2025 Volume 73 Issue 4 Pages 307-313
Sirtuin enzymes are interesting targets for developing new drug candidates. This study aims to design new indole-based sirtuin inhibitors, filtering through molecular docking alongside molecular dynamics and pharmacokinetic property prediction, synthesizing 4 compounds with an evaluation of their cytotoxic activity alongside the sirtuin inhibitor AGK2 against the breast cancer (MCF7) cell line via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The antibacterial activity of these compounds was evaluated by comparing the minimum inhibitory concentration (MIC) with ciprofloxacin against Staphylococcus aureus and Klebsiella pneumoniae using resazurin dye. The docking study showed a higher binding affinity for the synthesized compounds than sirtuin inhibitors AGK2 and selisistat against the sirtuin2 isoform. In addition, the molecular dynamics study showed good stability of the compound with the higher docking score in complex with sirtuin2 over 100 ns. The prediction of pharmacokinetic properties showed adherence to drug-likeness criteria. The MTT assay revealed comparable IC50 values for the compounds with AGK2, as compound AFJ1 showed the highest cytotoxic activity (IC50 = 2.6 μM). Among the synthesized compounds, AFJ2 showed the lowest MIC against K. pneumoniae (125 μg/mL) compared to ciprofloxacin (62.5 μg/mL).
The regulation of histone protein acetylation is carried out by specific enzymes known as histone acetyltransferases (HATs) and histone deacetylases (HDACs).1,2) HDACs are a group of enzymes that require zinc as a catalyst in classes I, II, and IV, while class III are nicotinamide adenine dinucleotide (NAD+)-dependent enzymes, known as sirtuins (SIRTs). SIRTs consist of 7 isoforms (SIRT1–7), with SIRT1–3 being the most thoroughly investigated.3) SIRT2 protein forms complicated signaling networks that make this enzyme a valuable target for many therapeutic applications, including anticancer action.4,5) One of the proposed mechanisms of SIRT2 inhibition is correlated with elevated α-tubulin acetylation levels, which ultimately destabilize microtubules, increase stress in cells, and cause cell death.6) Additionally, SIRT2 inhibition can also improve neurological conditions such as Parkinson’s disease.7)
Over the past 2 decades, researchers have synthesized, developed, and classified numerous SIRT inhibitors into various groups. Selective SIRT2 inhibitors have been found to have several beneficial effects. AGK2 is a selective SIRT2 inhibitor with an IC50 of 3.5 μM and an IC50 higher than 50 μM for both SIRT1 and SIRT3. AGK2 has been studied for various biological effects, including the reduction of toxicity mediated by α-synuclein in a cellular model of Parkinson’s disease.7,8) Studying the effect of SIRT2 inhibition by AGK2 on C6 glioma cells’ viability revealed the induction of cell necrosis and apoptosis via the caspase-3 pathway.9) Another study showed that AGK2 inhibits hepatitis B virus replication in vitro and in vivo.10) The cytotoxic effects on breast cancer cells were studied alone or in combination with paclitaxel. The 2 compounds showed cytotoxic effects with synergistic activity in MCF7 luminal cells.11)
The well-known SIRT inhibitor, selisistat, has selectivity toward SIRT1 over SIRT2 and SIRT3, with an IC50 of 0.09 μM for SIRT1, 19.6 μM for SIRT2, and 48.7 μM for SIRT3. Selisistat involves an indole moiety fused with an aliphatic 6-membered ring12) (Fig. 1). However, modifications in the chemical structure while preserving the indole moiety shift the selectivity toward SIRT2.13) An in silico study showed that many selisistat derivatives have a higher virtual affinity toward SIRT2 as well.14)
Indole-containing natural products, such as alkaloids and hormones, with different biological actions, are widely used.15) Panobinostat is an indole-based HDAC pan-inhibitor that gained approval for treating multiple myeloma.16) Therefore, in this study, we employed molecular modeling tools to design new indole-based molecules. The top-scoring molecules were synthesized (Fig. 2), and their antiproliferative and antibacterial activities were evaluated.
In the molecular docking study, 170 indole-based molecules were evaluated against SIRT1–3, showing varying binding affinities. The group consisting of 50 tryptamine-based molecules showed reasonable data. The design of these molecules was based on the indole moiety, which is present in many SIRT inhibitors, including selisistat.12) The SIRT inhibitors developed in the last 2 decades have shown high diversity in their chemical structures, making the design of new inhibitors unlimited to certain criteria. An extensive docking study using extra precision (XP) settings was performed, and top molecules with superior docking scores and proper interaction fitness within the binding site were nominated for organic synthesis.
Docking score values revealed that the final compounds have a higher affinity for SIRT2 than for SIRT1 and SIRT3. Additionally, these compounds showed a better affinity than AGK2 and selisistat regarding SIRT2 (Table 1). It is worth noting that realistic selisistat selectivity toward SIRT1 is kinetically derived rather than depending on the structural features of the 3 SIRT isoforms.17) Hence, the higher affinity toward SIRT3 in the docking study resulted from the structural diversity factor among the 3 isoforms. Another important point to consider is the effect of the compounds’ ionization state (AFJ2–AFJ4). The docking score values of the ionized form were higher than the unionized form against SIRT2. The visual inspection of the interaction poses of AFJ4(+), AGK2, and selisistat revealed that the interaction with SIRT2 occurred at the same binding site (Supplementary Fig. S1). The root mean square deviation (RMSD) values for the docking of co-crystallized ligands were 0.248, 0.2231, and 0.2235 Å for SIRT1, SIRT2, and SIRT3, respectively. These values indicated good validation regarding the XP docking algorithm.
| Compound | SIRT1 | SIRT2 | SIRT3 |
|---|---|---|---|
| AFJ1 | –9.53 | –12.17 | –7.42 |
| AFJ2 | –4.99 | –9.99 | –5.77 |
| AFJ2(+)* | –10.1 | –11.77 | –3.9 |
| AFJ3 | –8.03 | –9.63 | –5.39 |
| AFJ3(+)* | –7.12 | –12.16 | –9.43 |
| AFJ4 | –6.78 | –9.17 | –6.32 |
| AFJ4(+)* | –6.71 | –12.45 | –9.39 |
| AGK2 | –5.18 | –9.7 | –4.81 |
| Selisistat | –9.78 | –8.98 | –10.39 |
* The (+) sign indicates the ionized form of the tertiary amine atom in compounds AFJ2–AFJ4.
The 2-dimensional ligand interaction diagram was used to visualize and evaluate the type of interactions for each compound, as well as to compare the ionized and unionized forms of AFJ2–AFJ4. Generally, the final compounds showed interactions with 6 amino acid residues (PRO94, PHE96, PHE119, HID187, VAL233, and PHE235) with 3 types of interactions: H-bonding, π–π stacking, and π–cation interaction (Supplementary Fig. S2).
Most interestingly, the ionized tertiary amine is creating H-bonding and π–cation interactions that were not present in the unionized form. This is why the ionized form of these compounds had a higher docking score. AFJ4 in the ionized form was making 7 interactions with SIRT2: H-bonding with the carbonyl oxygen of PRO94, π–π stacking with the phenyl ring of PHE119, H-bonding with the NH in the imidazole ring of HID187, π–cation interaction with the imidazole ring of HID187, 2 H-bonding interactions with the carbonyl oxygen of VAL233, and π–cation interaction with the phenyl ring of PHE235 (Supplementary Fig. S3).
The molecular dynamics simulation for AFJ4(+) with SIRT2 was conducted to further assess the stability of the ligand–protein complex and docking results. The RMSD results showed that the major fluctuations of the complex were in the first 8 ns, after which the complex was highly stable until the end of the simulation (Supplementary Fig. S4a). The root mean square fluctuation (RMSF) of the protein showed that the interacting residues had fluctuations of less than 2 Å, while the RMSF values of the ligand were also less than 2 Å, except for 1 of the methyl groups (Supplementary Figs. S4b, S4c). The protein–ligand contact diagram showed interactions with 5 protein residues for more than 60% of the simulation time (Supplementary Fig. S4d). These results are compatible with the docking results of AFJ4(+) showing high affinity for SIRT2.18) To observe the direction of deviation for the target enzyme’s binding site and the ligand, 2 snapshots were taken at 1 and 100 ns of the simulation timeframe (Supplementary Fig. S5). The superimposition of these snapshots revealed the same direction of deviation for the enzyme and the ligand, as well as continuous fitting of the ligand within the binding site.
The predicted pKa values by ChemDraw Professional were 5.11, 7.15, 7.05, and 7.35 for AFJ1–AFJ4, respectively. Epik predictions were 4.93 ± 0.69, 8.03 ± 1.05, 8.61 ± 1.05, and 8.53 ± 1.05 for AFJ1–AFJ4, respectively. These values explain the presence of ionized forms of compounds AFJ2–AFJ4 in the docking study, as the pKa values nearly approach the physiological pH, resulting in a higher percentage of ionization for the compound. It is worth noting that a lower pH in the cancer microenvironment can increase the accumulation of these compounds through an ion-trapping principle, leading to better anticancer activity (Fig. 3).
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The predicted absorption, distribution, metabolism, excretion (ADME) descriptors revealed the adherence of the final compounds to drug-likeness criteria by the RuleOfFive, predictions for being orally active drugs by the RuleOfThree and HumanOralAbsorption, and all the predicted descriptors were within the acceptable ranges for 95% of known drugs by #stars. The predicted CNS activity was low, and the number of metabolites was within the acceptable range19) (Table 2).
| Compound | #starsa) | CNSb) | HumanOral Absorptionc) |
RuleOfFived) | RuleOfThreee) | #Metabf) |
|---|---|---|---|---|---|---|
| AFJ1 | 0 | −1 | 3 | 0 | 0 | 5 |
| AFJ2 | 0 | −1 | 3 | 0 | 0 | 4 |
| AFJ2(+) | 0 | −1 | 3 | 0 | 0 | 4 |
| AFJ3 | 0 | −1 | 3 | 0 | 0 | 4 |
| AFJ3(+) | 0 | −1 | 3 | 0 | 0 | 4 |
| AFJ4 | 0 | −1 | 3 | 0 | 0 | 4 |
| AFJ4(+) | 0 | 0 | 3 | 0 | 0 | 4 |
| AGK2 | 2 | 0 | 1 | 1 | 1 | 2 |
| Selisistat | 1 | 0 | 3 | 0 | 0 | 3 |
a) The #start shows the number of descriptors that fall outside the 95% range of similar values for known drugs with a normal range (0–5). b) The CNS shows the predicted central nervous system activity (−2 inactive to +2 active). c) The HumanOralAbsorption shows a prediction of oral absorption (1, low; 2, medium; 3, high absorption). d) The RuleOfFive shows the violation of Lipinski’s rule (maximum is 4). e) The RuleOfThree shows the violation of Jorgensen’s rule (maximum is 3). f) The (#Metab) shows the number of likely metabolic reactions (1–8).
The final compounds were synthesized through 4 chemical reaction steps: amide formation, N-tert-butyloxycarbonyl (Boc) deprotection, another amide formation, and the N-alkylation reaction (Chart 1). The first step of amide formation included the utilization of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC.HCl) as a coupling agent that activated the carboxylic acid group in the presence of 4-dimethylamino pyridine (DMAP) as a catalyst and diisopropyl ethylamine (DIPEA) as a base to remove the HCl moieties from EDC.HCl and tryptamine hydrochloride. Thionyl chloride was reacted with cold absolute ethanol for in situ generation of anhydrous HCl. The anhydrous acid was able to break the carbamate bond, forming an HCl salt by protonating the liberated amine and leaving the amide bond intact. The second amide formation step also included the use of DIPEA as a base to remove the HCl moiety from the amine and to prevent the reprotonation of the amine by liberated HCl molecules from the reaction. The final step involved using the liquid amines as a reactant and solvent at the same time. The large excess of amines compared to PAF3 accelerated the reaction rate and reduced the risk of quaternary ammonium side product formation.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results revealed comparable cytotoxic activity of final compounds with AGK2 against the breast cancer MCF7 cell line, with the best cytotoxicity shown by compound AFJ1, which contains an oxygen atom that might participate in some interactions with enzymes responsible for proliferation in MCF7 cells (Table 3). Although the final compounds were proposed to be SIRT inhibitors, the cytotoxicity might involve actions on more than 1 target in the MCF7 cells. This would explain the higher cytotoxicity of compound AFJ1 over other compounds. On the other hand, compounds AFJ2–AFJ4 showed a well-established relationship between their cytotoxicity and docking scores against SIRT2 in their ionized forms.
| Compound | IC50 (μM) |
|---|---|
| AFJ1 | 2.60 ± 0.50 |
| AFJ2 | 5.88 ± 1.14 |
| AFJ3 | 4.86 ± 1.18 |
| AFJ4 | 3.23 ± 0.37 |
| AGK2 | 3.43 ± 0.68 |
With the increasing bacterial resistance and the demand for new antibacterial agents, newly synthesized compounds could be assessed for the possibility of antibacterial actions. The final compounds showed better antibacterial activity against the Gram-negative Klebsiella pneumoniae than the Gram-positive Staphylococcus aureus. The compound AFJ2, which showed the least cytotoxic activity in the MTT assay, showed better antibacterial activity than other compounds (Table 4).
| Compound | MIC (μg/mL) | |
|---|---|---|
| K. pneumoniae | S. aureus | |
| AFJ1 | 500 | 1000 |
| AFJ2 | 125 | 1000 |
| AFJ3 | 500 | 1000 |
| AFJ4 | 500 | 1000 |
| Ciprofloxacin | 62.5 | 500 |
New indole-based compounds were designed, docked on SIRTs, synthesized, characterized by spectroscopy, and evaluated for anticancer and antibacterial activities. The docking study showed a higher affinity of compounds toward SIRT2, with docking scores reaching −12 kcal/mol, especially for the ionized forms of compounds AFJ2–AFJ4. The compound with the highest docking score showed good stability in complex with SIRT2 over 100 ns of dynamic simulation. The final compounds seemed to have comparable cytotoxic activity with the SIRT inhibitor AGK2 on the MCF7 cell line in the MTT assay, and the most active compound was AFJ1 with an IC50 = 2.6 μM. The determination of minimum inhibitory concentration (MIC) revealed that the final compounds were more active against the Gram-negative K. pneumoniae than the Gram-positive S. aureus. AFJ2 showed the highest activity among the final compounds with MIC = 125 μg/mL against K. pneumoniae, although ciprofloxacin showed better activity with MIC = 62.5 μg/mL. Further studies regarding other cancer cell lines and enzymatic assays would be conducted to assess the cytotoxicity profile and mechanism of action of the synthesized compounds.
All chemicals used were purchased from commercial providers. Solvents were subjected to prework drying. TLC plates from Merck (Darmstadt, Germany, silica gel 60 F254) were used in all TLC analysis processes, and spots of compounds were detected under UV light at 254 nm. Fourier transform IR (FT-IR) spectroscopy was conducted utilizing the Shimadzu IR Affinity-1 spectrometer from Shimadzu (Kyoto, Japan). The 1H-NMR and 13C-NMR analyses were conducted at frequencies of 400 and 100 MHz, respectively, using a Bruker (Billerica, MA, U.S.A.) Avance III 400 MHz spectrometer, and the solvent used was deuterated dimethyl sulfoxide DMSO-d6.
Molecular DockingUsing ACD/ChemSketch (Freeware 2022.2.3), about 500 new molecules were designed with various heterocycles, from which a group of indole-based molecules consisting of 170 molecules was selected. The indole moiety was situated in the middle of some molecules and linked to different moieties through 2 amide linkages, while it was placed at the edge of other molecules as a tryptamine moiety. The linking of these moieties to various functional groups and heterocyclic rings resulted in the described number of molecules. These molecules were prepared in Maestro (Version 13.0135) by the LigPrep wizard. Crystal structures of Homo sapiens SIRT1 (4I5I), SIRT2 (5D7Q), and SIRT3 (4BV3) proteins were downloaded from the Protein Data Bank, and prepared in Maestro by the protein preparation workflow.20–22) Standard precision (SP) docking was performed using grid-based ligand docking with energetics (Glide) program for all compounds on the 3 mentioned proteins.23,24) The top compounds with the best docking scores from SP docking were selected for XP docking. The XP docking was validated by re-docking of the co-crystallized ligands followed by the calculation of RMSD.
Molecular Dynamics SimulationThe molecular dynamics simulation study was conducted using the Desmond tool within the Maestro platform. The system was constructed using the system builder wizard with the incorporation of the SPC water model and limited within an orthorhombic periodic box (10 × 10 × 10 Å) using the OPLS_2005 force field. The pH was adjusted to be neutral by the addition of (Na+ and Cl–) ions. The simulation was conducted for 100 ns with a recording of data every 50 ps at 300 K and 1 bar.25)
Pharmacokinetic Properties PredictionThe final compounds were subjected to ADME descriptors prediction using QikProp software in Schrodinger Maestro.25) Additionally, ChemDraw Professional (17.1.0.105) and Epik within Schrodinger Maestro were utilized to predict the pKa value of each compound.
Chemistry Tert-Butyl (4-((2-(1H-Indol-3-yl)ethyl)carbamoyl)benzyl)carbamate (PAF1)In a round-bottom flask, the following compounds were added: N-Boc para-amino methyl benzoic acid (N-Boc PAMBA) (2.51 g, 10 mmol, 1 equiv.), tryptamine hydrochloride (2.36 g, 12 mmol, 1.2 equiv.), DMAP (1.22 g, 10 mmol, 1 equiv.), and EDC.HCl (2.29 g, 12 mmol, 1.2 equiv.). The mixture was dissolved in 30 mL of dimethyl formamide (DMF), and then DIPEA (5.16 g, 40 mmol, 4 equiv.) was added. The mixture was stirred at room temperature for 72 h with ongoing (TLC) monitoring. Afterward, the reaction mixture was combined with a solution of distilled water (DW) and acetic acid (AA) to create a solution in the following proportions: DW 7 : reaction mixture 2 : AA 1). The precipitate formed was filtered and washed with 5% aqueous sodium carbonate solution.26) Yield: 89% (3.49 g); melting point 151–159°C; FT-IR: 3414 (indole N-H), 3317 (amide and carbamate N-H), 1697 (carbamate C=O), 1620 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.94 (s, 1H), 8.63 (t, 1H), 7.82 (d, 2H), 7.59 (d, 1H), 7.49 (t, 1H), 7.35 (d, 1H), 7.31 (d, 2H), 7.18 (s, 1H), 7.07 (t, 1H), 6.98 (t, 1H), 4.18 (d, 2H), 3.54 (m, 2H), 2.96 (t, 2H), 1.40 (s, 9H); 13C-NMR (101 MHz, DMSO) δ: 166.42, 156.31, 143.78, 136.72, 133.61, 127.75, 127.63, 127.09, 123.10, 121.36, 118.77, 118.66, 112.34, 111.87, 78.38, 43.60, 40.68, 28.70, 25.69 (Supplementary Figs. S6–S8).
N-(2-(1H-Indol-3-yl)ethyl)-4-(Aminomethyl)benzamide Hydrochloride (PAF2)In an ice bath, thionyl chloride (2.97 g, 25 mmol, 5 equiv.) was added slowly to 50 mL of absolute ethanol with continuous stirring for 30 min. Subsequently, PAF1 (1.96 g, 5 mmol, 1 equiv.) was added to the solution and stirred at room temperature for 1 h to eliminate the Boc group. PAF2, as hydrochloride salt, was self-precipitated from the solution during the stirring process. The precipitate was filtered and rinsed with toluene several times.27) Yield: 80% (1.32 g); melting point 290–292°C; FT-IR: 3390 (indole N-H), 3286 (amide N-H), 1627 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.91 (s, 1H), 8.78 (t, 1H), 8.71 (s, 3H), 7.85 (d, 2H), 7.55 (d, 1H), 7.52 (d, 2H), 7.28 (d, 1H), 7.11 (s, 1H), 6.99 (t, 1H), 6.91 (t, 1H), 3.98 (s, 2H), 3.47 (m, 2H), 2.89 (t, 2H); 13C-NMR (101 MHz, DMSO) δ: 166.05, 137.51, 136.70, 134.92, 129.23, 127.79, 127.74, 123.10, 121.33, 118.77, 118.64, 112.29, 111.88, 42.16, 40.70, 25.63 (Supplementary Figs. S9–S11).
N-(2-(1H-Indol-3-yl)ethyl)-4-((2-chloroacetamido)methyl)benzamide (PAF3)In an ice bath, PAF2 (1.32 g, 4 mmol, 1 equiv.) was dissolved in 10 mL of DMF in the presence of DIPEA (1.29 g, 10 mmol, 2.5 equiv.). After that, chloroacetyl chloride (0.68 g, 6 mmol, 1.5 equiv.) was gradually added to the solution. The reaction mixture was stirred at room temperature for 4 h and then poured into a 3.5% HCl aqueous solution. The crude precipitate was filtered and purified through column chromatography using 100% ethyl acetate as a mobile phase.28) Yield: 40% (0.59 g); melting point 165–170°C; FT-IR: 3321 (amide N-H), 3294 (amide N-H), 1639 (amide C=O), 1624 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.83 (s, 1H), 8.83 (t, 1H), 8.60 (t, 1H), 7.83 (d, 2H), 7.60 (d, 1H), 7.35 (d, 3H), 7.18 (s, 1H), 7.08 (t, 1H), 6.99 (t, 1H), 4.37 (d, 2H), 4.16 (s, 2H), 3.55 (m, 2H), 2.96 (t, 2H); 13C-NMR (101 MHz, DMSO) δ: 166.63, 166.34, 142.44, 136.71, 133.86, 127.76, 127.70, 127.47, 123.09, 121.39, 118.77, 118.70, 112.38, 111.85, 43.12, 42.66, 40.69, 25.67 (Supplementary Figs. S12–S14).
General Procedure for the Synthesis of (AFJ1–AFJ3)Compound PAF3 (0.11 g, 0.3 mmol, 1 equiv.) was dissolved separately in preheated liquid amine (30 mmol, 100 equiv.). Specifically, 2.6 g of morpholine, 2.55 g of piperidine, and 2.09 g of pyrrolidine were used to obtain AFJ1, AFJ2, and AFJ3, respectively). The mixture was then stirred for 15 min at 80°C, and the completion of the reaction was checked by TLC. The remaining liquid amine was azeotroped with toluene and evaporated. Using a gradient mobile phase of dichloromethane (DCM) and methanol (100 : 0 – 97 : 3), the crude product was purified with column chromatography.29)
N-(2-(1H-Indol-3-yl)ethyl)-4-((2-morpholinoacetamido)methyl)benzamide (AFJ1)Yield: 66% (0.083 g); melting point 167–169°C; FT-IR: 3282 (N-H), 1670 (amide C=O), 1627 (amide C=O), 1114 (C-O); 1H-NMR (400 MHz, DMSO) δ: 10.84 (s, 1H), 8.59 (t, 1H), 8.41 (t, 1H), 7.81 (d, 2H), 7.59 (d, 1H), 7.36 (d, 1H), 7.33 (d, 2H), 7.18 (s, 1H), 7.07 (t, 1H), 6.99 (t, 1H), 4.35 (d, 2H), 3.62 (t, 4H), 3.54 (m, 2H), 3.00 (s, 2H), 2.95 (t, 2H), 2.44 (t, 4H); 13C-NMR (101 MHz, DMSO) δ: 169.75, 166.37, 143.36, 136.70, 133.62, 127.75, 127.63, 127.36, 123.08, 121.38, 118.78, 118.69, 112.37, 111.85, 66.52, 62.03, 53.83, 42.08, 40.67, 25.68 (Supplementary Figs. S15–S17).
N-(2-(1H-Indol-3-yl)ethyl)-4-((2-(piperidin-1-yl)acetamido)methyl)benzamide (AFJ2)Yield: 36% (0.045 g); melting point 182–184°C; FT-IR: 3282 (N-H), 1670 (amide C=O), 1627 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.83 (s, 1H), 8.59 (t, 1H), 8.31 (t, 1H), 7.81 (d, 2H), 7.59 (d, 1H), 7.36 (d, 1H), 7.33 (d, 2H), 7.18 (s, 1H), 7.07 (t, 1H), 6.99 (t, 1H), 4.35 (d, 2H), 3.54 (m, 2H), 2.96 (d, 2H), 2.94 (s, 2H), 2.39 (t, 4H), 1.54 (m, 4H), 1.38 (p, 2H); 13C-NMR (101 MHz, DMSO) δ: 170.26, 166.36, 143.45, 136.70, 133.60, 127.75, 127.62, 127.33, 123.08, 121.38, 118.77, 118.68, 112.37, 111.85, 62.65, 54.76, 42.04, 40.68, 25.89, 25.68, 24.01 (Supplementary Figs. S18–S20).
N-(2-(1H-Indol-3-yl)ethyl)-4-((2-(pyrrolidin-1-yl)acetamido)methyl)benzamide (AFJ3)Yield: 79% (0.095 g); melting point 173–178°C; FT-IR: 3275 (N-H), 1670 (amide C=O), 1624 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.84 (s, 1H), 8.60 (t, 1H), 8.41 (t, 1H), 7.81 (d, 2H), 7.59 (d, 1H), 7.36 (d, 1H), 7.33 (d, 2H), 7.18 (s, 1H), 7.07 (t, 1H), 6.98 (t, 1H), 4.34 (d, 2H), 3.54 (m, 3H), 3.18 (s, 2H), 2.96 (t, 2H), 2.59 (t, 4H), 1.74 (t, 4H); 13C-NMR (101 MHz, DMSO) δ: 170.08, 166.37, 143.34, 136.69, 133.61, 127.74, 127.62, 127.39, 123.08, 121.38, 118.77, 118.68, 112.36, 111.85, 59.22, 54.36, 42.07, 40.67, 25.68, 23.86 (Supplementary Figs. S21–S23).
N-(2-(1H-Indol-3-yl)ethyl)-4-((2-(diethylamino)acetamido)methyl)benzamide (AFJ4)Compound PAF3 (0.11 g, 0.3 mmol, 1 equiv.) was dissolved in a mixture of preheated diethyl amine (2.21 g, 30 mmol, 100 equiv.) and 1.5 mL acetonitrile. The mixture was then refluxed for 1 h at 60°C, and the completion of the reaction was checked by TLC. After that, the remaining mixture of diethyl amine and acetonitrile was evaporated. Using a gradient mobile phase of DCM and methanol (100 : 0 – 97 : 3), the crude product was purified with column chromatography.29) Yield: 75% (0.091 g); melting point 153–155°C; FT-IR: 3286 (N-H), 1670 (amide C=O), 1624 (amide C=O); 1H-NMR (400 MHz, DMSO) δ: 10.84 (s, 1H), 8.59 (t, 1H), 8.31 (t, 1H), 7.81 (d, 2H), 7.59 (d, 1H), 7.36 (d, 1H), 7.32 (d, 2H), 7.18 (s, 1H), 7.07 (t, 1H), 6.99 (t, 1H), 4.35 (d, 2H), 3.54 (m, 2H), 3.01 (s, 2H), 2.95 (t, 2H), 2.51 (q, 4H), 0.98 (t, 6H); 13C-NMR (101 MHz, DMSO) δ: 171.53, 166.36, 143.46, 136.70, 133.58, 127.75, 127.60, 127.34, 123.08, 121.38, 118.77, 118.68, 112.38, 111.84, 57.42, 48.34, 42.07, 40.68, 25.68, 12.41 (Supplementary Figs. S24–S26).
MTT Cell Viability AssayThe MCF7 cell line was cultured in minimum essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 IU penicillin, and 100 μg streptomycin. It was then incubated in a humidified atmosphere at 37°C. Exponentially growing cells were used for experiments.30)
The MTT assay was used to evaluate the antiproliferative activity of the final compounds. The breast cancer cell line MCF7 was exposed to the final compounds and the positive control AGK2 for 72 h at different concentrations (10, 5, 2.5, 1.25, 0.62, and 0.31 μg/mL). The MTT dye solution (28 μL) was added to each well and incubated for 3 h, then DMSO (100 μL) was added to each well and incubated for 15 min.31) A microplate reader was used to measure the optical density (OD) at 492 nm, and the cytotoxicity % was calculated using the following formula:
Cytotoxicity % = (OD control – OD sample)/OD control × 100%
MIC DeterminationTwo bacterial strains were isolated and cultivated from urine samples (S. aureus and K. pneumoniae), and then samples from each strain were diluted in normal saline to produce a bacterial suspension equivalent (equiv.) to the MacFarland standard (0.5). The final compounds and ciprofloxacin were dissolved in DMSO 1 : 1 DW to obtain stock solutions with a concentration of 2000 μg/mL. In microtiter plates, 100 μL of Mueller–Hinton broth was added to each well, and then 100 μL from each prepared stock solution was transferred to the first well in a separate row and mixed with the broth to get a concentration of 1000 μg/mL. The serial dilution (2-fold dilution) was carried out by transferring 100 μL from the first well to the second one and mixing it with the existing broth. Repeating this step for each well resulted in 2-fold dilutions in the range (1000–0.48 μg/mL). Ten microliters of the bacterial suspension was added to each well. Two rows were assigned as controls, 1 containing a mixture of the solvent and broth without the bacterial suspension or tested compounds, and the other containing the same mixture with the bacterial suspension. The plates were incubated at 37 ± 1°C for 18 h, then 20 μL of resazurin dye was added to each well, and the plates were incubated again for 30 min. The change in color was observed to determine the MIC for each compound.32)
The authors would like to thank Othman Makki, Osama Hassan, Nabeel Ahmed, and Rusul Mohammed for their support in conducting chemical synthesis, column chromatography, biological studies, and molecular modeling.
The authors declare no conflict of interest.
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