the aldolization occurred exclusively at C-29. The second difference was in the 13C NMR spectrum. This spectrum showed the presence of seven aromatic carbons at *δ*<sup>C</sup> 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.

#### *2.2. α-Glucosidase 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 IC50 value of 202 μM, compared with an acarbose-positive control (IC50 360 μM). Other compounds were inactive.

#### **3. Materials and Methods**

#### *3.1. 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 1H NMR and 125 MHz for 13C NMR. HRESIMS spectra were recorded on a Bruker MICROTOF-Q 10187.

**Extraction and Isolation**. The air-dried *Bombax ceiba* leaves (4 kg) were ground into powder and exhaustively extracted at room temperature with MeOH (2 × 10 L). The filtered solution was evaporated under reduced pressure to afford a residue (473.4 g). This crude extract was subsequently partitioned using solvents of *n*-hexane and EtOAc to yield *n*-hexane (40 g) and EtOAc (88 g) extracts. The *n*-hexane extract was fractionated by silica gel column chromatography (CC), eluted with *n*-hexane–EtOAc (isocratic, 10:1, *v*/*v*), to produce five fractions (H1-H5). Fraction H2 (15 g) was rechromatographed by silica gel CC using *n*-hexane–CHCl3 (isocratic, 12:1, *v*/*v*) as eluent to afford lupeol (**1**) (1.5 g).

#### *3.2. 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**.

Compound **2**. Isolated yield: 74.6 mg (37%), white solid. 1H and 13C NMR data were consistent with those reported previously [6].

**(3a***R***,5a***R***,5b***R***,7a***R***,9***S***,11a***R***,11b***R***,13a***R***,13b***S***)-1-((***S***)-1-(formyloxy)ethyl)-3a,5a,5b,8,8, 11a-hexamethylicosahydro-1***H***-cyclopenta[a]chrysen-9-yl acetate (2a)**. Isolated yield: 9.4 mg (4%), white solid. 1H NMR (500 MHz, CDCl3, *δ*, ppm): 2.05 (3H, *s*, CH3-2- ), 8.00 (1H, *s*, OCHO-29), 5.33 (1H, *m*, H-20), 4.48 (1H, *dd*, *J* = 10.5, 6.0 Hz, H-3), 2.13 (1H, *m*, H-3), 1.18 (3H, *d*, *J* = 6.5 Hz, CH3-30), 1.03 (3H, *s*, CH3-26), 0.90 (3H, *s*, CH3-27), 0.87 (3H, *s*, CH3-25), 0.85 (3H, *<sup>s</sup>*, CH3-23), 0.84 (3H, *<sup>s</sup>*, CH3-24), 0.79 (1H, *<sup>d</sup>*, *<sup>J</sup>* = 9.5 Hz, H-5), 0.76 (3H, *<sup>s</sup>*, CH3-28). 13C NMR (125 MHz, CDCl3, *<sup>δ</sup>*, ppm): 171.2 (C-6- ), 21.5 (C2- ), 161.6 (C-29), 81.1 (C-3), 73.4 (C-20), 55.5 (C-5), 50.2 (C-9), 48.8 (C-18), 43.5 (C-17), 43.0 (C-14), 42.6 (C-19), 41.0 (C-8), 40.5 (C-22), 38.5 (C-4), 38.0 (C-1), 37.3 (C-13), 37.2 (C-10), 35.5 (C-16), 34.4 (C-7), 29.9 (C-21), 28.1 (C-23), 27.3 (C-15), 27.1 (C-2), 23.8 (C-12), 21.0 (C-11), 18.4 (C-6), 18.1 (C-28), 16.7 (C-25), 16.4 (C-26), 16.1 (C-24), 14.4 (C-27), 14.2 (C-30). HRESIMS calcd C32H52NaO4 ([M+Na]+): 523.3732, found: 523.3763.

**(3a***R***,5a***R***,5b***R***,7a***R***,9***S***,11a***R***,11b***R***,13a***R***,13b***S***)-1-((***R***)-1-(formyloxy)ethyl)-3a,5a,5b,8,8, 11a-hexamethylicosahydro-1***H***-cyclopenta[a]chrysen-9-yl acetate (2b)**. Yield: 9.4 mg (5%), white solid. 1H NMR (500 MHz, CDCl3, *δ*, ppm): 2.04 (3H, *s*, H-2- ), 8.11 (1H, *s*, OCHO-29), 5.26 (1H, *m*, H-20), 4.48 (1H, *dd*, *J* = 11.5, 5.5 Hz, H-3), 2.31 (1H, *m*, H-19), 1.22 (3H, *d*, *J* = 6.5 Hz, CH3-30), 1.03 (3H, *s*, CH3-26), 0.86 (3H, *s*, CH3-25), 0.85 (3H, *s*, CH3-27), 0.85 (3H, *<sup>s</sup>*, CH3-23), 0.84 (3H, *<sup>s</sup>*, CH3-24), 0.77 (1H, *<sup>d</sup>*, *<sup>J</sup>* = 2.0 Hz, H-5), 0.75 (3H, *<sup>s</sup>*, CH3-28). 13C NMR (125 MHz, CDCl3, *<sup>δ</sup>*, ppm): 171.1 (C-6- ), 21.5 (C2- ), 163.7 (C-29), 81.1 (C-3), 72.7 (C-20), 55.5 (C-5), 50.0 (C-9), 47.1 (C-18), 44.4 (C-19), 43.2 (C-17), 43.0 (C-14), 41.0 (C-8), 40.1 (C-22), 38.5 (C-4), 38.0 (C-1), 37.5 (C-13), 37.3 (C-10), 35.3 (C-16), 34.4 (C-7), 29.9 (C-21), 28.1 (C-23), 27.4 (C-15), 26.9 (C-2), 23.9 (C-12), 21.0 (C-11), 20.1 (C-30), 18.4 (C-6), 18.1 (C-28), 16.7

(C-25), 16.3 (C-26), 16.1 (C-24), 14.4 (C-27). HRESIMS calcd C32H52NaO4 ([M+Na+H2O]+): 541.3870, found: 541.3869.

Synthesis of **3**: Compound **2** (70 mg, 0.163 mmol) together with NaOH (35 mg, 0.875 mmol) in ethanol (7 mL) was stirred at 55 ◦C for 15 min. Then, 4–bromobenzaldehyde (64.35 mg, 0.35 mmol) was added to the mixture. The reaction was performed at 55 ◦C for 2 h. The mixture was extracted with EtOAc–water (1:1, *v*/*v*) to gain the organic layer. This solution was applied to silica gel CC using the gradient system of *n*-hexane–EtOAc (10:1, *v*/*v*) to obtain compound **3**. Isolated yield: 68 mg (48%), white solid.

**(***E***)-3-(4-bromophenyl)-1-((1***R***,3a***R***,5a***R***,5b***R***,9S,11a***R***)-9-hydroxy-3a,5a,5b,8,8,11a-hexamethylicosahydro-1***H***-cyclopenta[a]chrysen-1-yl)prop-2-en-1-one (3):** 1H NMR (500 MHz, CDCl3, *δ*, ppm): 7.51 (2H, *d*, *J =* 8.5 Hz, H-3- ,7- ), 7.46 (1H, *d*, *J =* 16.0 Hz, H-6- ), 7.42 (2H, *d*, *J =* 8.5 Hz, H-4- ,6- ), 6.75 (1H, *d*, *J =* 16.0 Hz, H-29), 3.19 (1H, *dd*, *J =* 11.2, 4.8 Hz, H-3), 2.87 (1H, *td*, *J =* 11.5, 6.0 Hz, H-19), 1.02 (3H, *s*, CH3-26), 0.98 (3H, *s*, CH3-27), 0.96 (3H, *s*, CH3-23), 0.84 (3H, *s*, CH3-24), 0.80 (3H, *s*, CH3-25), 0.75 (3H, *s*, CH3-28). 13C NMR (125 MHz, CDCl3, *δ*, ppm): 204.1 (C-20), 141.0 (C-6- ), 133.9 (C-5- ), 132.3 (C-3- ,7- ), 129.8 (C-4- ,6 '), 126.9 (C-29), 124.7 (C-2- ), 79.0 (C-3), 55.4 (C-5), 50.4 (C-9), 50.1 (C-18), 43.3 (C-17), 42.9 (C-14), 40.9 (C-8), 40.3 (C-22), 39.0 (C-4), 38.8 (C-1), 37.3 (C-10), 35.2 (C-16), 34.3 (C-7), 28.7 (C-15), 28.1 (C-23), 27.9 (C-2), 27.5 (C-12), 21.1 (C-11), 18.4 (C-6), 18.3 (C-28), 16.2 (C-26), 16.0 (C-25), 15.5 (C-24), 14.6 (C-27). HRESIMS calcd C36H52BrO2 ([M−H]−): 595.3151, found: 595.3188.

#### *3.3. 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 Na2CO3 (130 μL). Enzymatic activity was quantified by measuring absorbance at 405 nm. All samples were analyzed in triplicate at five different concentrations around the IC50 values, and the mean values were retained. The inhibition percentage (%) was calculated as follows: Inhibition (%) = [1 − (Asample/Acontrol)] × 100.

#### **4. 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 (IC50 202 μM) than acarbose (IC50 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.

**Supplementary Materials:** The following are available online. Copies of HRESIMS and NMR spectra for compound **2a**, **2b**, and **3**.

**Author Contributions:** Conceptualization, T.-H.D. and J.S. methodology, T.-H.D. and J.S.; formal analysis, H.-T.-T.L., T.-H.D. and J.S.; investigation, T.-H.-T.N., H.-T.-T.L., Q.-C.C., T.-H.D. and Q.-T.-P.T.; data curation, N.-K.-T.P. and N.-H.N.; writing—original draft preparation, T.-H.D. and J.S.; writing—review and editing, T.-H.D. and J.S.; supervision, T.-H.D.; project administration, T.-H.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was funded by The Youth Incubator for Science and Technology Programme, managed by the Youth Development Science and Technology Center—Ho Chi Minh Communist Youth Union and Department of Science and Technology of Ho Chi Minh City (38/2020/HÐ-KHCNT-VƯ). This work was also supported by Thammasat University Research Unit in Natural Products Chemistry and Bioactivities.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data for the compounds presented in this study are available in the Supplementary Materials of this paper.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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