Synthesis and Characterization of Coumarins 1–10

Coumarins 1–5 were synthesized according to the methods of a previous report (Scheme 1) (Vega-Granados et al. 2021; Hersi et al. 2020). A mixture of aromatic aldehydes (10 mmol) and 4-hydroxycoumarin (20 mmol) was dissolved in 100 mL of EtOH. A few drops of piperidine were added, and the mixture was stirred for 3 h at room temperature. After reaction completion as determined by TLC, water was added until precipitation occurred. The solid was filtered off and then recrystallized from ethanol to give compounds 1–5.

Scheme 1

Synthetic route of compounds 1–5

3,3’-((4-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (1): m.p. 209–210 °C. IR (KBr pellet cm− 1): 2920, 1650, 1610, 1520, 1412, 1340, 1102, 761 cm. 1H NMR (CDCl3, δ, ppm): 11.581(s, 1H), 11.286(s, 1H), 8.021–8.053(t, 2 H), 7.606–7.649(m, 2 H), 7.398–7.419(d, 4 H), 6.852–6.873(d, 1H), 6.680–6.738(m, 2 H), 6.064(s, 1H), 5.579(s, 1H), 3.748(s, 3 H). HRMS (ESI+): m/z: calcd for C26H18O8: 481.0894 [M + Na]+; found: 481.0812.

3,3’-((3-hydroxy-4-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (2): m.p. 210–211 °C. IR (KBr pellet cm− 1): 2910, 1650, 1630, 1510, 1345, 1320, 1024, 761 cm. 1H NMR (CDCl3, δ, ppm): 11.612(s, 1H), 11.277(s, 1H), 8.034–8.070(d, 2 H), 7.623–7.666(m, 2 H), 7.414–7.435(d, 4 H), 6.799–6.819(t, 2 H), 6.708–6.729(t, 1H), 6.047(s, 1H), 5.612(s, 1H), 3.895(s, 3 H). HRMS (ESI+): m/z: calcd for C26H18O8: 481.0894 [M + Na]+; found: 481.0876.

3,3’-((3-fluorophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (3): m.p. 221–223 °C. IR (KBr pellet cm− 1): 2915, 1721, 1630, 1520, 1422, 1323, 1012, 761 cm. 1H NMR (CDCl3, δ, ppm): 11.616(s, 1H), 11.327(s, 1H), 8.023-8. 109(q, 2 H), 7.645–7.688(m, 2 H), 7.435-7. 456(d, 4 H), 7.301–7.340(m, 1H), 6.941–7.047(m, 3 H), 6.095(s, 1H). HRMS (ESI+): m/z: calcd for C25H15FO6: 453.0745 [M + Na]+; found: 453.0751.

3,3’-((3-iodophenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (4): m.p. 231–232 °C. IR (KBr pellet cm− 1): 2932, 1721, 1620, 1571, 1432, 1310, 1021, 761 cm. 1H NMR (CDCl3, δ, ppm): 11.576(s, 1H), 11.308(s, 1H), 8.028-8. 109(q, 2 H), 7.628–7.691(m, 3 H), 7.547–7.550(d, 1H), 7.397–7.457(q, 4 H), 7.215–7.235(d, 1H), 7.064–7.103(t, 1H), 6.074(s, 1H). HRMS (ESI+): m/z: calcd for C25H15IO6: 560.9806 [M + Na]+; found: 560.9828.

3,3’-((4-(trifluoromethyl)phenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (5): m.p. 221–222 °C. IR (KBr pellet cm− 1): 2930, 1712, 1634, 1560, 1330, 1310, 1014, 763 cm. 1H NMR (DMSO-d6, δ, ppm): 8.419–8.439(d, 2 H), 7.770–7.809(q, 2 H), 6.626–7.677(q, 4 H), 7.513–7.583(m, 4 H), 4.985(s, 1H). HRMS (ESI+): m/z: calcd for C26H15F3O6: 503.0713 [M + Na]+; found: 503.0731.

Coumarins (6–10) were also synthesized according to a reported procedure (Scheme 2) (Li et al. 2022). A mixture of 4-hydroxycoumarin (10 mmol), aromatic aldehydes (10 mmol), malononitrile (10 mmol) and 4-(dimethylamino)pyridine (DMAP) (1 mmol) in ethanol (100 mL) was refluxed for 2–3 h and then cooled to room temperature. The solid was filtered off and then recrystallized from ethanol to give compounds 6–10.

Scheme 2

Synthetic route of compounds 6–10

2-amino-4-(3,4-dimethylphenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (6): m.p. 232–233 °C. IR (KBr pellet cm− 1): 2970, 1821, 1745, 1612, 1400, 1310, 1014, 756 cm. 1H NMR (DMSO-d6, δ, ppm): 7.887–7.910 (q, 1H), 7.694–7.737 (m, 1H), 7.457–7.514 (q, 2 H), 7.379 (s, 2 H), 6.846–6.893 (q, 2 H), 6.731–6.757 (q, 1H), 4,411 (s, 1H), 3.714–3.717 (d, 6 H). HRMS (ESI+): m/z: calcd for C21H16N2O3: 367.1053 [M + Na]+; found: 367.1099.

2-amino-4-(3-methoxyphenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (7): m.p. 223–224 °C. IR (KBr pellet cm− 1): 2940, 1712, 1630, 1520, 1432, 1320, 1024, 761 cm. 1H NMR (DMSO-d6, δ, ppm): 7.890–7.913 (q, 1H), 7.697–7.736 (q, 1H), 7.424–7.516 (m, 4 H), 7.219–7.259 (t, 1H), 6.800-6.841 (q, 3 H), 4.432 (s, 1H), 3.726 (s, 3 H). HRMS (ESI+): m/z: calcd for C20H14N2O4: 369.0846 [M + Na]+; found: 369.0822.

2-amino-5-oxo-4-(3,4,5-trifluorophenyl)-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (8): m.p. 254–255 °C. IR (KBr pellet cm− 1): 2840, 1652, 1623, 1532, 1412, 1305, 1024, 761 cm. 1H NMR (DMSO-d6, δ, ppm): 7.884–7.907 (q, 1H), 7.709–7.753 (m, 1H), 7.463–7.520 (q, 4 H), 7.332–7.371 (q, 2 H), 4.569 (s, 1H). HRMS (ESI+): m/z: calcd for C19H9F3N2O3: 393.0457 [M + Na]+; found: 393.0421.

2-amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (9): m.p. 229–230 °C. IR (KBr pellet cm− 1): 2934, 1734, 1633, 1554, 1332, 1243, 1014, 760 cm. 1H NMR (DMSO-d6, δ, ppm): 8.075 (s, 2 H), 8.017 (s, 1H), 7.902–7.925 (q, 1H), 7.717–7.760 (m, 1H), 7.577 (s, 2 H), 7.468–7.535 (m, 2 H), 4.868 (s, 1H). HRMS (ESI+): m/z: calcd for C21H10F6N2O3: 475.0488 [M + Na]+; found: 475.0460.

2-amino-4-(4-(benzyloxy)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (10): m.p. 265–267 °C. IR (KBr pellet cm− 1): 2828, 1643, 1620, 1532, 1324, 1245, 1014, 760 cm. 1H NMR (DMSO-d6, δ, ppm): 7.879–7.899 (d, 1H), 7.707–7.747 (t, 1H), 7.451–7.519 (m, 4 H), 7.300-7.392 (m, 3 H), 7.112–7.149 (t, 1H), 6.978–7.041 (m, 5 H), 6.821–6.841 (d, 2 H), 4.477 (s, 1H). HRMS (ESI+): m/z: calcd for C26H18N2O4: 445.1159 [M + Na]+; found: 445.1198.

X-ray Crystallography

SuperNova diffractometer was utilized in acquiring XRD patterns. CrysAlisPro was employed to analyze intense data and converted the data to HKL files. SHELXS in the light of direct mean and SHELXL-2014 software based on least-squares strategy were employed respectively for the synthesis and refinement of original architectural modes. After the addition of non-H atoms, the anisotropic parameters could be mixed. Eventually, the entire H atoms could be fixed on the C atoms that bridged with AFIX commands in geometry. The as-prepared compounds’ refinement details and crystallography parameters are displayed in Table 1.

Table 1 Refinements details and crystallography parameters of the as-prepared compoundsStrains and Animals

Methicillin-resistant Staphylococcus aureus N315 (MRSA N315); Staphylococcus aureus ATCC 29,213 (S. aureus ATCC 29,213); Pseudomonas aeruginosa (P. aeruginosa); Klebsiella pneumonia (K. pneumoniae); Acinetobacter baumannii (A. baumannii) and Escherichia coli (E. coli) were purchased from the American Type Culture Collection (ATCC). All the clinical A. baumannii strains were provided by the Zibo Prevention and Treatment Hospital for Occupation Diseases (Zibo, China). The bacteria were cultured on Nutrient agar plates (Sigma-Aldrich, USA) at 37℃ in an incubator and then transferred into Nutrient broth for strain replication at the condition of 220 rpm, 37℃.

BALB/c mice (5-6weeks, 20–22 g) were used in this research, which were purchased form the Animal Research Center of the Fourth Military Medical University (Xi’an, China). All the mice were maintained at a specific pathogen-free (SPF) environment of 20–25℃, combined with a 12- hour light/dark schedule and free standard laboratory mouse chow and water supply. All the preformation in mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the Fourth Military Medical University, and conducted totally under the guidance of the health guidelines for the use of laboratory animals.

MIC Determination

The minimum inhibitory concentration (MIC) values of the synthesized compounds were determined with broth micro-dilution technique for the anti-bacterial activity evaluation. This preformation was conducted strictly under the guidance of the instructions with some modifications (Kowalska-Krochmal and Dudek-Wicher 2021). In brief, the bacterial strains were cultured in Nutrient broth and seeded into the microtiter plates at a final concentration of 1 × 108 CFU/ml overnight. Then, the synthesized compounds and reference antibiotic in 100 µl culture medium was added into the wells with serial concentrations (2–256 µg/mL) for another 24 h. The OD600 values of each well were measured, and the MIC values of the compounds were regarded as the lowest concentrations completely inhibit the bacterial growth.

A. baumannii Growth Curves

The inhibitory activity of the synthesized S1032 on A. baumannii growth was evaluated in this experiment under the guidance of the following descriptions. The A. baumannii strain was collected and cultured in Nutrient broth at the condition of 220 rpm, 37 °C. Then the bacterial cells were planted into microtiter plates (1 × 108 CFU/ml) with S1032 added for treatment, the same concentration of Ofloxacin and Gentamicin were used as control. The plates were placed in an automated Bioscreen C system in the automatic bacterial growth curve analyzer (Bioscreen, Finland) at 37 °C, and the absorbances of all the wells were determined every hour at 600 nm (Rivani et al. 2022).

qPCR Assay

The qPCR was performed in this research to measure the relative expression of the genes related with bacterial biofilm formation. In brief, the A. baumannii bacterial cells were grown in Nutrient broth at 37 ℃ to exponential phase and then treated with the S1032 compound with the indicated concentrations for 2 h. After the indicated treatment, the A. baumannii bacterial cells were collected, washed, and the total RNA in the bacterial cells was extracted with TRIZOL reagent. After measuring the quality and quantity of the total RNA, which was then reverse transcripted into cDNA with reverse transcription kit (Qiagen) in 20 µl total reaction system. Finality the relative expression of biofilm formation related genes (bfmR, bfmS, CsuA, CsuB) were determined by SYBR Green Master Mix after S1032 treatment, 16s was used as internal control. 2−ΔΔCt method was used for statistical analysis. This research was performed three times. All the primer sequences were listed in Table 2.

Table 2 Sequences of primers used in this researchMice Lung Infection Model

BALB/c mice (5-6weeks, 20–22 g) used in this present study were divided randomly into different groups: the negative control group (n = 7), positive control group (receive antibiotic treatment, n = 7) and S1032 treatment groups (1, 2 and 5 mg/kg, n = 7). Then the mice were challenged with 5 × 109 CFU A. baumannii in 20 µl Nutrient broth via nasal drops. After bacterial infection, S1032 (1, 2 and 5 mg/kg) and Ofloxacin (5 mg/kg) was given for 7 consecutive days, with saline used as negative control. The survival rates of the mice in each group were monitored since infection, and the cumulative percentage survival was plotted (Tansho-Nagakawa et al. 2021).

Therapeutic Activity Evaluation

To evaluate the therapeutic activity of the synthesized S1032, the A. baumannii bacterial CFU numbers in lung tissue was determined in this research. Briefly, after the construction of lung infection mice model, S1032 was given for treatment with indicated concentrations (1, 2 and 5 mg/kg) for three consecutive days. 7 days after infection, the animals were euthanized for the infected tissue collection. The tissue samples were grinded, diluted and dripped onto Nutrient agar plates for 24 h cultivation at 37℃ and 5%CO2 condition. The numbers of bacterial colony were counted and recorded. This experiment was repeated at least three times, and the results were presented as mean ± SD.

After the collection of infected mice lung tissue, which were then fixed in 10% neutral buffered formalin for 24 h. Then the tissue samples were dehydrated and processed into paraffin sections totally according to the standard procedure. The paraffin sections were then stained with hematoxylin and eosin (H&E) staining for histopathology analysis, and the microscope was used for observation of tissues morphologies.

Biofilm Formation Assay

To evaluate the inhibitory activity of the S1032 compound on bacterial biofilm formation, the crystal violet staining assay was performed in this research. All the conduction was finished strictly under the guidance with only a little modification (Khan et al. 2022). In short, A. baumannii strain were seeded in 96 well plates and cultured in the incubator at the condition of 37℃ for 24 h. After the bacterial biofilm formation, 0.1% crystal violet (CV) (Sigma) solution was added into wells for staining at 37 °C for 10 min. After that, PBS was used to wash the unattached CV and 95% ethanol was added to detainee the stained biofilm cells. The solution absorption was measured at 600 nm, each group has independent replicates, and this experiment was repeated at least three times.

Scanning Electron Microscope for Biofilm Observation

The bacterial culture solution was diluted to 1 × 108CFU/ml. 500 µl of the diluted bacterial solution was added to the 24-well cell culture plate wells that contained sterilized coverslips. The plate was incubated at 37℃ in a incubator for 36 h. The coverslips with adhered bacteria were gently washed with sterile PBS to remove any unattached free-floating bacteria. Then, 2.5% (v/v) glutaraldehyde solution was added, and the samples were fixed overnight at 4℃. The fixative was aspirated, and the samples were washed three times with sterile PBS, each time for 10 min. After washing with sterile water several times, the samples were dehydrated with 30%, 50%, 70%, and 90% ethanol for 15 min each, and finally dehydrated twice with 100% ethanol, each time for 15 min. The samples were then placed in a vacuum freeze-drying machine and freeze-dried for 2–3 h. The dried samples were taken out, attached to a sample holder with conductive adhesive, and observed under a scanning electron microscope (Gould et al. 2022).

Intracellular ROS Determination

The A. baumannii bacterial cells were grown in Nutrient broth at 37 ℃ to exponential phase and seeded into the microtiter plates at the final destiny of 5 × 107 CFU/ml overnight. Then, the Nutrient broth containing the active S1032 with the final concentrations of 0.25, 0.5 and 1 µg/m was added into the bacterial wells for 2 hours incubation. After that, the A. baumannii bacterial cells were collected, washed, centrifuged and re-suspended with PBS solution. 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 µl) was added to the above suspension to detect the content of overall intracellular ROS. After incubation 30 min in the dark, the bacteria cells were harvested, washed at least three times to remove the extra fluorescence probe. Fluorescence intensity of each sample was detected at an excitation wavelength of 484 nm and an emission wavelength of 525 nm (Wang et al. 2022). This research was repeated at least three times.

Cytotoxicity Evaluation

Cell Counting Kit-8 (CCK-8) assay was conducted in this research to evaluate the cytotoxicity of active S1032 against human umbilical vein endothelial cells (HUVEC) and human embryonic kidney (Hek) 293T cells. This research was conducted under the guidance of manufactures’ instruction. The HUVEC and 293T cells were collected and seeded into the 96 well plates at the destiny of 5000 cell/well. After the cell got the 70–80% confluence, the S1032 was added for 48 h incubation with serial different concentrations (2.5, 5, 10, 20, 40, 80, 100 and 200 µM). After that, the culture medium was discarded, and the cells were washed with PBS solution. Fresh culture medium containing 10 µL CCK-8 reagents was added into the well for 4 h incubation. Then, the absorbance of each well was measured at 490 mm. This preformation was conducted at least three times, and the results were presented as mean ± SD.

ADMET Predication Online

The ADMET parameter of the S1032 was predicated using SWISS ADME online tool (http://www.swissadme.ch/) (Daina et al. 2017). The SMILES of the compound were produced and was lodged into the SWISS ADME website, and the physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness and medicinal chemistry was predicated.

Molecular Docking

Four structures were considered as the target proteins for the docking studies, their structures were retrieved from Protein Data Bank (PDB) and the PDB IDs were 3TD3 (Park et al. 2012), 4JAS (Podgornaia et al. 2013), 5BUF (Sutton et al. 2016) and 6FJY (Pakharukova et al. 2018). The active sites of S1032 and predicated target proteins were measured with molecular docking study via AutoSite (v1.1) tool software (Ravindranath and Sanner 2016). Firstly, the 3D structures of target proteins in PDB format were downloaded from RCSB Protein Data Bank. Then, the ligand structure (S1032) was prepared by Avogadro (v1.2) (Hanwell et al. 2012), an energy minimization was performed through steepest descent algorithm adopting general Amber force field (GAFF) (Wang et al. 2004). After obtaining structures for both ligand and proteins, we used AutoDockTools (v1.5.7 patch 1) to prepare the docking simulations, and the docking studies were conducted using AutoDock4 (v4.2.6) (Morris et al. 2009). For the docking simulation, 50 potential docking poses are evaluated using the Lamarckian genetic algorithm (LGA). The binding affinity of the complexes was recorded with a unit of kcal/mol. The protein-ligand interactions were analyzed using PLIP (Adasme et al. 2021).

Reinforcement Learning Studies

The reinforcement learning approach that was implemented and performed in the current study is based on the widely used molecule deep Q-networks (MolDQN) (Zhou et al. 2019). During the reinforcement learning process, a known molecule that has been proved to have excellent bioactivities depending on demands and requirements of the study (in the current work, the desired molecule is the inhibitor of A. baumannii) is used as the training template (Almihyawi et al. 2022; Abdelaziz et al. 2022). Then, multiple actions are allowed to modify the template and generate novel structure which is expected to have similar or even better bioactivity than the given template, the allowed actions are atom addition, bond addition, bond removal and un-modification. After a certain number of actions has been performed. Three qualification characteristics are determined upon the optimized structure, these qualification characteristics are: (i). binding affinity energy between the optimized molecule and the protein; (ii). synthetic accessibility (SA) score; (iii) quantitative estimate of drug-likeness (QED) score. Explicitly, the binding affinity energy is evaluated by QuickVina 2 (Alhossary et al. 2015), which has been proven to have both accuracy and efficiency as AutoDock 4, as has been mentioned in the above docking study, the gym-molecule library has been employed to calculate the SA score, and the open source library rdkit (https://www.rdkit.org/) has been utilized to estimate the QED score. For these three qualification measurements, the Smile code of the molecular structure has been used as input, consequently, the 3-dimensional structure is effected using Open Babel.

For each of the ligand (S1032) – protein (3TD3, 4JAS, 5BUF and 6FJY) combination, the maximum number of allowed modifications (actions) is set to 20, during the modification, only C, N, O atoms are allowed, and up to 10,000 molecule structures are evaluated. After the reinforcement learning study, four molecules that are showing excellent binding affinity energies, SA and QED scores are selected, and a further molecular docking simulation has been performed using the same procedure described in the above Molecular Docking section.

Reinforcement Learning Studies

The reinforcement learning approach that was implemented and performed in the current study is based on the widely used molecule deep Q-networks (MolDQN) (Zhou et al. 2019). During the reinforcement learning process, a known molecule that has been proved to have excellent bioactivities depending on demands and requirements of the study (in the current work, the desired molecule is the inhibitor of A. baumannii) is used as the training template. Then, multiple actions are allowed to modify the template and generate novel structure which is expected to have similar or even better bioactivity than the given template, the allowed actions are atom addition, bond addition, bond removal and un-modification. After a certain number of actions has been performed. Three qualification characteristics are determined upon the optimized structure, these qualification characteristics are: (i). binding affinity energy between the optimized molecule and the protein receptor; (ii). synthetic accessibility (SA) score; (iii) quantitative estimate of drug-likeness (QED) score. Explicitly, the binding affinity energy is evaluated by QuickVina 2, which has been proven to have both accuracy and efficiency as AutoDock 4, as has been mentioned in the above docking study, the gym-molecule library has been employed to calculate the SA score, and the open source library rdkit (https://www.rdkit.org/) has been utilized to estimate the QED score. For these three qualification measurements, the Smile code of the molecular structure has been used as input, consequently, the 3-dimensional structure is effected using Open Babel.

For each of the ligand (S1032) – protein (3TD3, 4JAS, 5BUF and 6FJY) combination, the maximum number of allowed modifications (actions) is set to 20, during the modification, only C, N, O atoms are allowed, and up to 10,000 molecule structures are evaluated. After the reinforcement learning study, four molecules that are showing excellent binding affinity energies, SA and QED scores are selected, and a further molecular docking simulation has been performed using the same procedure described in the above Molecular Docking section.

Statistical Analysis

All the biological experiments in this research were conducted in triplicate and the results were presented as the mean ± standard deviation (SD). SPSS 20.0 statistical software (SPSSInc., Chicago, IL, USA) was recommended for the analysis. Two-tailed Student’s t-test was used to analyze the difference between two groups, and the one-way ANOVA method was carried out for the analysis among more than three groups. Statistical significance was considered as P < 0.05.

Ethics Statement

All the in vivo experiments were approved by the Committee of Animal Ethics of the Fourth Military Medical University (Xi’an, China). All the preformation was strictly in accordance with the manufactures’ instructions with some modifications.