Synthesis, characterization and biological applications of novel Schiffbases of 2-(trifluoromethoxy)aniline
Thierry Youmbi Fonkui1, Monisola Itohan Ikhile2*, Freddy Munyololo Muganza3, Marthe Carine Djuidje Fotsing2, Charmaine Arderne4, Xavier Siwe-Noundou5, Rui Werner Macedo Krause5, Derek Tantoh Ndinteh2*, Patrick Berka Njobeh1
1. Department of Biotechnology and Food Technology, P.O. Box 17011, Doornfontein Campus 2028, University of Johannesburg, South Africa
2. Department of Applied Chemistry, P.O. Box 17011, Doornfontein Campus 2028, University of Johannesburg, South Africa
3. Department of Chemistry, Sefako Makgatho Health Sciences University, Molotlegi Street, Ga-Rankuwa 0204, Pretoria, South Africa
4. Department of Chemistry, P. O. Box 524, Auckland Park, Johannesburg, 2006, University of Johannesburg, South Africa
5. Department of Chemistry, P.O. Box 94, Grahamstown 6140, Rhodes University, South Africa
Abstract: A series of five new Schiff bases (1–5) were synthesized by reacting 2-(trifluoromethoxy) aniline with different aromatic aldehydes. The Schiff base compounds were characterized by spectroscopic and analytical methods. Crystal structure of one new compound was also reported. The pharmacological properties, including antibacterial (14 bacterial species), antifungal (7 strains),antimalarial, anti-trypanosomal and anti-HIV activities of the compounds, were investigated. Cytotoxicity of the tested compounds was evaluated against human cervix adernocarcinoma cells (HeLa). Bacterial minimum inhibitory concentration (MIC) results by broth microdilution method showed that Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Proteus vulgaris, Klebsiella oxytoc and Klebsiella pneumonia were more sensitive in the presence of tested compounds with an MIC value of 15.6 µg/mL. All the tested compounds showed good to moderate activity against fungi. The sensitivity of Aspergillus fumigatus was higher than other strains with aminimum cell death concentration (MFC) of 15.6 µg/mL. Compound 1 showed greater antimalarial and anti-trypanosomal properties with very low to no cytotoxic effects against HeLa cells as compared with compound 5, while other compounds exhibited poor activity. Compounds 1–5 demonstrated good activity against HIV type-1. These Schiff base compounds could be used as good antimicrobial agents.
Keywords: Synthesis; Biological activity; Schiff bases; Antimicrobial; 2-(Trifluoromethoxy) aniline
CLC number: R916 Document code: A Article ID: 1003–1057(2018)5–307–17
Azomethine or imine compounds with the structural skeleton (-C=N-) are an important group of organic compounds often found in some naturally occurring molecules, and they can also be chemically synthesized. When obtained by the condensation of a carbonyl (ketone or aldehyde) and a primary amine as initially reported by Hugo Schiff in 1864, these carbonyl analog-based compounds were named “Schiff bases” after Hugo Schiff. Schiff bases represent a subgroup of imine compounds that have gained the attention of many scientists around the world due to their multifaceted applications. Schiff bases not only act in chemistry and physics as catalysts, anticorrosive agents but also continue to reduce the limitations of pharmacologists, microbiologists, and promote the medical sector at large. The pharmacological applications of Schiff bases include antibacterial, antifungal, antiviral, antimalarial, anticancer, anti-tuberculosis and anti-HIV activities, which are clearly documented[4–7]. Hugo’s discovery and findings have laid down the foundation and paved the way for the development of novel compounds, drug development, and antibiotics production.are already a major concern worldwide. In addition to these, increased microbial spoilage (25% global harvest dumped per year)due to drug-resistant microorganisms worsens the situation. The literature records severe economic losses associated with food contaminants and different cases of human and animal life loss linked to ingestion of foodborne pathogens. Drug tolerance enhanced by microbial adaptations and gene mutations promote the screening and continuous search for drugs against pathogen attacks. Physical, chemical and biological approaches have been adopted to address the issue, but there is still need for more. In the development of novel compounds with pharmaceutical properties, Schiff bases derived from aromatic carbonyls and amines promise a better tomorrow.
The current health issues and the need to feed the booming demographic population
Aniline, a prototypical aromatic amine in material sciences, is gaining interest in biological science[10,11]. Also known as phenylamine or amino benzene, aniline is a precursor of various industrial applications. It has been used in the fabrication of rubbers, organic resins, paints, leathers, plastics and petroleum[12,13]. Schiff base of 3,5-bis(trifluoromethyl)aniline and benzaldehyde has been recorded with good antibacterial and antifungal activities, showing minimum inhibitory concentration (MIC) values ranging from 32 to 128 µg/mL. Moreover, the antifungal, antibacterial, and antitumor activities of (E)-3-chloro-N-((5-nitrothiophen-2-yl)methylene) aniline Schiff bases have been reported.
The versatile properties of Schiff bases are ever increasing as they are beneficially used in metals chelation as inhibitors. Derivatives of aromatic Schiff bases with electron-rich nuclei like oxygen, nitrogen and sulfur are very important in coordination chemistry of transition elements. The presence of these electron donor atoms favors the coordination of Schiff base ligands with different transition metal ions and produces stable metal complexes with greater pharmacological potency. Ruthenium (III) complexes of 2-hydroxy-L-naphthaldehyde with aniline and its p-substituted derivatives with improved antibacterial activity compared with the ligands have been documented. Similar observations have been recorded with various transition metals. In this study, we described the synthesis, structural characterization and the biological applications of novel Schiff bases of 2-(trifluoromethoxy)aniline with various aromatic aldehydes. The pharmacological applications included antibacterial, antifungal, antimalarial, anti-trypanosomal and anti-HIV activities. To the best of our knowledge, we, for the first time, reported the synthesis and some pharmacological applications of Schiff bases with 2-(trifluoromethoxy)aniline scaffold.
2. Materials and methods
Reagents and chemicals recorded herein were used as received from commercial sources. Ethanol of HPLCgrade, methanol, 2,4-dihydroxybenzaldehyde, 2-hydroxy-5-bromobenzaldehyde, 2-hydroxy-5-chlorobenzaldehyde,methanol, acetone, 2-(trifluoromethoxy)aniline and 2-methoxy-4-hydroxybenzaldehyde were purchased from Sigma Aldrich, South Africa. The compounds prepared were characterized using optical and spectral measurements. The different transitions (π-π* & n-π*) were obtained on a Japanese Shimadzu UV-2540, UV-VIS absorption spectrophotometry equipped with an IRS 40 integratingsphere attachment. The melting point of the compounds was recorded on a 20 Watt, Electro-thermal digital melting point apparatus with a maximum temperature of 450 °C. Fourier transform infrared (FTIR) spectra were recorded on a Spectrum 100, PerkinElmer FTIR spectrometer. Samples were analyzed on a full length and expressed in wave number (cm–1). Elemental analysis (C, H, N) was collected on a Flash 2000 organic elemental analyzer. UV spectra were measured on an Agilent Technologies Cary 60 UV-Visible spectrophotometer. 1H and 13C NMRs (nuclear magnetic resonance) of the compounds were obtained in d6-DMSO on a 400 MHz Bruker spectrometer apparatus operating at room temperature. Values were reported with reference to TMS (tetramethylsilane) used as an internal solvent.
2.2. General procedure for synthesis
Schiff bases were prepared by mixing 2-(trifluoromethoxy)aniline and the corresponding carbonyls in an equimolar proportion following the method reported by Yousif et al. as shown in Scheme 1.
Scheme 1. Synthesis of Schiff bases 1–5.
Compound 1 was synthesized by mixing equimolar amounts (100 mg, 10 mL) of 2-(trifluoromethoxy)aniline with 2,4-dihydroxybenzaldehyde dissolved in ethanol. Upon constant refluxing for 4–6 h, the solution changed from a colorless solution into yellow solution. This solution was then allowed to evaporate at room temperature in the fume hood, and the yellow precipitate was thoroughly washed with methanol and kept dry in a desiccator for further analysis. Yellow powder; yield: 72%; M.P. 132–134 °C; UV/Vis (DMSO), λmax = 334 nm; FTIR-(cm–1): 3307 (-OH), 2919 (C-H), 1678 (C=N), 1458 (C=C),1080 (C-N), 884 (C-H). 1H NMR (400 MHz, DMSO-d6) δ: 10.40 (1H, s, Ar-OH), 9.92 (1H, s, Ar-OH), 8.87 (1H, s, C=NH), 7.57–7.52 (2H, m, Ar-H), 7.49–7.46 (2H, m, Ar-H), 7.38–7.35 (2H, m, Ar-H), 8.81–6.79 (1H, d, J = 8 Hz, Ar-H), 6.56–6.53 (1H, t, J = 8 Hz, Ar-H), 6.45–6.32 (2H, m, Ar-H), 5.32 (1H, s, N-H); 13C NMR (100 MHz, DMSO-d6)δ: 165.13 (C=NH), 164.34 (OHC=C), 163.21 (OHC=C), 141.48 (OHC=C-O),134.87 (C=C-N), 132.80 (C=C), 128.77 C=C), 127.81 (C=C), 122.66 (OCF3), 121.52 (C=C), 120.22 (C=C), 116.3 (C=C), 115.66 (C=C). Anal calcd for C14H10F3NO3%: C 56.57; H 3.39, N 4.71, O 16.15, F 19.18; found: C 57.01, H 3.59, N 4.54, O 15.89, F 19.08. In the same manner, the other compounds 2–5 were prepared.
2.2.2. 4-Bromo-2-[(2trifluoromethoxy-phenylimino)-methyl]-phenol (2)
Yellow powder;yield: 70%; M.P. 102–103.5 °C; UV/vis (DMSO), λmax = 353, 269 nm; FTIR-(cm–1): 3677 (-OH), 2995 (C-H), 1620 (C=N), 1475 (C=C), 1154 (C-N), 921 (C-H). 1H NMR (400 MHz, DMSO-d6) δ: 8.54 (1H, s, C=NH), 7.49–7.45 (1H, dd, J = 4 Hz, Ar-H), 7.31–7.35 (1H, m, Ar-H), 7.34–7.33 (1H, m, Ar-H), 7.32–7.30 (1H, dd, J = 4 Hz, Ar-H), 7.26–7.25 (1H, m, Ar-H), 7.25–7.24 (1H, m, Ar-H), 6.93–6.91 (1H, d, J = 8 Hz Ar-H). 13C NMR (100 MHz, DMSO-d6) δ: 163.09 (C=NH), 160.61 (OHC=C), 142.61 (OHC=C), 141.34 (C=C-O), 136.56 (C=C-N), 134.68 (C=C), 128.31 (C=C), 128.24 (C=C), 122.92 (O-CF3), 121.85 (C=C), 120.62 (C=C), 120.15 (C=C), 119.79 (C=C), 119.75 (C=C). Anal calcd for C14H9BrF3NO2%: C 46.69,H 2.52, N 3.89, O 8.89, F 15.83, Br 22.19; found: C 46.78,H 2.51, N 3.89, O 8.79, F 15.63, Br 21.90.
2.2.3. 4-Chloro-2-[(2trifluoromethoxy-phenylimino)-methyl]-phenol (3)
Yellow powder; yield: 63%; M.P.: 73.1–73.5 °C; UV/vis (DMSO), λmax = 345, 275 nm; FTIR-(cm–1): 3287 (-OH), 2965 (C-H), 1547 (C=N) 1463 (C=C), 1090 (C-N), 882 (C-H). 1H NMR (400 MHz, DMSO-d6) δ: 8.55 (1H, s, C=NH), 7.37–7.36 (1H, d, J = 4 Hz, Ar-H), 7.34–7.31 (1H, m, Ar-H), 7.27–7.26 (1H, d, J = 4 Hz, Ar-H), 7.25–7.24 (1H, m, Ar-H), 6.99–6.97 (1H, d, J = 8 Hz, Ar-H), 13C NMR (100 MHz, DMSO-d6) δ: 163.23 (C=NH), 160.16 (OHC=C), 142.60 (C=C-O), 141.41 (C=C-N), 133.80 (C=C), 131.67 (C=C), 128.30 (C=C), 128.24 (C=C), 123.98 (C=C), 122.94 (C=C), 121.85 (C=C), 120.19 (C=C-Cl), 119.98 (C=C), 119.35 (C=C). Anal calcd for C14H9ClF3NO2%: C 53.27, H 2.87, N 4.44, O 10.14, F 18.06, Cl 11.23; found: C 53.26, H 3.10, N 4.31, O 10.46, F 17.9, Cl 10.88.
2.2.4. 5-Diethylamino-2-[(2-trifluoromethoxy-phenylimino)-methyl]-phenol (4)
Dark red powder;yield: 34%; M.P.: 67.8 °C; UV/vis, (DMSO) λmax = 316, 277 nm; FTIR-(cm–1): 3378 (-OH), 2915 (C-H), 1559 (C=N), 1414 (C=C), 1047 (C-N), 879 (C-H).1HNMR (400 MHz, DMSO-d6) δ: 9.59 (1H, s, Ar-H), 8.74 (1H, s, C=NH), 7.57–7.55 (1H, d,J = 8 Hz, Ar-H), 7.45–7.40 (2H, m, Ar-H), 7.35–7.33 (1H, d, J = 8 Hz, Ar-H), 7.30–7.27 (1H, t, J = 8 Hz, Ar-H), 6.34–6.32 (2H, d, J = 8 Hz, Ar-H), 6.09 (1H, s, Ar-H), 6.03 (1H, s, Ar-H), 3.40–3.38 (4H, m, CH2-CH3),1.12–0.85 (6H, dd, J = 4 Hz, CH3-CH2-), 13C NMR (100 MHz, DMSO-d6) δ: 163.38 (C=NH), 163.36 (OHC=C), 162.89 (C=C-O), 153.79 (C=C-N), 151.99 (C=C-N), 141.65 (C=C), 141.39 (C=C), 134.57 (C=C), 133.98 (C=C), 128.68 (C=C), 126.31 (C=C), 122.63 (O-CF3), 119.73 (C=C), 44.09 (N-CH2CH3), 43.95 (N-CH2CH3), 12.48 (CH3CH2N), 12.37 (CH3CH2N).Anal Calcd for C18H19F3N2O2%: C 61.36, H 5.44, N 7.95, O 9.09, F 16.17; found C 61.14, H 5.94, N 7.22, O, 9.25, F 16.43.
2.2.5. 3-Methoxy-4-[(2trifluoromethoxy-phenylimino)-methyl]-phenol (5)
Yellow powder; yield: 51%; M.P.: 105 °C; UV/vis (DMSO), λmax = 334 nm; FTIR-(cm–1): 3317 (-OH), 2975 (C-H), 1584 (C=N), 1376 (C=C), 1092 (C-N), 878 (C-H). 1H NMR (400 MHz, DMSO-d6) δ: 10.10 (1H, s, Ar-OH), 8.64 (1H, s, C=NH), 7.87–7.85 (1H, d, J = 8 Hz, Ar-H), 7.57–7.55 (2H, d, J = 8 Hz, Ar-H), 6.80–6.78 (1H, d, J = 8 Hz, Ar-H), 6.53–6.51 (1H, m, Ar-H), 5.30 (1H, s, N-H), 13CNMR (100 MHz, DMSO-d6) δ: 165.26 (C=NH), 163.82 (C=C-O), 163.07 (OHC=C), 161.40 (C=C-O), 157.34 (C=C-N), 142.6 (C=C), 130.09 (C=C), 128.69 (C=C), 127.88 (C=C), 125.99 (C=C), 122.38 (O-CF3), 121.59 (C=C), 116.45 (C=C), 115,78 (C=C), 108.62 (C=C), 55.68 (O-CH3). Anal calcd for C15H12F3NO3%: C 57.88, H 3.89, N 4.50, O 15.42, F 18.31; found C 58.23, H 4.10, N 4.97, O 15.01,F 18.69.
2.2.6. Single crystal X-ray structure of Schiff base (2)
A yellow plate-like crystal with approximate dimensions 0.30×0.25×0.12 mm3 was selected under oil ambient conditions and attached to the tip of a MiTeGen MicroMount©. The crystal was mounted in a stream of cold nitrogen at 100(2) K and centered in the X-ray beam by using a video camera. The crystal evaluation and data collection were performed on a Bruker APEXII CCD diffractometer with Mo Kα (λ = 0.71073 Å) radiation and the diffractometer to crystal distance of 5.00 cm. The initial cell constants were obtained from three series of w scans at different starting angles. Each series consisted of 12 frames collected at intervals of 0.5° in a 5° range about w with an exposure time of 10 seconds per frame. The reflections were successfully indexed by an automated indexing routine built into the APEX2 program suite. The final cell constants were calculated from a set of 7269 reflections from the actualdata collection. The data were collected using a set of ω andφ scan collection routines determined by COSMO to survey the reciprocal space to a resolution of 0.75 Å. A total of 25,944 reflections were harvested up toθmax = 28.3°, and 3318 reflections were unique. The data were successfully processed with SAINT in the APEX2 program suite. The highly redundant data sets were corrected for Lorentz and polarization effects, and the absorption correction was based on fitting a function to the empirical transmission surface as samples by multiple equivalent measurements (SADABS 2016/2). The systematic absences in the diffraction data were consistent for a monoclinic crystalsystem, and the E-statistics strongly suggested the centrosymmetric space group P21/c. The crystal structure was solved by direct methods with the SHELXTprogram and refined by a full-matrix least-squares procedure based on F2. All non-hydrogen atoms were refined with anisotropic displacement coefficients unless otherwise specified. All hydrogen atoms were included in the structure factor calculation. C-boundH atoms were placed in geometrically idealized positions, and C-H = 0.93 – 0.99 Å and were constrained to ride on their parent atoms with Uiso (H) = 1.2 Ueq (C) for aromatic and methylene H atoms. The H atom on an O atom was also located in the difference Fourier map, and since this was involved in a hydrogen bonding interaction, the position and isotropic displacement parameters of these atoms were allowed to refine freely. Diagrams and publication material were generated using OLEX2.
2.3. Biological activity assays
2.3.1. Antifungal assay
18.104.22.168. Fungi static screening test
The responses or behaviors of Aspergillus carbonarious,Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus, Fusarium proliferatum and Fusarium verticillioides upon exposure to Schiff bases were analyzed. Disc diffusion assay according to Jogee et al. was used to screen the inhibitory property of the ligands. Briefly, 6-mm ? filter paper (Whatman No. 1) was cut using a puncher and autoclaved together with the potatodextrose agar at 121 psi for 30 min. The medium (20 mL) was poured on Petri dishes following microbiologists’ standard and inoculated with 100 µL of Ringer solution containing 1×105 spores obtained from a 7-day fungal growth and allowed to completely dry at ambient temperature. From stock solutions, 20 µL of 1 mg/mL of each compound in DMSO was placed in triplicate on the prepared sterile blank discs. DMSO impregnated discs were used as a negative control, while amphotericin B and nystatin were used as positive controls because of their broad spectrum in the damaging cell membrane. The diameters of inhibitory zones were measured, and their average values were presented and compared with the standards.
22.214.171.124. Fungicidal test
The killing power (minimum fungicidal concentration) of the synthesized ligands was tested against the same strains mentioned above. The inoculum was obtained as described by CSLI standard M38 A2. Suspensionswere diluted in RPMI 1640 to a concentration of 1×105 spores corresponding to 0.5 Mac Farland standards. Microdilution technique was used to seed 100 µL working solutions of 500, 250, 125, 62.5 31.2 and 15.6 µg/mL of drugs in 96-well plates. Subsequently, 100 µL of inoculum was then added to each well in duplicate and incubated at 30 °C for 3 d. Cell viability was verified colorimetrically using resazurin dye, which turns pink enzymatically in the presence of living cells and remains blue in cells that die. After 72 h, the plates were seeded with 10 µL (0.02%) dye and allowed to stand for more than 30 min in the incubator, and minimum fungicidal concentration was recorded.
2.3.2. Antibacterial analysis
All the ligands presented herein were investigated for their antibacterial properties against 12 bacterial strains. The bacteria tested including both Gram+ and Gram– were purchased from Davies Diagnostic, South Africa, and they were maintained in glycerol at –8 ºC. Gram-positive bacteria included Bacillus cereus (BC) (ATCC10876), B subtilis (BS) (ATCC19659),Enterococcus faecalis (EF) (ATCC13047), Mycobacteriumsmegmatis (MS) (MC2155),and S aureus (SA) (ATCC25923). Gram negative bacteria includedEnterobacter cloacae (ECL) (ATCC13047), Escherischiacoli (EC)(ATCC25922), Enterobacter aerogenes, (EA) (ATCC13048) Klebsiella oxytoca (KO) (ATCC8724), K pneumonia (KP) (ATCC13882), Proteus mirabilis (PM) (ATCC7002)and Pseudomonas aeruginosa (PA) (ATCC27853). The microdilution method was the method of choice to determine the MIC. Fresh bacterial growths (18 h) were standardized to the 0.5 Mac Farland standards in Muller-Hilton broth, which was then used as inoculum. In 96-well plates containing 100 µL of 500, 250, 125, 62.5 31.2 and 15.6 µg/mL of ligands, 100 µL of inoculum were seeded in duplicate and allowed to grow overnight with 5% CO2 flow at 37 ºC. Muller-Hilton broth was used as negative control. Streptomycin and nalidixic acid were used as positive controls. Living cells were confirmed in the presence of resazurin dye after 4-h incubation, and the MIC was reported for each ligand.
2.3.3. Antimalarial assay
Parasite lactate dehydrogenase enzyme assay (pLDH) was used to evaluate the antimalarial properties of the prepared ligands. Plasmodium falciparum strain 3D7 parasites were maintained in RPMI 1640 medium containing 2 mM L-glutamine and 25 mM Hepes (Lonza). The medium was supplemented with 5% Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 µg/mL gentamycin and 2%–4% hematocrit human red blood cells. The parasites were then allowed to grow at 37 ºC under an atmosphere of 5% CO2, 5% O2, 90% N2 in sealed T25 or T75 culture flasks for 48 h. A volume of 140 µL containing 20 µg/L of test compounds dissolved in DMSO was mixed with the corresponding volume cultures in 96-well plates and incubated for 48 h. After incubation, 20 µL of this mixture was then transferred to fresh 96-wells containing 125 µL of Malsat and NBT/PES solutions, which were used to measure microscopically (λ620) the amount of purple products formed in the presence of the pLDH. The pLDH activity in each compound treated-well was calculated together with untreated control-well. Chloroquine was used as a standard drug for comparative purpose, and yielded IC50 values ranged from 0.01 to 0.05 µM.
2.3.4. Cytotoxicity assay
To assess the cytotoxicity of the drugs, 20 µg/mL of test compounds were incubated in duplicate in 96-well plates containing HeLa (human cervix adenocarcinoma) cells for 48 h. The numbers of cells surviving drug exposure were determined by using the resazurin-based reagent and reading resorufin fluorescence in a multi-well plate reader. Emetine (which induces cell apoptosis) was used as a positive control. Results were expressed as % cell viability, based on fluorescence reading in treated wells against untreated control well. Compounds that induced less than 50% (<50%) death in HeLa cells were considered non cytotoxic.
2.3.5. Trypanosoma brucei assay
Resazurin-based reagent was used to evaluate the trypanocidal activity of the ligands. Test compounds were dissolved in DMSO to a concentration of 1 µg/mL. From this stock solution, 140 µL containing 20 µg/mL of each compound was seeded together with fresh parasite cultures of Trypanosoma brucei in 96-well plates. Following 48 h-incubation, the killing capacity of the ligands was verified using resazurin, which is reduced enzymatically into a fluorescent resorufin in the presence of living cells. The amount of fluorescence compound present in each well of a multiwall plate reader was quantified in a spectrofluorometer with an excitation an emission wavelength of 560 and 590 nm, respectively. Results were expressed as % parasite viability referring to the resorufin fluorescence in compound-treated wells relative to untreated controls with the aid of a Synergy MX (BioTek®) plate reader. This test was done in duplicate wells, and a standard deviation (SD) was also included. Pentamidine standard drug (an existing drug treatment for trypanosomiasis) at 1 µM was used as a positive control.
2.3.6. Anti-HIV activity
126.96.36.199. HIV-1 IN assay
The ability of the test compounds to inhibit the human immunodeficiency virus type 1 integrase (HIV-1 IN) activity was adapted from previously described methods.Briefly, 20 nM double-stranded biotinylated donor DNA (5′-5BiotinTEG/ACCCTTTTAGTCAGTGTGG-AAAATCTCTAGCA-3′ annealed to 5′-ACTGCTAGA-GATTTTCCACACTGACTAAAAG-3′) was immobilized in wells of streptavidin-coated 96-well microtiter plates (R&D Systems, USA). Following a maximum of 40-min incubation at room temperature and a stringent wash step, 5 µg/mL of purified recombinant HIV-1 subtype C IN in integrase buffer 1 (50 mm NaCl, 25 mM Hepes, 25 mM MnCl2, 5 mM β-mercaptoethanol, 50 µg/mL BSA, pH 7.5) was added to individual wells of a multi-well plate. The prepared ligands and chicoric acid were added to individual wells to a final concentration of 20 µg/mL. Recombinant HIV-1 subtype C IN was assembled onto the pre-processed donor DNA through incubation at room temperature for 45 min. Strand transfer reaction was initiated by the addition of 10 nM (final concentration) double-stranded FITC-labelled target DNA (5′-TGACCAAGGGCTAA-TTCACT/36-FAM/-3′ annealed to 5′-AGTGAATTA-GCCCTTGGTCA-/36-FAM/-3′) in integrase buffer 2 (same as buffer 1, except that 25 mm MnCl2 was replaced with 2.5 mm MgCl2). After an incubation period of 60 min at 37 °C, the plates were washed using PBS containing 0.05% Tween-20 and 0.01% BSA, followed by the addition of peroxidase-conjugated sheep anti-FITC antibody (Thermo Scientific, USA) diluted at 1:1000 in the same PBS buffer. Finally, the plates were washed, and peroxidase substrate (SureBlue Reserve™, KPL, USA) was added to allow for detection at 620 nm using a Synergy MX (BioTek®) plate reader. Absorbance values were converted to % enzyme activity relative to the readings obtained from control wells (enzyme without inhibitor).
188.8.131.52. HIV-1 PRO assay
The HIV protease assay was performed using the fluorogenic substrate Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg (Sigma Aldrich) as reported by Lam et al. A stock solution (500 µM) of the substrate was prepared in DMSO. The reaction buffer (0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA, 1 mM DTT and 0.1% BSA, 5% DMSO, pH 4.7) was used to dilute test compounds to desired concentrations in a separate plate. This was then seeded into a fluorescenceassay plate at 50 µL per well together with 25 µL substrate (8 µM) and 25 µL of enzyme (50 ng/µL) to a final working volume of 200 µL. The mixture was incubated at 37 °C for 40 min, after which fluorescence was read at an excitation wavelength of 340 nm and emission wavelength of 485 nm using a Synergy MX (BioTek®) plate reader. Under the same condition, Ritonavir, an HIV-1 PRO standard enzyme inhibitor, was used as positive control. Fluorescence values were converted to % enzyme activity relative to the readings obtained from control wells (enzyme without inhibitor).
3. Results and discussion
The preparation of Schiff bases (1–5) containing 2-(Trifluoromethoxy)aniline was achieved by refluxing 2-(Trifluoromethoxy)aniline (100 mg: 564 mmol) in ethanol with an equimolar amount of corresponding aldehydes for 4 h as shown in Scheme 1. The products crystallized at –4 °C after 48 h in ethanol. The excess chemicals were washed out with ethanol, and the compounds were rinsed with methanol twice. Methanolwas then dried out in a laminar airflow, and the crystals were kept from moisture under vacuum. The melting points of this series of Schiff bases ranged from 67 to 134 °C. FTIR spectroscopy, ultraviolet visible light (UV-vis), elemental analysis, and 1H and 13C NMRs were used to confirm the synthesis of the prepared Schiff bases. The melting point, molar mass, yield and the lipophilicity value (cLogP) of each compound are presented in Table 1.
Table 1. Chemical property of the synthesized Schiff bases.
Percentage yield, molecular weight (MW), melting point (mpt) and lipophilicity constant (cLogP) of the synthesized Schiff bases 1–5.The cLogP values were obtained experimentally on ChemDraw Ultra 7.0.
3.1.1. FTIR data
The absence of the double stretching absorption band characteristic of the primary amine (-NH2) stretch around 3500 wave numbers (cm–1) and the disappearance of the (-C=O) stretch around 1700–1800 (cm–1) in the spectra indicated the formation of the Schiff bases. The imine bond (-C=N) absorption band was identified between 1547–1678 wave numbers (cm–1), which was in agreement with similar reported compounds. The stretching vibrations around 2915–2995 (cm-1) were attributed to (C-H) of the phenyl ring. These observationswere correlated with those of Thangavel et al. The broad and not intense absorption band characterizes free (O-H) on the aromatic rings ranged from 3287–3677 cm–1 in the ligands as expected.
3.1.2. UV-VIS spectra
The UV-VIS spectra of the Schiff bases were obtained in DMSO, and the chromatograms are presented in Figure 1. The absorption spectra were below 400 nm for all Schiff bases (Table 2). Compounds 1 and 4 were absorbed maximally at 334 and 339 nm, respectively. Compounds 2, 3 and 5 had three absorption bands. Compounds 1 and 4 bands at 334 and 339 nm were attributed to the π-π* transition characteristic of the azomethine bond (C=N), while 2, 3 and 5 showed their n-π* transitions at 269, 273, 271 nm, respectively and were attributed to the non-bonding electron of the imine (-C=N) linkage and their π-π* transition appeared at 226, 223, 227 nm, respectively. The third bands (345, 353, 307 nm) observed in 2, 3 and 5could be assigned to C=N group charge transfer in the ligands or they may emerge as a result of the π-π* and n-π*transition of the phenolic chromophore (Ar-OH).
Figure 1. UV-VIS absorbance spectra of Schiff bases 1–5 in DMSO.
Table 2. UV-VIS spectroscopy of the synthesized Schiff bases.
3.1.3. Elemental analysis
The compounds prepared had the different physical appearance as a result of their different physical and chemical properties. The elemental chemical composition of the compounds was experimentally studied and compared with theoretical values. Results showed that the compounds were successfully prepared. This was sustained with the difference of ±0.5 in values observed for all the compounds. The calculated values agreed with experimental data and validated the method used for the synthesis.
3.1.4. NMR analysis
1H and 13C NMRs were used to further assess and confirm the structure of the ligands synthesized. In 13C NMR, azomethine resonances were seen between 163 and 164.34 ppm for all ligands analyzed, whereas imine proton apparition ranged from 8 to 9 ppm. The absence of any signal characteristic of the aniline amines (-NH2) on the spectra confirmed the successful formation of the (C=N-H) bond. The resonances observed above 9 ppm were assigned to the H-bonded intra-molecular -OH of the aromatic. Characteristic proton peaks of the aromatic rings ranged from 6.02 to 7.59 ppm. Proton (-N-H) peak was observed around 3.33–3.82 ppm for 1, 4 and 5, and it was completely absent in compounds 2 and 3.
3.1.5. The molecular structure of Schiff base 2
Schiff base 2 was structurally analyzed by X-ray diffraction studies. A summary of the crystal data and the refinement parameters of compound 2 are summarized in Table 3,and selected bond length and bond angles for compound 2 are summarized in Table 4.
Table 3. Crystal and refinement data for compound 2.
Table 4. Selected bond lengths (Å) and selected bond angles (º) for compound 2.
A complete table of geometrical parameters of the compound can be found in the supplementary information. The molecular structure diagram is shown in Figure 2. Compound 2 crystallized in a monoclinic crystal system in the space group P21/c with one molecule in the asymmetric unit. There was one intramolecularhydrogen bonding interaction evident in this compound, and the details of this interaction are recorded in the supplementary information. The bond lengths and bond angles in the compound all fall within standard ranges, where a full geometry check with Mogul Geometry analysis was performed within the CSD software. The Mogul analysis showed no other unusual bond lengths, bond angles, torsion angles or ring defects.
Figure 2. Molecular structure diagram of compound 2, atomic displacement ellipsoids are drawn at the 50% probability level.
3.2. Biological activity
3.2.1. Antifungal activity
To check the potency of the synthesized Schiff bases to inhibit fungal growth, 20 µg of the compound in DMSO placed on a sterile blank disc in triplicate was used against 1×105 fungal spores applied on potato dextrose agar medium. Table 5 shows the results of the disc diffusion assay used for screening purposes.The growth of the studied fungi was not affected by exposure to the tested compounds 1–5. No growth inhibition was observed in the vicinity of discs impregnated test compounds as it was the case with nystatin (NYT) positive control.
Table 5. Antifungal activity by disc diffusion assay of compounds 1–5.
~: No activity observed. AMB: amphotericin B, NYT: nalidixic acid.
The activity of amphotericin B was observed to be strain dependent because no clear zone of inhibition was observed against Aspergillus carbonarius and Aspergillus parasiticus. Of course negative control DMSO showed no antifungal property as expected. The absence of any inhibitory growth property could be attributed to low or weak drug’s potency linked to the amount of test materials loaded on the disc. The activity could be concentration dependent. It is also good to point out that the discs could mitigate the potency of the test compounds by adsorption due to a very low detection limit with regard to disc diffusion assay. We, therefore, suggested that an increase in drug’s concentration should result in a different observation with respect to the fungicidal test recorded.
The ability of the compounds to kill fungi was further investigated using microdilution technique, and the minimum fungicidal concentration (MFC) was recorded (Table 6). From the data obtained, it was clear that the use of microdilution technique revealed more on the pharmacological property of the compounds. In this test, the microorganisms were all sensitive to the test compounds in a concentration dependent way. Moderate to high toxicities were recorded for the compounds as compared with the standards. Aspergillus carbonarius was more affected in the presence of 2, 3 and 4 with MFC values of 62.5, 31.2, 31.2 µg/mL respectively lower than 125 µg/mL for amphotericin B. Aspergillus fumugatus was observed to be more sensitive in the presence of test compounds with an MIC value of 15.6 µg/mL. The killing ability of these ligands could be associated to their chemical properties and their structural chemical similarity. Compound 6 was the most cytotoxic compound with an MFC 15.6 µg/mL against more strains (Aspergillus flavus, Asperigillus fumigatus and Fusariumverticillioides) compared with other compounds because of the ortho and para hydroxyl substitution.
Table 6. MFC for the prepared Schiff bases.
AMB: amphotericin B, NYT: nystatin.
3.2.2. Antibacterial screening
Without preliminary screening of the antibacterial property of ligands by disc diffusion assay, broth dilution method helped determine the MIC of theligands. All tested materials showed good to high activity against Gram+ and Gram- bacteria (Table 7). Two bacterial representatives, Bacillus subtilis and Mycobaterium smegmatis, were very sensitive compared with all Gram+ tested in the presence of all the ligands. On the other hand, Klebsiella oxytoca, Proteus vulgarisand Staphylococcusaureous were more affectedstrains among all the Gram– strains. The vulnerability of Gram– negative bacteria was attributed to their thin membrane cell wall made of <10% peptidoglycan. A compromise of peptidoglycan layer affects its role as membrane protector that causes cell death due to osmosis.
Table 7. MIC of Schiff bases 1–5.
STM: streptomycin, NLD: nalidixic acid.
The ligands were very toxic to Enterococcus faecalis with an MIC value of 15.6 µg/mL, which was 8 and 32 times lower than that of streptomycin and nalidixic acid, respectively. Similar observations were noted with Proteus vulgaris (32 times), Staphylococcus aureous (16 times)and Enterobacter aerogenes (16 times) with reference to nalidixic acid. These observations suggestedthat the compounds prepared were preferable compared with nalidixic acid standard antibacterial drug for some of these microbial pathogens. The sensitivity of these strains to tested compounds should result from the ease interaction of the lipid content of the bacterial cell membrane and the substituted Schiff bases. Oneimportant parameter that controls cell membrane interaction with foreign molecules is their polarity. The hydrophobicity of the cell membrane will permeate hydrophobic compounds. Lipophilicity (cLogP) is an important factor for controlling antimicrobial activity. High lipophilicity constant generally leads to compounds with rapid turnover,resulting in good antimicrobial property. However, that was the observation made in this study. Compound 1 had the lowest cLopP value of 3.81 but stand out to be the most potent bactericidal agent possibly due to its chemical structure. Structure activity relationship showed that the hydroxyl substitution had greater activity than bromo, chloro and methoxy substitutions. All Schiff bases prepared here had at least one hydroxyl (-OH) functional group either in orthoor para position, but we could see that the dihydroxyl in compound 6 made the compound the most potent antimicrobial activity within this series. The bromo, chloro and diethylamino substituted Schiff bases (2, 3 and 4) had interchangeable activity against the tested bacteria.
3.2.3. Anti-malarial, anti-trypanosomal and cytotoxicity testing
Malaria is a deadly disease caused by protozoan parasites that invade human red blood cells after a mosquito’s bite. The anti-malarial and anti-trypanosoma properties of azomethine-containing compounds were also studied against Plasmodium falciparum andTrypanosoma brucei parasites. These investigations were coupled with the cytotoxicity effect of the test compounds on HeLa cells (human cervix adenocarcinoma cells). All ligands were incubated with HeLa cells in duplicate for 48 h, and the number of cells surviving compound exposure was expressed as percentage cell viability with reference to the blank. Data obtained (Fig. 3) in general showed that the parasites Plasmodium falciparum and Trypanosoma brucei were affected in the presence of the test compounds. Though the effect of the synthesized Schiff bases was not too pronounced, parasites were more sensitive in the presence of compound 1,which showed approximately morethan 20% reduction in cell growth compared withother test materials. All the tested compounds were not harmful against Hela cells. All ligands exhibited less than 50% cytotoxicity against Hela cells. The parasite growth was not altered when inoculated with compound 2, while HeLa cells suffered more from its presence, which resulted in more than 25% cell death when compared with other compound. Structure relationship activity (SAR) is pointed out here. The hydroxyl (-OH) at position 1 of the benzene ring was seen to be affecting parasite growth compared with 3 and 5. The presence of halogens (bromine and chlorine) at position 4 in the series of ligandsshowed a moderate effect on HeLa cells and the parasites. Substitution of (-OH) functional group at position 1 (compound 2) to (-OCH3) moiety (compound 5) slightly interrupted the metabolic pathway of HeLa cells. This suggested that in the development of new drugs against pathogenic microorganisms, substitution at position 1 was considerably advised.
Figure 3. Antimalarial, antitrypanosomal activities and cytotoxicity effect of compounds 1, 2, 3 and 5 on human cervix adenocarcinoma cells. The effect of the ligands on the microorganism was measured at a concentration of 20 µg/mL, and data were expressed as percentage of viability.
The calculated lipophilicity (cLogP) values for the ligands (Table 1) ranged from 3.81 to 5.59. cLogP values of compounds 2, 3 and 4 were greater than 5, and those of compounds 1 and 5 were below the value 5. The growth inhibitory property of 2, 3 and 4 on Plasmodium falciparum and Trypanosoma bruceiparasites was lower compared with compounds 1 and 5 with cLogP values lower than 5. The significant decrease in activity of the parasite lactate dehydrogenase (pLDH) enzymes should be linked to the decrease in lipophilicity of test compounds 1 and 5.With these remarks in mind, it was suggested that the antimalarial and antitrypanosomal activities of azomethine-based compounds should be linked also to their lipophilicity.
3.2.4. Anti-HIV assay
HIV, a retrovirus responsible for acquired immunodeficiency diseases (AIDS), exist in different types. Type-1 HIV contains an integrase (IN) a reverse transcriptase (RT) and a protease (PR) enzyme all in control of the life cycle of the virus. We assessed the ability of the prepared ligands to mitigate the activity of the protease and the integrase enzymes under standards laboratory conditions. Results showed the tested compounds had greater inhibitory properties against HIV-1 integrase (Fig. 4) compared with HIV-1 protease enzyme that was more resistant to all test compounds (results not shown).
Figure 4. Percentage inhibitory activities of Schiff bases (1, 2, 3 and 5) against HIV-1 integrase enzyme at a concentration of 8 µg/mL, and chicoric acid was used as a positive control (PC).
Up to 50%, the inhibitory activity of integrase enzyme was recorded in the presence of compound 5, while 39% and 40% inhibition were noted for compounds 1 and 3, respectively, all at a concentration of 8 µg/mL. Zero to no inhibitory activity against integrase was recorded in the presence of compound 2. Integrase and protease enzymes have different mechanisms of action. Integrase catalyzes the covalent insertion of the viral DNA into the chromosomes of infected cells. We suggested that the ligands should be interacting with the metal cofactors, thereby inhibiting the binding of integrase to the viral DNA since it is well recorded that Schiff base chelation is favored in compounds with hydroxyl (-OH) groups in position 2 in the aromaticrings. HIV-1 protease acts like other proteases catalyzing the hydrolysis of peptide bonds due to its nucleophilic properties at its aspartate residue. According to the literature, inhibitors of protease do this by binding to the enzyme active site or allosterically, by binding at a site different from the enzyme active. Moreover, this was not the case with this series of synthesized Schiff bases (compounds 1–5). The absence of activity could be associated with the chemical and physical properties of the ligands under the reaction conditions that limit the reactivity and/or their geometric representation.
In this study, we synthesized five novel Schiff bases derived from the condensation reaction between 2-(trifluoromethoxy)aniline and different carbonyls. The compounds were successfully prepared as confirmed by the FTIR, UV-vis, elemental analysis, 1H and13C NMR and single crystal X-ray crystallography. All tested compounds were of pharmacological importance as biological applications showed increase potency in mitigating and limiting the metabolic activity of different microbes even at a very low concentration. The ligands demonstrated growth inhibitory properties against Plasmodium falciparum 3D7 and Trypanosoma brucei with very poor cytotoxicity effect against HeLa cells. High anti-HIV by inhibition of the HIV-1 integrase enzyme was noted at a single analytical concentration of 8 µg/mL, and therefore further studies are required to determine the IC50 of the most potent ligands.
Crystallographic data in CIF format for the structural analysis of compound 2 have been deposited with the Cambridge Crystallographic Data Centre, CCDC number 1536006. Copies of this information may be obtainedfree of charge from: The Director, CCDC, 12 Union Road, Cambridge, CB2IEZ, UK, Fax: +44122336033,firstname.lastname@example.org. or/at http://www.ccdc.cam.ac.uk.
We thank the Department of Applied Chemistry, Department of Biotechnology and Food Technology, University of Johannesburg for availing its facilities for this project. The Department of Chemistry, X-ray Diffraction Unit, University of Johannesburg, is acknowledged for its facilities for collection of the X-ray data in this study. Malaria, trypanosomiasis, HIV, and cytotoxicity studies were supported by the South African Medical Research Council (MRC) with funds from the National Treasury under its Economic Competitiveness and Support Package, and Rhodes University “Sandisa Imbewu”.
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Received: 2018-01-13, Revised: 2018-03-22, Accepted: 2018-04-18.
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