Synthesis, Characterization, and Anti-TB Application of Redox-Active Ethyl Carbazate-Derivatized Phenanthroline and Its Silver Complexes

Abstract

Exploring the design and synthesis of new antibiotic compounds is important to treat multidrug-resistant bacterial infections for the already exposed drug molecules. In this work, a phenanthroline derivative, namely, 6-[2-(ethoxycarbonyl)­diazen-1-yl]-1,10-phenanthroline-5-one (ECDPO), and its mono- and bis-ligand silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) were synthesized. Single-crystal X-ray diffraction (XRD) structure of methanol-solvated ECDPO was studied, which crystallized in a monoclinic system, P2₁/n space group. ECDPO is a planar molecule, and supramolecular arrays are stabilized by various hydrogen bonding, namely, O–H···N, N–H···O, and C–H···O, and π–π interactions. The spectroscopic features of ECDPO and its silver complexes were thoroughly studied by high-resolution mass spectrometry (HRMS), IR, ¹H NMR, ¹³C NMR, UV–visible spectroscopy, and X-ray photoelectron spectroscopy (XPS). Electrochemical redox features were studied by cyclic voltammetry. The ECDPO molecule and its silver complexes were studied for antibacterial activity against Escherichia coli bacteria and Mycobacterium tuberculosis (Mtb). ECDPO shows a minimum inhibitory concentration (MIC) of 1.56 μg/mL against Mtb, which is comparable to that of one of the clinically used drug candidates, namely, ethambutol. Silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) showed enhanced anti-TB activities and MICs of 0.78 and 0.39 μg/mL, respectively.

1. Introduction

The development of new bioactive compounds remains a key research focus to address the growing need for effective treatments against bacterial infections. This demand is driven by bacterial and cellular mutations, along with the increasing drug resistance that common therapeutic drug molecules face. − Tuberculosis (TB) is an infectious disease caused by exposure to the bacterium Mycobacterium tuberculosis (Mtb). For treating the TB patients, combination drug therapy, including isoniazid, ethambutol, rifampicin, and pyrazinamide, is commonly recommended, typically administered over an extended period of 6 months to 1 year. , A major drawback of the current treatment is that prolonged exposure to these drugs can lead to undesirable side effects and the development of resistance to these existing medications, highlighting the need for new drug candidates. Bacteria can develop drug resistance to therapeutic agents either inherently or by acquiring new genetic materials through horizontal gene transfer processes, such as bacterial conjugation, transduction, and transformation from other organisms. , Drug-resistant bacterial infections can lead to a significant loss of human life and substantial economic impact worldwide. Developing new classes of compounds is essential to effectively treat drug-resistant bacteria. −

Phenanthroline (phen) is a nitrogen-containing heterocyclic compound containing two nitrogen atoms in juxtaposition suitable for metal ion binding and for forming hydrogen bonds with biomolecules. The planar molecule phen has shown interesting bioactivities against bacteria and fungi. − Functionalization of the phenanthroline backbone by substitution with alkyl, aryl, halide, and nitro groups has shown promising enhancement in anti-tuberculosis activities. The exceptional chelating properties of these bidentate nitrogen donor ligands to bioessential metal ions change homeostasis and inhibit metalloprotein-based enzymatic metabolisms, which are associated with their antimicrobial activity.

Functionalization of the phenanthroline backbone by substitution with alkyl, aryl, halide, and nitro groups has shown promising enhancement in anti-tuberculosis activities. A derivative of phen, namely, 1,10-phenanthroline-5,6-dione (PD), has more electron-donating exocyclic carbonyl groups at the fifth and sixth positions. The electrochemical redox properties as well as the antibacterial and anticancer properties of PD have been investigated. Derivatives of phenanthroline exhibit their bioactivity efficiency via disrupting the metal ion homeostasis in the cellular environment through interfering with the fundamental metal ion acquisition pathway in microorganisms as well as altering basic biological processes such as adhesion, proliferation, nutrition, invasion for infection, and survival. , Phenanthroline derivatives due to their planar aromatic nature are potentially known to interact with DNA through π-stacking intercalation, leading to DNA damage. −

Hydrazide-hydrazones are Schiff’s base compounds prepared from carboxylic acid, hydrazine, and various molecules possessing either aldehyde or ketone groups. Hydrazide-hydrazones have been studied for their potential applications as antibacterial and anticancer agents. − Hydrazide-hydrazones are also explored as anti-tuberculosis agents. − The bis­(1,10-phenanthroline)­silver­(I) acetate monohydrate shows promising bioactivity against lung cancer cells. A hybrid molecule derived from one of the first-line anti-tuberculosis drug isoniazid and 1,10-phenanthroline-5,6-dione as a class of hydrazide-hydrazones have been reported, and the study showed that the bioactivity was enhanced for the organic ligand when coordinated to silver and manganese metal ions. , Carbazate, which possesses an extra oxygen atom in comparison to hydrazide for derivatizing heterocyclic compounds, has not been much explored. −

Therapeutic agents containing metal ions in comparison to traditional organic molecular drugs possess potential advantages due to the flexibility shown by metal ions in forming varying pharmacophore geometries in addition to their redox properties and multiple mode of actions on microbial cells. , Presently, there has been significant research interest in developing silver complexes with antimicrobial properties. − Silver compounds have received considerable attention owing to their high toxicity to microorganisms while showing less toxic effects on human cells. − Because of these advantages, the synthesis of new metal complexes with new ligands is an attractive approach to develop alternative complex drug candidates.

Because of the wide usage of common antibiotic drugs, resistance toward the already exposed familiar drugs is evident in bacterial infection and in M. tuberculosis-infected patients worldwide, which necessitates the design and preparation of new drug molecules. Additional research and development of new phenanthroline derivatives could lead to the discovery of novel treatments for bacterial infections and potentially help combat the growing issue of antibiotic resistance. Hydrazones derived from alkyl hydrazines or alkyl carbohydrazines with various aldehydes or ketones have been reported. However, the hydrazone of ethoxycarbohydrazine with the heterocyclic compound phendione has not been explored. Here, we report the synthesis, spectroscopy, electrochemical, and single-crystal characterization of ethoxycarbohydrazone of phendione. In addition, synthesis and characterization of mono- and bis-ligand silver complexes of this ligand are also reported here along with the biological application of these ligand and silver complexes for anti-Escherichia coil (E. coli) and anti-Tuberculosis (TB) activities.

2. Results and Discussion

2.1. Synthesis and Characterization of the Compound ECDPO Ligand

The condensation reaction of 1 equiv of 1,10-phenanthroline-5,6-dione (PD) with 1 equiv of ethyl carbazate (EC) yielded 6-[2-(ethoxycarbonyl)­diazen-1-yl]-1,10-phenanthroline-5-one (ECDPO) as a light yellow color compound in around 90% good yield, Scheme S1.

The structure of the compound ECDPO is confirmed by HRMS, IR, and NMR studies. The high-resolution mass spectrum, HRMS-ESI­(+), shows a prominent m/z peak at 297.0956, which is assigned to the [M + H]⁺ molecular ion peak with an expected mass value for [C₁₅H₁₂N₄O₃ + H]⁺ of 297.0988. The other relatively smaller intensity m/z peaks with masses of 319.0769 and 615.1646 are assigned for formation of sodium complexes of ECDPO, such as [(C₁₅H₁₂N₄O₃)­Na]⁺ (theoretical mass 319.0807) and [(C₁₅H₁₂N₄O₃)₂Na]⁺ (theoretical mass 615.1717), respectively, Figure S1.

The solid-state neat IR spectra recorded in ATR mode of the synthesized compound ECDPO along with the precursor compounds PD and EC are displayed in Figures S2 and S3 for comparison. The carbonyl bond CO stretching band for precursor PD was observed at 1683 cm^–1. Similarly, the ester carbonyl CO bond stretching band for the other precursor molecule EC appeared at 1700 cm^–1. In ECDPO, the carbonyl CO stretching frequencies of ester group carbonyl and exocyclic carbonyl are observed at around 1764 and 1627 cm^–1, respectively. The appearance of the quinonoid exocyclic carbonyl group at a lower stretching frequency in ECDPO is attributable to the intramolecular hydrogen bonding interaction between the quinonoid exocyclic carbonyl oxygen with the NH group of the carbazate side chain. A broad IR appears at around 3380, which is assigned to the N–H stretching vibration. The aromatic and aliphatic C–H bond stretching features are observed at 3079, 3062, 2977, and 2930 cm^–1. The IR bands at 1579, 1559, 1479, and 1446 cm^–1 arise from the aromatic ring CN and CC bonds. The 1506 cm^–1 IR band of ECDPO is probably due to the partial enolic form amide CO stretching, and the corresponding band for the precursor ethyl carbazate (EC) molecule is observed at 1508 cm^–1. The 1032 and 1173 cm^–1 bands are correspondingly assigned to the single-bond C–O stretching frequencies of the ester group.

2.1.1. ¹H and ¹³C NMR of ECDPO

The ¹H NMR of ECDPO in D₆-DMSO, Figure S4, shows the ¹H NMR signal at 1.34 (t, J = 7.1 Hz, 3H) and 4.36 (q, J = 7.1 Hz, 2H) due to CH₃ and CH₂ protons, respectively, of the carbazate side chain ethyl group. The N–H proton of the carbazate side chain of the ECDPO molecule is observed at 13.91 ppm (s, 1H). The heterocyclic ring C–H protons appearing as doublet of doublets at 7.73 ppm (dd, J = 8 and 4.6 Hz, 1H) and 7.64 ppm (dd, J = 8.0 and 4.5 Hz, 1H) are assigned to C2–H and C7–H protons, respectively. The aromatic ring protons such as C3–H and C6–H appear at 8.54 ppm (dd, J = 8 and 1.8 Hz, 1H) and 8.47 ppm (dd, J = 8.2 and 1.6 Hz, 1H), respectively. The other two aromatic C–H protons adjacent to the N of the heterocyclic ring appearing at 9.08 (dd, J = 4.6 and 1.8 Hz, 1H) and 8.85 (dd, J = 4.5 and 1.6 Hz, 1H) are correspondingly assigned to C1–H and C8–H protons, respectively. The N–H proton of the carbazate side chain of the ECDPO molecule is observed at 13.91 ppm (s, 1H). This N–H in the ECDPO ligand (13.91 ppm) is comparatively more deshielded than that of EC (7.84 ppm), indicating the acidic nature or involvement of intramolecular hydrogen bonding in the ECDPO molecule (Figures S5 and S6; ¹H NMR spectra of PD and EC).

The ¹³C NMR spectrum of ECDPO measured in D₆-DMSO is illustrated in Figure S7. The carbonyl carbon of the quinonoid ring C5 appeared in the ¹³C NMR at a highly deshielded region at 181.45 ppm. The carbazate group carbonyl carbon C13 appears at 155.99 ppm. The C1 and C8 carbon atoms next to the nitrogen heterocyclic atoms appear at 151.07 and 153.18 ppm, respectively. The carbons C10 and C11 that connect the two pyridine rings are observed at 146.50 and 152.72 ppm, respectively. The other heterocyclic rings carbons C2, C3, C6, C7, C9, and C12 appear at 125.49, 132.73, 131.78, 125.17, 127.86, and 128.84 ppm, respectively. Imine carbon C4 is observed at 136.17 ppm. The peaks at 63.28 and 14.69 ppm are correspondingly assigned to the aliphatic CH₂ and CH₃ groups, respectively, of the ester ethyl group in ECDPO.

2.1.2. UV–vis Spectroscopy

The UV–vis spectra of the compound ECDPO and its precursor PD in MeCN solvent are illustrated in Figure S8. PD showed four electronic transitions at 196 nm (23,747 M^–1 cm^–1), 253 nm (35,136 M^–1 cm^–1), 294 nm (4154 M^–1 cm^–1), and 368 nm (660 M^–1cm^–1), where first three higher energy electronic transitions were assigned to π–π*-based electronic transitions, and the weak lower energy transition at 368 nm could be assigned for the n–π* electronic transition. The compounds ECDPO showed five distinct multiple electronic transitions, which are clearly unique compared to the precursor PD. The UV–vis spectra of ECDPO showed five multiple electronic transitions at 196 nm (27,713 M^–1 cm^–1), 247 nm (32,369 M^–1 cm^–1), 304 nm (12,590 M^–1 cm^–1), 337 nm (14,699 M^–1 cm^–1), and 385 nm (6606 M^–1 cm^–1). Based on their high absorbance coefficient values, these electronic transitions are assigned to intraligand π–π* electronic transitions.

2.1.3. Electrochemistry of ECDPO

Heterocyclic polyazine compounds are redox-active in nature due to their electron conjugation system. , The redox electrochemical nature of ECDPO is explored by studying cyclic voltammetry under a three-electrode setup using glassy carbon (GC) as the working electrode, Ag/AgCl as the reference electrode, and a Pt wire as the counter electrode in acetonitrile solution containing 0.1 M TBAPF₆ electrolyte. The cyclic voltammogram measured in the potential range from +2 to −2 V window at a 100 mV/s scan rate of the freshly polished GC electrode showed one oxidation event at +1.557 V and two reduction events at negative potentials −0.881 and −1.521 V (Figure S9). The one single-electron irreversible oxidation electrochemical event at +1.557 V is assigned to the formation of the one-electron-oxidized [ECDPO]¹⁺ cation. The one-electron reduction electrochemical events at negative potentials of −0.881 and −1.521 V are assigned to the reduction of the neutral molecule [ECDPO] to [ECDPO]^1– and [ECDPO]^2– anions, respectively (Scheme ).

1. Electrochemical Redox Nature of ECDPO in MeCN Solution .

^a [ECDPO]⁰, [ECDPO]¹⁺, [ECDPO]^1–, and [ECDPO]^2– are neutral, one-electron-oxidized, one-electron-reduced, and two-electron-reduced forms, respectively.

2.1.4. Single-Crystal XRD

Single crystals of ECDPO were grown as yellow blocks from the slow evaporation of methanol solution. ECDPO crystallized in a monoclinic system, P2₁/n space group, with the following unit cell parameters: a = 15.0450(3) Å, b = 13.1740(4) Å, c = 15.6436(4) Å, α = 90°, β = 93.585(2)°, γ = 90°, V = 3094.54(10) ų, and ρ_calc = 1.409 g/cm³ at T = 133 K (Table S1). Two molecules of ECDPO (C₁₅H₁₂N₄O₃) along with two methanol solvent molecules (Z′ = 2) are observed in an asymmetric unit in crystal structure solution with slightly different bonding lengths and angle parameters (Figure S10 and Tables S2 and S3). The ORTEP diagram of (ECDPO)₂(CH₃OH)₂ is illustrated in Figure .

1.

Crystal structure of the methanol-solvated ECDPO compound showing intramolecular and intermolecular hydrogen bondings. Thermal ellipsoids are drawn at a 50% probability level. CCDC 2379196.

The neutral ECDPO molecule is nearly planar in structure, including the phenanthroline ring and the carbazate group connected to the ring through a CN imine bond. For clarity, the bonding parameters for only one out of two molecules are described here (Tables S2 and S3). The planarity of the molecular structure of ECDPO is stabilized by intramolecular hydrogen bonding between the N–H of the hydrazinyl group and the exocyclic carbonyl group, N4–H4···O1 (1.90(3) Å), Figure S11. The C5O1 exocyclic carbonyl distance is 1.234(2) Å, which is slightly longer in comparison to the precursor compound PD, 1.209(3) Å. However, the cyclic ring C4–C5 bond distance of 1.481(2) Å in ECDPO is shorter in comparison to PD, 1.534(4) Å. The carbazate side chain carbonyl C13O2 bond distance is 1.201(2) Å. The imine group C4N3 and amide group C13–N4 bond lengths are 1.305(2) and 1.388(2) Å, respectively. The carbazate side chain N3―N4 bond distance of ECDPO is 1.340(2) Å, which is slightly short in comparison to that of 1.406(3) Å in ethyl carbazate (EC). Each ECDPO molecule is intermolecularly hydrogen-bonded to an individual methanol solvent molecule via the ring imine nitrogen atom and alcoholic proton with hydrogen bonding length of O4–H4A···N2 (2.17 Å) with the symmetry code (1/2 + x, 3/2 – y, 1/2 + z). The primitive crystal structure of methanol-solvated ECDPO consists of two solvated ECDPO moieties arranged in head to tail mode to form a dimeric structure stabilized through C6–H6···O101 (2.27 Å) and C106–H106···O1 (2.28 Å), forming a hydrogen bonding graph set R₂ ²(10) (Figure and Table S4). The 2D and 3D supramolecular molecular arrays are stabilized involving various C–H···O and C–H···N hydrogen bonding and π–π interactions (Figures S12 and S16).

2.2. Synthesis and Characterization of ECDPO-Coordinated Silver Complexes

Silver ions (Ag⁺) form coordination complexes with nitrogen donor ligands with varying coordination numbers based on the denticity and bulkiness of the ligands. , Silver forms [Ag­(phen)­(ONO₂)] and [Ag­(phen)₂]­(NO₃) complexes based on the stoichiometry of the bidendate phen ligands to the silver ion employed in the reaction. , Silver nitrate was reacted with 1 equiv and 2 equiv of light yellow color ligand ECDPO independently in methanol at room temperature protected from light to obtain dark yellow color silver complexes [Ag­(ECDPO)­(ONO₂)] and [Ag­(ECDPO)₂]­(NO₃), respectively (Scheme ).

2. Synthesis of [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) from the ECDPO Ligand and Silver Nitrate.

In the ESI­(+) high-resolution mass spectroscopy measurement, [Ag­(ECDPO)­(NO₃)] showed an m/z 402.9919 mass peak corresponding the [Ag­(ECDPO)]⁺ ion, which matched with the theoretical mass value of 402.9960 for the [C₁₅H₁₂N₄O₃Ag]⁺ ion (Figure S17). The C, H, and N elemental analysis of the complex [Ag­(ECDPO)­(NO₃)] showed 38.64­(% C), 2.50­(% H), and 15.00­(% N), which matched with the expected values for C₁₅H₁₂N₅O₆Ag as 38.65­(% C), 2.59­(% H), and 15.02­(% N). The silver complex [Ag­(ECDPO)₂]­(NO₃) showed an HRMS-ESI­(+) m/z value of 699.0805 attributed to the [Ag­(ECDPO)₂]⁺ ion, which very well matched with the theoretical value of 699.0869 for the (C₃₀H₂₄N₈O₆Ag)⁺ ion (Figure S18). CHN analysis of [Ag­(ECDPO)₂]­(NO₃) yielded 47.20­(% C), 3.17­(% H), and 16.50­(% N) in experimental elemental analysis, which matched with the expected 47.26­(% C), 3.17­(% H), and 16.53­(% N) for C₃₀H₂₄N₉O₉Ag.

2.2.1. IR of [(ECDPO)­Ag­(NO₃)] Complex

The infrared spectra of ECDPO and its silver complexes are compared for complex formation and structural elucidation (Figure S19). The slight shift in the IR peak of the complex when compared to ECDPO indicates the formation of [Ag­(ECDPO)­(NO₃)], and IR bands of two carbonyl functional groups, which appear at 1629 and 1765 cm^–1 in ECDPO, are found shifted to 1620 cm^–1 (quinoid ring carbonyl group (ν_CO) stretching frequency) and 1760 cm^–1 (side chain ester carbonyl group (ν_CO) stretching frequency), respectively. The IR bands at 1573, 1563, 1481, 1454, and 1442 cm^–1 are due to the CN and CC aromatic ring functional groups. The 1510 cm^–1 IR band of [Ag­(ECDPO)­(NO₃)] is assigned to the partial enolic form amide CO stretching. A slight shift of 1579 cm^–1 in the free ECDPO ligand to 1573 cm^–1 in the silver complex may indicate the coordination of aromatic imine nitrogen to the metal center. The presence of additional IR bands at 1280, 1362, and 1499 cm^–1 in [Ag­(ECDPO)­(NO₃)] suggests the coordination of nitrate in the monodentate mode to the silver center, Ag–O–NO₂. ,

2.2.2. IR of [Ag­(ECDPO)₂]­(NO₃) Complex

In the infrared spectrum of the bis-ligand silver complex [Ag­(ECDPO)₂]­(NO₃), two carbonyl stretching bands due to the ester group of the carbazate side chain and the hydrogen-bonded quinoid ring carbonyl group are, respectively, observed at 1760 and 1633 cm^–1. The IR bands at 1575, 1563, 1479, 1444, and 1417 cm^–1 are due to the CN and CC aromatic ring functional groups. A slight shift of 1579 cm^–1 in the free ECDPO ligand to 1575 cm^–1 in the silver complex may indicate the coordination of aromatic imine nitrogen to the metal center. A medium intensity IR band at 1339 cm^–1 appears for [Ag­(ECDPO)₂]­(NO₃) and corresponds to the uncoordinated nitrate ion.

2.2.3. ¹H and ¹³C NMR of the Monoligand Silver Nitrate Complex [Ag­(ECDPO)­(NO₃)]

In the ¹H NMR spectrum of the monoligand silver nitrate complex, [Ag­(ECDPO)­(NO₃)], the N–H proton of the coordinated ligand appears at 13.90 ppm as a singlet. A triplet at 1.36 ppm (3H) and a quartet at 4.39 ppm (2H) are due to the CH₃ and CH₂ protons, respectively, of the ethyl group. The six distinct proton signals of the aromatic ring protons of the coordinated ECDPO ligand in the [Ag­(ECDPO)­(NO₃)] complex are all observed at slightly downfield and deshielded chemical shift regions in comparison to those of the uncoordinated free ECDPO ligand (Table S5). 9.11 (dd, 1H) and 8.89 (dd, 1H) are, respectively, assigned for the ring C–H protons of C1 and C8, respectively, which are adjacent to coordinated nitrogen groups. The proton NMR signals at 8.78 (dd, 1H), 8.69 (dd, 1H), 7.96 (dd, 1H), and 7.89 (dd, 1H) are correspondingly assigned to C(3)–H, C(6)–H, C(2)–H, and C(7)–H ring protons, respectively (Figures and S20).

2.

¹H NMR spectra of the ECDPO ligand and [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) complexes in D₆-DMSO. For clarity, only the 9.2–7.5 ppm chemical shift range is plotted here.

The ¹³C NMR spectrum of the monoligand silver complex [Ag­(ECDPO)­(NO₃)] measured in D₆-DMSO is illustrated in Figures and S21. The carbonyl carbon of the quinonoid ring C5 appeared in the ¹³C NMR at a highly deshielded region at 180.22 ppm. The carbazate group carbonyl carbon C13 appeared at 156.15 ppm. The C1 and C8 carbon atoms next to the nitrogen heterocyclic atoms appeared at 151.68 and 153.05 ppm, respectively. Carbons C10 and C11 that connected the two pyridine rings were observed at 142.96 and 149.04 ppm, respectively. The other heterocyclic ring carbons C2, C3, C6, C7, C9, and C12 appeared at 127.26, 133.60, 132.10, 126.84, 128.81, and 130.25 ppm, respectively. Imine carbon C4 was observed at 137.92 ppm. The peaks at 63.54 and 14.69 ppm were correspondingly assigned to the aliphatic CH₂ and CH₃ groups, respectively, of the ester ethyl group of the coordinated ECDPO ligand. In the ¹H NMR spectral studies, general downfield chemical shifts were observed for coordinated ECDPO in the silver complexes in comparison to the uncoordinated free ECDPO ligand. But this generalization of chemical shift was not applicable for the ¹³C NMR spectrum, as a downfield chemical shift was observed for some carbons, while for some other carbons, an upfield shift was observed (Table S6).

3.

¹³C NMR spectra of the ECDPO ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) complexes in D₆-DMSO. For clarity, only the 160–120 ppm chemical shift range is plotted here.

2.2.4. ¹H and ¹³C NMR of the Bis-Ligand Silver Nitrate Complex [Ag­(ECDPO)₂]­(NO₃)

Similar to the monoligand silver nitrate complex, general downfield shifts are observed in the proton NMR chemical shift positions of the nine distinct proton NMR signals of the coordinated ligand in the bis-ligand silver nitrate complex [Ag­(ECDPO)₂]­(NO₃) (Figures and S22 and Table S5). The proton NMR signals at 9.10­(dd, 2H), 8.89­(dd, 2H), 8.78­(dd, 2H), 8.70­(dd, 2H), 7.95­(dd, 2H), and 7.88­(dd, 2H) ppm arise due the C(1)–H, C(8)–H, C(3)–H, C(6)–H, C(2)–H, and C(7)–H ring protons, respectively. The carbazate side chain N–H proton appears at 13.92­(s, 2H) ppm. The CH₂ and CH₃ protons of the side chain ethyl groups appear at 4.39­(q, 4H) and 1.36­(t, 6H), respectively.

The ¹³C NMR spectrum of the monoligand silver complex [Ag­(ECDPO)₂]­(NO₃) measured in D₆-DMSO is illustrated in Figures and S23. The carbonyl carbon of the quinonoid ring C5 appeared in the ¹³C NMR at a highly deshielded region at 180.29 ppm. The carbazate group carbonyl carbon C13 appears at 156.20 ppm. The C1 and C8 carbon atoms next to the nitrogen heterocyclic atoms appeared at 151.71 and 153.06 ppm, respectively. Carbons C10 and C11 that connect the two pyridine rings were observed at 143.27 and 149.37 ppm, respectively. The other heterocyclic rings carbons C2, C3, C6, C7, C9, and C12 appeared at 127.19, 133.54, 132.14, 126.77, 128.80, and 130.22 ppm, respectively. Imine carbon C4 was observed at 137.84 ppm. The peaks at 63.54 and 14.69 ppm were assigned to the aliphatic CH₂ and CH₃ groups, respectively, of the ester ethyl group of the coordinated ECDPO ligand (Table S6).

2.2.5. UV–vis Spectroscopy of ECDPO-Coordinated Silver Complexes

The UV–vis spectra of [Ag­(ECDPO)­(NO₃)] in CH₃CN showed five multiple electronic transitions, such as 197 nm (37,924 M^–1 cm^–1), 248 nm (33,441 M^–1 cm^–1), 305sh nm (13,462 M^–1 cm^–1), 336 nm (15,516 M^–1 cm^–1), and 384 nm (7369 M^–1 cm^–1). Similarly, the UV–vis spectra in MeCN of [Ag­(ECDPO)₂]­(NO₃) showed electronic transitions at 197 nm (51,780 M^–1 cm^–1), 248 nm (54,711 M^–1 cm^–1), 305 nm (21,297 M^–1 cm^–1), 336 nm (25,180 M^–1 cm^–1), and 384 nm (11,633 M^–1 cm^–1) but with an enhanced absorbance extinction coefficient due to the contribution of the apparently two coordinated ECDPO ligands in the bis-ligand coordinated silver complex (Figure and Table S7). The UV–visible spectral features of the silver complexes are distinct in comparison with the calculated addition of ECDPO ligand absorbance values and free silver nitrate absorbance values, indicating the stability of the silver complexes in solution (Figure S24). The stability of the ECDPO-bound silver complexes is also evident from the ¹H NMR and ¹³C NMR studies.

4.

UV–vis spectra of the ECDPO ligand (black line) and [Ag­(ECDPO)­(NO₃)] (red line) and [Ag­(ECDPO)₂]­(NO₃) (blue line) complexes in 15 μM acetonitrile solutions.

2.2.6. DFT Analysis of Solid-State Structures of ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺

Density functional theory (DFT) molecular modeling studies were performed using the B3LYP exchange–correlation functional to study the stability and geometries of neutral [Ag­(ECDPO)­(NO₃)] complex and [Ag­(ECDPO)₂]⁺ cation systems and compared them with those of the uncoordinated ECDPO organic ligand. We generated the initial guess geometry using GaussView software, and then optimized the geometry using DFT methods. We considered both the planar and tetrahedral geometries as initial guesses with respect to the Ag atom for all of the Ag complexes ([Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]⁺). However, we found that the tetrahedral geometries with respect to the Ag atom are energetically favorable for these two Ag complexes. In [Ag­(phen)₂]⁺ and [Ag­(py-pz)₂]⁺, (where phen and py-pz are bidentate nitrogen donating ligands similar to ECDPO), which are also crystallographically reported to adopt tetrahedral arrangements around the four-coordinated Ag⁺ center. ,, Optimized geometries of ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺ are presented in Figure . The silver complexes adopt distorted tetrahedral geometries with dihedral angles between chelating ligands of around 88 and 86° for [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]⁺, respectively. In the [Ag­(ECDPO)­(NO₃)] complex, silver is four-coordinated with bidentate mode binding of both ECDPO and nitrate ion. ECDPO is bound to silver through the two ring nitrogen atoms, and nitrate is bound to silver through the two oxygen atoms. For [Ag­(ECDPO)­(NO₃)], the two Ag–N bond lengths are 2.391 and 2.405 Å, and the corresponding two Ag–O bond lengths are 2.365 and 2.512 Å. The bond angles N–Ag–N and O–Ag–O are 70.11 and 53.11°, respectively. For [Ag­(ECDPO)₂]⁺, the Ag–N bond lengths are 2.392, 2.401, 2.411, and 2.416 Å with an average Ag–N bond distance of 2.405 Å. The N–Ag–N bidentate chelation bond angles are 69.81 and 69.85°. The intraligand N–Ag–N bond angles are 129.36, 130.96, 131.85, and 136.56° with an average N–Ag–N value of 132.18°. For ECDPO ligand, the energies of the HOMO and LUMO levels are −6.46 and −2.99 eV, respectively. For the monoligand complex [Ag­(ECDPO)­(NO₃)], the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energies are −6.31 and −3.19 eV, respectively, where the HOMO is centered on the silver nitrate part and LUMO is centered on the organic ligand ECDPO part. Similarly, HOMO and LUMO energies for the bis-ligand complex [Ag­(ECDPO)₂]⁺ are −6.70 and −3.22 eV, respectively. For [Ag­(ECDPO)₂]⁺, HOMO is centered on both coordinated ECDPO along with the bound silver, but LUMO is doubly degenerated centered on ECDPO ligands.

5.

Optimized geometry, highest occupied (HOMO), and lowest unoccupied (LUMO) molecular orbitals of the ECDPO ligand, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺.

2.2.7. Electrochemistry of the ECDPO Ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) Complexes

The cyclic voltammetry (CV) studies are performed for the ECDPO ligand and its silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) in DMSO solvent containing 0.1 M TBAPF₆ under a GC working electrode, a Ag/AgCl reference electrode, and a Pt wire counter electrode, a three-electrode setup at a 100 mV/s scan rate. The CV is performed starting from 0 to +0.8 to −2.0 to +0.8 V and to 0 V cyclic voltage scan conditions (Figure and Table ). For the initial 0 to +0.8 V voltammetry scan, no redox events were observed. The free ECDPO ligand showed two reduction events at −0.888 and −1.645 V due to the electroreduction of the neutral [ECDPO] molecule to one-electron-reduced [ECDPO]^1– and two-electron-reduced [ECDPO]^2– anion formation, respectively. The electro-reduced [ECDPO] tend to adsorb on the working electrode surface, which results in oxidation of adsorbed electro-reduced species at around −0.090 V. For the [Ag­(ECDPO)­(NO₃)] complex, an additional reduction event at −0.234 V occurred due to the Ag⁺ to Ag⁰ reduction event followed by the two coordinated ligand-based electrochemical reduction events at −0.779 and −1.509 V. Coordination of ECDPO to the silver center drive the reduction of ECDPO to corresponding [ECDPO]^1– and [ECDPO]^2– easier at less negative potentials in comparison reduction of uncoordinated free ligand. The anodic oxidation event at +0.474 V in the CV of [Ag­(ECDPO)­(NO₃)] is due to the reoxidation of Ag⁰ to Ag⁺ oxidation, as this event is not present in the uncoordinated free ECDPO ligand alone. For the [Ag­(ECDPO)₂]­(NO₃) complex, Ag⁺ → Ag⁰, [ECDPO]⁰ → [ECDPO]^1–, [ECDPO]^1– → [ECDPO]^2–, and Ag⁰ → Ag⁺ electrochemical events occurred at −0.224, −0.776, −1.477, and +0.386 V, respectively.

6.

Cyclic voltammograms of the ECDPO ligand (black line), [Ag­(ECDPO)­(NO₃)] (red line), and [Ag­(ECDPO)₂]­(NO₃) (blue line) in DMSO containing 0.1 M TBAPF₆ electrolyte solution at 100 mV/s.

1. Cyclic Voltammetry Based Redox Potentials and Electrochemical Events of the ECDPO Ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) Complexes in DMSO Solvents at a 100 mV/s Scan Rate .

redox events	ECDPO	[Ag(ECDPO)(NO3)]	[Ag(ECDPO)2](NO3)
Ag+ → Ag0	---	–0.234	–0.224
[ECDPO]0 → [ECDPO]1–	–0.888	–0.779	–0.776
[ECDPO]1– → [ECDPO]2–	–1.645	–1.509	–1.477
Ag0 → Ag+	---	+0.474	+0.386

^aA GC working electrode, a Ag/AgCl reference electrode, and a Pt wire counter electrode were used with a 0.1 M TBAPF₆ electrolyte.

2.2.8. XPS of the ECDPO Ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) Complexes

The broadband survey scan XPS spectra of the ECDPO ligand and its mono- and bis-ligand coordinated silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) are illustrated in Figure . The ECDPO ligand showed XPS peaks assignable to the presence of C 1s, N 1s, and O 1s. In the XPS spectra of the ECDPO ligand and its silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃), additional XPS peaks corresponding to Ag 3d_3/2 and Ag 3d_5/2 are observed in addition to the features corresponding to the presence of C 1s, N 1s, and O 1s binding energy peaks. Narrow-scan XPS spectra in the binding energy region of Ag 3d and N 1s are explored to learn the effect of ECDPO ligand coordination to the silver metal ion center.

7.

XPS survey scan spectra of the ECDPO ligand, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃).

In the Ag 3d XPS of silver nitrate, the peaks at binding energies of 368.68 and 374.68 eV with an expected Δmetal of 6 eV are observed, which are assignable to the Ag 3d_5/2 and Ag 3d_3/2 core electron binding energies, respectively (Figure ). For the monoligand silver complex [Ag­(ECDPO)­(NO₃)], Ag 3d_5/2 and Ag 3d_3/2 XPS peaks appear at 368.08 and 374.08 eV, respectively, which are 0.6 eV less than that of silver nitrate, suggesting that coordination of the ECDPO ligand to the silver ion increases the electron density at the silver metal ion center after the coordinated complex formation (Table ). A similar trend with a greater binding energy shift toward less energy is observed for the bis-ligand coordinated silver complex [Ag­(ECDPO)₂]­(NO₃). In the narrow band Ag 3d XPS of the complex [Ag­(ECDPO)₂]­(NO₃), the Ag 3d_5/2 and Ag 3d_3/2 peaks appeared at 367.38 and 373.38 eV, respectively. For comparison, the Ag 3d_5/2 XPS binding energies in AgNO₃, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) are 368.68, 368.08, and 367.38 eV, respectively. Clear binding energy shifts of −0.6 and −1.3 eV are noticeable for [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃), respectively, in comparison to silver nitrate alone. Each ECDPO ligand contributes additional electron densities around the silver ion center so that Ag 3d binding energy shifts around −0.6 eV for each additional ligand coordination to the silver center. ,

8.

Narrow band Ag 3d XPS spectra (normalized) of AgNO₃ (black line), monoligand [Ag­(ECDPO)­(NO₃)] (red line), and bis-ligand [Ag­(ECDPO)₂]­(NO₃) (blue line) silver complexes.

2. Effect of ECDPO Ligand Coordination on the Ag 3d XPS Spectra Data and Ag 3d_5/2 and Ag 3d_3/2 Binding Energy Shift in Silver Complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) in Comparison to AgNO₃ .

silver compound	Ag 3d XPS (eV)	Δmetal (eV)	Ag 3d XPS peak shift in comparison to AgNO3 (eV)
[AgNO3]	368.68 (Ag 3d5/2)	6
	374.68 (Ag 3d3/2)
[Ag(ECDPO)(NO3)]	368.08(Ag 3d5/2)	6	–0.6
	374.08 (Ag 3d3/2)		–0.6
[Ag(ECDPO)2](NO3)	367.38(Ag 3d5/2)	6	–1.3
	373.38 (Ag 3d3/2)		–1.3

The N 1s XPS spectra of the ECDPO ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) complexes along with silver nitrate are studied to understand the effect of coordination of the ligand to the silver metal ion center on the binding energies of the nitrogen atoms (Figure and Table ). In the N 1s narrow band XPS of the ECDPO ligand, two peak maxima are observed at 398.98 and 400.58 eV. The N 1s XPS of the ECDPO ligand is deconvoluted into four peaks with binding energies of 398.88, 388.18, 400.38, and 400.78 eV, which are respectively assigned to N1, N2, N3, and N4 atoms in the ECDPO molecule (Figure ). The N1 and N2 atoms are part of the heterocyclic ring system in the ECDPO molecule, atom N3 is an exocyclic imine group, and N4 is adjacent to a carbonyl group whose lone pair electron may be involved in resonance structure formation with carbonyl carbon.

11.

FE-SEM E. coli bacterial cells’ morphological images of untreated whole cells and E. coli cells treated with ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃), showing the morphological changes and cell wall disruptions induced by antibacterial compounds.

3. N 1s XPS Spectral Deconvoluted Data of the ECDPO Ligand, AgNO₃ Salt, and Silver Complexes [Ag­(ECDPO)­(ONO₂)] and [Ag­(ECDPO)₂]­(NO₃).

compounds	N 1s (eV)	N 1s (eV) deconvoluted	peak assignment	N 1s XPS peak shift with respect to uncoordinated ECDPO (eV)
PD	398.68	398.68	398.68 (ring CN)
EC	400.78	399.88	carbazate H2N–NH
		400.78	carbazate H2N–NH–CO
ECDPO	398.98	398.88	ring CN–
	400.58	398.98	ring CN–
		400.38	imine CN–NH
		400.78	amide N–NH–CO
AgNO3	406.88	406.88	nitrate
[Ag(ECDPO)(NO3)]	399.78	399.58	ring CN–	+0.7
	400.98	399.68	ring CN–	+0.7
	406.38	400.88	imine CN–NH	+0.5
		401.18	amide N–NH–CO	+0.3
		406.38	nitrate
[Ag(ECDPO)2](NO3)	399.38	399.08	ring CN–	+0.2
	400.68	399.28	ring CN–	+0.3
	405.98	400.48	imine CN–NH	+0.1
		400.88	amide N–NH–CO	+0.1
		405.98	nitrate

The monoligand silver complex [Ag­(ECDPO)­(NO₃)] showed three N 1s XPS peak maxima with binding energies of 399.78, 400.98, and 406.38 eV (Figure ). The 406.38 eV N 1s XPS peak is assigned to the coordinated nitrate group of the [Ag­(ECDPO)­(NO₃)] complex. The ligand part of N 1s XPS of [Ag­(ECDPO)­(NO₃)] is deconvoluted into 399.58, 399.68, 400.88, and 401.18 eV, which are respectively assigned to the N1, N2, N3, and N4 of the coordinated ECDPO ligand. Peak shifts of around +0.7 eV toward higher binding energies are observed for the N1 and N2 atoms of the coordinated ligand in comparison to that of the uncoordinated free ECDPO ligand.

9.

Narrow band N 1s XPS spectra of the ECDPO ligand, monoligand complex [Ag­(ECDPO)­(NO₃)], bis-ligand complex [Ag­(ECDPO)₂]­(NO₃), and AgNO₃.

The bis-ligand silver complex [Ag­(ECDPO)₂]­(NO₃) showed three N 1s XPS peak maxima with binding energies of 399.38, 400.68, and 405.98 eV (Figure ). The 405.98 eV N 1s XPS peak is assigned to the uncoordinated nitrate ion of the [Ag­(ECDPO)₂]­(NO₃) complex. The ligand part of N 1s XPS of [Ag­(ECDPO)₂]­(NO₃) is deconvoluted into 399.08, 399.28, 400.48, and 400.88 eV, which are respectively assigned for the N1, N2, N3, and N4 of the coordinated ECDPO ligand. Peak shifts of around +0.2 and +0.3 eV are observed for the N1 and N2 atoms, respectively, of the coordinated ligand in comparison to that of the uncoordinated free ECDPO ligand.

The coordination of the ligand through the nitrogen atom of the molecule through electron donation to the metal ion may influence the binding energy of N 1s core levels, which possibly may reflect a shift in N 1s binding energy toward the higher binding energy side shift. A shift of around +0.7 eV is observed for the monoligand coordinated [Ag­(ECDPO)­(NO₃)] complex, but around +0.2 to +0.3 eV shift is observed in the bis-ligand coordinated complex [Ag­(ECDPO)₂]­(NO₃), possibly due to the less demand for electrons from the individual ligands in the bis-ligand silver complex in comparison to the monoligand silver complex.

An interesting additional information may also be derived from the N 1s XPS spectra of AgNO₃, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) (Figure ). The N 1s XPS band due to the nitrate ion in AgNO₃, [Ag­(ECDPO)­(ONO₂)], and [Ag­(ECDPO)₂]­(NO₃) appeared at 406.88, 406.38, and 405.98 eV, respectively. All three oxygen atoms of the nitrate ion in silver nitrate salt are involved in coordination with silver metal ion centers, which may make the nitrogen atom of the nitrate ion in AgNO₃ relatively more deficient in electron density in comparison to the free uncoordinated nitrate ion. The silver center in [Ag­(ECDPO)­(ONO₂)] is expected to be three-coordinated, as reported for crystallographically established [Ag­(phen)­(ONO₂)], where phen = 1,10-phenanthroline. Only one out of three oxygen atoms of the nitrate ion in [Ag­(ECDPO)­(ONO₂)] is involved in coordination to the silver metal ion center. The electron density at the nitrogen of the monodentate nitrate ion in [Ag­(ECDPO)­(ONO₂)] is more than that at the nitrate ion of in AgNO₃, and hence, the N 1s XPS peak of nitrate ion in the monoligand silver complex is observed at a lower binding energy value of 406.38 eV in comparison to the nitrate ion in silver nitrate, which appeared at 406.88 eV. As the nitrate ion in the four-coordinated silver complex [Ag­(ECDPO)₂]­(NO₃) is not coordinated to any metal ion and possesses more electron density at the nitrogen in comparison to the three-coordinated [Ag­(ECDPO)­(ONO₂)] and AgNO₃, the N 1s XPS peak appears at a much lower binding energy (405.98 eV). Here, XPS may be very applicable for studying the coordination mode of nitrate ions in coordination complexes.

2.3. Antimicrobial Activity of the ECDPO Ligand and [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) Complexes

2.3.1. Anti-E. coli Bacterial Activities of ECDPO and Its [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) Complexes

The antibacterial properties of the synthesized ECDPO ligand and its mono- and bis-ligand silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) are evaluated by monitoring the E. coli bacterial cell growth, as the E. coli bacteria are found in various pathogenic health issues worldwide and are potentially known to develop drug resistance to exposed antibiotics.

The effect of different concentrations of ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) on DH5alpha E. coli bacterial growth kinetics is examined to assess the efficacy of the compounds in inhibiting bacterial growth (Figure A). The E. coli bacteria, 10⁷ cells/mL, in LB medium with an optical density of 0.04 at 600 nm (OD₆₀₀) were prepared as stock. The E. coli bacterial growth monitoring was performed using a 96-well plate containing 100 μL of final solutions containing different concentrations of compounds in the range of 0–100 μM. The cell growths were monitored at 600 nm for 8 h periods. The antibacterial activities of the ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) compounds and silver nitrate alone are evident from the reduction of E. coli growth kinetic curves, OD₆₀₀ versus time, in the presence of the different concentrations of the compounds (Figures S25–S28). The effects of the presence of the antibacterial compounds on the % inhibition as well as % survival of E. coli cells for the ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) compounds are plotted in Figure B–D, respectively. The % inhibition curves along with the concentration-dependent E. coli bacterial growth kinetics suggest that IC₅₀ values in the inhibition of E. coli growth by ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) are 20, 4, and 0.0064 μM, respectively.

$$\%\mathrm{survival}=[({\mathrm{OD}}_{\mathrm{sample}}-{\mathrm{OD}}_{\mathrm{pos}})/({\mathrm{OD}}_{\mathrm{neg}}-{\mathrm{OD}}_{\mathrm{pos}})]\times 100$$

$$\%\mathrm{inhibition}=[({\mathrm{OD}}_{\mathrm{neg}}-{\mathrm{OD}}_{\mathrm{sample}})/({\mathrm{OD}}_{\mathrm{neg}}-{\mathrm{OD}}_{\mathrm{pos}})]\times 100$$

OD_sample = OD of E. coli bacteria in the presence of PD-MC at different concentrations at 8 h.

10.

Concentrations and time-based E. coli bacterial growth/inhibition in LB medium at 37 °C and 8 h. (A) Comparison of time-based E. coli growth curves in the absence of an antibacterial agent and in the presence of the 100 μM ECDPO ligand and silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃). (B–D) Effect of the concentration of compounds ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) (0–100 μM) on the % inhibition and % survival of E. coli bacterial growth.

OD_pos = OD of E. coli bacteria in the presence of ampicillin at 8 h.

OD_neg = OD of E. coli bacteria at 8 h.

The FE-SEM images are monitored to study the influence of antibacterial agents on the morphology of the E. coli cell and for possible E. coli cell wall morphological change and disruption based on bacterial cell growth inhibition. The E. coli cell (10⁷ cells/L) and E. coli cells treated with 100 μM ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) in LB medium were independently incubated for 8 h at 37 °C. The above samples were further diluted 100 times, and 10 μL of each sample was drop-cast onto 0.13–0.16 mm thick and 9 mm × 9 mm sized microscopy cover glass plates individually and allowed to air dry. The samples were affixed to a brass stub using two-sided carbon tape and later subjected to sputter coating with 6 nm gold. Figure illustrates the FE-SEM E. coli bacterial cells’ morphological images of untreated whole cells and E. coli cells treated with ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃), showing the morphological changes and cell wall disruptions induced by antibacterial compounds. These FE-SEM studies suggest that the ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) antibacterial molecules effectively bind to the cell walls of E. coli and inhibit the multiplication of the bacterial cells, which leads to the inhibition of cell growth and induces cell death. The observed E. coli cell wall disruptions are more efficient in the bis-ligand silver complex [Ag­(ECDPO)₂]­(NO₃) in comparison to either the free ligand ECDPO or the monoligand silver complex [Ag­(ECDPO)­(NO₃)], most probably due to the enhanced amphiphilic nature of the bis-ligand silver complex, which binds and enters cell walls more efficiently.

The E. coli bacterial growth on agar plates in the absence and presence of test compounds was monitored through the naked eye, and the E. coli bacterial growth and inhibition properties of [Ag­(ECDPO)₂]­(NO₃) were determined. A preformed agar-coated Petri dish plate marked into four sections was used to monitor cell growth or inhibition. Sterile inoculation loops were used to pick E. coli (10⁷ cells/mL), and E. coli treated with 100 μM ECDPO, [Ag­(ECDPO)­(NO₃)], or [Ag­(ECDPO)₂]­(NO₃) individually were gently streaked on the surface of the agar plate in a zigzag pattern. The E. coli-streaked plate was capped and incubated at 37 °C for 24 h before capturing the picture. The E. coli bacterial colonies were found to grow more in E. coli cells alone and E. coli cells treated with compound ECDPO (Figure ). Inhibition or less growth is evident in the monoligand silver complex [Ag­(ECDPO)­(NO₃)]-treated section. Importantly, no visible cell growth and more efficient antibacterial cell growth inhibition are evident for the bis-ligand silver complex [Ag­(ECDPO)₂]­(NO₃)-treated E. coli.

12.

Photographic images of E. coli cells grown on an agar plate [A]only E. coli cells, and E. coli cells treated with [B]ECDPO, [C]–[Ag­(ECDPO)­(NO₃)], and [D]–[Ag­(ECDPO)₂]­(NO₃). The E. coli cells (10⁷ cells/mL) in LB broth in the presence of the respective compound (100 μM) are streaked on an agar plate and allowed to grow. The image is captured after incubation at 37 °C for 24 h.

2.3.2. Anti-Mycobacterial Tuberculosis (Anti-TB) Activity

Tuberculosis (TB) is an infectious disease caused by the bacterium M. tuberculosis (Mtb). Patients diagnosed with TB are typically treated with a combination drug therapy that includes isoniazid, ethambutol, rifampicin, and pyrazinamide. However, a significant drawback of this current treatment is that administering and exposure to these drugs over a prolonged period, usually lasting for six months to one year, can lead to adverse side effects and the development of drug resistance. Consequently, there is a pressing need to develop and identify new drug molecules to address these challenges. The anti-mycobacterial activity of compounds against M. tuberculosis (H37Rv) cell strains was evaluated using the Microplate Alamar Blue Assay (MABA). In this assay, the redox indicator resazurin changes color from blue to pink, indicating bacterial growth, as shown in Figures and S29. The minimum concentration of the compound required to completely inhibit bacterial growth is defined as the minimum inhibitory concentration (MIC).

13.

Microplate alamar blue-based mycobacterial growth/inhibition assay; blue color indicates the inhibition of cell growth and pink color indicates the cell growth, for comparing the anti-TB activities of compounds ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) along with precursor compounds PD and EC and a standard drug candidate, namely, ethambutol.

The organic ligand and its silver complexes are explored for their bioactivity against M. tuberculosis (H37Rv strain; Table ). Clinically recommended isoniazide (MIC, 0.05 μg/mL), rifampicin (MIC, 0.1 μg/mL), and ethambutol (MIC, 1.56 μg/mL) are used as references for evaluating the potential anti-TB activity of the synthesized compounds. The minimum inhibitory concentration (MIC) of the organic ligand ECDPO is 1.56 μg/mL, which is a good anti-TB activity value comparable to that of the clinically used drug candidate ethambutol. We found further enhancement of the anti-TB activity of the organic ligand in the silver-coordinated complexes. The monoligand silver complex [Ag­(ECDPO)­(NO₃)] and bis-ligand silver complex [Ag­(ECDPO)₂]­(NO₃), respectively, show MIC values of 0.78 and 0.39 μg/mL, which are in comparison better than the activity of ethambutol. The enhancement of the anti-TB activity of the ECDPO after metal complex formation may be attributable to the improved amphiphilic nature of silver-coordinated complexes, which assists in the cell membrane penetrating power and results in improved anti-TB efficiencies in [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) complexes. The ethyl carbazate-functionalized phenanthroline and its silver complexes may be promising molecules for anti-TB applications.

4. Anti-Mycobacterial Tuberculosis (Anti-TB) Activity of ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃).

s.no.	compound	MIC (μg/mL)
1	PD	>25
2	EC	>25
3	ECDPO	1.56
4	[Ag(ECDPO)(NO3)]	0.78
5	[Ag(ECDPO)2](NO3)	0.39
6	isoniazide	0.05
7	rifampicin	0.1
8	ethambutol	1.56

2.3.3. Molecular Docking Studies

The compounds ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺ were docked against the key therapeutic targets of Mtb, including gyrase A, enoylacyl carrier protein reductase (InhA), and DprE1. The inhibition of gyrase A, a potential therapeutic target for Mtb, results in the repression of DNA synthesis, and a previous study identified similar phenanthroline hits against Mtb gyrase A. On the other hand, the inhibition of InhA and DprE1 has been shown to inhibit Mtb cell wall synthesis.

The results of validation of the docking protocol revealed that the RMSD values between the docked and crystal poses were 1.1042, 1.7052, and 1.7118 Å for gyrase A, InhA, and DprE1, respectively, indicating that the docking algorithm was capable of predicting the native binding pose of ligands with high accuracy (Figure S30). The docking scores and ligand efficiency values of the standards are listed in Table .

5. Comparison of the Docking Scores and Ligand Efficiency Values of the Compounds with the Corresponding Standards for Gyrase A, InhA, and DprE1 .

		standard (crystal pose)		ECDPO		[Ag(ECDPO)(NO3)]		[Ag(ECDPO)2]+
Mtb target protein	PDB ID	docking score (kcal/mol)	ligand efficiency (kcal/mol)	docking score (kcal/mol)	ligand efficiency (kcal/mol)	docking score (kcal/mol)	ligand efficiency (kcal/mol)	docking score (kcal/mol)	ligand efficiency (kcal/mol)
Gyrase A	5BS8	–11.465 (moxifloxacin)	–0.395	–3.671	–0.167	–1.200	–0.044	–2.912	–0.065
InhA	4UVI	–5.173 (Q27462162)	–0.199	–6.353	–0.289	–2.765	–0.102	–1.631	–0.036
DprE1	4NCR	–1.904 (PBTZ169)	–0.063	–4.625	–0.210	–3.544	–0.131	–2.969	–0.066

^aThe docking metrics of the compounds that were superior to those of the corresponding standards are indicated in bold face.

The results of compound docking revealed that the docking scores and ligand efficiency values of all of the compounds were superior to those of the standard (PBTZ169) for DprE1 and followed the order ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺ (Table ). However, the docking scores and ligand efficiency values of the compounds with gyrase A and InhA were inferior to those of the corresponding standards, with the exception of ECDPO with InhA (Table ). The complexes formed by the compounds with gyrase A, InhA, and DprE1 are depicted in Figures S31–S33, respectively.

Analysis of the gyrase A–ligand interactions revealed that unlike moxifloxacin, none of the compounds formed hydrogen bonds with the DNA binding cleft of Mtb gyrase A. However, similar to moxifloxacin, the compounds formed hydrophobic interactions with Arg 482, Gly 483, Thr 500, and Glu 510 of gyrase A. Additionally, ECDPO formed four pi–pi stacking interactions with the bound DNA (Figure ). [Ag­(ECDPO)­(NO₃)] also formed hydrophobic interactions with Ala 90, Ser 91, Arg 128, and Asp 461, similar to moxifloxacin (Figure ). As illustrated in Figure , ECDPO formed a single hydrogen bond with Met 98 of the binding site of InhA, whereas both [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]⁺ form a π–π stacking interaction with Phe 97 of InhA. The compounds also formed hydrophobic interactions with Gly 96, Met 98, Pro 99, and Gln 100 (Figure ). As depicted in Figure , all three compounds formed a hydrogen bond with Lys 134 of the binding site of DprE1, similar to the PBTZ169 standard, indicating that Lys 134 played a key role in ligand binding. ECDPO and [Ag­(ECDPO)₂]⁺ also formed a hydrogen bond, each with His 315 and Arg 18, respectively. Residues Gly 117, Ser 228, Phe 313, Tyr 314, Pro 316, and Val 365 of DprE1 also formed hydrophobic interactions with the three compounds.

14.

Comparative analysis of the interactions of the three compounds (ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺) and moxifloxacin (standard) with the DNA binding cleft of Mtb gyrase A. The hydrogen bonds are indicated by purple arrows, while the π–π stacking interactions are denoted by green leader lines.

15.

Comparison of the interaction profiles of the three compounds (ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺) and standard with the active site of InhA. The hydrogen bonds are indicated by purple arrows, whereas the π–π stacking interactions are denoted by green leader lines.

16.

Analysis of the interactions of the three compounds (ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]⁺) and standard (PBTZ169) with the active site of DprE1. The hydrogen bonds are indicated by purple arrows, the π–π stacking interactions are denoted by green leader lines, and the salt bridges are indicated by solid blue-purple lines.

3. Experimental Section

Chemicals 1,10-phenanthroline, potassium bromate, ethyl carbazate, silver nitrate, tetrabutylammonium hexafluorophosphate (TBAPF₆), and HPLC-grade solvents were procured from commercial vendors. 1,10-Phenanthroline-5,6-dione was prepared following the literature room-temperature method.

CHN analysis was performed using an Unicube (Elementar). Detection of high-resolution mass (HRMS, m/z) was performed using an Agilent 6546 Q-TOF LC/MS in ESI mode. Spectroscopic studies were done using a UV–vis–NIR spectrophotometer, UV-670 (190–3200 nm) from Jasco. IR stretching frequencies were recorded in the Alpha II, Bruker, ATR mode (500–4000 cm^–1). A nuclear magnetic resonance Bruker AV NEO (400 MHz) was used to elucidate the structure of the molecule by ¹H NMR (400 MHz) and ¹³C NMR (100 MHz).

Antibacterial studies with E coli media used Miller Luria Bertani Broth (LB broth) brought from HiMedia, and for recording OD, the instrument used was a microplate reader (SpectraMax iD3, Molecular Devices, SpectraMax software).

3.1. Synthesis of ECDPO

The ethyl carbazate of phendione (ECDPO) is a yellow color crystalline compound synthesized by refluxing a clear solution of phendione (1 g, 0.00476 mol) and ethyl carbazate (0.496 g, 0.00476 mol) in a round-bottom flask. PD and EC were dissolved in 200 and 50 mL of methanol to get a clear solution, and then PD was added to EC. The reaction mixture was refluxed for 12 h under stirring. The soiled compound ECDPO was obtained by rota-evaporation and recrystallized in methanol, 1.27 g, yield = 90%. Melting point: 145 °C (decomposition to a dark solid). ECDPO, C ₁₅ H ₁₂ N ₄ O ₃ molecular weight 296.28 (theoretical). Mass spectroscopy: [M + H]⁺ (C₁₅H₁₂N₄O₃): 297 (experimental) matches the theoretical value of 297.29 (theoretical). [M + Na]⁺ (C₁₅H₁₅N₄O₃Na): 319 (experimental), 319.27 (theoretical). [2M + Na]⁺ (C₃₀H₁₄N₈O₆Na): 615 (experimental), 615.55 (theoretical). HRMS-ESI­(+): [M + H]⁺ (C₁₅H₁₂N₄O₃): 297.0983 (experimental), 297.0988 (theoretical) and [2M + Na]⁺ (C₃₀H₁₄N₈O₆Na): 615.1711 (experimental), 615.1717 (theoretical) match the theoretical value. The IR spectroscopy of ECDPO had a peak at 1633 cm^–1, resembling carbonyl stretch (CO) phendione, which was shifted to a lower wavenumber compared to the precursor PD from 1683 cm^–1. A new peak at 1759 cm^–1 was assigned for ester carbonyl and CO. The peaks at 1581, 1566, 3257, and 3170 are assigned to ring CN and CC and N–H of carbazate, respectively. ¹ H NMR (400 MHz, D ₆ -DMSO, ppm): δ 13.91 (s, 1H), 9.08 (dd, J = 4.6, 1.8 Hz, 1H), 8.85 (dd, J = 4.5, 1.6 Hz, 1H), 8.54 (dd, J = 8.0, 1.8 Hz, 1H), 8.47 (dd, J = 8.2, 1.6 Hz, 1H), 7.73 (dd, J = 8.0, 4.6 Hz, 1H), 7.64 (dd, J = 8.2, 4.5 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2H), and 1.34 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, D ₆ -DMSO, ppm): δ 181.45, 155.99, 153.18, 152.72, 151.07, 146.50, 136.17, 132.73, 131.78, 128.84, 127.86, 125.49, 125.17, 63.28, and 14.69 ppm. CHN analysis of ECDPO (C ₁₅ H ₁₂ N ₄ O ₃ ): 60.79­(%C), 4.05­(%H), and 18.90­(%N) match with the expected 60.81­(%C), 4.08­(%H), and 18.91­(%N).

3.2. Synthesis of [Ag­(ECDPO)­(NO₃)]

Silver nitrate (0.057 g, 0.338 mmol) was dissolved in methanol (10 mL) in a silver foil-wrapped round-bottom flask (100 mL). The ECDPO ligand (0.1 g, 0.338 mmol) in methanol (50 mL) was added slowly dropwise into the silver nitrate solution with stirring at room temperature. The reaction was stirred on a magnetic stirrer for 12 h to complete complex formation. The reaction was performed under dark conditions to avoid light-induced reduction of silver. After the completion of the reaction, the solvents were removed under rota-evaporation, and a dark yellow solid was washed with tetrahydrofuran (THF) solvent to remove any unreacted ligands to obtain complex [Ag­(ECDPO)­(NO₃)] in good yield (0.140 g, 89%). Melting point: 190 °C (decomposition to a dark solid). [Ag­(ECDPO)­(NO₃)]: C ₁₅ H ₁₂ AgN ₅ O ₆ molecular weight 466.15 (theoretical). HRMS-ESI­(+): [Ag­(ECDPO)]⁺ experimental 402.9919 (C ₁₅ H ₁₂ N ₄ O ₃ Ag) ⁺ and [Ag­(ECDPO)­(CH₃CN)]⁺ 444.0227 (C ₁₇ H ₁₅ N ₅ O ₃ Ag) ⁺ match with the theoretical values of 402.9960 and 444.0226, respectively. The IR spectroscopy of [Ag­(ECDPO)­(NO₃)] had a peak at 1760 cm^–1, which is shifted compared to the ECDPO peak at 1766 cm^–1, assigned for ester carbonyl and CO, and the peak at 1620 cm^–1 in comparison with the ECDPO peak at 1627 cm^–1 resembles carbonyl stretch (CO) phendione. The UV–vis spectroscopy peaks at 208, 251, 327, and 380 nm for [Ag­(ECDPO)­(NO₃)] are shifted when compared with ECDPO 202, 248, 334, 306, and 388 nm in methanol. ¹ H NMR (400 MHz, D ₆ -DMSO, ppm): δ 13.90 (s, 1H), 9.11 (dd, J = 4.8, 1.6 Hz, 1H), 8.89 (dd, J = 4.8, 1.6 Hz, 1H), 8.78 (dd, J = 8.0, 1.6 Hz, 1H), 8.69 (dd, J = 8.4, 1.6 Hz, 1H), 7.96 (dd, J = 8.0, 4.8 Hz, 1H), 7.89 (dd, J = 8.4, 4.8 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), and 1.36 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, D ₆ -DMSO, ppm): δ 180.22, 156.15, 153.05, 151.68, 149.04, 142.96, 137.92, 133.60, 132.10, 130.25, 128.81, 127.26, 126.84, 63.54, and 14.69 ppm. CHN analysis of [Ag­(ECDPO)­(NO₃)]. 38.64 (%C), 2.50­(%H), and 15.00­(%N) match with the expected 38.65 (%C), 2.59­(%H), and 15.02­(%N).

3.3. Synthesis of [Ag­(ECDPO)₂]­(NO₃)

Silver nitrate (0.057 g, 0.338 mmol) was dissolved in methanol (10 mL) in a silver foil-wrapped round-bottom flask (100 mL). The ECDPO ligand (0.2 g, 0.675 mmol) in methanol (50 mL) was added slowly dropwise into the silver nitrate solution with stirring at room temperature. The reaction was stirred with a magnetic stirrer for 12 h to complete the complex formation. The reaction was performed in the dark to avoid light-induced reduction of silver. After the completion of the reaction, the solvents were removed under rota-evaporation, and a dark yellow solid was washed with tetrahydrofuran (THF) solvent to remove any unreacted ligands to obtain complex [Ag­(ECDPO)₂]­(NO₃) in good yield (0.230 g, 89%). Melting point: 135 °C (decomposition to a dark solid). [Ag­(ECDPO)₂]­(NO₃): C ₃₀ H ₂₄ AgN ₉ O ₉ molecular weight of 762.44 (theoretical). HRMS-ESI­(+): [Ag­(ECDPO)]⁺ experimental 699.0805 (C ₃₀ H ₂₄ N ₈ O ₆ Ag) ⁺ matches the theoretical value of 699.0869. IR spectroscopy of [Ag­(ECDPO)₂(NO₃)] had a peak at 1762 cm^–1, which shifted compared to the ECDPO peak at 1766 cm^–1 assigned to ester carbonyl and CO, and the peak at 1633 cm^–1 in comparison with the ECDPO peak at 1627 cm^–1 resembles carbonyl stretch (CO) phendione. The UV–vis spectroscopy peaks at 208, 252, 334, and 380 nm for [Ag­(ECDPO)₂]­(NO₃) shifted when compared with ECDPO 202, 248, 334, 306, and 388 nm in methanol. ¹ H NMR (400 MHz, D ₆ -DMSO, ppm): δ 13.92 (s, 2H), 9.10 (dd, J = 4.8, 1.6 Hz, 2H), 8.89 (dd, J = 4.8, 1.6 Hz, 2H), 8.78 (dd, J = 8.0, 1.6 Hz, 2H), 8.70 (dd, J = 8.4, 1.6 Hz, 2H), 7.95 (dd, J = 8.0, 4.8 Hz, 2H), 7.88 (dd, J = 8.4, 4.8 Hz, 2H), 4.39 (q, J = 7.1 Hz, 4H), and 1.36 (t, J = 7.1 Hz, 6H). ¹³ C NMR (101 MHz, D ₆ -DMSO, ppm): δ 180.29, 156.20, 153.06, 151.71, 149.37, 143.27, 137.84, 133.54, 132.14, 130.22, 128.80, 127.19, 126.77, 63.54, and 14.69 ppm. CHN analysis of [Ag­(ECDPO)₂(NO₃)]: 47.20 (%C), 3.17 (%H), and 16.50 (%N) match with the expected 47.26 (%C), 3.17­(%H), and 16.53­(%N).

3.4. Single-Crystal XRD

The crystals suitable for single-crystal XRD were grown from a saturated solution of compound ECDPO in methanol solution using the slow evaporation crystal growth method. The compound ECDPO was crystallized as light yellow color blocks. A suitable single crystal of ECDPO coated with paratone oil was selected, mounted on a goniometer using a MiTeGen fiber loop, and frozen under a cold nitrogen gas stream. The X-ray diffraction intensity data were collected at 133(2) K using a single-crystal Rigaku Oxford XtaLab Pro Kappa dual home/near diffractometer with a micro focus sealed X-ray tube, and Cu Kα radiation (λ = 1.54184 Å) was used to collect the X-ray diffraction intensity at 133(2) K. The CrysAlisPro program was used for X-ray diffraction data collection, unit cell refinement, and data processing. The SCALE ABSPACK method was implemented for absorption correction using spherical harmonics. Using Olex2–1.5, the single-crystal structure of ECDPO was solved with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization. All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atom positions were determined by geometry and refined by a riding model. CCDC 2379196 contains supplementary crystallographic data of ECDPO deposited into the Cambridge Crystallographic Data Centre.

3.5. Computational Details

Geometry optimizations of all of the compounds are carried out using density functional theory (DFT) using the B3LYP exchange–correlation functional and the 6–31G­(d) basis set as implemented in the Gaussian 09 suite of programs. A LANL2DZ basis set with effective core potential (ECP) was used for Ag atoms. Electronic excitation calculations are performed using the time-dependent DFT (TD-DFT) method in an acetonitrile (ε = 37.5) solvent. The solvent was modeled using the integral equation formalism version of the polarizable continuum model (IEFPCM). The initial guess geometries were drawn using GaussView software for geometry optimization.

3.6. Cyclic Voltammetry (CV)

Cyclic voltammetry was used to record voltammograms using an Autolab PGSTAT128N potentiostat under a three-electrode setup. Glassy carbon (GC), a platinum wire, and Ag/AgCl were used as the working electrode, counter electrode, and reference electrode, respectively. 0.1 M TBAPF₆ in acetonitrile solution was used as the electrolyte. Cyclic voltammograms were recorded at a 100 mV/s scan rate for a 4 mL solution containing around 17 mM analyte in acetonitrile solution with TBAPF₆ (0.1 M) electrolytes under a nitrogen atmosphere.

3.7. Anti-E. coli Bacterial Kinetic Growth Inhibition Studies

The antibacterial activities of the ECDPO, [Ag­(ECDPO)­(NO₃)], and [Ag­(ECDPO)₂]­(NO₃) were evaluated by studying the impact and efficiency of the antimicrobial compounds on the inhibition of the growth of the bacterial sample. E. coli bacteria DH5alpha cells as a liquid culture in LB medium were incubated overnight at 37 °C. The E. coli cells were diluted to OD₆₀₀ = 0.04, which is approximately equivalent to 10⁷ cells/mL. The bacterial growth and inhibition study was carried out in Nunclon Thermo Scientific 96-well plates containing 90 μL of the bacterial sample and 10 μL of the ECDPO, [Ag­(ECDPO)­(NO₃)], or [Ag­(ECDPO)₂]­(NO₃) sample of different concentrations (0–100 μM). The bacterial growth/inhibition was studied by monitoring OD₆₀₀ values at every 1 h interval using SpectraMax iD3, Molecular Devices, and SpectraMax software.

3.8. E. coli Bacterial Growth/Inhibition Imaging of Agar Plate

Agar powder (1 g) was added to a freshly prepared Luria–Bertani (LB) broth powder (2.5 g) in 100 mL of distilled water medium, mixed well, and autoclaved under 15 psi at 121 °C for 30–60 min. This agar solution (15 mL) was poured into a sterile Petri dish (90 mm diameter) and allowed to form an agar layer coating. The E. coli cells (10⁷ cells/mL) in LB broth in the presence of 100 μM of the respective compound, ECDPO, [Ag­(ECDPO)­(NO₃)], or [Ag­(ECDPO)₂]­(NO₃), were streaked on an agar plate in a zigzag pattern using sterile inoculation loops and allowed for cell growth. The image was captured after incubation at 37 °C for 24 h.

3.9. Scanning Electron Microscopy Images of E. coli

The morphology of the E. coli samples was analyzed by the FEI Apreo Lo Vac field emission scanning electron microscope (FE-SEM) with a DBS detector. The E. coli bacterial samples (10⁷ cells/mL) in LB medium as a control and E. coli cells treated separately with 100 μM ECDPO, [Ag­(ECDPO)­(NO₃)], or [Ag­(ECDPO)₂]­(NO₃) were incubated for 8 h at 37 °C, followed by centrifugation at 5000 rpm for 5 min, and the supernatant was collected. The supernatants were further diluted 100 times. 10 μL of individual samples were drop-cast on a microscopy glass plate of 0.13–0.16 mm thickness and 9 mm × 9 mm width size and allowed to air dry. The samples were affixed to a brass stub using two-sided carbon tape and sputter coating with 6 nm gold/silver. The samples were sputter-coated with 6 nm gold for a duration of 1 min at a current of 20 mA utilizing a Leica Ultra Microtome EM UC7 Sputter coater. The acquisition of SEM images was done using an Apreo LoVac Field Emission Scanning Electron Microscopy (FEI, Thermo Scientific) instrument attached to a DBS detector with an acceleration voltage of 20 kV.

3.10. In Vitro M. tuberculosis MABA Assay

The in vitro anti-TB activity on M. tuberculosis H37Rv was studied by the Microplate Alamar Blue Assay (MABA). Briefly, inoculum was prepared from fresh Löwenstein–Jensen (LJ) medium resuspended in 7H9–S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted to an OD₅₉₀ of 1.0, and diluted 1:20; 100 μL was used as inoculum. The ECDPO, [Ag­(ECDPO)­(NO₃)], or [Ag­(ECDPO)₂]­(NO₃) [stock solutions were thawed and diluted in 7H9–S at 4-fold the final highest concentration tested. Serial 2-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 μL of 7H9–S. A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during incubation. The plate was covered, sealed in plastic bags, and incubated at 37 °C in a normal atmosphere. After 7 days of incubation, 30 μL of alamar blue solution was added to each well, and the plate was reincubated overnight. A change in color from blue (oxidized state) to pink (reduced state) indicated the growth of bacteria, and the minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that prevented this change in color. ,

3.11. Molecular Docking: M. tuberculosis (Mtb) Targets

The structures of gyrase A, InhA, and DprE1 were retrieved from the PDB (PDB entries 5BS8, 4UVI, and 4NCR, respectively). − The protein structures were prepared using the Protein Preparation Wizard module in Schrödinger, v2021–2. The original hydrogen atoms were removed, and explicit hydrogens were added to the structures. Any missing side chains and residues were modeled using Prime, and all water molecules beyond 5 Å from the heteroatoms were removed. The optimum protonation states of asparagine, histidine, and glutamine residues and hydroxyl groups were determined at pH 7.0 using PROPKA. The proteins were finally subjected to restrained minimization using the OPLS4 force field.

3.12. Grid Preparation and Validation

The grids for molecular docking were prepared based on the standard inhibitors, moxifloxacin, Q27462162, and PBTZ169, which were bound to the active sites of gyrase A, InhA, and DprE1, respectively, in the PDB structures 5BS8, 4UVI, and 4NCR, respectively. ,, The coordinates of the center of the grid were 40.181, 2.959, and 22.371 for gyrase A; 21.597, −25.688, and 24.008 for InhA; and 18.458, −22.013, and 0.767 for DprE1. The dimensions of the cubic grids were 25, 30, and 30 Å for gyrase A, InhA, and DprE1, respectively. The grid parameters and docking protocol were validated by redocking the bound inhibitors in the crystal structures. Typically, a low RMSD value (<2.0 Å) between docked and crystal poses indicates that the docking algorithm has a high predictive accuracy in determining the native binding pose of compounds. To this end, the bound inhibitors were redocked using Glide XP, and the RMSD between the docked conformation and crystal pose was determined to validate the predictive accuracy of the docking algorithm. The compounds were screened against the Mtb targets using Glide XP and scored using the XP empirical score. The protein–ligand interactions were visualized using Maestro v12.8.

4. Conclusions

In this work, 6-[2-(ethoxycarbonyl)­diazen-1-yl]-1,10-phenanthroline-5-one (ECDPO), a derivative of bidentate ligand 1,10-phenanthroline, was synthesized and characterized using HRMS, IR, UV–vis, and ¹H NMR, and ¹³C NMR techniques. The ECDPO is redox-active in nature; cyclic voltammetry study of ECDPO reflects the one-electron oxidation event (ECDPO¹⁺/⁰) and two consecutive one-electron reduction, (ECDPO⁰/^1–) and (ECDPO^1–/2–), electrochemical events. Single crystals of methanol-solvated ECDPO are grown and analyzed to reveal the planar nature of the ECDPO molecular moieties stabilized through intramolecular hydrogen bonding along with an intermolecular hydrogen bonding interaction to the methanol solvent. The extended supramolecular 3D packing arrays of the ECDPO molecules are found stabilized via various O- and N-based hydrogen bonding networks and π–π stacking interactions. Two silver complexes of the ECDPO ligand, [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃), were synthesized and characterized by HRMS, IR, UV–vis, ¹H NMR, ¹³C NMR, and electrochemical studies. The broadband narrow-scan N 1s and Ag 3d XPS study of the ECDPO ligand and its mono- and bis-ligand silver complex was carried out, and the N 1s and Ag 3d XPS binding energy shifts due to the coordination of the ligand to the silver metal center were explored. The ECDPO ligand and its silver complexes [Ag­(ECDPO)­(NO₃)] and [Ag­(ECDPO)₂]­(NO₃) shows a promising in vitro anti-TB and anti-E. coli bioactivity. ECDPO (MIC = 1.56 μg/mL) shows a comparable MIC value to one of the medically recommended drug candidates ethambutol against M. tuberculosis. Silver complexation, [Ag­(ECDPO)­(NO₃)] (MIC 0.78 μg/mL) and [Ag­(ECDPO)₂]­(NO₃) (MIC 0.39 μg/mL), interestingly improved the antibacterial activity of the ECDPO, which is importantly better in anti-TB activity in comparison to ethambutol. The molecular docking studies and SEM morphological studies infer the inhibition of cell wall synthesis and cell wall disruption-based antimicrobial activity.

Glossary

Abbreviations

Phen

1,10-phenanthroline

PD

1,10-phenanthroline-5,6-dione

EC

ethyl carbazate

ECDPO

6-[2-(ethoxycarbonyl)­diazen-1-yl]-1,10-phenanthroline-5-one

HRMS

high-resolution mass spectroscopy

FT-IR

Fourier transform infrared spectroscopy

NMR

nuclear magnetic resonance

UV–vis

ultraviolet–visible

XRD

X-ray diffraction

CV

cyclic voltammetry

FE-SEM

field emission scanning electron microscopy

XPS

X-ray photoelectron spectroscopy

DBS

directional back scatter

TB

tuberculosis

Mtb

Mycobacterium tuberculosis

MABA

microplate alamar blue assay

E. coli

Escherichia coli

OD₆₀₀

optical density at 600 nm

LB

Luria–Bertani

Bac

bacteria

MIC

minimum inhibitory concentration

GC

glassy carbon

TBAPF₆

tetrabutylammonium hexafluorophosphate (Bu₄NPF₆)

DMSO–D₆

deuterated dimethyl sulfoxide

ORTEP

oak ridge thermal ellipsoid plot

CCDC

Cambridge Crystallographic Data Centre

IC₅₀

half-maximal inhibitory concentration

DMSO

dimethyl sulfoxide

MeCN

acetonitrile

MHz

megahertz

μM

micromolar

nM

nanomolar

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c00871.

• HRMS, FTIR, UV–vis, NMR, CV, single-crystal data, and E. coli growth plots (PDF)

R.A.: conceptualization, investigation, methodology, formal analysis, data curation, validation, writingoriginal draft, and manuscript reviewing and editing. A.N.: in vitro biological characterization, data analysis, and writingoriginal draft. J.K.: in vitro biological characterization. D.S.: conceptualization, validation, supervision, and manuscript reviewing and editing. P.K.S.: Computation study and manuscript reviewing and editing. A.G.: conceptualization, computation study, and manuscript reviewing and editing. K.R.: conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources, supervision, writingoriginal draft, and manuscript reviewing and editing.

The authors declare no competing financial interest.