The Crystal Structure of 3-Amino-1-(4-Chlorophenyl)-9-Methoxy-1 H -Benzo[ f ]Chromene-2-Carbonitrile: Antimicrobial Activity and Docking Studies

: Compound 3-amino-1-(4-chlorophenyl)-9-methoxy-1 H -benzo[ f ]chromene-2-carbonitrile ( 4 ), was synthesized via the reaction of 7-methoxynaphthalen-2-ol ( 1 ), 4-chlorobenzaldehyde ( 2 ), and malononitrile ( 3 ) in an ethanolic piperidine solution under microwave irradiation. The synthesized pyran derivative 4 was asserted through spectral data and X-ray diffraction. The molecular structure of compound 4 was established unambiguously through the single crystal X-ray measurements and crystallized in the Triclinic, P-1, a = 8.7171 (4) Å, b = 10.9509 (5) Å, c = 19.5853 (9) Å, α = 78.249 (2) ◦ , β = 89.000 (2) ◦ , γ = 70.054 (2) ◦ , V = 1717.88 (14) Å 3 , Z = 4. The target molecule has been screened for antibacterial and antifungal functionality. Compound 4 exhibited favorable antimicrobial activities that resembled the reference antimicrobial agents with an IZ range of 16–26 mm. In addition, MIC, MBC, and MFC were assessed and screened for molecule 4 , revealing bactericidal and fungicidal effects. Lastly, a molecular docking analysis was addressed and conducted for this desired molecule.


Introduction
The crystalline configurations of drug candidates have amassed substantial appreciation as a critical criterion for rational drug design with the manipulation of their functional moieties impacting the drug's structure-activity relationship. Generally, the attained crystallographic data offer explicit/precise structural identification and absolute configuration [1][2][3][4][5][6], which accordingly elucidates the performance of the novel drugs without triggering the adverse response of the biological system stimuli. Of the drug candidates with an elevated disposition to forge crystallographic structures, chromene compounds are among the most notorious and prosperous [7][8][9]. The comprehensive biomedical features of chromene molecules have motivated scientific figures within the drug discovery biosphere to cultivate new derivatives of this class of materials and explore their novel biological characteristics. Chromenes have been renowned for their incredible biological functions, which assisted their assimilation into various applications such as antimicrobial activities [10][11][12][13][14], hypolipidemic [15], antileishmanial, antiviral, anti-HIV, antianaphylactic activities [16][17][18], insecticidal [19], targeting of c-Src kinase enzyme [20,21], anticancer and cytotoxic activities [22][23][24][25], cell cycle analysis, apoptotic effects, caspase 3/7, and inhibition of the topoisomerase enzyme [26][27][28][29][30][31][32][33]. Among the synthetic strategies to acquire chromene molecules, microwave irradiation is one of the most efficacious and eco-friendly procedures, which facilitates the isolation of the desired compounds in a short period of time and results in good yields [34][35][36][37]. Furthermore, the dihydrofolate reductase (DHFR) enzyme used as a therapeutic target in the treatment of infections through NADPH is used in the reduction of DHFR and is involved in the synthesis of cell proliferation raw material [38]. DHFR inhibitors are widely used in the treatment of fungal, bacterial, and mycobacterial infections through block DNA replication as well as in fighting cancer [39]. In addition, the chromene derivatives are used as potential agents against DHFR [40].
In continuation of our efforts to discover oxygen-heterocyclic derivatives with promising antimicrobial and antitumor activities [41-57], we present the synthesis of 3-amino-1-(4-chlorophenyl)-9-methoxy-1H-benzo[f ]chromene-2-carbonitrile and portray its crystallographic structure. Moreover, the antimicrobial behavior of the target molecule is evaluated and its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentrations (MFC) are appraised. Additionally, a molecular docking assessment of the novel compound is addressed and granted.

Optical Activity
Compound 4 has a chiral feature; consequently, this specific rotation was gauged, utilizing a Carl Zeiss polarimeter to attribute the stereochemistry of the 1-position to the 1H-benzo[f ]-chromene moiety. Results revealed that compound 4 has zero rotation (meaning the molecule is optically inactive) and is obtained in the form of a racemic (±) mixture [26-28], as illustrated in Scheme 1.

Spectroscopic Data
The structure and purity of compound 4 were substantiated through spectral analyses, including: IR, 1 H NMR, 13 C NMR, MS, and X-ray single crystal (see Supplementary Materials, Figures S1-S3).

Crystal Data
In the title compound 4, C 21 H 15 ClN 2 O 2 , the crystallographic data and purification information are outlined in Table 1. The asymmetric unit of molecule 4 incorporates two independent compounds, which is witnessed in Figure 1. All the bond lengths and angles are in normal ranges [58]. As displayed in the crystal packing (Figure 2), the molecular components of compound 4 were linked through two intermolecular hydrogen bonds and two intramolecular hydrogen bonds, as shown in Table 2.   2.5. Biological Activity 2.5.1. Antimicrobial Activity Assay Molecule 4 was estimated through a preliminary screening of its antibacterial activity via the agar diffusion methodology, employing a Mueller-Hinton agar medium for bacteria and a Sabouraud's agar medium for fungi [59]. The analyzed collections encompassed three Gram-positive species of pathogenic bacteria: Staphylococcus aureus (RCMB 000106), Bacillus subtilis (RCMB 000108), and Staphylococcus epidermtitis (RCMB 000107); three Gram-negative bacteria: Enterococcus cloaca (RCMB 000101), Escherichia coli (RCMB 000103), and Salmonella typhimurium (RCMB 000103), utilizing reference antibiotic drugs Ampicillin and Gentamycin (5 µg/mL). Compound 4 was also scrutinized against three fungi: Aspergillus fumigatus (RCMB 002003), Aspergillus flavus (002002), and Candida albicans (RCMB 005003), utilizing the reference antibiotic Ketoconazole (5 µg/mL). The minim zone of inhibition (IZ) in mm ± standard deviation beyond the well diameter (6 mm) was established, employing a 5 µg/mL concentration of compound 4. Dimethyl sulfoxide (DMSO) was utilized as a blank and exhibited no antimicrobial activity. The inhibitory impacts of the synthetic compound in evaluation against these organisms are illustrated in Table 3. Compound 4 showed lower IZ than reference drugs against most of the tested microorganisms (S. aureus, S. epid, E. cloaca, S. typhi and A. fumigates). Furthermore, its compound displayed the same IZ with reference inhibitors against B. subtili and A. flavus.

MIC, MBC/MFC Studies
MIC denotes the minimum inhibitory concentration (the lowest concentration required to inhibit bacterial growth), MBC to the mean bactericidal concentration (the lowest concentration of the synthesized drugs required to kill specific bacteria), and MFC to the minimum fungicidal concentration (the lowest concentration of the synthesized drugs required to kill specific fungi). MIC, MBC, and MFC were assayed for the active compound 4 in µg/mL. The examined antimicrobial data (MICs/MBCs and MICs/MFCs) of the desired molecule 4 and their standardized drugs are supplied in Table 4.

Structure-Activity Relationship (SAR) Study
The antimicrobial activity of molecule 4 is depicted in Table 3. The SAR study revealed that compound 4 with inhibitory effects ranging from 22-26 and 16-31 mm illustrated stronger vitality against the Gram-positive tested bacteria (S. aureus, B. subtilis, S. epidermtitis), Gramnegative tested bacteria (E. cloaca, E. coli, S. typhimurium), and inhibitory effects ranging from 15-21 mm against the tested fungi (A. fumigates, A. flavus, C. Albicans) in evaluation of the standard antibiotics Ampicillin (IZ = 24-28 mm), Gentamycin (IZ = 17-30 mm), and Ketoconazole (IZ = 16-20 mm), respectively. The presence of a hydrophobic electronwithdrawing substituent (the chlorine atom at the para-position on the phenyl group at the 1-position) alongside an electron-donating substituent (the methoxy group at the 9-position of the 1H-benzo[f ]chromene moiety) has enhanced the antimicrobial behavior significantly.

Molecular Docking Analysis
According to the inhibitory functionalities of molecule 4, the molecular docking was performed against dihydrofolate reductase "DHFR", and its positioning of the active compound within the substrate binding pocket assists in the comprehension of its mode of interaction. We selected two crystal structures for the hDHFR protein ((PDB): 4DFR [61]) and (PDB ID: 3NTZ [62]). Compound 4, the reference inhibitor Methotrexate, and reference drugs (Ampciline, Gentamicin, Ketocwere) were stationed in the binding pocket of enzyme s, and its binding interactions were illustrated in Figures 3 and 4.
The mGenTHERADER generated the 3D loop structure of DHFR and the applied docking framework. The biological behavior was represented as binding-interaction BI term for 3-amino-1-(4-chlorophenyl)-9-methoxy-1H-benzo[f ]chromene-2-carbonitrile over DHFR, then compared with Methotrexate and reference drugs. Compound 4 re-docked and showed promising (root mean square deviation, RMSD = 0.93, 0.52 Å) compared to other compounds against both enzymes. The reference inhibitor reported interaction with vital binding site of 4DFR (ASP27, ILE5, ILE94, ARG52, ARG57). Compound 4 showed BI = −7.69 Kcal/mol. compared to Methotrexate BI = −8.86 Kcal/mol (Table 5). BI was arranged as 4 > Gentamicin > Methotrexate > Ketoconazole > Ampicillin. Compound 4 formed a strong H-bond between methoxy and vital ASP27 with a distance 1.2 • and E = −1.69(kcal/mol), compared to Methotrexate, which showed an H-bond with ASP27 and formed a distance of 2.88 • and ∆E = −5.4 (kcal/mol. The interaction mode for compound 4 and reference drugs in the binding pocket had the same manner as the reference inhibitor and might be responsible for the high inhibitory activity for compound 4. In case of 3NTZ; Compound 4 showed the highest BI = −9.43 Kcal/mol. compared to other inhibitors (Table 5). BI was arranged for other inhibitors as Gentamicin > Ketoconazole > Ampciline > Methotrexate. The active site of 3NTZ comprises the following amino acid residues: Val 6, Ala 9, Leu 22, Pro 25, Asp 27, Leu 28, Glu30, Gln35, Phe 31, Ser 49, Ile 50, Thr56, Leu62, and Thr 111. Compound 4 formed a strong H-bond between the amino group and Ala9 and formed a π-π bond between Leu22. Methotrexate showed an H-bond with Glu30 and Arg70 ( Figure 4). Therefore, the interaction with vital amino acids of hDHFR plays an important role in the inhibitory effects of this compound. Furthermore, the activities of compound 4 were due to the presence of the amino and cyano groups.

Materials and Equipment's
All chemicals purchased and instruments used are mentioned in the Supplementary Material.

Biological Screening
Compound 4 was screened for its in vitro antimicrobial activities against Grampositive species of pathogenic bacteria: Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermtitis; three Gram-negative bacteria Enterococcus cloaca, Escherichia coli, and Salmonella typhimurium using the standard antibiotics Ampicillin and Gentamycin as reference drugs. The investigation also included three fungi: Aspergillus fumigatus, Aspergillus flavus, and Candida albicans using the standard antibiotic, Ketoconazole, as a reference drug [59]. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentrations (MFC) were determined as previously reported [60]. The antimicrobial activities were performed at the Regional Centre for Mycology & Biotechnology (RCMP), Al-Azhar University.

X-ray Crystallography Analysis
Compound 4 was obtained as single crystals by slow evaporation from an ethanol solution of the pure compound at room temperature with CCDC 2054799. Data were collected on a Bruker APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα and Cu Kα radiations at 293 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT. SHELXTL-2018/3 [65,66] was used to solve the structure.

Docking Assay
Small ligands (4 and methotrexate) were prepared using the DFT theory with the Becke3-Lee-Yang-parr (B3LYP) level using 6-311G ** basis as implemented in Gaussian 09W [67]. The optimization geometry for molecular structures was carried out and used in the docking experiment.
The 3D crystal-structure for the GHFR model was prepared using the glide-tool as described [68,69].

Conclusions
In an effort to develop potent antimicrobial agents, compound 4 was synthesized and characterized employing an X-ray diffraction technique. Subsequently, the antimicrobial behavior of molecule 4 was appraised against different pathogenic bacterial and fungal strains, which demonstrated promising antimicrobial activities in correspondence with the reference antimicrobial agents exhibiting an IZ range of 16-31 mm. Furthermore, the values of MIC, MBC, and MFC were ascertained for compound 4, disclosing its bactericidal and fungicidal activites. The molecular docking analysis was performed to relate our biological findings with the chemical structure and to show their ability to bind with the DHFR active site similar to Methotrexate.