Design and Synthesis of Hybrid Compounds for Potential Treatment of Bacterial Co-Infections: In Vitro Antibacterial and In Silico Studies

Abstract

Background: The need for innovative therapeutic strategies to enhance patient outcomes has increased due to the rise in bacterial co-infections associated with COVID-19. Methods: In this study, ten hybrid compounds were synthesized by combining two known pharmaceutical scaffolds to enhance antibacterial activity and overcome resistance mechanisms. The synthesized compounds were evaluated for their antibacterial activity against five Gram-negative and seven Gram-positive bacterial strains. In silico pharmacokinetic and drug-likeness properties of selected active compounds (12–16, 19, 21, and 23) were predicted using the SwissADME web tool. Results: Compounds 12–16, 19, 21, and 23 demonstrated significant antibacterial activity, with compound 16 (a ciprofloxacin-containing hybrid) exhibiting the most potent effect, showing a minimum inhibitory concentration (MIC) of 7.8125 µg/mL against all tested bacterial strains. The in silico analysis revealed favorable pharmacokinetic profiles, drug-likeness, lipophilicity, and water solubility of most hybrid compounds. Discussion: The synthesized hybrid compounds exhibited enhanced antibacterial activity and desirable pharmacokinetic properties, particularly compound 16. These findings suggest the potential of these molecules in combating bacterial pathogens, especially those implicated in co-infections in COVID-19 infections. Conclusions: The study presents promising hybrid antibacterial agents with potential application as adjunct therapies for treating COVID-19-associated bacterial co-infections. Further investigation is needed, which may lead to effective treatments for managing secondary bacterial infections in viral disease contexts.

1. Introduction

The COVID-19 pandemic demonstrated the complexity of viral infections, especially with the concurrent rise in bacterial co-infections that complicate clinical outcomes [1]. Bacterial co-infections in COVID-19 patients have been linked to prolonged hospital stays, increased mortality, and an elevated need for intensive care. The risk of bacterial co-infections alongside COVID-19 continues to be a significant concern, although the WHO downgraded the status of COVID-19 in 2023 [2]. These co-infections are often caused by opportunistic bacteria, which frequently lead to respiratory complications, bloodstream infections, and sepsis, especially in immunocompromised individuals or those requiring mechanical ventilation [3]. Bacillus subtilis [4] and Enterococcus faecalis [5] have been detected in infected individuals. Giacobbe et al. [6] reported that the predominant bacterial co-pathogens in SARS-CoV-2 infections include Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae. Interestingly, Zhong et al. [7] found that Staphylococcus epidermidis was among the most common respiratory microbial taxon in critically ill patients. A study by Basnet et al. on 49 hospitalized COVID-19 patients found that 6.12% were co-infected with uropathogenic Escherichia coli (95% confidence interval: 0.59–12.83) [8]. Additionally, Pintado’s research [9] highlighted an increased risk of carbapenemase-producing Enterobacter cloacae infections in COVID-19 patients, which often present as severe nosocomial infections with high mortality rates. Koupaei et al. [10] reported a Klebsiella spp. co-infection rate of 42.91% in ICU-admitted patients, primarily involving K. oxytoca (6.7%), K. aerogenes (9.4%), and K. pneumoniae (26.81%). Overall, bacterial infections were found in 15.5% of hospitalized COVID-19 patients, with a mortality rate of 32.5% among co-infected cases. The situation is further exacerbated by the widespread use of antibiotics, which has led to increased antimicrobial resistance (AMR), rendering many standard treatments ineffective [11].

Hybrid compounds have emerged as a promising class of therapeutics that combine two or more bioactive pharmaceutical scaffolds into a single molecule, potentially enhancing antibacterial potency while minimizing resistance [12,13]. This method takes advantage of the synergistic effects of the combined pharmacophores, which could result in molecules with reduced side effects and increased biological activity [13,14,15]. These compounds can act on multiple targets within bacterial cells, thus improving efficacy and reducing the likelihood of resistance development [14,16,17]. Moreover, hybrid molecules have been shown to exhibit unique pharmacokinetic and pharmacodynamic profiles [18], offering potential advantages in managing co-infections by broadening antibacterial coverage and reducing the dosage needed for effective treatment.

This study focuses on developing hybrid compounds as potential therapeutic drugs for bacterial co-infections associated with COVID-19. The antibacterial properties of these hybrid molecules were tested against specific bacterial pathogens reported in the literature as being related to bacterial co-infections in COVID-19 patients. By strategically combining pharmacophores (in Figure 1) with known pharmacological properties, we hypothesize that these hybrid molecules will exhibit enhanced antibacterial activity, particularly against multidrug-resistant strains commonly found in co-infected COVID-19 patients. We also conducted an in silico evaluation of the synthesized hybrid compounds to predict their pharmacokinetic profiles, drug-likeness, physicochemical properties, lipophilicity, water solubility, and medicinal chemistry to gain insights into their pharmacokinetic behaviors using SwissADME. The findings from this research could provide a basis for new therapeutic options that address the bacterial challenges posed by COVID-19.

In this study, compounds 1–10, as shown in Figure 1, were explored for hybridization due to their potent therapeutic properties, such as antibacterial, antifungal, and antiviral properties. Numerous research studies have observed essential oils extracted from various aromatic and medicinal plants to have potent antibacterial activity against resistant microbial strains [19,20,21,22]. This antibacterial effect is attributed to phenolic compounds, including thymol (3), carvacrol (2), and eugenol (1), which are recognized as key constituents of most essential oils [23]. Among the antifungal drugs, the azole class, particularly fluconazole (5), is the most commonly used treatment for various forms of candidiasis. However, resistance to fluconazole (5) and its associated side effects have become significant challenges in recent years [24,25], highlighting the urgent need for novel antimicrobial molecules with a wide range of pharmacological properties and reduced toxicity. Zidovudine (6), an approved oral antiviral drug, has emerged as one of the most repurposed antiviral drugs for combating multidrug-resistant Gram-negative bacterial infections. However, its therapeutic effect is hindered by the rapid development of bacterial resistance resulting from prolonged use, overuse, or misuse, underscoring the need for alternative therapeutic options [26]. Artesunic acid (7) is a semisynthetic derivative of artemisinin, a sesquiterpene extracted from Artemisia annua L., used for centuries to cure malaria in Chinese medicine. Research over the years has demonstrated the diverse potential of artemisinin compounds in various applications, including antitumor activity, immune regulation, osteoporosis prevention, and antibacterial and antiviral effects [27,28,29,30]. These findings have expanded the clinical utility of artemisinin, making it effective against a wide range of diseases. Ciprofloxacin, a fluoroquinolone antibiotic, has been used extensively for almost thirty years to treat many diseases, such as lower respiratory tract infections, urinary tract infections, gastric infections, endocarditis, skin infections, and chronic otorrhea. Its primary mechanism of action involves inhibiting DNA replication by targeting the A subunit of DNA gyrase while also influencing cell wall components. Ciprofloxacin (8) can be taken orally or intravenously, and it reaches therapeutic concentrations in the majority of body fluids and tissues; however, it is associated with potential adverse effects, including well-documented safety concerns and black box warnings related to tendon damage and other serious risks, particularly in specific patient populations [31,32,33].

Additionally, resistance to ciprofloxacin (8) has increasingly been reported in bacterial strains such as E. coli, S. typhi, S. aureus, and P. aeruginosa [34]. Oleanolic acid (9) and ursolic acid (10) are widely occurring isomeric triterpene phytochemicals known for their diverse pharmacological properties, such as anti-inflammatory, antitumor, antivirus, antibacterial, and antidiabetic properties [35,36,37,38]. However, their limited water solubility significantly restricts their clinical application [39]. We synthesized novel hybrid molecules 12–19, 21, and 23 directly linked with the abovementioned pharmaceutical scaffolds through esterification or amidation reactions or using linkers such as succinic anhydride or chloroacetyl chloride. Most of these hybrid molecules are linked with 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine. Heterocyclic 1,3,4-oxadiazoles, known as effective bioisosteres for amides and esters, have the potential to interact with receptors through hydrogen bonding, which may significantly enhance their pharmacological activities [40]. The literature also highlights the potential pharmacological activities of oxadiazoles and their derivatives, such as antibacterial, anti-inflammatory, antifungal, and anticancer properties [40,41,42,43]. Based on all these reported findings, we synthesized and evaluated the antibacterial activity of the hybrid compounds, with most of them incorporating 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine, as shown in Scheme 1.

2. Results and Discussion

2.1. Synthesis

2.1.1. Synthesis of Hybrid Compounds 12–16

The hybrid compounds, 12–16 were successfully synthesized through a two-step reaction sequence involving the chloroacetylation of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (4) followed by nucleophilic substitution with bioactive molecules such as eugenol (1), thymol (3), carvacrol (2), and ciprofloxacin (11). These reactions proceeded with low to good yields, ranging from 29% to 60%. The structures of the synthesized hybrid molecules were confirmed using spectroscopic techniques, such as NMR, IR, and HRMS. The characteristic peaks corresponding to the functional groups provided strong evidence of successfully synthesizing the desired compounds. The eugenol-based hybrid compound 12 was obtained in a 57% yield with the presence of peaks at 1710 cm⁻¹ (C=O stretching), 3554 cm⁻¹ (N–H stretching), and 1626–1481 cm (C=C stretching) in the IR spectrum, confirming the formation of the amide linkage. The ¹HNMR spectrum displayed signals at 7.81–7.79 ppm for aromatic protons, with peaks at 6.81–6.79 ppm and 5.33 ppm corresponding to the eugenol moiety. The ¹³C-NMR spectrum displayed a carbonyl carbon (C=O) at 171.16 ppm (C14), confirming the presence of an amide functional group. The peaks at 157.07 (C10) and 150.86 (C8) indicate electron-rich aromatic carbons, likely from the oxadiazole and eugenol moieties. The HRMS data further supported the proposed molecular formula. The thymol-based hybrid 13 was obtained with a 55% yield. A strong carbonyl absorption was visible at 1735 cm⁻¹ in the IR spectrum, confirming the presence of the amide group. N–H stretching (3354 cm⁻¹) suggested the presence of an amide. C=C stretching at 1500 cm⁻¹ confirmed the presence of aromatic and alkene systems. The ¹HNMR spectrum showed characteristic signals of thymol at 7.19–7.18 ppm and 6.37–6.35 ppm, confirming the presence of the oxadiazole and thymol moieties. The ¹³CNMR spectra indicated carbonyl carbon (C=O) at 1735 cm⁻¹ and 170.31 ppm (C14), which confirmed the presence of an amide functional group. The peaks at 164.46 (C18) and 157.08 (C10) suggest electron-rich aromatic carbons from the oxadiazole and thymol moieties. The HRMS analysis provided a molecular ion peak that agrees with the calculated mass. Hybrid 14 containing carvacrol was synthesized with a 52% yield, slightly lower than the yield obtained for hybrid 13. Hybrid 14 also demonstrated similar spectral characteristics with a C=O stretching band at 1694 cm⁻¹ and N–H stretching at 3451 cm⁻¹ in the IR spectrum. The ¹HNMR spectrum exhibited signals corresponding to the carvacrol moiety, including peaks at 6.97–6.95 ppm and 6.56–6.54 ppm. The carbonyl carbon (C=O) at 171.21 ppm (C14) in the ¹³CNMR spectrum confirmed the presence of an amide functional group. The peaks at 164.48 (C18) and 157.07 (C10) indicated electron-deficient aromatic carbons, consistent with the oxadiazole and carvacrol moieties. The HRMS data validated the proposed structure. The synthesis of hybrid 15 was achieved with a relatively low yield of 29%, indicating potential challenges in the reaction efficiency or purification process. The ¹³C NMR spectrum confirmed the successful formation of the hybrid compound, with characteristic peaks for carbonyl (C=O) at 172.21 ppm and aromatic carbons ranging from 151.27 to 113.22 ppm. The ¹H NMR spectrum further supported the structure, showing distinct signals for aromatic protons and the methylene group at δ 3.75 ppm, corresponding to the chloroacetyl moiety. The IR spectrum displayed key functional groups, including N–H stretching (3544–3352 cm⁻¹), C=O stretching (1708 cm⁻¹), and aromatic C=C stretching (1623–1493 cm⁻¹), confirming the expected molecular framework. Additionally, the HRMS analysis provided a mass-to-charge ratio (m/z) of 430.0329, closely matching the calculated value of 430.0348, further validating the proposed structure. The low yield may be attributed to steric hindrance, incomplete conversion, or difficulties in purification, which could be optimized in future modifications. Hybrid 16 was successfully synthesized with a yield of 60%. The ¹³CNMR spectrum showed key signals for various carbon atoms, including the carbonyl (C=O) at 172.81 ppm, characteristic of the chloroacetyl group, and aromatic carbons ranging from 164.48 to 104.98 ppm, suggesting the presence of a complex aromatic system. The ¹H NMR spectrum further confirmed the structure, with notable peaks such as a singlet at 9.02 ppm for the amide proton (H-4), doublets at 7.81–7.80 ppm and 7.61–7.59 ppm for aromatic protons, and additional singlets and multiplets corresponding to other hydrogen atoms in the structure. The IR spectrum showed significant functional groups, including OH stretching (3302 cm⁻¹), C=O stretching (1688 cm⁻¹), and aromatic C=C stretching (1667–1557 cm⁻¹), which supported the presence of the anticipated functional groups. The HRMS analysis with a mass-to-charge ratio (m/z) of 566.1481 closely matched the calculated value (566.1521), confirming the molecular composition of hybrid 16. Overall, the spectroscopic data support the successful synthesis of the hybrid compounds, with the yields indicating efficient reactions, although optimization may be considered to improve the yields further.

2.1.2. Synthesis of Hybrid Compounds 17 and 18

The obtained yields for both molecules were moderate, with 42% for compound 17 and 38% for compound 18, indicating effective but not highly efficient conversions, likely due to steric hindrance or side reactions during amide bond formation. The structures of hybrids 17 and 18 were confirmed using various spectroscopic techniques. The IR spectra displayed characteristic peaks for NH stretching (3357 cm⁻¹) and C=O stretching (1581 cm⁻¹), confirming the amide bond formation. The ¹HNMR spectra exhibited downfield shifts at 7.81 ppm (singlet, H-35) and 5.28 ppm (triplet, H-28) for hybrid 17, and 7.95 ppm (singlet, H-36) and 5.14 ppm (triplet, H-29) for hybrid 18, corresponding to aromatic and olefinic protons, respectively. The ¹³C NMR spectra further confirmed the structures with 172.31 ppm (C=O) peaks for hybrid 17 and 177.81 ppm (C=O) for hybrid 18. The HRMS analysis validated the molecular compositions, with the observed masses matching the calculated values, indicating successful synthesis.

2.1.3. Synthesis of Hybrid Compound 19

Compound 19 was obtained with a 48% yield. The moderate yield could be attributed to possible steric effects or partial hydrolysis during purification. The IR spectrum exhibited NH stretching (3357 cm⁻¹) and C=O stretching (1709 cm⁻¹), confirming amide bond formation. The ¹H NMR spectrum showed a characteristic singlet at 8.00 ppm (H-27), corresponding to the oxadiazole moiety, while the artesunate protons appeared in the range of 2.13–0.69 ppm. The ¹³CNMR data further supported the structural integrity, showing a characteristic carbonyl signal at 178.67 ppm (C26). HRMS confirmed the similarity of the calculated molecular weight (561.1878) with the observed value (561.1902), affirming successful hybridization.

2.1.4. Synthesis of Hybrid Compound 21

Hybrid 21, a succinylated oxadiazole–zidovudine hybrid, was synthesized via a two-step process involving the formation of an N-succinylated intermediate (20) followed by coupling with zidovudine (6) using DCC/DMAP in dichloromethane. The reaction proceeded smoothly, yielding a final product that was isolated and purified using column chromatography. The IR analysis revealed NH stretching (3357 cm⁻¹) and C=O stretching (1709 cm⁻¹), supporting the formation of the desired conjugate. The ¹H NMR spectrum displayed downfield shifts at 8.00 ppm (singlet, H-27), while the zidovudine (6) protons appeared in the expected region. The ¹³C NMR spectrum confirmed the presence of characteristic carbonyl and aromatic signals, consistent with the expected structure. The HRMS analysis validated the molecular weight, confirming the successful synthesis of hybrid 21.

2.1.5. Synthesis of Hybrid Compound 23

Compound 23 was obtained with a yield of 53%, indicating moderate efficiency. The ¹³C NMR spectrum revealed key peaks for various carbon atoms, including the carbonyl (C=O) stretching at 172.86 ppm of the ester functional group, and signals for aromatic carbons in the range of 154.59 to 104.39 ppm. This confirmed the presence of the aromatic structures derived from eugenol (1) and fluconazole (5). The ¹H NMR spectrum displayed characteristic signals, including a singlet at 8.33 ppm for the protons attached to the aromatic rings, as well as a complex multiplet in the range of 6.70–6.58 ppm, indicating the presence of several aromatic protons from the coupled components. The peaks for protons at 5.98–5.90 ppm and 5.04–5.03 ppm were likely due to the allyl and methoxy groups, further confirming the structure. The IR spectrum showed typical functional group vibrations, such as C=O stretching at 1710 cm⁻¹, indicative of the ester group, and C=C stretching at 1471 cm⁻¹, a characteristic aromatic group. The presence of C–O stretching signals between 1180 and 500 cm⁻¹ also confirmed the ester linkage and the aromatic ring systems.

2.2. Antibacterial Evaluation

MIC

All the synthesized targeted compounds (12–19, 21, and 23) were assessed for their antibacterial potential against twelve bacterial strains, with DMSO as the negative control (

Table 1. Antibacterial activity of the synthesized hybrid molecules.

MIC (µg/mL)
Gram-Positive Bacteria
Bacterial Strains	12	13	14	15	16	17	18	19	21	23	AMP	STM	NLD	8
Bacillus subtilis (ATCC19659)	7.81	7.81	7.81	7.81	7.81	125	125	7.81	7.81	7.81	26	16	16	7.81
Enterococcus faecalis (ATCC13047)	7.81	7.81	7.81	7.81	7.81	15.6	15.6	7.81	7.81	7.81	26	128	>512	7.81
Staphylococcus epidermidis (ATCC14990)	125	125	125	250	7.81	125	62.5	125	250	500	26	8	64	31.25
Staphylococcus aureous (ATCC25923)	7.81	125	125	250	7.81	125	125	125	125	500	26	256	64	125
Mycobaterium smegmatis (MC2155)	250	250	250	250	7.81	125	125	250	250	250	26	4	512	250
Gram-Negative Bacteria
Enterobacter cloacae (ATCC13047)	7.81	7.81	7.81	7.81	7.81	125	31.2	7.81	7.81	7.81	26	512	16	7.81
Proteus vulgaris (ATCC6380)	7.81	7.81	7.81	7.81	7.81	125	31.2	7.81	7.81	7.81	416	128	128	7.81
Klebsiella oxytoca (ATCC8724)	7.81	7.81	7.81	7.81	7.81	7.81	15.6	7.81	7.81	7.81	26	16	8	7.81
Pseudomonas aeruginosa (ATCC27853)	7.81	7.81	7.81	7.81	7.81	125	31.2	7.81	7.81	7.81	64	128	128	7.81
Proteus mirabilis (ATCC7002)	125	125	125	125	7.81	125	31.2	125	125	125	26	128	32	125
Escherischia coli (ATCC25922)	125	250	125	250	7.81	250	250	250	250	250	26	64	512	250
Klebsiella pneumonia (ATCC13882)	250	250	250	250	7.81	125	62.5	250	250	250	26	512	256	250

). The most potent compounds against Gram-positive bacteria (B. subtilis, E. faecalis, S. epidermidis, S. aureus, and M. smegmatis) were compounds 12–16, 19, 21, and 23, which exhibited MIC values as low as 7.8125 µg/mL against various strains. Compound 16 was particularly noteworthy, as it exhibited MIC values of 7.8125 µg/mL for all the Gram-positive strains used in the study. Compounds 17 and 18 showed moderate activity against E. faecalis (MIC = 15.625 µg/mL), the most susceptible strain. Compound 16 again demonstrated the highest antibacterial activity, with an MIC of 7.8125 µg/mL against all Gram-negative bacteria. Compounds 12–16, 19, 21, and 23 exhibited similar activities against Gram-negative strains, with MIC values of 7.8125 µg/mL against E. cloacae, P. vulgaris, K. oxytoca, and P. aeruginosa. Compounds 17 and 18 showed the highest activity with MIC values of 7.8125 and 15.625 µg/mL, respectively, against K. oxytoca, while compound 18 showed slightly higher MIC values, particularly against E. cloacae, P. vulgaris, P. aeruginosa, P. mirabilis, and K. aerogenes (31.25–62.5 µg/mL).

In view of the structure-activity relationship, the parent compounds used for hybridizing these compounds played an important role in their antibacterial activities. Among the tested hybrids, compound 16, which incorporates ciprofloxacin via a chloroacetyl linker, demonstrated the most potent and broad-spectrum antibacterial activity. It exhibited MIC values of 7.81 µg/mL against all tested Gram-positive and Gram-negative bacterial strains, closely mirroring or surpassing the efficacy of the parent ciprofloxacin. This suggests that hybridization did not compromise the activity of ciprofloxacin but rather retained or enhanced its potency.

Hybrid compounds containing essential oil components, such as compound 12 containing eugenol, compound 13 containing thymol, compound 14 containing carvacrol, and compound 19 containing eugenol, exhibited the highest antibacterial activities, particularly against (B. subtilis, E. faecalis, E. cloacae, P. vulgaris, P. aeruginosa, and K. oxytoca) with MIC values of 7.8125 µg/mL. However, these compounds were less effective against S. epidermidis, S. aureus, M. smegmatis, P. mirabilis, E. coli, and K. aerogenes, where their MIC values ranged from 125 to 250 µg/mL. This variation in activity may be attributed to differences in the bacterial cell wall composition and membrane permeability [44].

Terpenoid-based compounds such as compound 17, containing ursolic acid, and compound 18, containing oleanolic acid, exhibited weaker antibacterial activity, with MIC values ranging from 125 to 250 µg/mL. Interestingly, the oleanolic acid-based hybrid compound 18 showed moderate activity against Gram-negative strains, such as E. cloacae, P. vulgaris, K. oxytoca, P. aeruginosa, and P. mirabilis, with MIC values ranging from 15.6 to 31.3 µg/mL, suggesting some degree of membrane penetration capability. However, these compounds were generally less effective against Gram-positive bacteria, likely due to steric hindrance and a lower interaction with bacterial targets [45,46].

Other hybrid compounds, such as the fluconazole-based compound 23 and zidovudine-based compound 21, demonstrated similar antibacterial trends, effectively inhibiting B. subtilis, E. faecalis, P. aeruginosa, P. vulgaris, and K. oxytoca with MIC values of 7.81 µg/mL, while being less potent against S. epidermidis, S. aureus, M. smegmatis, and K. aerogenes. The dimer of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (compound 15) followed a similar pattern, reinforcing the notion that certain bacterial strains are more resistant to these compounds.

The antibacterial activities of hybrid compounds 12–19, 21, and 23 were compared to the standard ampicillin, streptomycin, and nalidixic acid. Among the tested compounds, compound 16 showed superior or equal activity to ciprofloxacin across all tested strains, making it the most potent hybrid. This activity was notably superior to the other standard drugs, particularly against resistant strains, where AMP, STM, and nalidixic acid showed much higher MIC values (e.g., NLD > 512 µg/mL against E. faecalis and S. aureus, STM = 512 µg/mL against B. subtilis and K. aerogenes). Compounds 12–15, 19, 21, and 23 also displayed notable antibacterial activities, with MIC values ranging from 7.8125 to 250 µg/mL. In contrast, compounds 17 and 18 exhibited moderate antibacterial activities, with MIC values mostly between 15.6 and 250 µg/mL. Notably, compounds 12–15, 19, 21, and 23 were more effective against Gram-negative bacteria (e.g., E. cloacae, P. vulgaris, K. oxytoca, and P. aeruginosa) compared to the standard antibiotics, particularly when compared to AMP and STM, which had significantly higher MIC values against these strains. Overall, this comparative analysis highlights the promising potential of ciprofloxacin-based and phenolic monoterpenoid-based hybrids as effective antibacterial agents, warranting further exploration and optimization.

According to the literature [47,48], the most prevalent bacterial strains in COVID-19 patients are E. coli and K. pneumonia. The current study demonstrated that compound 16, the ciprofloxacin-based hybrid, exhibited the strongest activity against both E. coli and K. pneumoniae, with a MIC value of 7.81 µg/mL, far surpassing the activity of the standard ciprofloxacin (MIC = 250 µg/mL). This highlights the hybrid’s enhanced potency and potential for overcoming resistance mechanisms. All other hybrids, including those with natural monoterpenoids (eugenol, thymol, and carvacrol) and bioactive scaffolds like artesunic acid and zidovudine, demonstrated weak to moderate activity (MIC = 125–250 µg/mL), indicating limited effectiveness against these critical Gram-negative pathogens. The oleanolic acid hybrid (compound 18) showed slightly improved activity against K. pneumoniae (MIC = 62.5 µg/mL) compared to its performance against E. coli, suggesting some scaffold-dependent variation in activity. Notably, standard antibiotics such as streptomycin and nalidixic acid showed high MIC values, reinforcing the challenge of treating E. coli and Klebsiella infections in the context of increasing resistance.

2.3. SwissADME Predictions

Drug-like chemical entities are extensively explored in drug discovery and candidate selection. Compounds with pharmacokinetic characteristics that enable them to remain viable throughout human clinical trials are classified as drug-like chemical species. The SwissADME database was utilized to assess the physicochemical properties, lipophilicity, water solubility, pharmacokinetic profile, drug-likeness, and medicinal chemistry of hybrid compounds 12–16, 19, 21, and 23.

2.3.1. Physicochemical Properties

The physicochemical properties of a molecule play a crucial role in determining its efficacy, safety, and metabolism. These properties can be predicted using Lipinski’s rule of five, Veber’s rule, or Muegge’s rule [49]. The physicochemical properties of the hybrid compounds in

Table 2. The physicochemical properties of the synthesized hybrid compounds (12–16, 19, 21 and 23).

Physicochemical Properties
Hybrids	Formula	Molecular Weight	Num. of Heavy Atoms	Num. of Arom. Heavy Atoms	Fraction Csp3	Num. of Rotatable Bonds	Num. of H-Bond Acceptors	Num. of H-Bond Donors	Molar Refractivity	TPSA
12	C20H18ClN3O4	399.83 g/mol	28	17	0.15	9	6	1	105.67	86.48 Å2
13	C20H20ClN3O3	385.84 g/mol	27	17	0.25	7	5	1	104.62	77.25 Å2
14	C20H20ClN3O3	385.84 g/mol	27	17	0.25	7	5	1	104.62	77.25 Å2
15	C18H12Cl2N6O3	431.23 g/mol	29	22	0.06	7	7	2	106.19	118.97 Å2
16	C27H24ClFN6O5	566.97 g/mol	40	21	0.30	8	8	2	152.28	133.80 Å2
19	C27H32ClN3O8	562.01 g/mol	39	11	0.63	8	10	1	137.98	131.24 Å2
21	C22H21ClN8O7	544.90 g/mol	38	17	0.36	11	12	2	128.41	208.16 Å2
23	C27H26F2N6O5	552.53 g/mol	40	22	0.26	15	11	0	136.87	123.25 Å2

reveal distinct structural and molecular characteristics that influence their drug-like behaviours. Compounds 12, 13, 14, and 15 have moderate molecular weights (ranging from 385.84 to 431.23 g/mol) and a balanced number of hydrogen bond acceptors (5–7) and donors (1–2), indicating a potential for good drug-like properties. Their molar refractivity values range from 104.62 to 106.19, and they exhibit total polar surface areas (TPSA) between 77.25 and 118.97 Ų, which suggests a reasonable balance between permeability and solubility. In contrast, compounds 16, 19, 21, and 23 have significantly higher molecular weights (above 540 g/mol), exceeding Lipinski’s rule for optimal oral bioavailability. These compounds also have higher numbers of heavy atoms (38–40) and rotatable bonds (8–15), which may negatively affect their conformational flexibility and bioavailability. Compound 21 has an exceptionally high TPSA (208.16 Ų), suggesting poor membrane permeability. Moreover, compounds 19 and 23 possess a higher fraction of sp³-hybridized carbons (0.63 and 0.26, respectively), which may influence their three-dimensional complexity. Overall, while compounds 12–15 exhibit more favourable physicochemical properties for drug-likeness, compounds 16, 19, 21, and 23 may require modifications to improve their bioavailability and permeability.

2.3.2. Lipophilicity

As shown in

Table 3. The characteristics of the lipophilicity of the hybrid compounds (12–16, 19, 21 and 23).

Lipophilicity
Hybrids	Log Po/w (iLOGP)	Log Po/w (XLOGP3)	Log Po/w (WLOGP)	Log Po/w (MLOGP)	Log Po/w (SILICOS-IT)	Consensus Log Po/w
12	3.36	4.29	3.95	2.54	4.34	3.70
13	3.40	4.74	4.65	3.15	4.63	4.11
14	3.45	4.74	4.65	3.15	4.63	4.12
15	3.00	3.71	3.76	2.68	3.15	3.26
16	3.03	3.29	3.04	2.24	2.84	2.89
19	4.43	4.53	4.53	3.43	2.73	3.93
21	3.83	1.37	1.96	0.14	1.14	1.69
23	3.76	3.45	4.14	3.23	3.87	3.69

, the lipophilicity of the hybrid compounds, as measured by their consensus Log P_o/w values, reveals variations in their hydrophobicity, which directly impacts their absorption, distribution, and bioavailability. Compounds 12, 13, 14, and 15 exhibit moderate lipophilicity, with consensus Log P_o/w values ranging from 3.26 to 4.12. Among these, compounds 13 and 14 have the highest lipophilicity (4.11 and 4.12, respectively), suggesting good membrane permeability but a potential risk of reduced aqueous solubility. Compound 16 has the lowest lipophilicity (Log P_o/w = 2.89), indicating better solubility but potentially lower membrane permeability. Meanwhile, compound 19 shows relatively high lipophilicity (Log P_o/w = 3.93), suggesting favourable permeability characteristics. In contrast, compound 21 has the lowest lipophilicity (Log P_o/w = 1.69), which may lead to challenges in crossing lipid membranes and reduced oral bioavailability. Compound 23 falls in the moderate range (Log P_o/w = 3.69), suggesting balanced permeability and solubility. Overall, while most compounds have lipophilicity values within an acceptable range for drug-likeness, compounds with very high or very low Log P_o/w values may require further modification to enhance their pharmacokinetic properties.

2.3.3. Water Solubility

The water solubility of the hybrid compounds varies across different prediction methods. Based on the ESOL model, compounds 12, 13, 14, 15, 16, 19, and 23 exhibit moderate solubility, with solubilities ranging from 7.77 × 10⁻⁴ mg/mL to 7.69 × 10⁻³ mg/mL, as shown in

Table 4. The characteristics of the water solubility of the hybrid compounds (12–16, 19, 21 and 23).

Water Solubility
Hybrids	Log S (ESOL)	Solubility	Class	Log S (Ali)	Solubility	Class	Log S (SILICOS-IT)	Solubility	Class
12	−4.88	5.31 × 10−3 mg/mL; 1.33 × 10−5 mol/L	Moderately soluble	−5.82	6.07 × 10−4 mg/mL; 1.52 × 10−6 mol/L	Moderately soluble	−7.656	1.09 × 10−5 mg/mL; 2.74 × 10−8 mol/L	Poorly soluble
13	−5.22	2.31 × 10−3 mg/mL; 5.99 × 10−6 mol/L	Moderately soluble	−6.09	3.12 × 10−4 mg/mL; 8.09 × 10−7 mol/L	Poorly soluble	−7.81	6.03 × 10−6 mg/mL; 1.56 × 10−8 mol/L	Poorly soluble
14	−6.09	2.31 × 10−3 mg/mL; 5.99 × 10−6 mol/L	Moderately soluble	−6.09	3.12 × 10−4 mg/mL; 8.09 × 10−7 mol/L	Poorly soluble	−7.81	6.03 × 10−6 mg/mL; 1.56 × 10−8 mol/L	Poorly soluble
15	−4.95	4.83 × 10−3 mg/mL; 1.12 × 10−5 mol/L	Moderately soluble	−5.90	5.44 × 10−4 mg/mL; 1.26 × 10−6 mol/L	Moderately soluble	−8.47	1.46 × 10−6 mg/mL; 3.40 × 10−9 mol/L	Poorly soluble
16	−5.29	2.92 × 10−3 mg/mL; 5.15 × 10−6 mol/L	Moderately soluble	−5.78	9.52 × 10−4 mg/mL; 1.68 × 10−6 mol/L	Moderately soluble	−7.08	4.69 × 10−5 mg/mL; 8.28 × 10−8 mol/L	Poorly soluble
19	−5.86	7.77 × 10−4 mg/mL; 1.38 × 10−6 mol/L	Poorly soluble	−7.01	5.52 × 10−5 mg/mL; 9.82 × 10−8 mol/L	Poorly soluble	−5.83	8.24 × 10−4 mg/mL; 1.47 × 10−6 mol/L	Moderately soluble
21	−3.69	1.12 × 10−1 mg/mL; 2.06 × 10−4 mol/L	Soluble	−5.34	2.47 × 10−3 mg/mL; 4.53 × 10−6 mol/L	Moderately soluble	−6.12	4.11 × 10−4 mg/mL; 7.55 × 10−7 mol/L	Poorly soluble
23	−4.86	7.69 × 10−3 mg/mL; 1.39 × 10−5 mol/L	Moderately soluble	−5.72	1.05 × 10−3 mg/mL; 1.91 × 10−6 mol/L	Moderately soluble	−7.57	1.48 × 10−5 mg/mL; 2.69 × 10−8 mol/L	Poorly soluble

. Compound 21, however, is classified as soluble, with a solubility of 1.12 × 10⁻¹ mg/mL. According to the Ali model, the solubility trend is similar, with most compounds classified as moderately soluble, except for compound 21, which remains moderately soluble but with a lower value of 2.47 × 10⁻³ mg/mL. In the SILICOS-IT model, most of the compounds are poorly soluble, with solubility values ranging from 1.46 × 10⁻⁶ mg/mL to 1.48 × 10⁻⁵ mg/mL. Compound 21, however, shows moderate solubility, at 4.11 × 10⁻⁴ mg/mL. Overall, solubility predictions indicate that while most hybrids are moderately soluble, their solubility decreases significantly when evaluated through the SILICOS-IT model.

2.3.4. Pharmacokinetics

In

Table 5. The pharmacokinetic properties of the synthesized hybrid compounds calculated with the SwissADME database.

Pharmacokinetics
Hybrids	GI Absorption	BBB Permeant	P-gp Substrate	CYP1A2 Inhibitor	CYP2C19 Inhibitor	CYP2C9 Inhibitor	CYP2D6 Inhibitor	CYP3A4 Inhibitor	Log Kp (Skin Permeation)
12	High	No	No	Yes	Yes	Yes	No	Yes	−5.69 cm/s
13	High	No	No	Yes	Yes	Yes	No	Yes	−5.29 cm/s
14	High	No	No	Yes	Yes	Yes	No	Yes	−5.29 cm/s
15	High	No	No	Yes	Yes	Yes	No	Yes	−6.30 cm/s
16	High	No	Yes	No	Yes	Yes	Yes	Yes	−7.42 cm/s
19	Low	No	Yes	No	No	No	Yes	Yes	−6.51 cm/s
21	Low	No	Yes	No	No	No	No	Yes	−8.65 cm/s
23	High	No	No	Yes	Yes	Yes	No	Yes	−7.22 cm/s

, the pharmacokinetics of the hybrid compounds indicate diverse absorption, permeability, and metabolic profiles. Compounds 12, 13, 14, 15, and 23 demonstrate high gastrointestinal (GI) absorption, suggesting good systemic availability upon oral administration. These compounds also show the potential to inhibit several cytochrome P450 enzymes, including CYP1A2, CYP2C19, CYP2C9, and CYP3A4, which could influence the metabolism of co-administered drugs. However, none of these compounds are substrates for P-glycoprotein (P-gp), and most are not permeable to the blood–brain barrier (BBB), which limits their potential central nervous system effects. In contrast, compounds 19 and 21 exhibit lower GI absorption, with compound 19 showing reduced permeability (low GI absorption, BBB non-permeant, and P-gp substrate). These compounds also exhibit different inhibition profiles, with compound 16, in particular, showing broader inhibitory activity toward various CYP enzymes (CYP2C19, CYP2C9, CYP2D6, and CYP3A4). Skin permeation, as indicated by Log Kp values, was also evaluated, with all compounds showing low skin permeability, ranging from −5.69 cm/s to −8.65 cm/s, suggesting that they may not be suitable for transdermal delivery.

2.3.5. Drug-Likeness

The drug-likeness of the hybrid compounds in

Table 6. Drug-likeness scores of the synthesized hybrid compounds calculated with the SwissADME database.

Drug-likeness
Hybrids	Lipinski	Ghose	Veber	Egan	Muegge	Bioavailability Score
12	Yes; 0 violations	Yes	Yes	Yes	Yes	0.55
13	Yes; 0 violations	Yes	Yes	Yes	Yes	0.55
14	Yes; 0 violations	Yes	Yes	Yes	Yes	0.55
15	Yes; 0 violations	Yes	Yes	Yes	Yes	0.55
16	No; 2 violations: MW > 500, NorO > 10	No; 2 violations: MW > 480, MR > 130	Yes	No; 1 violation: TPSA > 131.6	Yes	0.56
19	No; 2 violations: MW > 500, NorO > 10	No; 3 violations: MW > 480, MR > 130, #atoms > 70	Yes	Yes	Yes	0.17
21	No; 2 violations: MW > 500, NorO > 10	No; 1 violation: MW > 480	No; 2 violations: Rotors > 10, TPSA > 140	No; 1 violation: TPSA > 131.6	No; 2 violations: TPSA > 150, H-acc > 10	0.17
23	No; 2 violations: MW > 500, NorO > 10	No; 2 violations: MW > 480, MR > 130	No; 1 violation: Rotors > 10	Yes	No; 1 violation: H-acc > 10	0.17

reveals a range of profiles based on different drug-likeness rules. Compounds 12, 13, 14, and 15 all adhere to Lipinski’s rule of five, with no violations, suggesting they possess good oral bioavailability potential. These compounds also pass the Ghose, Veber, Egan, and Muegge filters, further supporting their drug-likeness. These compounds show a relatively high bioavailability score of 0.55, indicating moderate to good bioavailability. However, compounds 16, 19, 21, and 23 display significant deviations from the drug-likeness rules. For example, compound 16 violates Lipinski’s rule with a molecular weight (MW) >500 and the number of rotatable bonds (NorO) >10 and violates Muegge’s criteria with a topological polar surface area (TPSA) >131.6. Compound 19 exhibits more significant violations in terms of MW, molecular rigidity (MR), and atom count. Compounds 21 and 23 also show multiple violations, including TPSA and hydrogen bond acceptors (H-acc), resulting in lower bioavailability scores (0.17). Overall, while compounds 12–15 demonstrate strong drug-likeness, others may require further modification to improve their drug-like properties.

2.3.6. Medicinal Chemistry

The medicinal chemistry analysis of the hybrid compounds demonstrated in

Table 7. Medicinal chemistry of the synthesized hybrid compounds calculated with the SwissADME database.

Medicinal Chemistry
Hybrids	PAINS	Brenk	Leadlikeness	Synthetic Accessibility
12	0 alerts	1 alert: isolated_alkene	No; 3 violations: MW > 350, Rotors > 7, XLOGP3 > 3.5	3.28
13	0 alerts	0 alerts	No; 2 violations: MW > 350, XLOGP3 > 3.5	3.29
14	0 alerts	0 alerts	No; 2 violations: MW > 350, XLOGP3 > 3.5	3.29
15	0 alerts	0 alerts	No; 2 violations: MW > 350, XLOGP3 > 3.5	3.12
16	0 alerts	0 alerts	No; 2 violations: MW > 350, Rotors > 7	3.87
19	0 alerts	1 alert: peroxide	No; 3 violations: MW > 350, Rotors > 7, XLOGP3 > 3.5	7.12
21	1 alert: azo_A	3 alerts: azido_group, diazo_group, quaternary_nitrogen_3	No; 2 violations: MW > 350, Rotors > 7	5.03
23	0 alerts	2 alerts: isolated_alkene, phenol_ester	No; 2 violations: MW > 350, Rotors > 7	3.97

indicates varying profiles in terms of potential alerts, lead likeness, and synthetic accessibility. Compounds 12, 13, 14, and 15 show no PAINS (pan-assay interference compounds) alerts, indicating that they do not contain functional groups likely to interfere with assay results. These compounds also demonstrate relatively favourable synthetic accessibility, with compound 15 showing the lowest accessibility score (3.12), suggesting that it may be slightly more challenging to synthesize. Compounds 16, 19, 21, and 23, however, exhibit more complex profiles. Compound 16 has no PAINS alerts but shows two violations related to molecular weight (MW) and rotatable bonds. Compound 19 contains a peroxide alert, which could pose stability issues, and it has a higher synthetic accessibility score (7.12). Compound 21 features multiple PAINS alerts, including azo and diazo groups, which could reduce its drug-likeness and increase its synthetic complexity (accessibility score of 5.03). Compound 23 has alerts related to isolated alkenes and phenol esters, which may affect its stability and reactivity, and it has a moderate synthetic accessibility score (3.97). Overall, compounds 12–15 are more favourable in terms of medicinal chemistry, while the others may require careful optimization to address their alerts and synthetic challenges.

3. Materials and Methods

3.1. Materials

All reactions were carried out using anhydrous solvents and reagents purchased from Sigma-Aldrich, Merck Pty Ltd., Johannesburg, South Africa), and were used without further purification. All reactions were performed under a nitrogen atmosphere. The synthesized hybrid molecules were characterized using ¹H NMR and ¹³C NMR spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), and High-Resolution Mass Spectrometry (HRMS). NMR spectra were recorded using a Bruker Avance III 500 MHz.

NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), with residual solvent peaks as internal references. NMR spectra were recorded using a Bruker Avance III 500 MHz NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), with residual solvent peaks as internal references. Chemical shifts (δ) are reported in parts per million (ppm). FTIR spectra were obtained using a PerkinElmer Spectrum Two FTIR spectrometer (PerkinElmer Inc., Waltham, MA, USA), while HRMS data were acquired using a Thermo Scientific Q Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Thin-layer chromatography (TLC) was performed on silica gel 60 F254 aluminium-backed plates (Merck KGaA, Darmstadt, Germany). Compounds were visualized using UV light at both 365 nm (long wavelength) and 254 nm (short wavelength), and with vanillin–sulfuric acid spray reagent (prepared from 100 mL methanol, 1 g vanillin, and 0.5 mL sulfuric acid; all from Sigma-Aldrich, Merck Pty Ltd., Johannesburg, South Africa). The synthesized compounds were purified using column chromatography on silica gel 60 (230–400 mesh ASTM, 0.04–0.063 mm particle size) (Merck KGaA, Darmstadt, Germany). Data processing and spectral analysis were performed using MestReNova software (version 14.2.1).

3.2. Chemistry

3.2.1. General Synthetic Procedure for Hybrid Compounds 12–16

Bulleted compounds 12–16 were synthesized by following the procedure outlined in Scheme 1 using a modified version of the synthetic procedures established by Anthwa et al. [50], Salama et al. [51], and Ayyash et al. [52]. 5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-amine (4) (1 g, 5.11 mmol, 1 equivalent) was reacted with chloroacetyl chloride (0.577 g, 5.11 mmol, 1 equivalent) in the presence of TEA (0.85 mL, 6.13 mmol, 1.2 equivalents) using anhydrous DCM (25 mL) as a solvent to form N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (11). The base (TEA) neutralized the hydrochloric acid formed during the reaction. The resulting chloroacetylated intermediate (11) was then reacted with compounds 1–4 and 8 in the presence of TEA to form the final products 12–16 through nucleophilic attack by the hydroxyl groups of eugenol (1), thymol (3), carvacrol (2) or by the NH group of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine or ciprofloxacin on the electrophilic chloroacetyl group. After each reaction step, the organic phase was washed with brine and water, dried using Na₂SO₄, and evaporated under low pressure. The final products were then purified by column chromatography.

2-(4-Allyl-2-methoxyphenoxy)-N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (12): N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (11) (0.200 g, 0.732 mmol, 1.0 equivalent), eugenol (1) (0.120 g, 0.732 mmol, 1.0 equivalent), TEA (0.12 mL, 6.13 mmol, 1.2 equivalent), DCM(20 mL). Column eluent: EtOAc/Hex(4:10). Yield: 57%. R_f = 0.56.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—171.16 (C-1, 157.07 (C-10), 150.86 (C-8), 136.49 (C-23), 135.37 (C-18), 129.81 (C-1, C-3), 127.22 (C-4, C-5, C-6), 123.75 (C-21), 109.96 (C-20), 84.49 (C-19), 83.96 (C-28), 61.32 (C-22), 60.67 (C-15), 36.68 (C-25), 12.62 (C-26) (Figure S1).

¹H-NMR (500-MHz, recorded in DMSO-d₆): ppm(δ) = 7.80 (H-6, doublet, 1H, J = 10.0 Hz), 7.80 (H-4, doublet, 1H, J = 10.0 Hz), 7.59 (H-1, doublet, 1H, J = 10.0 Hz), 7.59 (H-3, doublet, 1H, J = 10.0 Hz), 7.28 (H-22, singlet, 1H), 7.21 (H-20, doublet, 1H, J = 5.0 Hz), 6.80 (H-19, doublet, 1H, J = 10.0), 6.68–6.58 (H-27, multiplet, 1H), 5.33 (H-13, singlet, 1H), 4,85 (H-15, singlet, 1H), 4.42–4.39 (H-28, doublet of a doublet, 2H), 3.32 (H-25, singlet, 3H), 3.10 (H-26, doublet, 1H, J = 10.0 Hz) (Figure S1).

FT–IR (KBr ν in cm⁻¹): 3554 (N–H stretching), 2931–2855 (C–H stretching from alkanes), 1710 (C=O stretching), 1626–1481 (C=C stretching from aromatic or alkene), 1381–1156 (C–H bending (alkanes or aromatics)), 1000–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S11).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₀H₁₈ClN₃O₄]⁺: calculated 399.0986, observed 399.0958 (Figure S21).

2-(2-Isopropyl-5-methylphenoxy)-N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (13): N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (11) (0.200 g, 0.732 mmol, 1.0 equivalent), thymol (3) (131.9 mg, 0.878 mmol, 1.2 equivalents), TEA (0.153 mL, 1.098 mmol, 1.5 equivalents). Column eluent: EtOAc/Hex (7:3). Yield: 55%. R_f = 0.58.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—170.31 (C-14), 164.46 (C-18), 157.08 (C-10), 155.09 (C-8), 136.50 (C-2), 135.39 (C-20), 134.46 (C-23), 129.79 (C-3), 129.05 (C-4), 127.23 (C-22), 123.69 (C-21), 115.86 (C-19), 68.09 (C-15), 25.97 (C-24), 23.06 (C-27), 22.36 (C-25), 21.72 (C-26) (Figure S2).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—7.80 (H-6, doublet, 1H, J = 10.0 Hz), 7.80 (H-4, doublet, 1H, J = 10.0 Hz), 7.58 (H-1, doublet, 1H, J = 10.0 Hz), 7.58 (H-3, doublet, 1H, J = 10.0 Hz), 7.28 (H-19, singlet, 1H), 7.19 (H-22, doublet, 1H, J = 5.0 Hz), 6.37 (H-21, doublet, 1H, J = 10.0 Hz), 5.73 (H-13, singlet, 1H), 4.62 (H-15, singlet, 2H), 3.69–3.58 (H-24, multiplet, 1H), 3.39 (H-25, singlet, 3H), 1.10 (H-27, doublet, 6H, J = 10.0 Hz) (Figure S2).

FT–IR (KBr, ν in cm⁻¹): 3354 (N–H stretching), 2933–2872 (C–H stretching from alkanes), 1735 (C=O stretching), 1500 (C=C stretching from aromatic or alkene), 1388 (C–H bending of alkanes or aromatics), 1157–500 (C–O stretching, C–Cl bond, or skeletal vibrations from aromatic rings) (Figure S12).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₀H₂₀ClN₃O₃]⁺: calculated 385.1193, observed 385.1185 (Figure S22).

2-(5-Isopropyl-2-methylphenoxy)-N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (14): N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (11) (0.200 g, 0.732 mmol, 1.0 equivalent), carvacrol (2) (131.9 mg, 0.878 mmol, 1.2 equivalents), TEA (0.153 mL, 1.098 mmol, 1.5 equivalents). Column eluent: EtOAc/Hex (3:7). Yield: 52%. R_f = 0.63.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—171.21 (C-14), 164.48 (C-18), 157.07 (C-10), 154.61 (C-8), 135.71 (C-3), 135.38 (C-1), 131.68 (C-22), 129.82 (C-5), 127.23 (C-4), 126.08 (C-6), 123.75 (C-23), 120.10 (C-21), 116.06 (C-19), 71.02 (C-15), 26.51 (C-25), 23.05 (C-26), 23.05 (C-27), 21.10 (C-24) (Figure S3).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—7.81 (H-6, doublet, 1H, J = 5.0 Hz), 7.81 (H-4, doublet, 1H, J = 5.0 Hz), 7.60 (H-1, doublet, 1H, J = 5.0 Hz), 7.60 (H-3, doublet, 1H, J = 5 Hz), 6.97–6.95 (H-22, doublet, 1H, J = 10.0 Hz), 6.56 (H-19, singlet, 1H), 6.55 (H-21, doublet, 1H, J = 10.0 Hz), 4.88 (H-13, singlet, 1H), 3.33 (H-15, singlet, 1H), 3.18–3.12 (H-25, multiplet, 1H), 2.17 (H-24, doublet, 3H, J = 5.0 Hz), 1.13 (H-26, doublet, 3H, J = 10.0 Hz), 1.13 (H-27, doublet, 3H, J = 10.0 Hz) (Figure S3).

FT–IR (KBr, ν in cm⁻¹): 3451 (N–H stretching), 2942–2890 (C–H stretching from alkanes), 1694 (C=O stretching), 1468–1391 (C=C stretching from aromatic or alkene). 1389–1033 (C–H bending (alkanes or aromatics)), 1000–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S13).

HRMS (ESI) mass spectrometry analysis analysis (m/z) of [C₂₀H₂₀ClN₃O₃]⁺: calculated 385.1193, observed 385.1179 (Figure S23).

2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylamino)-N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)acetamide (15): N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (11) (0.300 g, 1.098 mmol, 1.0 equivalent), 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (260.5 mg, 1.318 mmol, 1.2 equivalents), TEA (0.23 mL, 1.647 mmol, 1.5 equivalents). Column eluent: EtOAc/Hex (3:5). Yield: 29%. R_f = 0.25.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—172.21 (C-14), 151.27 (C-26), 147.97 (C-10), 145.58 (C-8), 145.24 (C-24), 138.66 (C-19), 130.95 (C-21), 121.02 (C-5), 115.90 (C-6), 115.67 (C-22), 113.22 (C-20), 56.09 (C-15) (Figure S4).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm) = 6.73 (H-4, doublet, 1H, J = 10.0 Hz), 6.73 (H-6, doublet, 1H, J = 10.0 Hz), 6.70 (H-22, doublet, 1H, J = 5.0 Hz), 6.70 (H-20, doublet, 1H, J = 5.0 Hz), 6.58 (H-1, doublet, 1H, J = 10.0 Hz), 6.58 (H-3, doublet, 1H, J = 10.0 Hz), 6.58 (H-17, doublet, 1H, J = 10.0 Hz), 6.58 (H-19, doublet, 1H, J = 10.0 Hz), 5.08 (H-13, singlet, 1H), 3.75 (H-15, doublet, 2H, J = 10.0 Hz), 3.32 (H-29, singlet, 1H) (Figure S4).

FT–IR (KBr, ν in cm⁻¹): 3544–3352 (N–H stretching) 2930–2856 (C–H stretching from alkanes), 1708 (C=O stretching), 1623–1493 (C=C stretching (aromatic or alkene)), 1455–1361 (C–H bending (alkanes or aromatics)), 1156–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S14).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₁₈H₁₂Cl₂N₆O₃]⁺: calculated 430.0348, observed 430.0329 (Figure S24).

7-(4-(2-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-ylamino)acetyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (16): N-(chloroacetyl)-5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (200 mg, 0.732 mmol, 1.0 equivalent), ciprofloxacin (290.7 mg, 0.878 mmol, 1.2 equivalents), TEA (0.153 mL, 1.098 mmol, 1.5 equivalents). Column eluent: EtOAc/Hex 2:8. Yield: 60%. R_f = 0.62.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—172.81 (C-22), 164.48 (C-25), 157.07 (C-2), 154.61 (C29), 148.72 (C-31), 147.30 (C-12), 145.79 (C-14), 135.71 (C-4), 135.38 (C-37), 131.68 (C-9), 129.82 (C38), 127.23 (C-38), 126.08 (C-36), 123.75 (C-34), 120.10 (C-39), 116.06 (C-35), 110.99 (C-10), 107.99 (C-11), 104.98 (C-3), 53.79 (C-21), 52.89 (C-16), 47.45 (C-20), 46.33 (C-19), 43.14 (C-17), 26.51 (C-26), 23.95 (C-6), 23.95 (C-7), 21.10 (C-8) (Figure S5).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—8.33 (H-4, singlet, 1H), 7.49 (H-34, doublet, 1H, J = 5.0 Hz), 7.49 (H-38, doublet, 1H, J = 5.0 Hz), 7.45 (H-35, doublet, 1H, J = 10.0 Hz), 7.43 (H-37, singlet, 1H), 6.35 (H-21, singlet, 1H, J = 5.0 Hz), 5.36 (H-27, singlet, 1H), 3.73 (H-20, triplet, 2H, J = 5.0 Hz), 3.51 (H-16, triplet, 2H, J = 5.0 Hz), 3.23 (H-25, singlet, 2H), 3.01–2.94 (H-6, multiplet, 1H), 2.79 (H-19, triplet, 2H, J = 5.0 Hz), 2.66 (H-17, triplet, 2H, J = 5.0 Hz), 0.88–0.77 (H-7, multiplet, 2H), 0.77–0.67 (H-8, multiplet, 2H) (Figure S5).

FT-IR (KBr, ν in cm⁻¹): 3302 (OH stretching), 2942–2891 (C–H stretching from alkanes), 1688 (C=O stretching), 1667–1557 (C=C stretching from aromatic or alkene), 1465–1373 (C–H bending (alkanes or aromatics)), 1043–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S15).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₇H₂₄ClFN₆O₅]⁺: calculated 566.1481, observed 566.1521 (Figure S25).

3.2.2. General Synthetic Procedure for Hybrid Compounds 17 and 18

Two ursolic acid or oleanolic acid derivatives containing the 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine moiety were synthesized as outlined in Scheme 1 by following a modified procedure [53]. UA or OA was dissolved in anhydrous DMF, and then DCC and DMAP were added to activate their carboxyl groups. 5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-amine (4) was then introduced, allowing the amine group to react with the activated carboxyl group of ursolic acid or oleanolic acid, forming an amide bond. The reaction mixture was stirred at room temperature for 1 h and refluxed at 80 °C overnight. After the reaction, the solvent was removed, and the product was purified using column chromatography.

Hybrid 17: ursolic acid (300 mg, 0.657 mmol, 1.0 equivalent), 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (155.2 mg, 0.789 mmol, 1.2 equivalents), DCC (149.2 mg, 0.723 mmol, 1.1 equivalents), DMAP (8.1 mg, 0.066 mmol, 0.1 equivalent), DMF (10 mL). Column eluent: EtOAc/Hex (3:7). Yield: 42%. R_f = 0.72.

¹³C-NMR (500 MHz, recorded in CDCl-d₃): chemical shifts (δ, ppm)—172.31 (C-1), 157.09 (C-36), 156.02 (C-39), 138.33 (C-27), 136.07 (C-44), 132.15 (C-45), 130.67 (C-43), 128.77 (C-41), 127.60 (C-46), 125.96 (C-42), 124.54 (C-28), 77.29 (C-2), 55.22 (C-9), 52.41 (C-25), 49.65 (C-30), 47.98 (C-18), 47.35 (C-14), 42.13 (C-12), 39.08 (C-23), 38.85 (C-6), 38.66 (C-21), 36.95 (C-5), 33.81 (C-31), 31.62 (C-11), 30.31 (C-20), 29.45 (C-4), 28.70 (C-15), 25.80 (C-29), 24.92 (C-7), 23.76 (C-8), 21.24 (C-10), 17.46 (C-22), 17.37 (C-24), 16.52 (C-13), 15.50 (C-32) (Figure S6).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—7.97 (H-33, singlet, 1H), 7.71 (H-41, doublet, 1H, J = 5.0 Hz), 7.71 (H-45, doublet, 1H, J = 5.0 Hz), 7.66 (H-42, doublet, 1H, J = 5.0 Hz), 7.66 (H-44, doublet, 1H, J = 10.0 Hz), 5.40 (H-48, triplet, 1H, J = 5.0 Hz), 3.01 (H-2, triplet, 1H, J = 5.0 Hz), 2.12 (H-44, doublet, 1H, J = 10.0 Hz), 1.93–0.69 (aliphatic protons from the ursolic acid moiety) (Figure S6).

FT-IR (KBr, ν in cm⁻¹): 3357 (NH stretching), 3265 (OH stretching), 2941–2871 (C–H stretching from alkanes), 1581 (C=O stretching), 1557–1436 (C=C stretching from aromatic or alkene), 1375–1138 (C–H bending (alkanes or aromatics)), 902–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S16).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₃₈H₅₂Cl₂N₃O₃]⁺: calculated 633.3697, observed 633. 3665 (Figure S26).

Hybrid 18: oleanolic acid (300 mg, 0.657 mmol, 1.0 equivalent), 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (155.2 mg, 0.789 mmol, 1.2 equivalents), DCC (149.2 mg, 0.723 mmol, 1.1 equivalents), DMAP (8.1 mg, 0.066 mmol, 0.1 equivalent). Column eluent: EtOAc/Hex (4:6). Yield: 38%. R_f = 0.3.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—177.81 (C-1), 157.13 (C-34), 154.08 (C-2), 140.33 (C-25), 136.56 (C-42), 134.34 (C-43), 134.08 (C-41), 129.25 (C-39), 126.93 (C-44), 126.93 (C-40), 80.39 (C-2), 52.53 (C-9), 51.58 (C-28), 45.47 (C-22), 44.37 (C-18), 40.85 (C-23), 37.47 (C-14), 37.36 (C-12), 36.39 (C-6), 36.30 (C-5), 34.73 (C-29), 34.22 (C-20), 30.81 (C-11), 26.04 (C-21), 25.92 (C-46), 25.92 (C-47), 24.72 (C-19), 24.55 (C-19), 20.35 (C16), 13.17 (C-13), 12.72 (C-30) (Figure S7).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—7.95 (H-36, singlet, 1H), 7.03 (H-43, doublet, 1H, J = 10.0 Hz), 7.03 (H-47, doublet, 1H, J = 10.0 Hz), 6.67 (H-44, doublet, 1H, J = 10.0 Hz), 6.67 (H-46, doublet, 1H, J = 10.0 Hz), 5.14 (H-29, triplet, 1H, J = 5.0 Hz), 3.0 1 (H-27, triplet, 1H, J = 5.0 Hz), 2.48–2.47 (H-2, doublet of a doublet, 1H, J = 5.0 Hz), 2.21–0.69 (aliphatic protons from the oleanolic acid moiety) (Figure S7).

FT-IR (KBr, ν in cm⁻¹): 3357 (NH stretching), 3265 (OH stretching), 2941–2871 (C–H stretching from alkanes), 1581 (C=O stretching), 1557–1436 (C=C stretching from aromatic or alkene), 1375–1138 (C–H bending (alkanes or aromatics)), 902–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S17).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₃₈H₅₂Cl2N₃O₃]⁺: calculated 633.3697, observed 633.3635 (Figure S27).

3.2.3. Synthetic Procedure for Hybrid Compound 19

The synthesis of a 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine-artesunate hybrid (19) was carried out by activating artesunic acid (7) to react with the amine group of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine. First, artesunic acid (7) was dissolved in anhydrous DMF and activated using NHS to form an ester. Next, the activated artesunate was reacted with the amine group of 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine in DMF at room temperature, and the reaction was allowed to proceed for 24 h. After completion, the solvent was removed, and the crude product was extracted with dichloromethane and purified with column chromatography.

Artesunic acid (250 mg, 0.621 mmol, 1.0 equivalent), 5-(4-chlorophenyl)-1,3,4-oxadiazol-2-amine (122.1 mg, 0.261 mmol, 1.0 equivalent), NHS (78.6 mg, 0.683 mmol, 1.1 equivalent). Column eluent: EtOAc/Hex (3:7). Yield: 48%. R_f = 0.47.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—178.67 (C-26), 173.18 (C-22), 166.56 (C-32), 164.30 (C-29), 138.67 (C-37), 132.25 (C-38), 129.85 (C-36), 126.83 (C-34), 125.05 (C-39), 123.97 (C-35), 77.34 (C-5), 55.27 (C-6), 52.89 (C-11), 47.32 (C-18), 42.32 (C-1), 38.97 (C-13), 38.91 (C-14), 36.79 (C-25), 30.38 (C-7), 28.73 (C-24), 23.74 (C-12), 21.50 (C-15), 16.51 (C-20), 15.68 (C-8) (Figure S8).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—8.00 (H-27, singlet, 1H), 7.99 (H-35, doublet, 1H, J = 5.0 Hz), 7.99 (H-39, doublet, 1H, J = 5.0 Hz), 7.63 (H-36, doublet, 1H, J = 5.0 Hz), 7.60 (H-38, doublet, 1H, J = 5.0 Hz), 6.04 (H-19, doublet, 1H, J = 5.0 Hz), 5.14 (H-2, doublet, 1H, J = 5.0 Hz), 4.26 (H-41, singlet, 1H), 3.15 (H-24, triplet, 2H, J = 5.0 Hz), 2.82 (H-25, triplet, 2H, J = 5.0 Hz), 2.13–0.69 (aliphatic protons from artesunate) (Figure S8).

FT–IR (KBr, ν in cm⁻¹): 3357 (NH stretching), 3550–3371 (NH stretching), 2930–2858 (C–H stretching from alkanes), 1709 (C=O stretching), 1627 (C=C stretch from aromatic or alkene), 1383–1209 (C–H bending (alkanes or aromatics)), 1035–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S18).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₇H₃₂ClN₃O₈]⁺: calculated 561.1878, observed 561.1902 (Figure S28).

3.2.4. Synthetic Procedure for Hybrid Compound 21

5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-amine (250 mg, 1.272 mmol, 1.0 equivalent) was reacted with succinic anhydride (152.7 mg, 1.527 mmol, 1.2 equivalents) in pyridine under reflux. After 4 h, the product was precipitated in cold water, filtered, and dried to yield the N-succinylated intermediate (20). The N-succinylated intermediate was dissolved in dichloromethane, followed by the addition of zidovudine (6), coupling reagents, and DCC and DMAP as catalysts. The mixture was stirred at 0 °C for 15 min and left overnight at room temperature. After workup and purification, the final succinylated oxadiazole-zidovudine (21) hybrid was obtained.

(3-Azido-tetrahydro-5-(3,4-dihydro-5-methyl-2,4-dioxopyrimidin-1(2H)-yl)furan-2-yl)methyl 3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-ylcarbamoyl)propanoate (21): 3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-ylcarbamoyl)propanoic acid (20) (152 mg, 0.475 mmol, 1.0 equivalent), zidovudine (6) (127.0 mg, 0.475 mmol, 1.0 equivalent), DCC (107 mg, 0.522 mmol, 1.1 equivalents), DMAP (5.8 mg, 0.0475 mmol, 0.1 equivalent). Column eluent: DCM/EtOAc (15:3). Yield: 51%. R_f = 0.53.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—178.67 (C-14), 169.28 (C-17), 155.60 (C-10), 154.44 (C-8), 151.02 (C-28), 138.67 (C-32), 134.42 (C-2), 130.36 (C-3), 129.59 (C-1), 128.36 (C-5), 125.06 (C-4), 107.85 (C-31), 77.34 (C-25), 55.28 (C-22), 52.89 (C-21), 47.52 (C-15), 47.32 (C-16), 30.67 (C2-4), 27.48 (C-23), 15.80 (C-33) (Figure S9).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—8.67 (H-29, singlet, 1H), 8.33 (H-13, singlet, 1H), 7.80 (H-32, singlet, 1H), 7.53 (H-6, doublet, 1H, J = 10.0 Hz), 7.51 (H-4, doublet, 1H, J = 10.0 Hz), 7.43 (H-1, doublet, 1H, J = 10.0 Hz), 7.43 (H-3, doublet, 1H, J = 10.0 Hz), 6.73–6.69 (H-24, doublet of a doublet, 2H), 6.57 (H-21, doublet, 2H, J = 10.0 Hz), 5.98–5.90 (H-22, multiplet, 1H), 5.08–5.01 (H-24, multiplet, 1H), 3.32 (H-33, singlet, 3H), 3.27 (H-25, doublet, 1H, J = 10.0 Hz), 2.72 (H-16, triplet, 2H, J = 5.0 Hz), 2.63 (H-15, triplet, 2H, J = 5.0 Hz) (Figure S9).

FT–IR (KBr, ν in cm⁻¹): 3535–3393 (NH stretching), 2932–2855 (C–H stretching from alkanes). 1704–1626 (C=O stretching), 1494–1452 (C=C stretching (aromatic or alkene)), 1379–1253 (C–H bending (alkanes or aromatics)), 1038–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S19).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₂H₂₁ClN₈O₇]⁺: calculated 544.1222, found 544.1254 (Figure S29).

3.2.5. Synthetic Procedure for Hybrid Compound 23

Compound 23 was synthesized by following a modified procedure based on the method reported by Hou et al. [54]. Eugenol (1) (500 mg, 3.056 mmol, 1.0 equiv.) was first esterified with succinic anhydride (365.9 mg, 3.655 mmol, 1.2 equiv.) in anhydrous DMF (15 mL) using a catalytic amount of DMAP (55.9 mg, 0.457 mmol, 0.15 equiv.). The reaction mixture was stirred at room temperature under an inert atmosphere overnight. The progress of the reaction was monitored by TLC, and the product was purified by column chromatography. The obtained eugenol–succinic acid intermediate (22) was then dissolved in anhydrous DMF, and DCC and TEA were added. The mixture was stirred in an ice bath for 15 min before adding fluconazole (5), followed by overnight stirring at room temperature. After completion, the reaction mixture was filtered to remove by-products, and the crude product was extracted with ethyl acetate, washed with brine and water, dried over Na₂SO₄, and purified by column chromatography.

4-Allyl-2-methoxyphenyl 2-(3,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)propan-2-yl succinate(23): fluconazole (150 mg, 0.4900 mmol, 1.0 equivalent), 3-(4-allyl-2-methoxyphenoxy)carbonyl)propanoic acid (22) (129.05 mg, 0.4900 mmol, 1.00 equivalent), DCC (101.0 mg, 0.4900 mmol, 1.00 equivalent), TEA (49.6 mg, 0.4900 mmol, 1.00 equivalent), DMF (10 mL). Column eluent: EtOAc/Hex (6:10). Yield: 53%. R_f = 0.67.

¹³C-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—172.86 (C-15), 170.54 (C-11), 154.59 (C-37), 154.44 (C-32), 152.74 (C-6), 151.27 (C-5), 145.58 (C-23), 145.58 (C-27), 135.70 (C-35), 131.66 (C-34), 130.28 (C-21), 126.08 (C-18), 123.94 (C-27), 120.09 (C-2), 120.09 (C-19), 116.05 (C-20), 111.42 (C-4), 111.28 (C-28), 104.60 (C-1), 104.39 (C-22), 74.15 (C-7), 55.42 (C-29), 29.93 (C-8), 26.50 (C-26), 23.06 (C-14), 21.10 (C-13) (Figure S10).

¹H-NMR (500 MHz, recorded in DMSO-d₆): chemical shifts (δ, ppm)—8.33 (H-32, singlet, 1H), 8.33 (H-37, singlet, 1H), 7.80 (H-34, singlet, 1H), 7.80 (H-35, singlet, 1H), 6.73 (H-4, singlet, 1H), 6.70 (H-2, doublet, 1H, J = 5.0 Hz), 6.70 (H-19, doublet, 1H, J = 5.0 Hz), 6.58 (H-22, singlet, 1H), 6.57 (H-1, doublet, 1H, J = 10.0 Hz), 6.57 (H-20, doublet, 1H, J = 10.0 Hz,), 5.98–5.90 (H-27, multiplet, 1H), 5.04–5.03 (H-28, doublet, 2H, J = 5.0 Hz), 3.75 (H-8, singlet, 2H), 3.75 (H-29, singlet, 2H), 3.32 (H-25, singlet, 3H), 3.26 (H-26, doublet, 1H, J = 10.0 Hz), 2.89 (H-14, triplet, 2H, J = 5.0 Hz), 2.73 (H-13, triplet, 2H, J = 5.0 Hz) (Figure S10).

FT–IR (KBr, ν in cm⁻¹): 2945–2905 (C–H stretching from alkanes). 1710 (C=O stretching), 1471 (C=C stretching (aromatic or alkene)), 1431–1258 (C–H bending (alkanes or aromatics)), 1180–500 (C–O stretching, C–Cl bonds, or skeletal vibrations from aromatic rings) (Figure S20).

HRMS (ESI) mass spectrometry analysis (m/z) of [C₂₇H₂₆F₂N₆O₅]⁺: calculated 552.1933, observed 552.1919 (Figure S30).

3.3. Biological Evaluation

Antibacterial Activity

The minimum inhibitory concentration (MIC) determination of the hybrid compounds was conducted as outlined by Fonkui et al. (2018) [55]. Each compound was initially dissolved in DMSO at a concentration of 1 mg/mL. These solutions were serially diluted seven times in 100 µL of nutrient broth within a 96-well plate to achieve final concentrations of 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 µg/mL. Following this, 100 µL of each solution was plated in duplicate with 100 µL of a 0.5 McFarland bacterial suspension. Streptomycin (STM), ampicillin (AMP), nalidixic acid (NLD), and ciprofloxacin (8) were included as positive controls, while a DMSO and nutrient broth solution served as the negative control. The MIC values for all the evaluated hybrid molecules are presented in Table 1.

3.4. In Silico Studies

SwissADME Web Tool

In recent years, in silico studies have become highly popular in drug discovery and the development of small molecules. The SwissADME web tool allows the prediction of key pharmacokinetics, physicochemical properties, drug-likeness, and related characteristics for one or more molecules [56,57,58]. The free online software was accessed directly from the webpage http://www.swissadme.ch (accessed on 25 March 2025). In this study, the web was utilized to evaluate the physicochemical properties, lipophilicity, water solubility, pharmacokinetic profile, drug-likeness, and medicinal chemistry of hybrid compounds with promising antibacterial activity, such as compounds 12–16, 19, 21, and 23. The 2D structure of the synthesized compound was drawn using ChemDraw Ultra version 8.0. The structure was then imported, and their SMILES notation was entered. A SwissADME drug design analysis was performed, and the results were recorded. The physicochemical properties, lipophilicity, water solubility, pharmacokinetic profile, drug-likeness, and medicinal chemistry of these hybrid molecules are presented in Table 2, Table 3, Table 4, Table 5 and Table 6, respectively.

4. Conclusions

This study reports the successful synthesis and characterization of hybrid compounds (12–19, 21, and 23) developed through esterification or amidation reactions. The in vitro antibacterial evaluation revealed varying activity among the compounds, with compound 16 (ciprofloxacin-based) demonstrating the most potent and broad-spectrum efficacy, particularly against pathogens commonly associated with COVID-19 co-infections. Essential oil-derived hybrids (e.g., 12–14) showed moderate activity, while terpenoid-based hybrids were less effective, emphasizing the impact of structural features on antibacterial potency. These findings support the potential of hybridization strategies to enhance the efficacy of existing antibacterial agents.

In silico analyses further indicated favourable pharmacokinetic and drug-likeness profiles for most hybrids, with good GI absorption, oral bioavailability, and a low risk of CYP450 inhibition. These properties highlight the potential of the selected compounds as lead candidates for future drug development. Moving forward, experimental validation of the pharmacokinetic predictions, toxicity profiling, and structural optimization will be essential to advance these compounds toward therapeutic application.