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Novel Green Synthesis of Polyfunctionally Substituted Phthalazines Promoted by Visible Light, DFT Studies and Molecular Docking with Antimicrobial and Antibiofilm PotencyA
RamadanA.Mekheimer1✉Emailrmekh@yahoo.com
BasmaA.Khalifa2
ZeinabShawkyHashem3
SamarM.R.Allam1
KamalUsefSadek1
MohamedR.Eletmany4
1Chemistry Department, Faculty of ScienceMinia University61519MiniaEgypt
2Botany and Microbiology Department, Faculty of ScienceMinia University61519MiniaEgypt
3Microbiology and Immunology Department, Faculty of PharmacyMinia UniversityMiniaEgypt
4Department of Chemistry, Faculty of ScienceSouth Valley University83523QenaEgypt
Ramadan A. Mekheimer,1∗ Basma A. Khalifa,2 Zeinab Shawky Hashem,3 Samar M. R. Allam,1 Kamal Usef Sadek1 & Mohamed R. Eletmany4
1 Chemistry Department, Faculty of Science, Minia University, Minia 61519, Egypt.
2 Botany and Microbiology Department, Faculty of Science, Minia University, Minia 61519, Egypt.
3 Microbiology and Immunology Department, Faculty of Pharmacy, Minia University, Minia, Egypt.
4 Department of Chemistry, Faculty of Science, South Valley University, Qena 83523, Egypt.
∗ Corresponding author, Ramadan A. Mekheimer; Email: rmekh@yahoo.com
Abstract
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Visible-Light-Mediated synthesis is a green and efficient technique which garnered interest from organic chemists. A series of new polyfunctionally substituted phthalazine derivatives 3a-j have synthesized via the reaction of pyridazines 1a-d with arylidenes 2a-d in the presence of a catalytic amount of piperidine in abs. ethanol as a solvent and under white LED lamb irradiation in open air. All synthesized products 3a-j have screened as potential antimicrobial and antibiofilm agents. Both compounds 3g and 3j are the most promising derivatives. Compound 3g displayed outstanding activity against P. aeruginosa and K. pneumoniae with a MIC value of 3.12 and 12.5 µg/mL, respectively. Compound 3j gave a MIC value of 6.25 µg/mL for P. aeruginosa. As prospective antibiofilm agents against P. aeruginosa, compounds 3g and 3j exhibited inhibition with 81% and 75%, respectively. The geometrical structures were optimized using Density Functional Theory (DFT), frontier molecular orbitals (HOMO-LUMO), along with global reactivity descriptors, such as chemical potential, electronegativity and electrophilicity, were computed to assess the compounds' chemical reactivity and stability. Spectral characteristics were interpreted to support the theoretical findings, while Molecular Electrostatic Potential (MEP) mapping was employed to visualize charge distribution and potential reactive sites. Furthermore, topological analyses, including Electron Localization Function (ELF) and Reduced Density Gradient/Non-Covalent Interaction (RDG/NCI) analysis, offered insights into intra- and intermolecular interactions and the nature of non-covalent bonding. Molecular docking simulations were performed and revealed favorable binding affinities and key interactions within the active sites, indicating potential antimicrobial and enzyme-inhibitory properties, especially with 3j-3LD6 with binding energy of -9.5 kcal/mol.A
1. Introduction
Phthalazine derivatives are remarkably important nitrogen containing heterocycles with a significant prevalence in the contemporary drug discovering arena. This was evidenced by a high proportion of Food and Drug Administration (FDA) approved drugs.1 Phthalazines with its unique structure either natural products or designed synthetical scaffolds display a wide variety of biological activities as antidiabetic,2 vasorelaxant,3 antiallergic,4 PDE4 inhibitors,5 drug molecules like hydralazine,6,7 antitumor,8–10 zopolrestat,11 anti-asthmatic antimicrobial,12 antihypertensive13 and anti-inflammatory agent.14 Representative examples of biologically active phthalazines illustrated in (Fig. 1).
Fig. 1
Biologically active phthalazine derivatives.
Commonly, phthalazine derivatives could be synthesized by ring closure of hydrazine or arylhydrazines with dicarbonyl compounds,15,16 phthalic anhydride,17 Diels-Alder reaction,4,18 Ullmann-Goldberg and Buchwald-Hartwig cross coupling or Chan-Evans-Lam coupling.19–22 Although, these reactions show tremendous improvements, sometimes require harsh reaction conditions; use of toxic reagents and limiting accessibility to the targeted scaffolds.4,23,24 Few reports concerning the synthesis of phthalazine derivatives promoted by visible light were developed. Brachet et al.25 developed a new cascade reaction through a radical hydroamination reaction and subsequent Smiles rearrangement initiated by visible light. Same principle author et al.26 reported for the first time visible light assisted 6-exo-dig cyclization of phosphonohydrazones (derivatived from phosphoramidates) on alkynyl group. A palladium-catalyzed acylation and nucleophilic cyclocondensation with dinucleophilic reagents was developed by Satyanarayana and Suchand for the synthesis of phthalazines.27 Thus, looking for efficient, high yielding green strategy for the synthesis of polyfunctionally substituted phthalazines is highly demanded. Taking into account that the main gools of green chemistry is to minimize synthetic steps, by-products, wastes and to increase energy efficiency, as well as, utilizing green solvents and catalysts.28,29 To address such advantages, the green approaches for the synthesis of polyfunctionally substituted phthalazine scaffolds with improved and desired biological activities remaining inspired goal. Nowadays, thanks to visible light reactions, a plethora of biologically relevant heterocycles are easily synthesized.30–32
In the past, photochemical processes were dependent on ultraviolet (UV) light, which necessitated high-energy sources and specialized tools and frequently produced unpredictable and unselective results.30 A more sustainable and eco-friendly method has been proposed, nevertheless, with the invention of photocatalysts that can be triggered by low-energy photons.33 In photocatalysis, metallaphotocatalysts and organophotocatalysts use light as an energy source to propel a variety of chemical reactions. Through the use of mechanical pathways like energy transfer and single-electron transfer, photonic energy is transformed into chemical energy.34 By selectively activating particular functional groups or bonds, visible light catalysis has made a substantial advancement in this sector and made it possible to perform a variety of difficult synthetic transformations and functionalization.35
Antimicrobial therapy is facing a significant problem due to the emergence of multidrug resistance (MDR) in microbial infections, which makes many traditional treatments ineffective. One of the main processes causing this resistance is the creation of biofilms, which turns infections into chronic, hard-to-treat illnesses.36 Organized microbial communities called biofilms are encased in a self-produced extracellular polymeric substance, which protects bacteria from antibiotics and increases their survival. This allows the bacteria to stay in human tissues and fosters resistance to antimicrobial drugs. Quorum sensing (QS) is a chemical communication system that bacteria utilize to coordinate group behavior once a critical population density is reached.37
Because biofilm-related illnesses are linked to indwelling medical devices such implants and catheters, they pose a special risk in clinical settings.38 For example, two of the most important bacteria that cause device-associated infections are Staphylococcus aureus and S. epidermidis. Their capacity to create biofilms on the surfaces of devices leads to heightened resistance to antibiotics and evasion of host immune responses, which frequently results in tissue damage, chronic infections, and persistent inflammation.39,40 In addition to negatively impacting patient outcomes, this places a heavy financial strain on healthcare systems.41 Recent research has investigated different approaches that focus on biofilm inhibition in response. Significantly, heterocyclic compounds containing nitrogen have demonstrated encouraging antibiofilm action, making them viable options for the creation of innovative treatments to combat infections linked to biofilms and those that are resistant to multiple drugs.
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Several studies have reported that newly synthesized phthalazine derivatives exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacterial strains at various MIC.42–44 Glišić et al.45 previously demonstrated that phthalazine (Cu-II) complexes modulate quorum sensing signalling pathways, effectively disrupting biofilm formation in P. aeruginosa (PAO1), with an IC₅₀ of 0.97 µg/mL. More recently, they proved that phthalazine (Ag) complex exhibited broad-spectrum activity against both Gram-positive bacteria (S. aureus and E. faecalis) and Gram-negative bacteria (E. coli and P. aeruginosa PAO1), as well as the fungal pathogen C. albicans. Notably, this compound demonstrated a 7.2-fold lower MIC against P. aeruginosa PAO1 compared to its IC₅₀ value, indicating enhanced efficacy at lower concentrations.45 Also, Zaheer et al.46 reported that newly synthesized phthalazinone derivatives demonstrated notable biofilm inhibitory and antimicrobial activities, underscoring their potential in addressing multidrug-resistant pathogens. Certain derivatives exhibited strong inhibition of biofilm formation in Pseudomonas aeruginosa and Candida albicans, with IC₅₀ values in the low micromolar range. Moreover, some compounds showed significant antibacterial and antifungal activity, with MIC values comparable to those of standard antibiotics such as ciprofloxacin and fluconazole. In silico pharmacokinetic evaluations also indicated favourable oral bioavailability profiles, further supporting their promise as future therapeutic candidates.46We were previously engaged in a program aimed at the green synthesis of diverse substituted heterocycles. In continuation of our efforts to synthesize new bioactive heterocyclic compounds in our laboratory,47–51 we report herein, for the first time, a general, milder and efficient green synthesis of polyfunctionally substituted phthalazine via visible light photocatalytic reaction of methylpyridazines with ethyl 2-cyano-3-arylacrylates. Moreover, the biological evaluation reinforces the therapeutic significance of new synthesized phthalazines as promising candidates for antimicrobial and antibiofilm applications.
2. Results and discussions
2.1. Chemistry
We started our studies through the reaction of equimolar amounts of ethyl 1-(4-chlorophenyl)-5-cyano-4-methyl-6-oxo-1,6-dihydropyridazine-3-carboxylate (1a) with ethyl 3-(4-chloro-phenyl)-2-cyanoacrylate (2a) in abs. ethanol in the presence of a few drops of piperidine, as a base catalyst, promoted by convential or microwave heating at 70°C. The reactants were recovered almost unchanged even after heating for prolonged time. This is in contrast to the previously reported formation of the phthalazines by reaction of methyl-pyridazine 1 with arylidene-malononitriles.52 This could be explained by the less reactivity of ylidenic double bond in 2 as a result of weaker electron-withdrawing ability of ester function compared to cyano group.53 Delightly, performing the reaction under visible light in EtOH/pip in oxygen atmosphere for 16 h at ambient temperature afforded the corresponding diethyl 5-amino-7-(4-chlorophenyl)-4-oxo-3-(4-chlorophenyl)-3,4-dihydrophthalazine-1,6-dicarboxylate (3a) in 93% yield. Structure of 3a was established based on spectral and analytical data. Mass spectra of 3a revealed molecular ion peak [M+] at 526 (100%). 1H NMR showed two triplets at δ = 0.82 and 1.29 ppm (J = 7.2 Hz) for two esters CH3 groups, two quartets at δ = 3.99 and 4.36 ppm (J = 7.2 Hz) for two esters CH2 groups and NH2 function at δ = 7.97 ppm appeared, in addition to aromatic protons. 13C NMR further confirm the structure assigned for the reaction product (see Experimental).
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Afterwards, we examined the model reaction with different solvents such as dimethyl formamide, acetonitrile, tetrahydrofuran and dioxane. A lower yield of reaction products was obtained. Other bases such as sodium carbonate and triethylamine were tested, however, piperidine proved to be the optimal catalyst. Meanwhile, catalyst free protocol afforded no product. Signifying the crucial role of the catalyst carrying out the reaction under nitrogen atmosphere which serves as protective shield preventing air oxygen afforded a trace amount of product 3a implying its necessity for the reaction. The reaction temperature was monitored by inserting thermometer in the reaction mixture which did not exceed 25°C (room temperature) indicating a photochemical pathway rather than thermal one.A controlled experiment was conducted to clarify the reaction mechanism. A radical inhibition experiment was performed by adding 1.0 mol% of 1,4- benzoquinone, as a radical scavenger, to the reaction mixture under the same standard reaction conditions. No product was detected after 8 h exposure to visible light indicating the involvement of radical intermediate in the reaction course. It is worth mentioning that in the metal catalyst-free visible light synthesis at least one of the starting materials is able to absorb the light and activate the single electron transfer pathway generating the radical species.30
With optimizing visible light conditions, the scope and limitation of the reaction was investigated utilizing variety of pyridazines 1b-d and arylidenes 2b-d with electron-donating and electron-withdrawing aryl groups. A diversity of polyfunctionally substituted phthalazines were obtained in pure and excellent yields (Scheme 1).
Scheme 1
Synthesis of phthalazine derivatives 3a-j.
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A plausible mechanism based on control experiments and observation is proposed in Scheme 2. Firstly, pyridazine 1 with its active methyl group undergoes visible light, catalyst free, single electron transfer (SET), providing the methylene radical I which is in equilibrium with II. Visible light absorbance converted arylidene 2 to its excited state III. This was followed by coupling of radical II with III forming intermediate IV which abstracts hydrogen radial forming intermediate V which undergoes tautomerization and base catalyzed cyclization forming intermediate VII. Aromatization of VII via hydrogen cyanide loss afforded the final isolable product 3.
2.2. Biological assay
The cup-plate method was used to assess the phthalazines' antibacterial efficacy against multiresistant isolates of S. aureus, K. pneumoniae, P. aeruginosa and C. albicans. Table 1 displays the results, which showed different levels of inhibition. Compound 3g demonstrated the best activity among the compounds studied with inhibition zones of 26 mm against P. aeruginosa, 21 mm against K. pneumoniae, and 17 mm against C. albicans. Compound 3j also demonstrated significant inhibition, reaching 20 mm for K. pneumoniae and 25 mm for P. aeruginosa. With inhibition zones measuring 21–23 mm against P. aeruginosa and 18–19 mm against K. pneumoniae, 3b, 3e and 3i had moderate activity. On the other hand, 3h and 3c showed weak or no detectable activity, with inhibition zones ≤ 16 mm across all tested microorganisms. All tested strains showed least activity against S. aureus strain.
Compound | Diameter of zone of inhibition (mm) | |||
|---|---|---|---|---|
S. aureus | K. pneumoniae | P. aeruginosa | C. albicans | |
3a | 7 | 15 | 18 | 7 |
3b | 11 | 19 | 21 | 16 |
3c | 7 | 14 | 16 | 7 |
3d | 8 | 16 | 20 | 13 |
3e | 10 | 18 | 23 | 15 |
3f | 10 | 14 | 19 | 7 |
3g | 12 | 21 | 26 | 17 |
3h | 7 | 12 | 14 | 7 |
3i | 9 | 18 | 21 | 14 |
3j | 11 | 20 | 25 | 16 |
DMSO | ||||
Ciprofloxacin | 25 | 28 | 30 | |
Fluconazole | 26 | |||
* Ciprofloxacin was used as a control for bacterial strains and Fluconazole was used as control for C. albicans.
To further quantify the antimicrobial potency, the broth microdilution method was used to obtain the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) (Table 2). The findings confirmed that 3g exhibited the strongest activity, with a MIC of 3.12 µg/mL against P. aeruginosa and 12.5 µg/mL against K. pneumoniae, followed by 3j, which showed a MIC of 6.25 µg/mL for P. aeruginosa. Compounds 3b and 3e demonstrated moderate inhibitory effects, with MIC values ranging between 12.5 and 25 µg/mL for Gram-negative bacteria and C. albicans. On the other hand, 3h displayed the weakest antimicrobial activity, with MIC values exceeding 200 µg/mL across nearly all tested strains, indicating negligible inhibition. According to these results, 3g and 3j are the most promising candidates for further antimicrobial development.
Compounds | Determination MIC and MBC/MFC (µg/ml) | |||||||
|---|---|---|---|---|---|---|---|---|
S. aureus | K. pneumonia | P. aeruginosa | C. albicans | |||||
MIC | MBC | MIC | MBC | MIC | MBC | MIC | MFC | |
3a | > 200 | > 200 | 100 | > 200 | 50 | 100 | 50 | 100 |
3b | 100 | > 200 | 25 | 50 | 12.5 | 25 | 12.5 | 25 |
3c | > 200 | > 200 | 100 | > 200 | 50 | 100 | 50 | 100 |
3d | > 200 | > 200 | 50 | 100 | 25 | 50 | 25 | 50 |
3e | 100 | > 200 | 25 | 50 | 12.5 | 25 | 12.5 | 25 |
3f | > 200 | > 200 | 50 | 100 | 50 | 100 | 50 | 100 |
3g | 100 | > 200 | 12.5 | 25 | 3.12 | 6.25 | 25 | 50 |
3h | > 200 | > 200 | > 200 | > 200 | 100 | > 200 | > 200 | > 200 |
3i | > 200 | > 200 | 50 | 100 | 25 | 50 | 25 | 50 |
3j | 100 | > 200 | 12.5 | 50 | 6.25 | 25 | 25 | 50 |
Ciprofloxacin | 3.12 | 6.25 | 1.56 | 3.12 | 1.56 | 3.12 | ||
Fluconazole | 3.12 | 6.25 | ||||||
The microtiter plate method was used to measure the percentages of biofilm inhibition against biofilm forming strains S. aureus, K. pneumoniae, P. aeruginosa, and C. albicans in order to evaluate the antibiofilm activity of the phthalazine tested derivatives. The results revealed variations in antibiofilm efficacy among the compounds. Compound 3g exhibited the highest biofilm inhibition, achieving 81% against P. aeruginosa, 65% against K. pneumoniae, and 60% against C. albicans. Compound 3j also demonstrated strong activity, achieving 75% inhibition for P. aeruginosa and 72% for K. pneumoniae. These findings suggest that 3g and 3j are the most promising candidates for further exploration in antibiofilm applications, particularly against Gram-negative bacteria and fungal pathogens. Among the other active compounds, 3b and 3e showed moderate antibiofilm activities, with inhibition percentages of 55 and 52% respectively for C. albicans and inhibition levels ranging between 58 to 71% for Gram-negative bacteria. On the other hand, 3h showed no detectable antibiofilm activity against all tested strains (Table 3).
Compound | % Biofilm Inhibition | |||
|---|---|---|---|---|
S. aureus | K. pneumoniae | P. aeruginosa | C. albicans | |
3a | 17 | 43 | 48 | |
3b | 28 | 60 | 71 | 55 |
3c | 41 | 50 | ||
3d | 25 | 52 | 60 | 47 |
3e | 26 | 58 | 67 | 52 |
3f | 52 | 45 | 35 | |
3g | 35 | 65 | 81 | 60 |
3h | ||||
3i | 22 | 49 | 55 | 43 |
3j | 33 | 72 | 75 | 58 |
2.3. Theoretical studies
The designed phthalazine derivatives were subjected to comprehensive computational analysis via density functional theory (DFT) to achieve fully optimized geometrical and electronic parameters, employing the hybrid B3LYP functional.54,55
2.3.1. Geometrical structure and Frontier molecular orbitals (FMOs)
Figures 2 and 3 depict the optimized ground-state geometry of the compounds 3g and 3j, calculated at the same theoretical level. Two key structural parameters, bond lengths and angles, are discussed to gain deeper insight into its conformational behavior. The deviation from planarity in the molecule is primarily attributed to the orientation of the two aromatic rings and the outward projection of the ester group (CO2CH2CH3) from the molecular plane. The higher similarity in the structural properties makes the two studied molecules 3g and 3j, mostly carry similar geometrical parameters and hence electronic features. The presence of Cl atom in 3g structure makes its related phenyl group take the horizontal plane, intersecting the vertical plane of N1-N2. This finding is the best way to describe the far-located Cl from O1, preventing steric hindrance.
The comparison between geometrical parameters of the two compounds focuses on the bond distances of the H-bond formed with several oxygen atoms. In case of 3g, H1—O2 bond distance was estimated with a value of 2.194Å, while in 3j, the value is 2.166Å. This slight difference may be attributed to the effect of the PhNO2 group of 3j, which was classified as a strong electron-withdrawing group, resulting in further interaction of H with O atom. The hydrogen bonds of the amino groups with O1 and O5 (1.013Å and 1.006Å, respectively) are similar in both 3g and 3j. However, there is a very small difference in the NH2 bond angle (121.46o and 122.67o, for 3g and 3j, respectively). This point can also be noticed for the O4-C24-O5 angle in both compounds, where it was calculated with 122.04o for 3g, and 121.54o, for 3j.
Fig. 2
Geometrical structure of the designed compound 3g (a) labeled with, (b) bond lengths, (c) bond angles.
Fig. 3
Geometrical structure of the designed compound 3j (a) labeled with, (b) bond lengths, (c) bond angles.
To evaluate the stability and reactivity of molecular systems, analysis of frontier molecular orbitals (FMOs) is essential.56 Figs. 4 and 5 illustrate the energy distribution of key molecular orbitals—including HOMO-2, HOMO-1, HOMO, LUMO, LUMO + 1, and LUMO + 2—for the optimized structure in the gas phase. A fundamental indicator of chemical stability, the HOMO-LUMO energy gap (∆E), was calculated to be 3.755 eV for 3g, and 3.564 eV for 3j suggesting that 3g compounds possess a relatively stable electronic configuration compared with 3j. The molecular orbital distributions across all examined levels are delocalized throughout the structure, reinforcing the stability of both ground and excited electronic states. Notably, significant orbital contributions are localized around the phthalazine ring for both 3g and 3j, indicating prominent donor-acceptor interactions. These localized interactions further support the compound’s potential for intramolecular charge transfer in excited states.
Fig. 4
Energy excitation levels of 3g with energy values (eV).
Fig. 5
Energy excitation levels of 3j with energy values (eV).
2.3.2. Quantum chemical calculations
For compounds 3g and 3j, quantum chemical descriptors such as chemical potential (µ), nucleophilicity (ɛ), electrophilicity (ω), and electronegativity (χ) are vital for understanding their electronic reactivity and stability. These parameters offer insight into how each compound may behave in chemical environments, including potential interactions with other species. The chemical potential (µ) indicates the energy change associated with the addition of an electron, reflecting the compound's tendency to release or accept electrons. A higher µ (less negative value) suggests a stronger driving force for electron donation, such as in the case of 3g (-4.245 eV). Electrophilicity (ω), which quantifies a molecule’s ability to accept electron density, helps predict the compound’s behavior as an electrophile; higher ω values imply a greater capacity to interact with electron-rich species, as described for 3j with a higher value (17.636 eV). Electronegativity (χ), representing the ability of a compound to attract electrons, is another key factor in assessing its reactivity. Compound 3j can attract electrons slightly more than 3g based on the ω of each compound. In other words, these descriptors enable a comparative evaluation of compounds 3g and 3j, aiding in the prediction of their chemical behavior and guiding the design of molecules with tailored electronic properties (Table 4).
Structure | EHOMO (eV) | ELUMO (eV) | EGAP (eV) | I (eV) | A (eV) | µ (eV) | Χ (eV) | ω (eV) | Η (eV) | Σ (eV− 1) |
|---|---|---|---|---|---|---|---|---|---|---|
3g | -6.122 | -2.367 | 3.755 | 6.122 | 2.367 | -4.245 | 4.245 | 16.912 | 1.878 | 0.533 |
3j | -6.231 | -2.667 | 3.564 | 6.231 | 2.667 | -4.449 | 4.449 | 17.636 | 1.782 | 0.561 |
2.3.3. IR spectral analysis (comparing with experimental)
The vibrational infrared (IR) spectra of the studied ligands were computed to validate their optimized molecular geometries. Since quantum mechanical calculations predominantly employ harmonic oscillator models, it is essential to apply a correction factor to align the theoretical vibrational frequencies with experimental data. Table 5 presents the scaled computational frequencies alongside the corresponding experimental values for each ligand. Representative vibrational bands for the two ligands are depicted in Figs. 6 and 7. As ab initio methods typically neglect anharmonic effects, the application of a functional-specific scale factor is critical to improve the accuracy of the theoretical predictions. For both compounds, two vibrational modes observed at about 3552 cm⁻¹ and 3375 cm⁻¹ correspond to N–H stretching vibrations. A weaker absorption band around 3145 cm⁻¹ is attributed to = C–H stretching, and another at 3015 cm⁻¹ corresponds to aliphatic C–H stretching. Sharp absorption peaks assigned to C = O stretching vibrations were computed at 1729 cm⁻¹ and 1717 cm⁻¹ for both 3g and 3j, respectively. the other predicted peaks were tabulated in Table 5 for the studied compounds.
Fig. 6
Computational IR spectra of 3g.
Fig. 7
Computational IR spectra of 3j.
Functional group | Frequency B3LYP/6-311G(d,p) (cm− 1) | Frequency x scale factor (cm− 1) |
|---|---|---|
3g | ||
NH (primary) | 3674 − 3491 | 3552 − 3375 |
=C-H (aromatic) | 3253 − 3173 | 3145 − 3068 |
C-H (aliphatic) | 3118 − 3037 | 3015 − 2936 |
C = O | 1788 | 1729 |
C = C | 1649 | 1594 |
C = N | 1594 | 1541 |
C-F | 1255 | 1213 |
C-O | 1107 | 1070 |
C-Cl | 699 | 675 |
3j | ||
NH (primary) | 3673 − 3486 | 3551 − 3370 |
=C-H (aromatic) | 3251 − 3168 | 3143 − 3063 |
C-H (aliphatic) | 3118 − 3037 | 3015 − 2936 |
C = O | 1776 | 1717 |
C = C | 1663 | 1608 |
N = O | 1602 | 1549 |
C = N | 1598 | 1545 |
C-O | 1116 | 1079 |
2.3.4. UV–Vis electronic spectra by TD-DFT method
The solvation model based on Time-Dependent Density Functional Theory (TD-DFT) combined with the Conductor-like Polarizable Continuum Model (CPCM) was employed to investigate the electronic behavior of the derivatives. All TD-DFT calculations were carried out using the default parameters of the Gaussian 09 software package. The number of excited states was set to NStates = 6 to examine the lowest six electronic transitions. Figures 8 and 9 display three prominent electronic transition bands. According to the Gaussian output log file for the two designed compounds, the first strong singlet absorption for each compound corresponds to an n → π⁎ electronic transition. This transition is primarily characterized by a HOMO → LUMO excitation with a contribution of 79.7%, and 70.5% for 3g and 3j, respectively. The excitation energy values of 3.113 eV, and 2.856 eV for 3g and 3j, respectively, indicate the best electronic conjugation with further stability of 3g molecule compared with 3j. High excitation energy corresponds to absorption at a shorter wavelength, as shown in Table 6. The associated oscillator strength (f) is 0.218 for 3g, indicating a high probability of electronic excitation to the LUMO. As summarized in Table 6, subsequent transitions involve excitations such as HOMO → LUMO + 1 and HOMO–1 → LUMO, and HOMO→LUMO + 2 with comparable percentage contributions for both 3g and 3j. However, the oscillator strengths vary depending on the transition pathway and the electronic overlap between the involved molecular orbitals, reflecting differences in the likelihood of each excitation.
Fig. 8
UV–Vis electronic absorption spectra for 3g.
Fig. 9
UV–Vis electronic absorption spectra for 3j.
Spectral line number | Excitation energy (eV) | λmax (nm) | F | Type of transition | % orbital contribution |
|---|---|---|---|---|---|
3g | |||||
1 | 3.113 | 398 | 0.218 | HOMO→LUMO | 69.7 |
2 | 3.562 | 348 | 0.121 | HOMO→LUMO + 1 | 68.2 |
3 | 3.841 | 322 | 0.047 | HOMO-1→LUMO | 67.8 |
3j | |||||
1 | 2.856 | 434 | 0.005 | HOMO→LUMO | 70.5 |
2 | 3.046 | 407 | 0.358 | HOMO→LUMO + 1 | 69.9 |
3 | 3.548 | 349 | 0.097 | HOMO→LUMO + 2 | 67.9 |
2.3.5. 1H NMR and 13C NMR Spectra
The computational 1H NMR spectrum of compounds 3g and 3j, simulated using the GIAO method, Figs. 10a and 11a display characteristic chemical shifts ranging from 0 to 14 ppm, with distinct signal groupings indicating the presence of aromatic and aliphatic hydrogen environments. A prominent peak around 8.0 ppm suggests a set of equivalent aromatic protons. Additional peaks in the 3.5–4.0 ppm region point to methylene or methine protons adjacent to electronegative atoms, while signals between 1.0 and 2.0 ppm correspond to aliphatic methyl or methylene groups. The highly shifted protons to about 9.3 ppm corresponded to the NH2 group. Overall, the spectrum supports the structural presence of an aromatic ring, electronegative substituents, and aliphatic chains within compounds 3g and 3j.
Figures 10b and 11b display a simulated 13C NMR spectrum of 3g and 3j derivatives ranging approximately from 0 to 180 ppm, with multiple sharp peaks indicating distinct carbon environments. The majority of the signals appear in the aromatic and unsaturated carbon region (110–170 ppm), suggesting the presence of aromatic or conjugated structures. Signals near 60 ppm and below 20 ppm are indicative of aliphatic carbons or possibly carbon atoms attached to electronegative groups.
Fig. 10
Computational (a) 1H‑NMR and (b) 13C‑NMR spectra of 3g compound using GIAO method.
Fig. 11
Computational (a) 1H‑NMR and (b) 13C‑NMR spectra of 3j compound using GIAO method.
2.3.6. Electron localization function (ELF)
The electron localization function (ELF) provides a quantitative approach to analyzing electron pair localization, aligning with the conceptual framework of Lewis structures. It identifies regions in atomic space where electron localization is maximized, offering insights into the nature of chemical bonding and electron confinement.57 In this study, several key two-dimensional planes, O4-C24-O5, H2-N3-H3, C7-C8-C10, and C8-C7-C24, were analyzed for both 3g and 3j molecules to elucidate bonding characteristics. All atoms of interest lie within the same plane, as depicted in Figs. 12 and 13, enabling precise evaluation of electron density distribution. O4–C24–O5 plane, in both designed compounds, displays pronounced electron localization between carbon and oxygen atoms, as indicated by the concentrated red regions near O atoms and the bonding basins extending toward the central carbon, suggesting strong, polar covalent C = O or C–O bonds. The H2–N3–H3 plane shows a more diffuse ELF distribution along the N–H bonds, indicative of moderately localized bonding with potential for hydrogen bonding interactions due to the lone pair on N3. However, NH2 plane in, 3j, takes a more significant planarity with the surrounding atoms such as O1 and related carbon atoms (appear in Fig. 13b). The C7–C8–C10 plane, reveals significant ELF delocalization across the carbon atoms, with bonding basins shared between them, characteristic of conjugated or delocalized π-systems. The C8–C7–C24 plane illustrates an intermediate case where electron localization is observed in C–C bonds but with slight asymmetry in the ELF distribution, possibly due to substituent effects or hybridization differences along the C7–C24 axis. The 3g plane includes an ethyl group attached to O5, which describes a more planar structure.
Fig. 12
Electron localization function (ELF) colored map of 3g (a) O4-C24-O5, (b) H2-N3-H3, (c) C7-C8-C10, and (d) C8-C7-C24.
Fig. 13
Electron localization function (ELF) colored map of 3j (a) O4-C24-O5, (b) H2-N3-H3, (c) C7-C8-C10, and (d) C8-C7-C24.
2.3.7. Molecular electrostatic potential (MEP)
The molecular electrostatic potential (MEP) 3D map serves as a valuable tool for visualizing the distribution of electrostatic potential across a molecule, thereby highlighting regions of varying electron density. This topological descriptor is instrumental in elucidating non-covalent interactions, molecular recognition, and reactivity, as electrostatic forces predominantly govern long-range intermolecular interactions.58 In the present study, the MEP surfaces of the target compounds were analyzed using a color-coded scheme—red, orange, yellow, green, and blue-corresponding to regions of decreasing electrostatic potential. Electron-rich (nucleophilic) regions are indicated by red zones, while electron-deficient (electrophilic) regions appear in blue. The analysis revealed that oxygen atoms exhibit the highest negative electrostatic potential, indicating a strong nucleophilic character. As shown in Figs. 14 and 15, the electron-rich parts in both compounds contributed to the carbonyl (C = O) group, while NO2 is the best electron-rich group present in 3j compound. F and Cl atoms also exhibit some electronic-rich character in 3g. whereas hydrogen atoms of amino and ethyl groups represent potential electrophilic sites. The phenyl rings exhibited relatively neutral potential, with a moderate π-electron density reflected by a yellow color. This distribution suggests a propensity for nucleophilic attack by oxygen atoms on electrophilic centers, particularly hydrogen atoms of primary and secondary amines, thereby facilitating electron transfer within the molecular framework.
Fig. 14
3D-colored map of molecular electrostatic potential of 3g.
Fig. 15
3D-colored map of molecular electrostatic potential of 3j.
2.3.8. Reduced density gradient/non-covalent-interactions (RDG/NCI)
Noncovalent interactions (NCIs) within the studied molecules were characterized using the reduced density gradient (RDG) approach, which enables the visualization of weak intermolecular forces based on the electron density and its derivatives. Color-mapped RDG isosurfaces were employed to distinguish various types of NCIs.59 As shown in Figs. 16 and 17, hydrogen bond interactions are represented in blue color, van der Waals (vdW) interactions are represented in green, while steric (repulsive) interactions appear in red. The quantity sign(λ₂)ρ, derived from the product of the electron density (ρ) and the sign of the second eigenvalue (λ₂) of the Hessian matrix, was used to evaluate the nature and intensity of these interactions, particularly hydrogen bonding (H-bond). In the present analysis, prominent H-bond interactions were identified in the molecular regions between hydrogen atoms of the amino group and the adjacent oxygen atoms.
Also, significant vdW interactions are present in the cages of aromatic rings and acetate groups in both studied heterocyclic compounds. This type indicates a weak attractive force stabilizing the molecular conformation. Additional electrostatic interactions were observed between the two phenyl rings, suggesting π–π stacking or weak dispersion interactions. Steric repulsion, predominantly located inside the phenyl rings, is a result of spatial crowding within the constrained six-membered aromatic systems. However, such destabilizing interactions may be partially mitigated by favorable, strong H-bonds and electrostatic attractions elsewhere in the studied molecules, balancing the overall intermolecular interaction landscape.
Fig. 16
3D-NCI map and RDG plot of the designed compound 3g.
Fig. 17
3D-NCI map and RDG plot of the designed compound 3j.
2.4. Molecular Docking analysis
The bioactive potential of the designed ligand system was investigated through in silico molecular docking simulations.60 To evaluate the binding affinity and interaction profile, the ligand was docked into the active sites of two selected target macromolecules: S. aureus (ID:2XCT) and the key enzyme in cholesterol biosynthesis, CYP51 (ID: 3LD6). The docking simulations were performed to identify the most stable ligand–enzyme complexes and to predict the inhibitory potential of the compound. The results exported from the docking analysis of 3g and 3j against 2XCT are shown in Figs. 18 and 19. The binding affinity property is the best control to understand the docked ligand-protein stability in its predicted active site. The total binding energy for both 3g and 3j was estimated with values of -6.8 kcal/mol and − 7.7 kcal/mol, respectively. The predicted inhibition showed a similar impact of both ligands with common noncovalent interactions with several amino acids of the selected active site. The hydrogen bond interaction types (conventional and carbon-hydrogen bonds) are the strongest interaction type present within docked complex parts. GLN 1267 is the common interacting amino acid with 3g and 3j complexed against 2XCT. Besides, GLN 1095 is present within 3g and interacts with the O-acetate group. While the C-hydrogen type present in 3j with ARG 1092, and GLU 1088 increases the binding affinity of the docking score, π-cation type involves ARG 1092 in both docked ligands through the aromatic rings. Other polar contacts present with PHE 1097 for both compounds, besides PHE 1266 in the 3g-2XCT complex.
Additionally, there are other amino acid residues within both docked complexes, interacting with the surface of the molecule via van der Waals (vdW) interactions. Unfavorable donor-donor interaction (GLN 1095, Fig. 19) may destabilize the analyzed conformer within the active site, but other favorable interactions, such as H-bond types, can eliminate this unfavorable character. The higher inhibition was evaluated with human CYP51 (3LD6); the results are mapped in Figs. 20 and 21. The total binding energy for both docked conformers is estimated with values − 8.6 kcal/mol for 3g, and − 9.5 kcal/mol for 3j. Several interactions appear in this analysis involving the most proper H-bond type. While the interacting amino acids differ in their interaction type from one conformer to another, with the absence of common residues for the specific type. In case of 3g, the covalent H-bond involves ILE 450, while HIS 489 is present in the 3j predicted inhibitor. Halogen (fluorine) interaction was found with ARG 448 in 3g-3LD6, while π-sigma type is present within MET 487 of 3j-3LD6 complex, π-sulfur type present in both complexes and several other polar interactions. vdW interactions mostly cover the surface of the designed inhibitor. H-bond donor and acceptor character were mapped for each docked structure to understand the probable domains that accept and donate on the surface of the target protein. The docking results, summarized in Table 7, compare the performance of the designed predicted ligands against both target proteins, including interaction types, and total binding energies.
Fig. 18
Molecular docking simulation of the best pose 3g ligand in the predicted active sites of 2XCT, H-bond interaction with other polar contacts using 3D and 2D maps.
Fig. 19
Molecular docking simulation of the best pose 3j ligand in the predicted active sites of 2XCT, H-bond interaction with other polar contacts using 3D and 2D maps.
Fig. 20
Molecular docking simulation of the best pose 3g ligand in the predicted active sites of 3LD6, H-bond interaction with other polar contacts using 3D and 2D maps.
Fig. 21
Molecular docking simulation of the best pose 3j ligand in the predicted active sites of 3LD6, H-bond interaction with other polar contacts using 3D and 2D maps.
Model | B.E (kcal/mol) | Ligand interacted part | Residue interacted part | Type of interaction |
|---|---|---|---|---|
3g-2XCT | -6.8 | NH | GLN 1095 | Conventional H-bond |
O of the acetate group | GLN 1267 | Conventional H-bond | ||
Aromatic rings | ARG 1092 | π-cation | ||
Chloro-Phenyl ring | PHE 1266, PHE 1097, GLN 1267 | π-π T shaped, amide π-stacked, π-alkyl | ||
3j-2XCT | -7.7 | C = O | GLN 1267 | Conventional H-bond |
Ethyl group | GLU 1088 | Carbon-hydrogen bond | ||
Ethyl group | ARG 1092 | Carbon-hydrogen bond | ||
Pyridazine ring | ARG 1092 | π-cation | ||
Phenyl rings | GLN 1267, PHE 1097, ARG 1092 | π-π T shaped, amide π-stacked, π-alkyl | ||
3g-3LD6 | -8.6 | F | ILE 450 | Conventional H-bond |
F | ARG 448 | Halogen (Fluorine) | ||
Fluoro-aromatic ring | CYS 449 | π-sulfur | ||
π-electrons of aromatic rings and ethyl groups | MET 380, ILE 377, TYR 131, MET 487, TYR 145, ALA 311 | π-π T shaped, amide π-stacked, π-alkyl | ||
3j-3LD6 | -9.5 | C = O | HIS 489 | Conventional H-bond |
Pyridazine ring | MET 487 | π-sigma | ||
Aromatic rings | MET 487 | π-sulfur | ||
π-electrons of aromatic rings and ethyl groups | ILE 377, MET 378, HIS 236, TRP 239, LEU 134, PHE 234, TYR 131 | π-π T shaped, amide π-stacked, π-alkyl |
3. Experimental
A
General. Melting points were recorded with an electrothermal melting point apparatus. 1H- and 13C NMR were run with Bruker DPX instrument (400, 600 MHz) for 1H NMR and (100, 150 MHz) for 13C NMR spectrometer in DMSO-d6 as solvent with tetramethylsilane (TMS) an internal standard. Chemical shifts are expressed in δ ppm. Mass spectra were measured on a VG Autospec Q MS 30 and MS 9 (AEI) spectrometer, with electron ionization (70 eV) mode. Microanalyses were performed at the Microanalytical Data Unit, Cairo University. All reactions were monitored by thin layer chromatography (TLC) as aluminum sheets pre-coated Merck Silica gel (60F254) until the completion of the reaction. All chemicals were purchased from Aldrich or Merck companies and used without any further purification. The reaction mixture was exposed to visible light in open air using LED lamp (30w) at ambient temperature.General procedure for the synthesis of phthalazine derivatives 3a-j. To a mixture of pyridazines 1a-d (1 mmol) and arylidenes 2a-d (1 mmol) in abs. ethanol (10 mL), piperidine (30 mol%) was added. The reaction mixture was irradiated with white LED lamb (30W) in open air for 16 h. The reaction progress was monitored by TLC. The resulting solid product was collected by filtration, washed with warm EtOH and dried to afford pure samples of the products 3a-j.
Conclusion
A series of new phthalazine derivatives 3a-j was prepared and characterized with different spectroscopic and elemental activity against analyses techniques. Using Visible-Light-Mediated synthesis proved to be a simple and environmentally friendly approach with excellent yields (90–93%). The targeted compounds 3a-j experienced antimicrobial and antibiofilm activity, compound 3g gave the strongest activity, with a MIC value of 3.12 µg/mL against P. aeruginosa and 12.5 µg/mL against K. pneumoniae compared to Ciprofloxacin as a reference drug with a MIC value of 1.56 µg/mL. Compounds 3g and 3j displayed biofilm inhibition with 81% and 75%, respectively. Our protocol can pave the way for synthesis of novel heterocycles with several potential applications in pharmaceutical sciences. The DFT study reveals that 3g is electron electron-donating system slightly more than 3j with a less reactive structure (∆E = 3.755 eV). Molecular orbital and MEP analyses revealed electron-rich and electron-deficient regions, guiding predictions of possible interaction sites. Topological analyses through ELF and RDG/NCI further confirmed the presence of non-covalent interactions critical to molecular stability and function. Molecular docking studies demonstrated strong binding affinities of both compounds with Staphylococcus aureus (ID:2XCT) and human CYP51 enzyme (3LD6), suggesting potential antibacterial and enzyme-inhibitory activity. Human CYP51 enzyme was subjected to more inhibition behavior using the designed 3g (-8.6 kcal/mol) and 3j (-9.5 kcal/mol) structures.
Supplementary information
The supplementary information file contains characterization data for all compounds, and methodology for DFT and molecular docking.
A
Data Availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
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Funding:
No Funding
Electronic Supplementary Material
Below is the link to the electronic supplementary material
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Author Contribution
A,F: Reviewed the manuscript.B,C: Did the biology part and wrote it.D: Did the Org. experimental part.E: Wrote the main manuscript.
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