Phytochemical Investigation of Carex praecox Schreb. and ACE-Inhibitory Activity of Oligomer Stilbenes of the Plant

Phenolic compounds are the main special metabolites of Cyperaceae species from phytochemical, pharmacological, and chemotaxonomical points of view. The present study focused on the isolation, structure determination, and pharmacological investigation of constituents from Carex praecox. Twenty-six compounds, including lignans, stilbenes, flavonoids, megastigmanes, chromenes, and phenylpropanoids, were identified from the methanol extract of the plant. Five of these compounds, namely, carexines A–E, are previously undescribed natural products. All compounds were isolated for the first time from C. praecox. The ACE-inhibitory activity of seven stilbenoid compounds was tested, and (–)-hopeaphenol proved to be the most active (IC50 7.7 ± 0.9 μM). The enzyme–kinetic studies revealed a mixed-type inhibition; therefore, domain-specific studies were also conducted. The in silico docking of (–)-hopeaphenol to the ACE affirmed some favorable interactions. In addition, the antiproliferative and antibacterial effects of some compounds were also evaluated.


Introduction
Cardiovascular diseases (ischaemic heart disease and stroke) are still the leading causes of death responsible for approximately 27% of the total deaths worldwide [1][2][3].The angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is one of the main targets in treating hypertension, heart failure, myocardial infarction, and other related diseases.The ACE is composed of two independent catalytic domains: The C-domain is mainly responsible for the conversion of angiotensin I to angiotensin II and, thus, regulates blood pressure and hydrolyzing bradykinine, while the N-domain hydrolyzes other peptides, including the hemoregulatory peptide, AcSDKP [4,5].There are several available inhibitors of the ACE; however, most of them cause unpleasant adverse effects (e.g., dry cough, angioedema).Selective domain inhibitors might have potency in the treatment of hypertension without the undesirable adverse effects and in utilizing the different physiological effects of each ACE domain in clinical use [6].
The Carex genus, belonging to the family Cyperaceae (sedges), comprises approximately 2000 species that dominate wetlands, pastures, prairies, tundra, and the herb layer of temperate forests [7].Sedges are rich sources of phenolic secondary metabolites, like stilbenes, flavonoids, and lignans, but other types of plant metabolites, such as coumarins, quinones, alkaloids, and terpenoids have also been isolated from species of this plant family [8][9][10][11].However, up to now only a limited number of species (approx.20) have been investigated from phytochemical and pharmacological points of view [12].Previous studies revealed that Carex species are an abundant source of stilbene-type metabolites, among them monomers, dimers, trimers, and tetramers [12][13][14][15].Cyperaceae stilbenes are mainly oligomers of piceatannol and resveratrol.A 1,2-diaryl-dihydro benzofuran skeleton with trans-oriented aryl rings is the most important framework in stilbene oligomers of this family, and it is considered to be biosynthesized by regio-and stereoselective pathways [16].Stilbenes possess noteworthy biological activities and have been isolated from other heterogeneous and phylogenetically unrelated plant families, e.g., Dipterocarpaceae, Gnetaceae, Leguminosae, Polygonaceae, and Vitaceae, etc. [12,17].Our study aimed to isolate and identify specialized metabolites, especially stilbenes, from Carex praecox.C. praecox Schreb.(early sedge, spring sedge) is a perennial, 8-30 cm plant native to Europe and western Asia and is commonly found in moist to wet habitats, forests, or mountain grasslands.There is no available information on the ethnomedicinal importance of C. praecox and, according to the literature, it has not been investigated either from phytochemical or pharmacological points of view.
We describe here the isolation and structure determination of carexins A-E (1-5) and the identification of 21 other compounds , among them lignans, flavonoids, stilbenes, and megastigmanes, as well as the ACE-inhibitory activity of the isolated stilbenes.

Isolation and Structure Determination of the Compounds
Dried and ground C. praecox plant material was extracted with methanol at room temperature.After evaporation, the extract was dissolved in 50% aqueous methanol and subjected to solvent-solvent partitioning with n-hexane, chloroform, and ethyl acetate.Both the chloroform and the ethyl acetate phases of the plant were further purified by different chromatographic techniques, including column chromatography (CC), vacuum liquid chromatography (VLC), flash chromatography (FC), rotational planar chromatography (RCP), preparative thin-layer chromatography (TLC), and HPLC to afford altogether 26 compounds, among them 5 previously undescribed natural products (carexines A-E, 1-5) (Figure 1).
Molecules 2024, 29, x FOR PEER REVIEW 2 of 20 such as coumarins, quinones, alkaloids, and terpenoids have also been isolated from species of this plant family [8][9][10][11].However, up to now only a limited number of species (approx.20) have been investigated from phytochemical and pharmacological points of view [12].Previous studies revealed that Carex species are an abundant source of stilbenetype metabolites, among them monomers, dimers, trimers, and tetramers [12][13][14][15].Cyperaceae stilbenes are mainly oligomers of piceatannol and resveratrol.A 1,2-diaryldihydro benzofuran skeleton with trans-oriented aryl rings is the most important framework in stilbene oligomers of this family, and it is considered to be biosynthesized by regio-and stereoselective pathways [16].Stilbenes possess noteworthy biological activities and have been isolated from other heterogeneous and phylogenetically unrelated plant families, e.g., Dipterocarpaceae, Gnetaceae, Leguminosae, Polygonaceae, and Vitaceae, etc. [12,17].Our study aimed to isolate and identify specialized metabolites, especially stilbenes, from Carex praecox.C. praecox Schreb.(early sedge, spring sedge) is a perennial, 8-30 cm plant native to Europe and western Asia and is commonly found in moist to wet habitats, forests, or mountain grasslands.There is no available information on the ethnomedicinal importance of C. praecox and, according to the literature, it has not been investigated either from phytochemical or pharmacological points of view.
We describe here the isolation and structure determination of carexins A-E (1-5) and the identification of 21 other compounds , among them lignans, flavonoids, stilbenes, and megastigmanes, as well as the ACE-inhibitory activity of the isolated stilbenes.

Isolation and Structure Determination of the Compounds
Dried and ground C. praecox plant material was extracted with methanol at room temperature.After evaporation, the extract was dissolved in 50% aqueous methanol and subjected to solvent-solvent partitioning with n-hexane, chloroform, and ethyl acetate.Both the chloroform and the ethyl acetate phases of the plant were further purified by different chromatographic techniques, including column chromatography (CC), vacuum liquid chromatography (VLC), flash chromatography (FC), rotational planar chromatography (RCP), preparative thin-layer chromatography (TLC), and HPLC to afford altogether 26 compounds, among them 5 previously undescribed natural products (carexines A-E, 1-5) (Figure 1).The structure elucidation of the isolated compounds was carried out by extensive spectroscopic analysis, applying 1D ( 1 H and JMOD) and 2D ( 1 H-1 H COSY, HSQC, HMBC, and NOESY) NMR and HRMS measurements, and a comparison of the spectral data with the values in the literature.
Compound 1 was isolated as yellowish oil and exhibited a brownish-purple color on the TLC plate by spraying with vanillin sulfuric acid and then heating.From these data, the aglycone suggested a 2,5-diaryl-tetrahydrofuranoid-type lignan, and based on the comparison of its NMR data with the literature values, it was identified as icariol A 2 [18].HMBC spectrum determined the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between the anomeric proton (δ H 4.25) and C-9 (δ C 69.9) of the aglycone, as shown in Figure 2.This derivative is also known in nature [19].Moreover, the HMBC between the methyl protons (δ H 1.94) and methylene protons of H 2 -6 ′′ (δ H 4.33 and 4.15) with carbonyl carbon (δ C 172.7) determined that the glucose moiety was acetylated at C-6 ′′ .as icariol A2 [18].HMBC spectrum determined the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between the anomeric proton (δH 4.25) and C-9 (δC 69.9) of the aglycone, as shown in Figure 2.This derivative is also known in nature [19].Moreover, the HMBC between the methyl protons (δH 1.94) and methylene protons of H2-6″ (δH 4.33 and 4.15) with carbonyl carbon (δC 172.7) determined that the glucose moiety was acetylated at C-6″.The NOESY correlations confirmed the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between H-1″ and H-9a and H-9b.The NOESY correlations were detected between H-7 and H-9a/9b and H-7′ and H-9′a/9′b, proving H-7 to be on the same side as 9-methylene, and H-7′ on the same side as 9′-methylene.This is in agreement with the coupling constants of H-7 (J7,8 = 8.4 Hz) and H-7′ (J7′,8′ = 8.5 Hz) in trans position with H-8 and H-8′, respectively.Thus, the structure of 1 was assigned as icariol A2 9-O-β-D-(6″-acetyl)-glucopyranoside, a new natural compound, and named carexine A (Figure 1).as icariol A2 [18].HMBC spectrum determined the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between the anomeric proton (δH 4.25) and C-9 (δC 69.9) of the aglycone, as shown in Figure 2.This derivative is also known in nature [19].Moreover, the HMBC between the methyl protons (δH 1.94) and methylene protons of H2-6″ (δH 4.33 and 4.15) with carbonyl carbon (δC 172.7) determined that the glucose moiety was acetylated at C-6″.The NOESY correlations confirmed the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between H-1″ and H-9a and H-9b.The NOESY correlations were detected between H-7 and H-9a/9b and H-7′ and H-9′a/9′b, proving H-7 to be on the same side as 9-methylene, and H-7′ on the same side as 9′-methylene.This is in agreement with the coupling constants of H-7 (J7,8 = 8.4 Hz) and H-7′ (J7′,8′ = 8.5 Hz) in trans position with H-8 and H-8′, respectively.Thus, the structure of 1 was assigned as icariol A2 9-O-β-D-(6″-acetyl)-glucopyranoside, a new natural compound, and named carexine A (Figure 1).The NOESY correlations confirmed the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between H-1 ′′ and H-9a and H-9b.The NOESY correlations were detected between H-7 and H-9a/9b and H-7 ′ and H-9 ′ a/9 ′ b, proving H-7 to be on the same side as 9-methylene, and H-7 ′ on the same side as 9 ′ -methylene.This is in agreement with the coupling constants of H-7 (J 7,8 = 8.4 Hz) and H-7 ′ (J 7 ′ ,8 ′ = 8.5 Hz) in trans position with H-8 and H-8 ′ , respectively.Thus, the structure of 1 was assigned as icariol A 2 9-O-β-D-(6 ′′ -acetyl)-glucopyranoside, a new natural compound, and named carexine A (Figure 1).
Compound 1 was isolated as yellowish oil and exhibited a brownish-purple color on the TLC plate by spraying with vanillin sulfuric acid and then heating.(Table 1).The 13  From these data, the aglycone suggested a 2,5-diaryl-tetrahydrofuranoid-type lignan, and based on the comparison of its NMR data with the literature values, it was identified as icariol A2 [18].HMBC spectrum determined the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between the anomeric proton (δH 4.25) and C-9 (δC 69.9) of the aglycone, as shown in Figure 2.This derivative is also known in nature [19].Moreover, the HMBC between the methyl protons (δH 1.94) and methylene protons of H2-6″ (δH 4.33 and 4.15) with carbonyl carbon (δC 172.7) determined that the glucose moiety was acetylated at C-6″.The NOESY correlations confirmed the position of the β-D-glucopyranosyl group to be at C-9 by showing correlations between H-1″ and H-9a and H-9b.The NOESY correlations were detected between H-7 and H-9a/9b and H-7′ and H-9′a/9′b, proving H-7 to be on the same side as 9-methylene, and H-7′ on the same side as 9′-methylene.This is in agreement with the coupling constants of H-7 (J7,8 = 8.4 Hz) and H-7′ (J7′,8′ = 8.5 Hz) in trans position with H-8 and H-8′, respectively.Thus, the structure of 1 was assigned as icariol A2 9-O-β-D-(6″-acetyl)-glucopyranoside, a new natural compound, and named carexine A (Figure 1).The molecular formula and molecular weight of carexine C (3) were found to be the same as those of compound 2, based on the HRESIMS data.Only slight differences could be observed in the 1D and 2D NMR spectra (Table 2).The only difference between the two compounds was the cis orientation of H-8/H-8 ′ , as shown by the NOESY correlation between H-8 ′ and H-8 and H-8 ′ and H-7 (Figure 4).Previously, a lignan (vibruresinol) with the same skeleton was reported from the stems of Viburnum erosum [20].
According to our phytochemical results, C. praecox is a rich source of polyphenolic compounds.Polyphenols, including flavonoids, stilbenes, phenolic acids, lignans, and others, possess different health benefits [37].They are secondary plant metabolites implicated in protection against pathogens and ultraviolet radiation and have allelopathic effects [38].Due to their known antioxidant activity, they have been attributed a probable role in preventing various diseases associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases [39].It is hypothesized that the increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms) and potentially enhances antioxidant activity and antifungal capacities [40].
Megastigmanes are identified as phytotoxic compounds.Several compounds inhibited the germination of Lactuca sativa seeds [41], e.g., 5,7-dihydroxy chromone (22) inhibited the germination of velvetleaf seeds; therefore, it has allelopathic activity [42].p-Cresol (25) also possessed an allelopathic effect [43].Tricin (9) has been previously isolated from other Cyperaceae species, such as Cyperus exaltatus var.iwasakii [44], Rhynchospora corymbose [45], and Cyperus rotundus [46].Tricin (9) exerts unique biological activities over other flavonoids, such as antileishmanial [47], and antihistaminic [48] effects, and has a protective effect against UV-B-irradiation-caused skin damage [49].According to our phytochemical results, C. praecox is a rich source of polyphenolic compounds.Polyphenols, including flavonoids, stilbenes, phenolic acids, lignans, and others, possess different health benefits [37].They are secondary plant metabolites implicated in protection against pathogens and ultraviolet radiation and have allelopathic effects [38].Due to their known antioxidant activity, they have been attributed a probable role in preventing various diseases associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases [39].It is hypothesized that the increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms) and potentially enhances antioxidant activity and antifungal capacities [40].
For a better understanding of the actual interaction between (-)-hopeaphenol ( 16) and the ACE, a domain-specific assay was performed.The inhibitory activity of compound 16 compared to bradykinin-potentiating peptide b (BPPb) on the C-and Ndomain of a rabbit lung ACE using the FRET substrates was investigated.Based on the results, (-)-hopeaphenol ( 16) inhibits the N-domain favorably (IC50 = 35.67 ± 2.3 μM), while it has a 10 times lower affinity for the C-domain (IC50 > 300 μM) (Table 5).Hopeaphenol (16) was the first oligostilbene to be isolated in 1951 from Hopea odorata (Dipterocarpaceae) [50].(-)-Hopeaphenol ( 16), was identified as a selective inhibitor of HIV transcription that targets, in part, PKC-and NF-κB-mediated HIV transcription and CDK9 activity in T cells, resulting in the inhibition of virus production in vitro and infectious virus replication in peripheral blood mononuclear cells (PBMCs) [51].The compound also inhibited cellular entry of SARS-CoV-2 USA-WA1/2020, B.1.1.7,and B.1.351variants [52].

Pharmacological Assays
The isolated stilbenes (12)(13)(14)(15)(16)(17)(18) were subjected to different pharmacological studies.To investigate the potential cardioprotective effect of the isolated stilbenes, an ACE-inhibitory assay was performed, and the IC 50 values of the compounds were determined.All the tested stilbenes (except resveratrol ( 12)) exerted notable activity at a concentration of 90 µM; among them, the tetramer (-)-hopeaphenol ( 16) was the most active, with an IC 50 value of 7.7 ± 0.9 µM (Table 4).For a better understanding of the actual interaction between (-)-hopeaphenol ( 16) and the ACE, a domain-specific assay was performed.The inhibitory activity of compound 16 compared to bradykinin-potentiating peptide b (BPPb) on the C-and N-domain of a rabbit lung ACE using the FRET substrates was investigated.Based on the results, (-)hopeaphenol ( 16) inhibits the N-domain favorably (IC 50 = 35.67 ± 2.3 µM), while it has a 10 times lower affinity for the C-domain (IC 50 > 300 µM) (Table 5).
Since selective inhibition of the N-domain will result in the accumulation of AcSDKP, it might be promising for treating fibrosis without affecting blood pressure [53].Furthermore, inhibition of the N-terminal ACE-active site may have important clinical applications in facilitating hematopoietic recovery after aggressive cancer chemotherapy by controlling the hematopoietic cycle and stem cell proliferation [54].

Molecular Docking
In silico docking was applied to characterize the binding behavior of (-)-hopeaphenol ( 16).The energy-minimized model of compound ( 16) was docked using AutoDock4 into both domains of the ACE crystal structure retrieved from the Protein Data Bank (PDB ID: 1O86 for the C-domain and 2C6N for the N-domain) to explain the chemical interactions between (-)-hopeaphenol ( 16) and both ACE active binding sites.This program uses the Lamarckian genetic algorithm (LGA) to generate a range of potential conformations from a starting ligand in an arbitrary conformation and then searches for favorable dockings at the protein-binding site [55].The docking results revealed a network of hydrogen bonds and electrostatic and hydrophobic interactions between (-)-hopeaphenol ( 16) and the N-domain of the ACE with a strong binding energy (E = −9.83kcal/mol).Importantly, interaction with residues Tyr 369 , Arg 381 , and Thr 496 could be detected; these residues were previously connected to the N-domain selectivity [56].The most important interactions are the π-π interactions between (-)-hopeaphenol ( 16) and Tyr 369 and the carbon-hydrogen bond with Arg 381 (Figure 7).On the other hand, (-)-hopeaphenol ( 16) bound to the C-domain with a very poor affinity (binding energy: E = +41.42kcal/mol).Several unfavorable interactions were identified as the reason for the low binding efficiency (Figure 8).This is well in line with the results of the in vitro domain-specific studies, suggesting that (-)-hopeaphenol ( 16) is an N-domain-selective ACE inhibitor.On the other hand, (-)-hopeaphenol ( 16) bound to the C-domain with a very poor affinity (binding energy: E = +41.42kcal/mol).Several unfavorable interactions were identified as the reason for the low binding efficiency (Figure 8).This is well in line with the results of the in vitro domain-specific studies, suggesting that (-)-hopeaphenol ( 16) is an N-domain-selective ACE inhibitor.
On the other hand, (-)-hopeaphenol ( 16) bound to the C-domain with a very poor affinity (binding energy: E = +41.42kcal/mol).Several unfavorable interactions were identified as the reason for the low binding efficiency (Figure 8).This is well in line with the results of the in vitro domain-specific studies, suggesting that (-)-hopeaphenol ( 16) is an N-domain-selective ACE inhibitor.

Other Pharmacological Assays
The antiproliferative capacity of compounds 12-18 was tested against human colon adenocarcinoma cells (Colo 205 sensitive and the resistant Colo 320/MDR-LRP expressing ABCB1).A thiazolyl blue tetrazolium bromide (MTT) assay was used for each compound

Other Pharmacological Assays
The antiproliferative capacity of compounds 12-18 was tested against human colon adenocarcinoma cells (Colo 205 sensitive and the resistant Colo 320/MDR-LRP expressing ABCB1).A thiazolyl blue tetrazolium bromide (MTT) assay was used for each compound to assess the concentration required for 50% inhibition of viability of the cell population (IC 50 ), and cisplatin and doxorubicin were used as positive controls.Interestingly, only the monomer resveratrol (12) and the tetramer (-)-hopeaphenol ( 16) exerted notable antiproliferative activity against both cell lines with IC 50 values comparable to those of the two positive controls, while the other stilbenes were found inactive (Table 6).The antibacterial effect of the n-hexane, chloroform, and ethyl acetate fractions of the methanol extract of C. praecox was investigated against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Moraxella catarrhalis, MRSA, Pseudomonas aeruginosa, Staphylococcus aureus, S. epidermidis, and Streptococcus pyogenes using the disk diffusion method.The ethyl acetate fraction showed remarkable activity against S. epidermidis (inhibitory zone 18 mm), S. aureus (14 mm), MRSA (14 mm), M. catarrhalis (9 mm), and B. subtilis (12 mm), respectively.Therefore, the antibacterial effect of the isolated stilbenes (12)(13)(14)(15)(16)(17)(18) was also in-vestigated against these bacterial strains, but none of the compounds possessed remarkable antibacterial activity (data not indicated).

General Experimental Procedures
NMR spectra were recorded in methanol-d 4 , chloroform (CDCl 3 ), and DMSO-d 6 on a Bruker Avance DRX 500 spectrometer (Bruker, Ettlingen, Germany) at 500 MHz ( 1 H) and 125 MHz ( 13 C).The signals of the deuterated solvents were chosen as references.The chemical shift values (δ) were given in ppm, and the coupling constants (J) are in Hz.The two-dimensional (2D) experiments were conducted using standard TopSpin 3.6.1 Bruker software.Gradient-enhanced versions were applied in correlation spectroscopy ( 1 H-1 H COSY), nuclear Overhauser effect spectroscopy (NOESY), heteronuclear single quantum coherence spectroscopy (HSQC), and heteronuclear multiple bond correlation (HMBC) experiments.The high-resolution MS spectra were acquired with a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI ion source in positive ionization mode.The data were acquired and processed with MassLynx software 4.1 (SCN805).Optical rotation measurements were carried out by a Jasco-P2000 digital polarimeter (JASCO Corporation, Tokyo, Japan).

Plant Material
The whole plants of Carex praecox Schreb.(1.8 kg of dried plant material) were collected during the flowering period in Besenyőtelek, Hungary (GPS coordinates: 47.691670, 20.438270) in July 2019 and were identified by László Bakacsy (Department of Plant Biology, University of Szeged, 6726 Szeged, Hungary).A voucher specimen (No. 899) was deposited in the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary.

ACE-Inhibitory Assay
Angiotensin-converting enzyme inhibition was determined using the Angiotensin-I Converting Enzyme (ACE) Activity Assay Kit (CS0002, Sigma-Aldrich, USA) with modification of the volume of enzyme and samples added to the wells.In summary, a 96-well black plate (655096, F-bottom, Grenier bio-one, Frickenhausen, Germany) containing 25 µL of samples diluted in methanol-assay buffer was filled with 25 µL of enzyme solution (each 25 µL contained 1.5 mU of the ACE: ACE from rabbit lung, A6778, Sigma-Aldrich, USA).The solution was incubated for 5 min at 37 • C with shaking, and then 50 µL of the substrate (a 100-fold dilution of the substrate provided) was added.A plate reader (BMG Lambtech GmbH, Ortenberg, Germany) was used to monitor the fluorescence for 5 min in kinetic mode at Ex/Em 290/450 nm as soon as the substrate was added.At the end of the measurement, the percentage inhibition by each compound was calculated as follows: % inhibition = (values without samples − sample values)/(values without samples) × 100. ( Dose-effect studies on the compounds 12-18 were used to determine the concentration that inhibits 50% of the ACE.ACE-inhibitory kinetic studies were performed on compound 16, the most potent compound, to determine its inhibition mechanism.Similarly, 25 µL of enzyme was added to plate wells containing 25 µL of several concentrations of compound 16 (0-10 µM).Following a 5 min incubation period at 37 • C, 50 µL of the substrate Abz-Gly-Phe (NO 2 )-Pro (4003531, Bachem, Bubendorf, Switzerland) was added at varying concentrations (125-500 µM).The plate reader was then used to monitor the fluorescence in kinetic mode at extinction values of Ex/Em 290/450 nm.The Michaelis constant (Km) and maximal velocity (Vmax) of ACE were determined via Lineweaver-Burk plots, using the pharmacological and biochemistry transform and simple linear regression functions of the software, GraphPad Prism 8.0 (La Jolla, CA, USA).

Domain-Specific Studies
ACE domain-specific inhibition studies were performed based on previously reported methods (Carmona et al., 2006 [57]; Lunow et al., 2015 [58]).Fluorescence resonance energy transfer (FRET) substrates, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH were used for the N-domain and C-domain, respectively.The initial velocity of the reaction was determined using various concentrations (1-128 µM) of the FRET substrates.Briefly, 40 µL of assay buffer and 60 µL of the FRET substrate solutions were preincubated for 10 min at 37 • C, with the reaction started by adding 20 µL of diluted ACE solution (5 µL ACE + 15 µL 0.1 mol/L TRIS buffer) and fluorescence measured at λex/λem = 290/450 nm every minute at 37 • C for 30 min.The corresponding K M of the FRET substrates, determined using the Michaelis-Menten equation, was used as the substrate concentration of the FRET substrates used in percent inhibition studies.The inhibitory activity of 16 on both domains was determined as described above with the 40 µL solution containing the inhibitor (inhibitor in DMSO-Assay buffer, 1:9).The control samples, which correspond to 100% enzyme activity, were prepared by replacing the inhibitor solution with TRIS buffer.Dose-effect studies on 16 using the FRET substrates were used to determine the IC 50 of this compound on both ACE domains.All experiments were performed in triplicates.The ACE-inhibitory activity was calculated using the following equation: (%) = (Ab − Aa) − (Cb − Ca)/(Ab − Aa) × 100 (2) where Aa is the absorbance of control wells at 0 min; Ab is the absorbance of control wells at 15 min; Ca is the absorbance of the inhibitor wells at 0 min; and Cb is the absorbance of inhibitor wells at 15 min.

Molecular Docking
The structure of the compound was drawn using a ChemDraw 12.0.2software (ACD/LABS, Advanced Chemistry Development, Inc., Toronto, ON, Canada), and the energy of the compound ( 16) was minimized at the default mode, using a minimum RMS gradient of 0.010 in the software, Chem3D Pro 12.0 (ACD/LABS, Advanced Chemistry Development, Inc.).The energy-minimized compound was subsequently saved in PDB format before using it in the docking procedure.The X-ray crystallographic structures of the C-and N-domains of the human angiotensin I-converting enzyme complexed with lisinopril were obtained from the RCSB Protein Data Bank (PDB ID: 1O86 and 2C6N, respectively) [56,59].PDB files for the enzyme and compounds were converted to the PDBQT format using the graphical user interface, AutoDock4 (The Scripps Research Institute, La Jolla, CA, USA) [60].
Before the docking analysis, water molecules and the lisinopril were eliminated from the 1O86 ACE protein model (C-domain) using AutoDock 4.2 (The Scripps Research Institute), while the zinc and chlorine atoms were retained in the ACE protein model, as these have been reported to be essential for the activity of ACE.By adding polar hydrogens, combining non-polar hydrogens, and adding Kollman charge to 1O86 using AutoDockTools, the final receptor for docking was created.A grid box (X: 43.817, Y: 38.308, and Z: 46.652, with 50 × 70 × 50 grid points of 0.375 Å spacing) created to include all active residues around the Zn(II) prosthetic group was used to calculate the zinc-centered map for ACE 1O86 [61].
The N-domain enzyme, 2C6N, was prepared using an identical method to the one used in preparing the C-domain enzyme, except that the protein's water molecules and sugar moieties were eliminated along with the lisinopril.To encompass all active residues and Zn(II) heteroatom in the A chain of this domain [62], a grid box (X: −28.034,Y: −24.612, and Z: −33.992; number of grid points in the three dimensions [npts]: X: 70, Y: 70, and Z: 60; spacing: 0.375 Å) was defined.
The docking procedure was performed with 10 docking runs.The Lamarckian algorithm was used to dock ligands once the docking parameters were set to their default settings.The binding energies were obtained from the resulting DLG files, and visualization of the interactions was achieved via Biovia (Discovery Studio visualizer version 21.1.0.20298;Dassault Systèmes, Vélizy-Villacoublay, France) after conversion of the docked PDBQT files into PDB files using OpenBabel GUI software version 2.4.1 [63].

Antiproliferative Assay
The antiproliferative effects of the compounds were tested in decreasing serial dilutions (2-fold dilutions starting from 100 µM) of human cancer cell lines (Colo 205, Colo 320) in

Table 4 .
Results of the ACE-inhibitory assay.