Synthesis, Antimicrobial, and Anticancer Activities of Novel Nitrofuran Derivatives

Abstract

Keeping in view the varying therapeutic attributes of 5-nitrofuran and isatin derivatives, novel 5-nitrofuran‒isatin molecular hybrids (2, 5–7) were synthesized by standard protocols, characterized by various spectroscopic techniques, and eventually evaluated against a group of pathogenic bacteria and fungi. Greater potency against Methicillin-resistant Staphylococcus aureus (MRSA) was exhibited by hybrids 5 and 6 with minimum inhibitory concentration values of 8 and 1 μg/mL, respectively. Cytotoxicity against both human embryonic kidney cells (HEK-293) and human red blood cells (RBCs) was investigated for the hybrids in hand. All hybrids appeared to have good safety; all of them were devoid of cytotoxicity, and none displayed hemolytic activity at the highest test concentration (CC₅₀ and HI10 > 32 μg/mL). To support the postulation that these hybrids would be analogous to drugs containing the 5-nitrofurn ring system, molecular docking was carried out to streamline the binding affinity of the investigated hybrids towards the E. coli nitroreductase (NTR). Compared to the standard drug nitrofurazone, hybrid 6 demonstrated a higher affinity and better binding interactions with the NTR binding pocket. Therefore, it could be concluded that 6 displays its antibacterial action through a mechanism similar to that of nitrofurazone. Nonetheless, further wet investigations are to be conducted to confirm this finding. Encouraged by the well-established anticancer activity of isatin derivatives, 2, 5–7 were assessed for their potential antitumor activity, and they well demonstrated potent inhibitory activity against the human colon cancer cell line HCT 116 (IC₅₀ = 1.62–8.8 μM) with isatin hybrid 3 being the best (IC₅₀ = 1.62 μM). Thus, it is herein reported that these 5-nitrofuran‒isatin molecular hybrids could represent an ideal starting point for future studies to develop potent antimicrobial agents.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

The development of microbial resistance is considered one of the most serious public health concerns [1, 2]. This resistance along with patient noncompliance, multiple disease coinfections, drug-drug interactions, and drug toxicity are all issues that put a strain on the drug discovery process [3, 4]. This strain is further exacerbated by economic issues, such as the high prevalence of the disease among the poorest of the poor, the availability and accessibility of medicine, and the cost of drugs [4]. Globally, about 700,000 deaths occur every year as a result of resistant microorganisms. If these current issues continue, this number is anticipated to reach 10 million after three decades [5]. Certain Gram-negative and Gram-positive bacteria such as Klebsiella pneumonia and Staphylococcus aureus have been classified by the US Center for Disease Control and Prevention (CDC) as «High Priority Antibiotic Resistant Bacteria» [6].

The treatment of microbial infections by antibiotics is considered one of the main successes in history. Nonetheless, the irrational use of antibiotics over the last 10 decades has contributed to the development of resistance to currently used antibiotics [7]. Moreover, there are no antibiotics in the clinical pipeline, and the number of novel compounds under development are very limited [8]. Therefore, it is an urgent priority to develop new antimicrobial agents with a broad spectrum of activity against resistant microbes [9].

Nitrofuran derivatives have been used as antibacterials since nearly 8 decades ago, and they are characterized by their potent activity against a wide range of pathogenic organisms [6, 10]. Nitrofurantoin, nifuroxazide, and furazolidone (Figure 1) are a few antibacterial drugs from the heterocyclic compounds containing the nitrofuran ring system [6, 11]. Besides, there is also published work on the promising antitubercular properties of nitrofuran derivatives [12]. To exert their antimicrobial action, nitrofuran derivatives are metabolically activated inside the microorganism. This is achieved through the microbial enzyme nitroreductase which reduces the nitro group (‒NO₂) to produce diverse free radicals which eventually damage the microorganism [13].

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The principal pharmacophoric features of the 5-nitrofurans are a nitro group (‒NO₂) directly linked to a furan ring in addition to a hydrazone moiety (Figure 2) which contributes to the overall stability of the nitrofuran ring, facilitating free radical formation [4].

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Isatin (Figure 3) is a privileged heterocyclic scaffold with versatile molecular architecture, rendering it an ideal platform for chemical modifications [14]. A wide spectrum of biological activities including chemotherapeutic ones have been reported to be exhibited by isatin and its derivatives [15–18]. Recently, in vitro and in vivo antibacterial properties have been reported by several isatin derivatives [19]. Various reports in the literature have revealed that modifications of the isatin ring at positions 1 (N‒alkylation or aminoalkylation) and 3 can improve their antimicrobial activities [3, 20–24].

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Recently, several drug discovery strategies were successfully implemented to develop antimicrobials with enhanced activity against drug-resistant microbes (DRMs) [3]. It has been reported that pharmaceutical agents acting on multiple molecular targets display higher potency and lower resistance compared to those acting on a single molecular target [3, 25, 26]. Recently, the approach of molecular hybridization has been successfully applied to develop novel drug entities with improved biological activity relative to the parents’ bioactive units. It is an approach in which more than one bioactive entity is combined into a single molecular hybrid [27]. This hybrid is expected to exhibit significant antimicrobial activity, lower toxicity, and a reduced rate of multiple drug resistance development [3, 27]. In 2019, Guo has reviewed several hybrid molecules in which isatin was hybridized with a wide range of antimicrobial entities [19]. These hybrids have been reported to demonstrate enhanced therapeutic properties against diverse microorganisms (Figure 4).

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Considering the widespread resistance to the antimicrobial drugs used in current treatment protocols, we report herein the design and synthesis of new efficacious nitrofuran hybrids containing isatin for antimicrobial studies (Figure 5). Furthermore, several isatin and nitrofuran derivatives have been documented to display potent anticancer activity [28–31]. Therefore, the hybrid molecules were also screened as potential anticolon cancer agents.

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2. Results and Discussion

2.1. Chemistry

The 5-nitrofuran-isatin hybrids (2, 5–7) were synthesized as outlined in Scheme 1. Step one involved the condensation of the commercially available isatin with hydrazine hydrate to provide the intermediate (1) in 91% global yield. Diverse precursors have been used to prepare different (Z)-3-hydrazonoindolin-2-ones [28, 30]. In step 2, compound 1 underwent a second condensation step by refluxing with 5-nitro-2-furaldehyde using EtOH acidified with glacial acetic acid as a solvent to afford the Schiff base (2). With 2 in hand, we performed Mannich reaction using 40% formalin and different secondary amines in EtOH to furnish Mannich bases (5–7) in 70–85% yields. To investigate the impact of the 5‒nitrofuran functionality on antibacterial activity, hydrazone 1 was allowed to react with 2‒furaldehyde and 4‒nitrobenzaldehyde under the same experimental conditions to give the Schiff bases 3 and 4, respectively (Figure 6).

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Various spectroscopic techniques such as IR, NMR (¹H and ¹³C), and mass spectrometry were employed to verify the chemical structures of the prepared derivatives and were found to be consistent with the proposed structures. IR spectra of hydrazones 2–4 and Mannich bases 5–7 confirmed the existence of the carbonyl groups as shown by the stretching vibration bands in the region 1720–1737 cm⁻¹. Only compounds 2–4 showed the absorption band of NH functionality in the region 3280–3288 cm⁻¹. In addition, the IR spectra of 2, 4–7 revealed two stretching bands at 1513 and 1305–1348 cm⁻¹ attributed to the nitro group linked to the aromatic system. The (C=N) stretching vibrations in the range of 1596–1612 cm⁻¹ were observed for compounds 2–7. The IR spectra of Mannich bases 5–7 exhibited characteristic bands at 2919–2977 cm⁻¹ corresponding to stretching absorption of aliphatic (‒CH₂‒) protons. The ¹H and ¹³C-NMR spectra of compound 6 are discussed in detail for the reader as an example of the designed hybrids. The singlet at 2.15 ppm shown in the ¹H NMR spectrum of 6 corresponds to the ‒CH₃ group. The eight protons of the piperazine ring appeared as two separate signals at 2.32 and 2.58 ppm. The first set of piperazine protons corresponding to the first peak was attributed to CH₃‒NCH₂ protons, while the second set resonating at 2.58 ppm was ascribed to CH₂‒N‒CH₂‒protons. The characteristic singlet signal at 4.48 ppm was attributed to the methylene protons attached to the N1 of the isatin ring (‒N‒CH₂‒N‒). The six aromatic protons of the benzene and furan rings were observed at δ 7.11–7.60 ppm. In addition, the signal displayed as a singlet at 8.60 ppm integrated for one proton was assigned to the azomethine ‒CH=N‒ proton. ¹³C-NMR spectrum of 6 showed two carbon signals at δ = 161.30 and 151.94 ppm, referring to the carbon of carbonyl (C=O) and azomethine (CH=N) groups, respectively. In addition, the presence of four carbon signals at the aliphatic region of the spectrum δ = 56.51, 55.12, 54.59, and 45.86 ppm indicated the existence of (‒N‒CH₂‒N‒), two sets of piperazine carbons and the ‒N‒CH₃ groups, respectively. Further, characteristic carbon signals belonging to the aromatic and C-3 of the isatin ring (=C) carbons were observed in the region δ = 150.57 − 112.68 ppm. One more piece of evidence for the formation of compound 6 was obtained by recording the mass spectra. The molecular ion peak was observed at m/z = 397.2 corresponding to [M + H]⁺. Further, the other fragmentation peaks of 6 were observed at 289.1 (12%), 154.1 (100%), 136.1 (82%), and 113.1 (23%).

2.2. Antimicrobial Activity

The potentiality of the target compounds 2–7 as antimicrobials was appraised for their in vitro antimicrobial studies against representative pathogenic Gram-positive strains (Methicillin-resistant S. aureus (MRSA) and Gram-negative strains (E. coli, P. aeruginosa, A. baumannii, and K. pneumoniae (MDR KP) in addition to two fungal strains, namely, C. albicans and C. neoformans (Table 4). The activity results of the target compounds were described in Table 1 as MIC values. It is evident from this table that aminoalkylated derivatives 5–7 proved to be more potent antibacterial agents than the parent compound 2. In contrast, compounds 3 and 4 demonstrated reduced potency compared to 2 against most of the tested organisms, highlighting the importance of 5‒nitrofuran moiety for better antimicrobial activity [32]. In order to ascertain this hypothesis and to establish a meaningful structure-activity relationship (SAR), an appreciable number of diversified 5-nitrofuran‒isatin hybrids need to be synthesized and investigated. Compound 6, bearing N-methylpiperazine periphery, showed much higher activity against methicillin-resistant S. aureus (MRSA) with a low MIC of only 1 μg/mL (D_max = 98.5%). MRSA is one of the most prominent and widespread pathogenic organisms responsible for serious hospital and community-acquired infections and is therefore considered a high priority for new antibiotic discovery [33–35]. The obtained MIC value for compound 6 against MRSA (ATCC 43300) is superior relative to the reference oxacillin (MIC = 2 μg/mL) when examined against the same strain [36]. Interestingly, it was equipotent to vancomycin, the last line of defense against MRSA infection [37, 38]. However, moderate activity was observed for 6 when tested against E. coli, A. baumannii, and MDR KP (MICs = 32 μg/mL, D_max = 90–99%). On the other hand, compound 6 at the highest tested concentration (MIC > 32 μg/mL) was devoid of activity against both fungal pathogens. Replacement of the N-methylpiperazine ring in 6 with morpholine in 5 resulted in 8‒fold reduction in potency against MRSA (MIC = 8 μg/mL, D_max = 95.5%). While the same potency was conserved against E. coli and A. baumannii (MIC = 32 μg/mL), compound 5 was found inactive against MDR KP (MIC > 32 μg/mL). Additionally, replacement of the methyl group in 6 with the phenyl ring in 7 led to a great reduction in anti-MRSA activity (MIC = 32 μg/mL). Notwithstanding, compound 7 was found equipotent to 6 against A. baumannii. Since Mannich bases of isatin exhibit more antibacterial activity relative to their corresponding Schiff bases, the findings in the present study are consistent with this fact [24]. Regarding the activity against the tested fungal pathogens, all of the prepared derivatives were found to be inactive. In an attempt to verify the therapeutic value of the target compounds in the current work, they were investigated for their cytotoxicity against mammalian cells and hemolytic properties against RBCs. All the compounds are devoid of any considerable cytotoxicity at the highest test concentration (CC₅₀ > 32 μg/mL). Notably, all target compounds didn’t result in RBC hemolysis at the maximum test concentration (HC₁₀ > 32 μg/mL), as well. Obviously, the promising in vitro antibacterial potency of compounds 5 and 6 along with their revealed safety, provide a platform for future development of potent antimicrobial drugs.

Table 1

Biological evaluation (MICs, μg/mL) of the synthesized derivatives against selected microorganisms.

	MIC (μg/mL)
	Species/compound	2	3	4	5	6	7
Gram-positive bacteria	Staphylococcus aureus (MRSA)	>32	>32	>32	8 (Dmax 95.5%)	1 (Dmax 98.5%)	32 (Dmax 93.7%)
Gram-negative bacteria	Escherichia coli	>32	>32	>32	32 (Dmax 97.6%)	32 (Dmax 98.9%)	>32
	Pseudomonas aeruginosa	>32	>32	>32	>32	>32	>32
	Acinetobacter baumannii	>32	>32	>32	32 (Dmax 99.9%)	32 (Dmax 89.9%)	32 (Dmax 85.3%)
	Klebsiella pneumoniae (MDR KP)	>32	>32	>32.0	>32	32 (Dmax 96.1%)	>32
Fungi	Candida albicans	>32	>32	>32.0	>32	>32	>32
	Cryptococcus neoformans	>32	>32	>32.0	>32	>32	>32

^∗p < 0.05.

2.3. Anticancer Activity

The anticancer activity of synthesized compounds was evaluated against the colon cancer cell line HCT 116. Potent inhibitory activity with IC₅₀ values ranging from 1.62 to 8.8 μM was demonstrated by the hybrid molecules except hybrid 4 which bears a nitrobenzene moiety (Table 2). Notably, 3 revealed prominent inhibitory activity (IC₅₀ = 1.62 μM) that was two times more potent than the reference drug sunitinib (IC₅₀ = 3.4 μM) against the same cancer cell line [39]. Therefore, compound 3 exhibited as a potential candidate for developing a novel therapeutic agent against colon carcinoma.

Table 2

The inhibitory activity on the colon cancer cell of compounds 2–7.

Compounds	IC50μM (HCT 116 colon cancer cell line)
2	2.6∗
3	1.62∗
4	Inactive
5	5.28
6	8.8
7	2∗

^∗p < 0.05.

2.4. Computational Studies

2.4.1. Molecular Docking

Being a designed hybrid, the most active compound 6 is expected to affect its antibacterial activity via inhibition of enzymes known to be targets for compounds having a nitrofuran moiety and/or isatin scaffold. In order to confirm this, we docked compound 6 with the nitroreductase (NTR) enzyme using autodock vina. The nitrofuran moiety has to go through activation reactions catalyzed by NTR to produce free radicals that react with cellular macromolecules, leading to a series of cellular events that eventually end in microbial death. Interestingly, compound 6 was proved to have a good affinity to the NTR judged by binding energy when compared to that of the known native ligand nitrofurazone (−9.2 kcal/mol and −7.3 kcal/mol, respectively). Compound 6 docked nicely between the two subunits A and B of NTR occupying almost the same area of docked nitrofurazone (Figure 7). It exhibited hydrogen bonds with SER12, LYS14, and LYS205. Two more hydrogen bonds were observed uniquely for compound 6 with ASN200 and ARG107 further stabilizing the 6-NTR complex. Moreover, the pi-cation and pi-alkyl interactions with ARG207 and Pro163, respectively, helped anchor 6 to NTR increasing its affinity as evidenced by the observed high binding energy.

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2.4.2. In-Silico Calculation of Physicochemical and ADME Descriptors

Theoretical estimation of the pharmacokinetic properties was performed for compounds 3 and 6 using the QikProp module of Schrödinger. The default parameters in normal mode were used for ADME prediction. Table 3 illustrates the ADME properties for the topmost compounds 3 and 6. Overall, ADME values were within the specified limits recommended for a compound to act as a suitable drug candidate [40]. However, the hybrid 6 showed low lipophilicity and consequently low oral absorption (49%) along with poor penetration to the CNS (QPPMDCK < 25). Therefore, hybrid 6 should be subjected to further optimization steps to improve its pharmacokinetic properties. On the other hand, hybrid 3 has the requisite physicochemical properties to have the potential to be an anticancer drug lead.

Table 3

Drug-like properties of the most potent hybrids 3 and 6 obtained from QikProp.

Properties	Permissible ranges	3	6
#stars	(0–5)	0	0
#amide	(0-1)	0	0
#rotor	(0–15)	3	6
#rtvFG	(0–2)	1	1
mol_MW	(130–725)	239.233	396.405
SASA	(300–1000)	479.921	711.627
FOSA	(0–750)	24.431	267.141
FISA	(7–330)	99.275	167.889
PISA	(0–450)	356.215	276.597
WPSA	(0–175)	0	0
donorHB	(0–6)	1	0
accptHB	(2–20)	5.5	11
QPlogPo/w	(−2–6.5)	1.672	0.168
QPPCaco	(<25 poor, >500 great)	1133.639	15.762
CIQPlogS	−6.5–0.5	−2.8	−0.527
QPlogBB	−3.0–1.2	−0.518	−0.811
QPPMDCK	<25 poor >500 great	566.54	6.822
#metab	(1–8)	2	4
QPlogKhsa	−1.5–1.5	−0.392	−0.965
Human oral absorption	1, 2, or 3 for low, medium, or high	3	2
Percent human oral absorption	>80% is high	91.404	49.365
PSA	7–200	75.305	116.317
Rule of five	Maximum is 4	0	0
Rule of three	Maximum is 3	0	1

3. Experimental

3.1. General

The reacting materials included 5-nitrofuran-2-carbaldehyde, isatin, and secondary amines, all of which were commercially accessible from Sigma-Aldrich in the United States. Analytical grade solvents and reagents were employed in this study and did not require further purification steps. The reactions progress was monitored using precoated silica gel TLC plates, while visualization of the spots was carried out using a UV lamp. TLC was used to verify the purity of the products using DCM and methanol (9: 1) as the mobile phase. Melting points (mp) were measured uncorrected using a Stuart melting point instrument. Shimadzu IR apparatus was used to record infrared (IR) spectra on KBr disk, and the data are presented in $\overline{\nu }$ (cm⁻¹). The ¹H and ¹³C spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d6) at 400 and 100 MHz, respectively, and were performed at the NMR unit Faculty of Pharmacy -Mansoura University. Chemical shifts (δ_H) are recorded relative to the internal standard, tetramethyl silane (TMS). Fast atom bombardment mass spectrometry in positive-ion mode was used to provide mass spectra.

The antimicrobial screening was carried out against certain pathogenic bacteria and fungi (Table 4). This screening was done by open-access antimicrobial screening program, University of Queensland, Australia [41]. Molecular docking was performed using Autodock Vina, while the ADME analysis was obtained using the Qikprop module of Schrodinger.

Table 4

Microbial strains and cell lines.

Organism	Strain	Description
Escherichia coli	ATCC 25922	FDA control strain
Klebsiella pneumoniae	ATCC 700603	MDR
Acinetobacter baumannii	ATCC 19606	Type strain
Pseudomonas aeruginosa	ATCC 27853	Quality control strain
Staphylococcus aureus	ATCC 43300	MRSA
Candida albicans	ATCC 90028	CLSI reference
Cryptococci neoformans	ATCC 208821	H99, type strain
Homo sapiens embryonic kidney cells	ATCC CRL-1573	HEK-293
Homo sapiens	ARCBS 5400 00150	Whole blood

3.2. Synthesis of Hydrazone 1

Hydrazone 1 was synthesized according to the reported procedure [23]. This procedure involved dissolving both isatin (1 mmol) and hydrazine hydrate (99%, 0.055 g, 1.1 mmol) in absolute methanol (25 mL). Then this mixture was heated under reflux for 1 h, and the resultant solid product was collected after cooling, dried and recrystallized from EtOH/DMF to furnish hydrazone 1 in 91% yield.

3.2.1. (Z)-3-Hydrazonoindolin-2-one (1)

Mp. = 228–230°C (Lit. [42] Mp. = 230–231°C), yield is 91%, and physical appearance is pale yellow, odorless powder. IR (KBr): $\overline{v}$ cm⁻¹ 3349 − 3168 (NH, NH₂), 1681 (C=O), 1585 (C‒N). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ6.87 (d. 1H, J = 7.60 Hz, ArH), 6.97 (t, 1H, J = 7.6 Hz, ArH), 7.13 (t, 1H, J = 7.6 Hz, ArH), 7.37 (d. 1H, J = 7.2 Hz, ArH), 9.54 (d. 1H, J = 14.4 Hz., amino H), 10.56 (d. 1H, J = 14.0 Hz, amino H), 10.7 (s, 1H, NH).

3.3. Synthesis of Schiff Bases 2–4

In pure ethanol (30 mL), hydrazone 1 (5 mmol) was added to a stirred solution of the suitable aldehyde (5 mmol), acidified with 4 drops of glacial acetic acid, and then refluxed for 5 hours. Schiff bases 2–4 were obtained in 85–92% yield following recrystallization of the collected solid products using EtOH/DMF as solvent.

3.3.1. (Z)-3-((E)-((5-Nitrofuran-2-yl)methylene)hydrazono)indolin-2-one (2)

Mp. = 255–258°C, yield: 91%, physical appearance: pale yellow, odorless powder. IR (KBr): $\overline{v}$ cm⁻¹3286 (N—H), 1737 (C=O), 1612 (C=N), 1513, 1348 (N—O). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 6.91 (d, 1H, J = 7.6 Hz, ArH), 7.04 (t, 1H, J = 7.6, ArH), 7.42–7.48 (m, 1H, ArH), 7.65 (d, 1H, J = 3.6 ArH), 7.84–7.88 (m, 2H, ArH), 8.58 (s, 1H, CH=N), 10.94 (s, 1H, NH). ¹³C-NMR (DMSO-d6, 100 MHz) (ppm) δ 164.6, 153.38, 151.01, 150.74, 147.80, 145.88, 134.90, 129.58, 122.99, 119.65, 116.62, 114.68 and 111.50. FAB–MSm/z = 285 [M + H]⁺.

3.3.2. (Z)-3-((E)-(Furan-2-ylmethylene)hydrazono)indolin-2-one (3)

Mp. = 207–210°C, yield: 90%, physical appearance: pale orange, odorless powder. IR (KBr): $\overline{v}$ cm⁻¹ 3280 (N—H), 1733 (C=O), 1616 (C=N). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 6.81 (d, 1H, J = 1.2 Hz, ArH), 6.92 (t, 1H, J = 8, ArH), 7.05 (t, 2H, J = 8, ArH), 7.34–7.43 (m, 1H, ArH), 8.08 (d, 2H, J = 10.8, ArH), 8.57 (s, 1H, CH=N), 10.85 (s, 1H, NH).¹³C-NMR (DMSO-d6, 100 MHz) (ppm) δ 165.16, 151.39, 150.74, 149.54, 148.57, 145.38, 134.18, 129.54, 122.78, 120.31, 117.18, 113.68 and 111.27. FAB–MS m/z = 240 [M + H]⁺.

3.3.3. (Z)-3-((E)-(4-Nitrobenzylidene)hydrazono)indolin-2-one (4)

Mp. = 280–283°C, yield: 90%, physical appearance: pale orange, odorless powder; IR (KBr): $\overline{v}$ cm⁻¹ 3288 (N—H), 1729 (C=O), 1596 (C=N), 1513, 1338 (N—O).¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 6.91 (d, 1H, J = 8 Hz, ArH), 7.03 (t, 1H, J = 7.6, ArH), 7.43 (t, 1H, J = 7.6, ArH), 7.74 (d, 1H, J = 7.6, ArH), 8.21 (d, 2H, J = 8.8, ArH), 8.39 (d, 2H, J = 8.8, ArH), 8.69 (s, 1H, CH=N), 10.94 (s, 1H, NH).¹³C-NMR (DMSO-d6, 100 MHz) (ppm) δ 164.42, 156.83, 150.20, 149.51,145.78, 139.56, 134.66, 130.20, 129.23, 124.79, 122.98, 116.53, and 111.53. FAB–MS m/z = 295 [M + H]⁺.

3.4. Synthesis of Mannich Bases 5–7

Mannich bases 5–7 were synthesized using the method reported by Aboul-Fadl et al. [43]. This method involved reacting the Schiff’s base 2 (0.5 mmol) with formaldehyde (0.02 g, 0.75 mmol) at RT in 30 mL ethanol. Then the appropriate secondary amine (0.75 mmol) was gradually added with continuous stirring for 24 hours. Reaction progress was monitored using TLC. Once completed, the reaction solvent was concentrated and the resultant solid product was filtered, extensively washed and air dried to give Mannich bases 3–5 in 70–85% yield.

3.4.1. (Z)-1-(Morpholinomethyl)-3-((E)-((5-nitrofuran-2-yl)methylene)hydrazono)indolin-2-one (5)

Mp. = 215–218°C, yield: 74%, physical appearance: pale orange, odorless powder; IR (KBr): $\overline{v}$ cm⁻¹2977 (aliphatic C—H), 1729 (C=O), 1604 (C=N), 1515, 1342 (N—O). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 2.57 (m, 4H, morpholine CH₂N), δ 3.56 (m, 4H, morpholine CH₂O), 4.48 (s,2H, NCH₂N), 7.11 (m, 1H, ArH), 7.28 (d, 1H, J = 7.6, ArH), 7.53 (t, 1H, J = 7.6, ArH), 7.58 (d, 1H, J = 4, ArH), 7.88 (d, 1H, J = 3.6, ArH), 7.91 (d, 1H, J = 7.6, ArH), 8.60 (s, 1H, CH=N).¹³C-NMR (DMSO-d6, 100 MHz) (ppm) δ 164.64, 153.43, 151.02, 150.74, 147.93, 145.89, 134.90, 129.58, 122.99, 119.64, 116.63, 114.68, 111.65, 66.47, 62.01, and 51.08. FAB–MS m/z = 381.3 [M‒2]⁺.

3.4.2. (Z)-1-((4-Methylpiperazin-1-yl)methyl)-3-((E)-((5-nitrofuran-2-yl)methylene)hydrazono)indolin-2-one (6)

Mp. = 135–138°C, yield: 80%, physical appearance: red, odorless powder; IR (KBr): $\overline{v}$ cm⁻¹ 2927 (Aliphatic C—H), 1722 (C=O), 1604 (C=N), 1519, 1344 (N—O). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 2.15 (s, 3H, CH₃),2.32 (m, 4H, piperazine CH₃NCH₂), 2.58 (m, 4H, piperazine CH₂NCH₂), 4.48 (s, 2H, NCH₂N), 7.11 (m, 1H, ArH), 7.25 (d, 1H, J = 7.6, ArH),7.53 (t, 1H, J = 7.6, ArH), 7.58 (d, 1H, J = 3.6, ArH), 7.88 (d, 1H, J = 3.6, ArH), 7.91 (d, 1H, J = 7.6, ArH), 8.60 (s, 1H, CH=N). ¹³C-NMR (DMSO-d6, 100 MHz) (ppm) δ 161.30, 151.94, 150.57, 147.77, 145.89, 134.91, 129.58, 125.17, 122.99, 119.66, 116.63, 114.68, 112.68, 56.51, 55.12, 54.59 and 45.86. FAB–MS m/z = 397.2 [M + 1]⁺.

3.4.3. (Z)-3-((E)-((5-Nitrofuran-2-yl)methylene)hydrazono)-1-((4-phenylpiperazin-1-yl)methyl)indolin-2-one (7)

Mp. = 200–204°C, yield: 85%, physical appearance: orange, odorless powder; IR (KBr): $\overline{v}$ cm⁻¹3284 (Aromatic C—H), 2919 (Aliphatic C—H), 1720 (C=O), 1602 (C=N), 1544, 1305 (N—O). ¹H NMR (DMSO-d6, 400 MHz) (ppm), δ 2.74 (m, 4H, piperazine CH₂NCH₂), 3.13 (m, 4H, piperazine PhNCH₂), 4.58 (s, 2H, NCH₂N), 6.77 (t, 1H, J = 7.2, ArH), 6.91 (d, 1H, J = 8, ArH), 7.14 (t, 1H, J = 7.6, ArH), 7.20 (t, 2H, J = 7.9, ArH), 7.32 (d, 1H, J = 8, ArH), 7.88 (d, 1H, J = 4, ArH), 7.92 (d, 1H, J = 7.6, ArH), 8.60 (s, 1H, CH=N).¹³C-NMR (TFA, 100 MHz) (ppm) δ 163.34, 153.24, 151.70,147.00, 141.90, 133.33, 132.44, 131.99, 127.91, 126.76, 124.63, 121.95, 120.86, 119.85, 117.04, 114.22, 111.40, 68.95, 53.65, 43.47. FAB–MS m/z = 458.2 [M]⁺.

3.5. Biological Investigations

3.5.1. Test Microorganisms and Cell Lines

See Table 4.

3.5.2. Sample Preparation

Sample preparation was carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [34, 41]: “Samples for antibacterial, antifungal, cytotoxicity, and hemolysis assays were prepared in DMSO and water to a final testing concentration of 32 μg/mL or 20 μM and serially diluted 1: 2 fold for 8 times, then stored at 4°C until use. Each sample concentration was prepared in 384-well plates, nonbinding surface plate (NBS; Corning 3640) for each bacterial/fungal strain, tissue-culture-treated (TC-treated; Corning 3712/3764) black for mammalian cell types, and polypropylene 384-well (PP; Corning 3657) for hemolysis assays, all in duplicate (n = 2), keeping the final DMSO concentration to a maximum of 0.5%. All sample preparation was done using liquid handling robots.”

3.5.3. Standards Preparation and Quality Control

Standards preparation and quality control were carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [34, 41]: “Colistin (Sigma; C4461) and vancomycin (Sigma; 861987) were used as positive bacterial inhibitor standards for Gram-negative and Gram-positive bacteria, respectively. Fluconazole (Sigma; F8929) was used as a positive fungal inhibitor standard for C. albicans and C. neoformans. Tamoxifen (Sigma; T5648) was used as a positive cytotoxicity standard. Melittin (Sigma: M2272) was used as a positive hemolytic standard. Each antibiotic standard was provided in 4 concentrations, with 2 above and 2 below its Minimum Inhibitory Concentration (MIC) or CC₅₀ value, and plated into the first 8 wells of column 23 of the 384-well NBS plates. Tamoxifen and melittin were used in 8 concentrations in 2-fold serial dilutions with 50 μg/mL highest concentration. The quality control (QC) of the assays was determined by Z′-Factor, calculated from the Negative (media only) and Positive Controls (bacterial, fungal, or cell culture without inhibitor), and the Standards. Plates with a Z′-Factor of ≥ 0.4 and Standards active at the highest and inactive at the lowest concentration, were accepted for further data analysis. All values of the standard compounds: MIC (μg/mL) of the antibiotic and antifungal standards, susceptibility profile of human embryonic kidney cell lines (CC₅₀, μg/mL) and the susceptibility profile of human washed red cells (HC₁₀ and HC₅₀, μg/mL) are the average of ≥ 6 independent biological replicates.”

3.5.4. Cytotoxicity Assay

The cytotoxicity assay was carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [1, 41]: “HEK-293 cells were counted manually in a Neubauer hemocytometer and then plated in the 384-well plates containing the compounds to give a density of 5,000 cells/well in a final volume of 50 μL. DMEM supplemented with 10% FBS was used as growth media, and the cells were incubated together with the compounds for 20 h at 37°C in 5% CO₂. Cytotoxicity (or cell viability) was measured by fluorescence, ex: 560/10 nm, em: 590/10 nm (F_560/590), after the addition of 5 μL of 25 μg/mL resazurin (2.3 μg/mL final concentration) and after incubation for a further 3 h at 37°C in 5% CO₂. The fluorescence intensity was measured using a Tecan M1000 Pro monochromator plate reader using automatic gain calculation. CC₅₀ (concentration at 50% cytotoxicity) was calculated by curve fitting the inhibition values versus log (concentration) using a sigmoidal dose-response function, with variable fitting values for bottom, top and slope. In addition, the maximal percentage of cytotoxicity is reported as D_max, indicating any compounds with partial cytotoxicity. The curve fitting was implemented using Pipeline Pilot’s dose-response component, resulting in similar values to curve fitting tools such as GraphPad’s Prism and IDBS’s XlFit. Any value with > indicates a sample with no activity (low D_max value) or samples with CC₅₀ values above the maximum tested concentration (higher D_max value). Cytotoxic samples were classified by CC₅₀ ≤ 32 μg/mL or CC₅₀ ≤ 10 μM in either replicate (n = 2 on different plates). In addition, samples were flagged as partial cytotoxic if D_max ≥ 50%, even with CC₅₀ > the maximum tested concentration.”

3.5.5. Hemolysis Assay

The hemolysis assay was carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [1, 41]: “Human whole blood was washed three times with 3 volumes of 0.9% NaCl (saline) and then resuspended in saline to a concentration of 0.5 × 10⁸ cells/mL, as determined by manual cell count in a Neubauer hemocytometer. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 μL. After a 10 min shake on a plate shaker, the plates were then incubated for 1 h at 37°C. After incubation, the plates were centrifuged at $1000g$ for 10 min to pellet cells and debris, 25 μL of the supernatant was then transferred to a polystyrene 384-well assay plate. Hemolysis was determined by measuring the supernatant absorbance at 405 mm (OD₄₀₅). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC₁₀ and HC₅₀ (concentration at 10% and 50% hemolysis, respectively) were calculated by curve fitting the inhibition values versus log (concentration) using a sigmoidal dose-response function with variable fitting values for top, bottom, and slope. Hemolysis samples were classified by HC₁₀ ≤ 32 μg/mL or HC₁₀ ≤ 10 μM in either replicate (n = 2 on different plates). In addition, samples were flagged as partial hemolytic if D_max ≥ 50%, even with HC₁₀ > the maximum tested concentration.”

3.5.6. Antimicrobial Activity

(1) Antibacterial Assay. The antibacterial assay was carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [9, 41]: “All bacteria were cultured in cation-adjusted Mueller Hinton broth (CaMHB) at 37°C overnight. A sample of each culture was then diluted 40-fold in fresh broth and incubated at 37°C for 1.5–3 h. The resultant mid-log phase cultures were diluted (CFU/mL measured by OD₆₀₀), then added to each well of the compound-containing plates, giving a cell density of 5 × 10⁵ CFU/mL and a total volume of 50 μL. All the plates were covered and incubated at 37°C for 18 h without shaking. Inhibition of bacterial growth was determined by measuring the absorbance at 600 nm (OD₆₀₀) using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The MIC was defined as the lowest concentration at which the growth was fully inhibited, determined by an inhibition ≥80%. In addition, the maximal percentage of growth inhibition is reported as D_max, indicating any compounds with partial activity.”

(2) Antifungal Assay. The antifungal assay was carried out by CO-ADD, the Community for Open Antimicrobial Drug Discovery, according to the following method [33, 41]: “Fungi strains were cultured for 3 days on yeast extract-peptone dextrose (YPD) agar at 30°C. A yeast suspension of 1–5 × 10⁶ CFU/mL (as determined by OD₅₃₀) was prepared from five colonies. The suspension was subsequently diluted and added to each well of the compound-containing plates giving a final cell density of fungi suspension of 2.5 × 10³ CFU/mL and a total volume of 50 μL. All plates were covered and incubated at 35°C for 36 h without shaking. Growth inhibition of C. albicans was determined by measuring the absorbance at 630 nm (OD₆₃₀), while the growth inhibition of C. neoformans was determined by measuring the difference in absorbance between 600 and 570 nm (OD_600–570), after the addition of resazurin (0.001% final concentration) and incubation at 35°C for 2 h. The absorbance was measured using a BiotekMultiflo Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well using the negative control (media only) and positive control (fungi without inhibitors) on the same plate. The MIC was defined as the lowest concentration at which the growth was fully inhibited, determined by an inhibition ≥80% for C. albicans and an inhibition ≥70% for C. neoformans. Due to a higher variance in growth and inhibition, a lower threshold was applied to the data for C. neoformans. In addition, the maximal percentage of growth inhibition is reported as D_max, indicating any compounds with marginal activity. Inhibition assays of active compounds (Hit confirmation) against tested bacteria and fungi by whole cell growth were conducted as an 8-point dose response in duplicate (n = 2) to determine the MICs. The inhibition of growth is measured against those microorganisms that showed susceptibility to the compounds tested in the primary screen.”

3.5.7. Anticancer Activity

The cytotoxicity was assessed according to our previous report [44–46]. HCT 116 cells, a human colon cancer cell line, were cultured with DMEM containing 10% FBS. HCT 116 were seeded at a density of 5 × 10³ cells/well/100 μL in a 96-well plate for 24 h. Then, 100 μL of each compound prepared at 2-fold concentration series added to each well. After 72 h incubation, cells were washed out with PBS (-). Then, 100 μL of 10% Cell counting kit-8 Dojindo® was added to each well. After 30–60 minutes of incubation, the absorbance was measured using a microplate reader at 450 nm against the reference wavelength of 630 nm. The data was normalized by GraphPad Prism 9, and the concentration which induces 50% cell death was plotted out.

3.6. Computational Studies

3.6.1. Molecular Docking Study

Autodock Vina was employed to conduct the molecular docking studies [47] and the resultant complexes were visualized with BIOVIA Discovery Studio Visualizer [48]. The crystal structure of the E. coli nitroreductase (NTR) was downloaded from the archive of macromolecules structural data “Protein Data Bank” (PDB ID: 1YKI)[49]. The grid box was generated to include all the residues interacting with the cocrystallized ligand. The grid size was established as 60 × 60 × 60 xyz points; meanwhile, the grid point spacing of 0.35 Å was employed. The grid box’s dimensions were set at (x, y, z): 5.937, 12.311, 44.855.

3.6.2. In Silico Calculation of Physicochemical and ADME Descriptors

For ADME predictions, the QikProp module by Schrodinger, LLC, NY was employed [50]. Pharmaceutically relevant characteristics of the most potent hybrids 3 and 6 were delineated in Table 3.

4. Conclusion

In this study, some 5-nitrofuran-isatin hybrids were synthesized and screened for potential antimicrobial and anticancer activities. Greater potency was exhibited by compounds 5 and 6 against methicillin-resistant Staphylococcus aureus (MRSA) with the minimum inhibitory concentration values of 8 and 1 μg/mL, respectively. Molecular docking results on the E. coli nitroreductase (NTR) suggested that compound 6 might display its antibacterial action by a mechanism similar to that of nitrofurazone. Cytotoxicity and hemolytic activity studies revealed that the tested compounds were neither cytotoxic nor hemolytic at the highest test concentration (CC₅₀ and HI10 > 32 μg/mL), reflecting the potentiality of the designed hybrids to serve as future leads for further investigations. With compound 4 as an exception, the designed hybrids demonstrated significant anticancer activity against the HCT 116 colon cancer cell line (IC₅₀ = 1.62‒8.8 μM). Interestingly, compound 3 (IC₅₀ = 1.62 μM) was found to be superior to the reference anticancer drug sunitinib. Therefore, the designed 5-nitrofuran-isatin hybrids would be a potential platform for the discovery of potent chemotherapeutic agents in the future.

Acknowledgments

This study was funded by the Deanship of Scientific Research at Jouf University under grant no. (40/59).

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