An ef
ficient, catalyst-free, one-pot synthesis of 4H-chromene
derivatives and investigating their biological activities and mode of
interactions using molecular docking studies
Leila Dinparast
a, Salar Hemmati
b, Ali Akbar Alizadeh
a, Gokhan Zengin
c,
Hossein Samadi Ka
fil
b, Mir Babak Bahadori
d, Siavoush Dastmalchi
a,e,f,* aBiotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, IranbDrug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran cDepartment of Biology, Science Faculty, Selcuk University, Konya, Turkey
dMedicinal Plants Research Center, Maragheh University of Medical Sciences, Maragheh, Iran eSchool of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
fFaculty of Pharmacy, Near East University, POBOX:99138, Nicosia, North Cyprus, Mersin 10, Turkey
a r t i c l e i n f o
Article history: Received 23 July 2019 Received in revised form 13 November 2019 Accepted 14 November 2019 Available online 19 November 2019 Keywords: Chromene Ionic liquid Green chemistry Cytotoxicity Molecular docking Enzyme inhibition
a b s t r a c t
In the present study, the design and development of an efficient and green method for the synthesis of dialkyl 4-hydroxy-4H-chromene-2,3-dicarboxylate derivatives together with their biological evaluation are reported. A series of 4H-chromenes were synthesized in the presence of 1-hexyl-3-methylimidazolium bromide ([HMIM]Br) as an environmentally friendly media, without using any organic and toxic solvent and catalyst. The reaction was rapid and was conducted at room temperature with high-to-excellent yields. The antiproliferative potential of the synthesized compounds was evalu-ated against human lung (A549), breast (MCF-7), and colon (HT-29) cancerous cell lines by adopting MTT method. The tested chromenes showed cytotoxicity in the range of 8.8e82.3% against A549 cells at 200mg/mL. Also, chromene derivatives were assessed for tyrosinase anda-glucosidase inhibitory ac-tivities. Based on IC50values (2.99e4.39 mM), all chromenes exhibited significanta-glucosidase inhi-bition compared with acarbose (IC50¼ 7.90 mM). Furthermore, the ability of the studied compounds to inhibit tyrosinase was evaluated and found to be moderate (IC50¼ 3.50e12.20 mM). In silico studies were performed to explore the binding modes of the chromenes at the binding site of a-glucosidase and tyrosinase. Molecular docking results revealed the importance of hydrogen bonding, hydrophobic,p-p stacking,p-cation, and metal interactions between the target enzymes and the synthesized compounds. Collectively, the results obtained in the current work indicated that the studied chromenes may be regarded as lead compounds for designing new chemicals potentially effective in conditions such as skin disorders and diabetes mellitus.
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
Chromenes are heterocyclic compounds which consist of ben-zene ring fused to the pyran [1]. Chromene and its derivatives are important organic compounds distributed widely in the nature [2]. Several pharmaceutical and biological properties such as anticancer [3e6], antimicrobial [7e13], anti-neurodegenerative [14], and
anti-HIV [15] effects were reported for these compounds [16e20]. In addition, they are applied as the natural insecticides [21]. Also, chromene derivatives are widely used as the intermediates for the synthesis of natural and synthetic products [22]. The design and development of novel synthetic strategies and introducing efficient and green methodology for the synthesis of such valuable
hetero-cycles is a significant challenge for organic/medicinal chemists.
Nowadays, synthetic chemists are interested in developing envi-ronmentally benign, solvent-free, catalyst-free, pot, and one-step synthesis procedures [23e30]. The shorter reaction times as
well as the easy work-up and purification steps are some of the
advantages for the solvent-free methods [31].
* Corresponding author. Biotechnology Research Center and School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
E-mail address:[email protected](S. Dastmalchi).
Contents lists available atScienceDirect
Journal of Molecular Structure
j o u r n a l h o m e p a g e : h t t p : / / w w w . e l se v i e r . c o m / l o c a t e / m o l s t r u c
https://doi.org/10.1016/j.molstruc.2019.127426
Different synthetic methods have been reported for the
syn-thesis of chromenes [32]. 2H-chromene derivatives were
synthe-sized via the reaction of salicylaldehydes with dienophiles in the presence of CsF [33]. Also, the preparation of chromenes was re-ported by the cycloaddition reaction between N-tosylimines or salicylaldehydes and diethyl acetylenedicarboxylate in the pres-ence of DABCO or dimethylphenylphosphine under mild conditions in excellent yields [34]. The selective synthesis of 4H-chromene derivatives through the reaction of salicylaldehyde and dialkyl acetylene dicarboxylate in the presence of ZnO nanoparticles has been also reported [35].
The drug design and discovery processes in general involve the synthesis and identification of novel lead compounds as well as the introduction of new synthetic routes for the improvement of yield
and specificity. In most cases, the synthesized compounds are
screened for various biological endpoints to isolate the chemicals with desired activities. Cancer is one of the critical health problems in the world. Despite the existence of numerous reports about the synthesis of anti-cancer drugs, there is still a considerable demand
for the design, synthesis, and discovery of efficient anti-cancer
drugs to overcome the problems associated with current com-mercial drugs including toxicity and drug resistance [36e39].
Today, a promising technique for discovery of novel drugs for treatment or prevention of the public human disorders is the in-hibition of key enzymes involved in based biochemical processes of pertinent diseases. For instance,
a
-glucosidase,a
-amylase, chol-iesterases, tyrosinase, cyclooxygenase, and lipase are examples of such enzymes whose inhibitions are regarded as the therapeutic strategies for alleviating conditions such as diabetes mellitus, Alz-heimer’s disease, skin disorders, inflammation, and obesity [40].Here, the synthesis of some known 4-hydroxy-4H-chromene-2,3-dicarboxylate derivatives 3(a-l) and 4(a-c) using a new, ef fi-cient, rapid, one-pot, and solvent-free method is reported. In the present synthetic method, 1-hexyl-3-methylimidazolium bromide ([HMIM]Br) was used as a green media. These chemically important o-containing heterocycles were synthesized employing green pro-cedure in high-to-excellent yields without using catalyst (Scheme 1). The antiproliferative activity of chromenes 3(a-l) and 4(a-c) was evaluated on A549, MCF-7, and HT-29 cancerous cells. Addi-tionally, the enzyme inhibitory activity, along with antioxidant and antimicrobial effects of these compounds were studied. Finally, the interactions and binding modes of the synthesized chromenes 3(a-l) and 4(a-c) with the binding site of
a
-glucosidase and tyrosinase were evaluated using molecular docking studies. To the best of our knowledge, this is thefirst report about the catalyst-free synthesis and biological evaluation of these 4H-chromene derivatives.2. Materials and methods 2.1. Chemistry
The reagents were purchased from Merck (Germany) and used
without further purification. The IR spectra were obtained on a
Shimadzu FTIR-8400S spectrophotometer (Japan) using KBr pellets. NMR spectra were recorded on Bruker Avance 300 and 400 MHz spectrometers (Bruker, Rheinstatten, Germany), operating at 300 and 400 MHz for1H, as well as 75.4 and 100 MHz for13C. The CDCl3 was used as the deuterated solvent and TMS as an internal stan-dard. The elemental analysis for C, H, and N atoms was carried out using the Costech elemental analyzer. Melting points were measured in open glass capillaries using Electrothermal melting point apparatus.
2.1.1. General procedure for the synthesis of 1-hexyl-3-methylimidazolium bromide ([HMIM]Br)
1-Bromohexane (44 mL, 0.313 mmol) was added dropwise into
a 250 mL round bottomflask containing 1-methylimidazole (25 mL,
0.313 mmol) during 1 h at room temperature. Then, the reaction
mixture was refluxed at 70C for 24 h. The unreacted materials
were washed by diethyl ether (3 30 mL). Finally, the diethyl ether was removed under reduced pressure at 40C to yield a yellowish viscous liquid.
2.1.2. General procedure for the synthesis of chromene derivatives 3(a-l) and 4(a-c)
Salicylaldehyde derivatives (1 mmol) and 1 mL freshly synthe-sized [HMIM]Br were mixed thoroughly. Then, 1 mmol of dimethyl or diethyl acetylenedicarboxylate was added. The reaction mixture was stirred at room temperature. The reaction process was detected by TLC (n-hexane:ethyl acetate, 3:1). After the completion of the reaction, the mixture was extracted using ethyl acetate (3 20 mL). Afterward, ethyl acetate was evaporated by rotary evaporator at 45C and the crude product was purified by recrystallization from chloroform/n-hexane.
2.2. Biological assays 2.2.1. Cell culture
The human lung (A549), breast (MCF-7), and colon (HT-29) cancer cell lines were purchased from national cell bank of Iran, Pasteur Institute (Tehran, Iran) and cultured in DMEM supplement with 10% FBS (fetal bovine serum), and antibiotics (streptomycin
(50
m
g/mL) and penicillin (5 unit/mL)). The cells were grown in75 cm2 flasks at 37C under an atmosphere of 5% CO2and 95%
humidity [41].
2.2.2. In vitro cytotoxicity assay
The cytotoxicity of synthesized chromenes 3(a-l) and 4(a-c) was investigated against A549, MCF-7, and HT-29 cells using MTT colorimetric assay [42,43]. The cells were seeded into the 96-well plates and incubated at 37C, 5% CO2, and 95% humidity for 24 h, before the test compounds were added. The stock solutions were
prepared by dissolving chromenes in DMSO. Thefinal
concentra-tion of DMSO in the cell culture medium was less than 0.1% in all
experiments, which is far below the scientifically-approved maximum concentration of DMSO in the medium. The cells were
treated in triplicates with 50
m
L of samples with knownconcen-trations. After further incubation for 48 h, 20
m
L of the freshly prepared MTT solution (5 mg/mL in PBS) was added into each welland the plate was incubated for another 3 h in a humidified CO2
incubator. Finally, the media were replaced by 100
m
L DMSO todissolve the formazan crystals formed during the reduction of tetrazolium salts by metabolically viable cells. The UV absorbance of produced purple color was measured by microplate reader (Epoch-Biotek) at 570 nm. Three blank wells (the medium without tested compounds) were used as the negative controls and the wells with cisplatin were used as the positive controls.
2.2.3. Enzyme inhibitory activity
The possible effectiveness of chromene derivatives in skin dis-orders and diabetes mellitus were investigated by evaluating their
inhibitory effects against tyrosinase and
a
-glucosidase,respec-tively, using the previously reported standard methods [44], as outlined below.
2.2.3.1. Tyrosinase inhibitory activity. Tyrosinase inhibitory activity
of chromenes 3(a-l) and 4(a-b) was measured using the modified
dopachrome method with L-DOPA as a substrate [45]. Different
concentrations of chromene solutions (25
m
L) were mixed withtyrosinase solution (from mushroom, EC 1.14.18.1, 40
m
L) andphosphate buffer (100
m
L, pH¼ 6.8) in a 96-well microplate incu-bated for 15 min at room temperature. Then, the reaction was initiated with the addition ofL-DOPA (40m
L). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The optical density of the sample and blank was measured at 492 nm after incubation at25C (10 min). The absorbance of the blank was subtracted from
that of the sample and the tyrosinase inhibitory activity was expressed as IC50values.
2.2.3.2.
a
-Glucosidase inhibitory activity. The sample solution(50
m
L) with known concentrations was mixed with glutathione(50
m
L),a
-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50m
L) in phosphate buffer (pH¼ 6.8), and PNPG (p-nitrophenyl-a
-D-glucopyranoside) (50m
L) in a 96-well micro-plate and incubated for 15 min at 37C [46]. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (a
-glucosidase) solution. Due to the addition ofsodium carbonate (50
m
L, 0.2 M), the reaction was quenched.Finally, the UV absorbance of the mixture and blank were deter-mined at 400 nm. The absorbance of the blank was subtracted from
that of the sample and the
a
-glucosidase inhibitory activity wasexpressed as IC50values. Acarbose was applied as a positive control in this assay.
2.2.4. Antimicrobial assay
2.2.4.1. Disc diffusion method. The antimicrobial activity of syn-thesized 4H-chromenes 3(a-l) and 4(a-c) was screened against gram-positive (Bacillus subtilis, Staphylococcus aureus, and Listeria monocytogenes), gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, and two fungal strains (Candida albicans and Candida krusei) by agar disk diffusion method [47]. Bacterial and fungal strains were cultured overnight at 37C in Mueller Hinton agar. The solution of compounds at the concentration of 500
m
g/mL was prepared in DMSO. The paper discs (6 mm in diameter) were impregnated with 10m
L of the sample solution and were placed on the inoculated agar. In this assay, gentamycin was used as a stan-dard drug. Finally, the plates were incubated for 24 h at 37C.2.2.5. Antioxidant assay
Chromenes 3(a-l) and 4(a-b) were evaluated for their radical scavenging ability against DPPH (1,1-diphenyl-2-picrylhydrazyl radical) according to the previously reported method [48], where the discoloration of free radical solution through the tested
com-pounds was measured by employing UVeVis techniques. In brief,
the synthesized compounds solutions with various concentrations
(20
m
L) were prepared and combined with DPPH (180m
L, 0.1 mM).After shaking the mixture and incubation at ambient condition in dark room (30 min), the absorbance of the sample was recorded at 517 nm. Trolox was used as a standard free radical scavenger. 2.3. Molecular docking
The molecular docking of chromene derivatives 3(a-l) and
4(a-c) were performed using AutoDock 4.2 software [49]. The 3D
structure of the ligands were built by HyperChem 7.5 software [50]. The energy minimization of the structures were conducted initially
using the empirical method (i.e., MMþ) [51] followed by
semi-empirical technique AM1 [52] using the Polak-Ribiere algorithm
included in HyperChem 7.5 software. For experimental determi-nation of the 3D structure of Saccharomyces cerevisiae
a
-glucosi-dase (EC 3.2.1.20), the homology model for this enzyme was buildusing SWISS-MODEL web server (https://swissmodel.expasy.org/).
The sequence of Saccharomyces cerevisiae
a
-glucosidase (accesscode P53341) was retrieved from UniProt in FASTA format. The crystallographic structure of Saccharomyces cerevisiae isomaltase
(PDB ID: 3AXH) was identified as the most suitable template (with
72.6% identity and 84% similarity with the target enzyme) for ho-mology modeling by SWISS-MODEL web server and BLAST search of PDB database. The quality of the homology model was verified by QMEAN method. The binding site of the target enzyme was
pre-dicted using 3DLigandSite program available at http://www.sbg.
bio.ic.ac.uk/3dligandsite/[53].
The crystal structure of tyrosinase from Agaricus bisporus mushroom (PDB ID: 2Y9X, chain A [54]) was retrieved from protein data bank (https://www.rcsb.org) [55]. Before docking, the water molecules and tropolone (the co-crystalized ligand) were removed from the enzyme structure and the hydrogen atoms were added. The grid box was set at the center of trobolon’s binding site at x, y,
and z coordinates of9.447, 27.417, and 43.278 with dimensions
of 46 40 40. We included copper atoms in the binding pocket
for docking study. PoseView (version 1.1.2) and PyMOL (version 1.7.5.0) were used to visualize the interactions between ligand and macromolecule in 2D and 3D illustrations [56,57].
3. Results and discussion 3.1. Chemistry
Initially, the reaction between salicylaldehyde (1) and dimethyl acetylenedicarboxylate (DMAD) (2) was studied as a model reac-tion. For this purpose, salicylaldehyde (1) (1 mmol) was mixed with [HMIM]Br (2 mL) for 2 min. Then, 1 mmol of dimethyl acetylene-dicarboxylate (DMAD) (2) was added to the mixture and stirred at room temperature. The TLC which was used to monitor the reaction progress revealed that the reaction was started immediately after adding DMAD (2) at room temperature and was completed after 15 min. Work-up of the reaction mixture provided the dimethyl 4-hydroxy-4H-chromene-2,3-dicarboxylate (3a) in high yield (80%) as the main product and dimethyl 2-hydroxy-2H-chromene-2,3-dicarboxylate (4a) as the side product (10%). The crud product
was purified using re-crystallization in chloroform/n-hexane. The
chemical structure of these compounds was confirmed adopting
and reaction time when the reaction was performed at higher temperatures. The reaction of several salicylaldehyde derivatives (1) with dimethyl and diethyl acetylenedicarboxylate (2) was examined in [HMIM]Br as a reaction medium under solvent- and catalyst-free condition (Table 1). In all of the reactions,
4H-chro-menes (3) were isolated and purified as the main products. There
were extremely low yields of 2H-chromene derivatives (4) and the isolation, purification, characterization, and biological evaluation were performed only for three of them (4a, 4b, and 4c). The pre-dicted mechanism for the formation of chromenes was shown in
Scheme 2. The nucleophilic attack of salicylaldehyde (1) OH group on DMAD or DEAD (2) and subsequent interamolecular cyclization led to the formation of intermediate I. Then, the reactive interme-diate II was produced from dehydration of intermeinterme-diate I. Finally, products 3 and 4 were formed by the addition of H2O to II through two plausible pathways of A and B, respectively. All of the syn-thesized compounds in this work (3a-3l and 4a-4c) have been previously reported [32e35,58,59]. However, the synthetic route used for the preparation of these compounds in the current study is novel and different from those in the previous reports where various catalysts (nano-ZnO, silica gel, cesium fluoride, PPhMe2, imidazole-functionalized nano-silica, and K2CO3) were employed in the presence of organic solvents (benzene, dimethyl sulfoxide, and N,N-dimethylformamide). The quick catalyst- and organic solvent-free synthesis of 4H-chromene derivatives at ambient temperature with high-to-excellent yields are the major benefits of the proposed novel procedure compared with the previously re-ported ones.
3.2. Biological assays
3.2.1. In vitro cytotoxicity assay
Synthetic and natural chromenes are attractive compounds with the chemotherapeutic anticancer potential [3,5]. Firstly, chromenes have been identified as apoptosis inducers in cell-based anticancer screening but then, the majority of these compounds have been
detected as tubulin inhibitors [4,60e62]. Patil et al. synthesizedfive
4H-chromene derivatives by the cyclocondensation of
3-dimethyaminophenol, substituted naphthaldehydes, and melano-nitrile. They evaluated the antiproliferative activity of synthesized chromenes against two human metastatic melanoma cell lines (A375 and WM164), two human prostate cancer cell lines (LnCap and PC3), as well as Taxol resistant prostate cancer cell line
PC3-TxR. Their results showed that allfive compounds have activities
in nanomolar range (IC50¼ 7.4e640 nM) against these cell lines [5]. In another study, C4-active methine-substituted 4H-chromenes were synthesized and their cytotoxicity was evaluated against laryngeal carcinoma (Hep2), lung adenocarcinoma (A549), colon carcinoma (HT-29), and cervical cancer (HeLa) cells. The results suggested that 4H-chromenes with C4-malononitrile substitution are the most efficient inducers of apoptosis [63]. In the present work, MTT method was adopted to determine the antiproliferative activity of chromenes against three human cancer cell lines (A549,
MCF-7, and HT-29). The IC50of 11.20
m
g/mL was determined forcisplatin which was used as the positive control in this assay. The
cytotoxicity of chromenes 3(a-l) and 4(a-c) (at 200
m
g/mL) areshown in Fig. 1, represented as percentages of decreased cell
viability compared to the untreated cells using A549 cancerous cells. As shown in thefigure, the activity percentage of 3d, 3i, and 4a is more than 50%. The other compounds have cytotoxicity lower than 50% against A549 cells at this concentration. Also, the results
of MTT revealed that these compounds have no significant
cyto-toxic effects on MCF-7 and HT-29 cell lines (Fig. 2). 3.2.2. Enzyme inhibitory assays
3.2.2.1. Tyrosinase inhibitory activity. Tyrosinase inhibitors are uti-lized in cosmetic products as anti-hyperpigmentation agents. Also, they are used in food industries for preventing the enzymatic browning of fruits and vegetables [64]. Despite the introduction of various synthetic and natural compounds with tyrosinase inhibi-tory potential likeflavonoids, hydroquinones, chalcones, and stil-benes, only a few are used as drugs, cosmetics, and food additives
Table 1
The rapid and efficient synthesis of 4H-chromenes 3(a-l) and 4(a-c) in the presence of ionic liquid without using any catalyst and solvent at room temperature.
Entry R1 R2 Product Time (min) Yield (%)
1 H Me 3a 15 80 2 H Et 3b 20 85 3 5-Br Me 3c 15 80 4 5-Br Et 3d 15 85 5 3-OMe Me 3e 10 85 6 3-OMe Et 3f 10 85 7 4-OMe Me 3g 20 75 8 5-OMe Me 3h 10 85 9 5-OMe Et 3i 10 85 10 5-NO2 Me 3j 25 75 11 5-NO2 Et 3k 25 75 12 5-Cl Me 3l 15 80 13 5-Br Me 4a 20 10 14 H Me 4b 20 10 15 H Et 4c 15 15
due to safety problems [65]. Thus, it is essential to design and discover novel tyrosinase inhibitors without side effects. To this aim, chromenes (3a-3l, 4a-4b) were screened for their in vitro tyrosinase inhibitory activity. The IC50values are summarized in Table 2. As seen, the compounds have moderate potential for the inhibition of tyrosinase compared with kojic acid as the standard
drug (IC50 ¼ 0.91 mM). Dimethyl
6-bromo-4-hydroxy-4H-chro-mene-2,3-dicarboxylate (3a) is the most active compound among the chromenes. Earlier studies show limited data available about the tyrosinase inhibitory effects of 4H-chromenes. Safety assess-ment of these dialkyl 4-hydroxy-4H-chromene-2,3-dicarboxylate derivatives could be useful in designing new tyrosinase inhibitor agents for possible applications in cosmetics and food sciences.
3.2.2.2.
a
-Glucosidase inhibitory activity. As a metabolic disorder,Diabetes mellitus is a major health problem worldwide.
a
-Gluco-sidase is one of the key enzymes which hydrolyses non-reducing 1e4 linked
a
-glucose residues and releases the singlea
-glucose in blood. One of the approaches for the control of blood glucose level in diabetic patients is the inhibition of carbohydrate-hydrolyzing enzymes, which requires the production of safe andefficient drugs for
a
-glucosidase inhibition. Literature reviewdemonstrates that synthetic and natural compounds including
carbohydrates,flavonoids, steroids, and chromenes have
a
-gluco-sidase inhibitory capability. For example, Perumal et al. evaluated
the
a
-glucosidase inhibitory activity of2-amino-phenyldiazenyl-4H-chromene derivatives [66]. The inhibitory potentials of
com-pounds at 5 mM were about 30e80%. In the present study, possible
Scheme 2. The plausible mechanism for the formation of chromene derivatives 3(ael) and 4(aec).
anti-diabetic effects of the tested compounds 3(a-l) and 4(a-b)
were evaluated by determining their
a
-glucosidase inhibitoryac-tivities (IC50values) as shown inTable 2. The IC50values of chro-menes ranged from 2.99 to 4.39 mM. Activities of all compounds (with the exception of 4a which is 2H-chromene as a side product) are higher than acarbose as a reference compound. Diethyl 6-nitro-4-hydroxy-4H-chromene-2,3-dicarboxylate (3k) is the most active compound which is nearly 3 times more active than acarbose. 3.2.3. Antimicrobial assay
The antibacterial and antifungal activities of chromenes 3(a-l) and 4(a-c) were screened against gram-positive and gram-negative bacteria and fungal strains. The obtained results revealed that none of these compounds have antimicrobial effects at the tested con-centration (Fig. S1). Previously, the antibacterial activity of
syn-thesized 2-amino-tetrahydro-4H-chromene-3-carbonitrile
derivatives was evaluated against both positive and gram-negative bacterial strains by Moshafi et al. [67]. Their results showed that the two chromenes have antibacterial effects only against 2 g-positive bacterial strains including Micrococcus luteus and Bacillus subtilis.
3.2.4. Antioxidant assay
The antioxidative property of 4H-chromenes 3(a-l) and 4(a-b)
was investigated in this work. Findings showed that the tested
chromenes have no significant antiradical activity up to the
maximum tested concentration (Table S1). Antioxidant and radical scavengers are useful agents that protect the body against oxidant compounds which are responsible for oxidative stress and many other disorders in the human body. The antioxidant properties of several chromenes were reported by adopting various methods. Recently, 2-Phenyl-4H-chromen-4-one derivatives were synthe-sized and evaluated for free radical scavenging activity by DPPH assay [68]. These compounds showed IC50values ranging between 20.5 and 100 nM which had strong activities in comparison to ascorbic acid as a standard antioxidant (IC50¼ 20 nM). In another study, the radical scavenging capacity (RSCDPPH) of hydrazone de-rivatives bearing coumarin and chromene moiety were tested by DPPH method using vitamin C as a positive control [69]. The ob-tained result exhibited that the hydrazone derivatives containing 2H-chromene moiety have higher radical scavenging capacity than derivatives with coumarin moiety. In general, it could be concluded that this activity is mainly related to hydrazide/hydrazone functionalities.
3.3. Molecular docking
The molecular docking studies were performed to analyze the plausible binding modes of synthesized chromenes 3(a-l) and
4(a-c) against
a
-glucosidase and tyrosinase. The results of dockingstudy were represented inTables 3 and 4. The analysis of the
docking results for 3k as the most active compound against
a
-glucosidase showed that oxygen atom of nitro group on the ben-zene ring of 3k is involved in hydrogen bonds with amine group of Lys155. Also, the hydrogen bond was observed between carboxylic group of Asp408 and hydroxyl functional group of 3k (Figs. 3 and 4). The hydrophobic interactions and
p
-p
stacking were observed with Arg312, Asp349, and Phe157, respectively. All of these interactions stabilize the binding of 3k to the active site of thea
-glucosidase andthe binding energy of5.72 kcal/mol was calculated for
a
-gluco-sidase-3k complex. The molecular interactions between 3c (as the most active compound against tyrosinase) and binding site resi-dues of tyrosinase were represented inFigs. 5 and 6. As seen, the hydrogen bonds were formed between the oxygen atoms of hy-droxyl and ester functional groups of 3c and side chain amine groups of Asn260 and His244. Furthermore, the hydrophobic interaction was observed between the aromatic moiety of 3c and Val283. The estimated binding energy for these interactions
was6.17 kcal/mol.
Fig. 2. Cell viability percent of the synthesized chromenes 3(a-l) and 4(a-c) against MCF-7 and HT-29 cell lines at the concentration of 150mg/mL.
Table 2
Tyrosinase anda-glucosidase inhibitory activity of chromenes 3(a-l) and 4(a-b) (mM± SEM).
Compound Tyrosinase a-Glucosidase 3a 10.82± 0.04 4.39± 0.04 3b 9.58± 0.10 4.17± 0.17 3c 3.50± 0.03 3.15± 0.03 3d 5.09± 0.03 3.18± 0.03 3e -a 3.74± 0.03 3f 9.47± 0.31 3.41± 0.06 3g 9.45± 0.44 3.98± 0.01 3h 12.20± 1.52 4.38± 0.03 3i 8.98± 0.37 3.40± 0.03 3j 7.79± 1.26 3.75± 0.06 3k -a 2.99± 0.03 3l 4.43± 0.10 4.09± 0.07 4a 8.01± 1.70 na 4b -a 4.36± 0.08 Kojic acid 0.91± 0.07 nt Acarbose nt 7.90± 0.03 na: not active, nt: not tested.
Table 3
The molecular interactions between the synthesized chromenes 3(a-l) and 4(a-c) anda-glucosidase resulted from the docking analysis.
Entry Compound H-bond p-pinteraction Hydrophobic interaction
1 3a Gln350 e e
2 3b Arg439, Arg212, Glu276, Asp214 Phe177 Phe300, Thr215, Phe157, Tyr71, Phe177
3 3c Arg312, Gln350 Phe157 Phe157
4 3d Arg312, Gln350, Asp408 Phe157 Phe157, Arg312
5 3e Arg312 Phe157 Phe157
6 3f e Phe157 Thr215, Phe157, Phe300
7 3g Gln350, Asn347 e e
8 3h Arg312, Gln350 Phe157 Phe157
9 3i Asp408, Tyr313,Glu304,Arg312 Phe157 Phe157, Phe300 10 3j Gln350, Asp408, Lys155, Arg312 Phe157 Phe157, Arg312
11 3k Asp408, Lys155 Phe157 Arg132, Asp349
12 3l Arg312, Gln350 Phe157 Phe157
13 4a Arg312 Phe157 e
14 4b Asp349 Phe177 Thr215, Phe177
15 4c Arg312, Tyr313, Gln350 Phe157 e
Table 4
The molecular interactions between the synthesized chromenes 3(a-l) and 4(a-c) and tyrosinase resulted from the docking analysis.
Entry Compound H-bond Hydrophobic interaction Metal interaction p-Cation interaction
1 3a Asn260 His61, Phe292 e e
2 3b Asn260, His244 His61, His263 e e
3 3c Asn260, His244 Val283 e e
4 3d Asn260, His244 Val283 e e
5 3e Asn260, His244 Val283, His263 e e
6 3f Asn260, His244 Val283, His263, His85, Ser282 e e
7 3g His244 Val283, His85 Cu e
8 3h Asn260 His61, His263 Cu e
9 3i Asn260 His61, His263 Cu e
10 3j e e Cu e
11 3k Asn260, His244 Val283, His85 Cu e
12 3l Asn260, His244 Val283 e e
13 4a Asn260, His244 Val283, His263 e Cu
14 4b Asn260 Val283, His244 e e
15 4c Asn260 His263 Cu e
4. Conclusion
In the present study, a novel environmentally benign method-ology was developed for the synthesis of previously known
4-hydroxy-4H-chromene-2,3-dicarboxylate derivatives 3(a-l) and 4(a-c) based on the green chemistry principles. To this end, the compounds were synthesized in high yield, with short reaction times, and straightforward work-up, using ionic liquid ([HMIM]Br)
Fig. 4. Two-dimensional interaction diagram of the most active chromene (3k). The black dashed lines indicate H-bonds formed between Asp408 and Lys155 ofa-glucosidase and hydroxyl and nitro functional groups of 3k, respectively. The green dashed and solid lines represent thep-pstacking and hydrophobic interactions between the side chain residues of tyrosinase and 3k, respectively.
as a green media, in organic solvent-free condition, without using any catalyst, by one-pot and one-step reaction. Also, the obtained
products were purified only by recrystallization method without
employing any chromatographic techniques. In the next step, for
the first time, some biological properties such as cytotoxicity,
enzyme inhibitory potential, antioxidant, and antimicrobial effects
of these 4H-chromenes were investigated. Findings confirmed the
potential of this class of 4H-chromenes to be regarded as new
in-hibitors of
a
-glucosidase with possible anti-diabetic activity.Chromene derivatives were docked into the active sites of
a
-glucosidase and tyrosinase. The results revealed the existence of H-bond, hydrophobic,
p
-p
stacking,p
-cation, and metal interactions between the enzymes and the studied chromenes. More structuralmodifications on the studied chromenes are needed to further
improve their biological activities at the desired targets. The results obtained in this work can pave the way for future drug design studies, green synthesis, and pharmacological investigations on chromene derivatives.
Author contribution sections
Leila Dinparast: Designing of work, Synthesis, Characterization, Biological assays, Molecular docking studies. Salar Hemmati: Synthesis. Ali Akbar Alizadeh: MTT assay. Gokhan Zengin: Enzyme inhibitory assays. Mir Babak Bahadori: Antioxidant assay.
Hossein Samadi Kafil: Antimicrobial assay. Siavoush Dastmalchi:
Designing of work, Data analysis, Editing of paper. Declaration of competing interest
The authors declare no conflict of interest. Acknowledgment
The authors would like to acknowledge the Ministry of Health and Medical Education, and also, Biotechnology Research Center at Tabriz University of Medical Sciences for thefinancial support. Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127426.
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