Synthesis, Biological, and Molecular Docking Studies on 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives and Their Nanoparticles Targeting Colorectal Cancer

Abstract

Initiation of colorectal carcinogenesis may be induced by chromosomal instability caused by oxidative stress or indirectly by bacterial infections. Moreover, proliferating tumor cells are characterized by reprogrammed glucose metabolism, which is associated with upregulation of PDK1 and LDHA enzymes. In the present study, some 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives in addition to Fe₃O₄ and Fe₃O₄/SiO₂ nanoparticles (NPs) supported with a new Schiff base were synthesized for biological evaluation as PDK1 and LDHA inhibitors as well as antibacterial, antioxidant, and cytotoxic agents on LoVo and HCT-116 cells of colorectal cancer (CRC). The results showed that compound 1b is the most active as PDK1 and LDHA inhibitor with IC₅₀ values (μg/mL) of 57.10 and 64.10 compared to 25.75 and 15.60, which were produced by the standard inhibitors sodium dichloroacetate and sodium oxamate, respectively. NPs12a,b and compound 1b exhibited the strongest antioxidant properties with IC₅₀ values (μg/mL) of 80.0, 95.0, and 110.0 μg/mL, respectively, compared to 54.0 μg/mL, which was produced by butylated hydroxy toluene. Moreover, NPs12a and carbamate derivative 3b exhibited significant cytotoxic activities with IC₅₀ values (μg/mL) of 57.15 and 81.50 (LoVo cells) and 60.35 and 71.00 (HCT-116 cells). Thus, NPs12a and compound 3b would be considered as promising candidates suitable for further optimization to develop new chemopreventive and chemotherapeutic agents against these types of CRC cell lines. Besides, molecular docking in the colchicine binding site of the tubulin (TUB) domain revealed a good binding affinity of 3b to the protein; in addition, the absorption, distribution, metabolism, and excretion (ADME) analyses showed its desirable drug-likeness and oral bioavailability characteristics.

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer death globally.¹ In Saudi Arabia, it is ranked as the first cancer type in males and the third in females.²

CRC is a complex disease that results from accumulated genetic and epigenetic changes, which inactivate tumor suppressor genes and activate oncogenes and eventually initiate tumorigenesis.³

Several studies have shown that microbial dysbiosis of certain bacterial species such as Streptococcus bovis, Helicobacter pylori, enterotoxigenic Bacteroides fragilis (ETBF), Escherichia coli (E. coli), and Enterococcus faecalis (E. faecalis) may indirectly promote colorectal carcinogenesis via epigenome dysregulation.⁴⁻⁷

Further, throughout tumor progression, the neoplastic cells preferentially rely on glycolysis instead of oxidative phosphorylation (OXPHOS), which is known as the “Warburg effect”, in order to fuel their increased bioenergetic demands for survival, infinite proliferation, invasion, and metastasis.⁸ This metabolic switch is associated with overexpression of pyruvate dehydrogenase kinase (PDK-1)⁹ and lactate dehydrogenase A (LDHA).¹⁰ PDK-1 inhibits the multi-enzyme pyruvate dehydrogenase complex (PDC) to decrease the level of pyruvate in mitochondria, which in turn limits the OXPHOS and blocks the production of mitochondrial reactive oxygen species (ROS), leading to survival of cancerous cells.¹¹ Similarly, LDHA restrains mitochondrial supply of pyruvate and catalyzes its conversion to lactate.¹² The produced lactate facilitates tumor survival, growth, and metastasis via suppression of host immune surveillance, tumor resistance, and upregulation of various angiogenesis-stimulating molecules.¹³ Accordingly, inhibitions of PDK-1¹⁴ and LDHA¹⁵ have been emerged as therapeutic strategies against cancer.^16,17

It is noteworthy to indicate that the enhancement of the Warburg effect is attributed to accumulated abnormalities resulting from mutations in genomic DNA¹⁸ and mitochondrial DNA.¹⁹ These mutations augment the generation of ROS, which interfere with inflammatory signaling leading to activation of inflammation,²⁰ inducing chromosomal instability (CIN), oxidative stress, and eventually the development of CRC and drug resistance.²¹

Taking together, the optimal reduction of CRC incidence, morbidity, and mortality will require concerted efforts to develop novel drug candidates that are capable of eliminating oxidative stress, eradicating bacteria, and/or inhibiting protumorigenic LDHA and PDK1 enzymes. In this regard, thiophene derivatives are extensively explored in the field of drug design and development as antioxidant,²² antibacterial,²³ and anticancer agents.²⁴⁻²⁹ In particular, 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives have been recognized as promising anticancer agents.³⁰ Many of them (Figure 1) have produced their antiproliferative effects through targeting metalloproteinases 2 and 9 (MMP 2 and 9), HIF-1α and the vascular endothelial growth factor receptor,³¹ the epidermal growth factor receptor (EGFR), human EGFR-related receptor 2 (HER2),³² and the colon cancer-related genes, namely, collagen type X α1 (COL10A1) and collagen type XI α1 (COL11A1).³³

Figure 1.

Examples of reported 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives as anticancer agents against various therapeutic targets.

Moreover, nanotechnology has gained much interest in the field of medicinal chemistry because of advantages such as reduced doses of bioactive molecules, targeted delivery,³⁴ and enhanced bioavailability.³⁵ Indeed, thiophene derivatives loaded onto nanoparticles (NPs) displayed improved solubility and selectivity for cancerous cells compared to unmodified controls.^34,36

Based on the promising anticancer activities of various tetrahydrobenzo[b]thiophene derivatives and as a continuation to our recent investigations to develop new anti-CRC agents,^37,38 herein, we aim to describe the synthesis and characterization of a new series of 4,5,6,7-tetrahydrobenzo[b]thiophene-based carbamates, amides, acetamides, a cyclic imide, a formamidine, and a Schiff base. The later product was used as a capping agent to prepare thiophene-Fe₃O₄ and thiophene-Fe₃O₄/SiO₂ NPs. All the synthesized compounds were evaluated as PDK-1 and LDHA inhibitors as well as antioxidant and antibacterial agents. The most bioactive candidates were screened as cytotoxic agents against LoVo and HCT-116 cells of CRC. Target identification and in silico molecular docking were carried out based on structural similarity between the previously reported antitumor compounds and the promising cytotoxic candidate to elucidate the mechanism underlying the observed antineoplastic effects.

Results and Discussion

Synthesis of 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives

The synthetic routes toward the target compounds are outlined in Scheme 1. Initially, the starting materials 2-amino-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid ethyl ester 1a and 2-amino-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile 1b were allowed to react with some active carbonyl compounds, namely, ethyl chloroformate 2a, chloroacetyl chloride 2b, thiophene-2-carbonyl chloride 2c, and 4-chlorobenzoyl chloride 2d under different reaction conditions. These reactions produced a series of 6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene derivatives tethered with carbamate 3a,b, chloroacetamide 3c,d, and amide 3e–f functionalities, respectively.

Scheme 1.

Reagent and conditions: (i) ClCOOEt, reflux 10 h; (ii) dry CHCl₃, 0 °C, TEA, R′COCl, stirring 1 h, warm up to r.t., reflux 16 h; (iii) CH₃OH, reflux 10 h; (iv) NaOAc, oil bath at 200 °C 2 h; (v) dry xylene, reflux 10 h.

Subsequent condensation of chloroacetamides 3c,d with morpholine 4a and 1-(pyridin-2-yl)piperazine 4b led to nucleophilic displacement of the chloride anion by these secondary amines with the formation of the corresponding new thiophene-based morphino and piprazino derivatives 5a–c.

Moreover, compound 1b was converted to the thieno-pyrroldindione 7 and N,N-dimethylformamidine derivative 9 via reaction with succinic anhydride 6 and DMF-DMA 8, respectively.

Characterization of 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives

The structures of all the newly synthesized products were deduced on the basis of their spectroscopic data as explained using representative examples.

For example, the infra-red (IR) spectrum of carbamate 3b revealed the presence of the stretching absorption bands at ν_max/cm^–1 = 3221, 2219, and 1725 cm^–1 attributable to NH, CN, and CO groups, respectively. The ¹H NMR (300 MHz, DMSO-d₆) spectrum lacked the signal at δ_H = 7.01 ppm due to the primary amino group of the starting material, and it exhibited three new characteristic signals at δ_H = 1.22, 4.21, and 11.18 ppm as follows: a three-proton triplet and a two-proton quartet with coupling constants J = 7.1 Hz in addition to a one-proton singlet, which are attributed to the methyl, methylene, and amidic NH groups, respectively. Moreover, the ¹³C NMR (75 MHz, DMSO-d₆) spectrum displayed 13 signals as expected due to the 3-cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene core in addition to 3 signals at δ_C = 14.31, 61.78, and 153.57 ppm due to the CH₃, CH₂, and CO groups, respectively. Last, the mass spectrum of this compound showed the molecular ion peak [M⁺ + 2] at m/z = 328.41 (26%) for C₁₈H₁₈N₂O₂S in addition to the base peak at m/z = 123.84.

Furthermore, the IR spectrum of 5b exhibited absorption bands at ν_max/cm^–1 = 3222, 1724, 1662, and 1597 attributable to NH, C=O-ester, C=O-amidic, and C=N groups, respectively. The ¹H NMR (300 MHz, DMSO-d₆) spectrum confirmed the insertion of the (pyridin-2-yl)piperazine moiety, which exhibited a characteristic apparent singlet signal integrating to four protons at δ_H = 3.60 ppm due to two of the four CH₂–N groups of the piperazine ring. The four protons of the remaining CH₂–N groups are overlapped with the five protons of the cyclohexene ring at δ_H = 2.46–3.04 ppm. In addition, the four protons of the pyridine ring exhibited four signals as follows: a one-proton multiplet, a one-proton doublet with coupling constant J = 8.1 Hz, an apparent one-proton triplet of doublet with coupling constant values J = 8.1 and 2.4 Hz, and a one-proton multiplet at δ_H = 6.60–6.70, 6.84, 7.56, and 8.10–8.20 ppm, respectively. Similarly, the ¹³C NMR (75 MHz, DMSO-d₆) spectrum indicated the piprazinopyridinyl moiety as two signals at δ_C = 45.00 and 52.62 ppm attributable to the four methylene groups of the piperazine ring in addition to five signals belonging to the five sp²-carbons of the pyridine ring, which were detected among the fifteen signals in the aromatic region at δ_C = 107.16, 111.30, 113.11, 125.61, 126.24, 126.83, 128.37, 130.22, 137.56, 145.67, 146.06, 147.57, 158.92, 164.86 and 167.95 ppm. Moreover, its mass spectrum showed the molecular ion peak [M⁺ + 2] at m/z = 506.41 (24%) for C₂₈H₃₂N₄O₃S and the base peak at m/z = 369.47.

Analogously, the IR spectrum of the fromamidine derivative 9 missed the absorption band due to the primary amino group of its precusror 1b and displayed a new stretching absorption band at ν_max/cm^–1 = 1628 cm^–1 corresponding to the C=N group. The ¹H NMR (300 MHz, DMSO-d₆) spectrum confirmed the presence of the dimethyl formamidine moiety [=CHN(CH₃)₂], which exhibited three singlet signals at δ_H = 2.98, 3.08, and 7.96 ppm attributable to the two methyl groups and the azomethine group, respectively. ¹³C NMR (75 MHz, DMSO-d₆) disclosed the presence of the two methyl groups at δ_C = 34.62 and 38.81 ppm and the azomethine carbon at δ_C = 155.28 ppm. Additionally, the mass spectrum of this compound revealed the presence of the molecular ion peak [M⁺ + 2] at m/z (%) = 311.49 (57), corresponding to the molecular formula of C₁₈H₁₉N₃S, and the base peak at m/z = 93.18.

Synthesis and Characterization of Thiophene-Fe₃O₄ and Thiophene-Fe₃O₄/SiO₂ NPs

It is well established that iron NPs possess enhanced antibacterial, antioxidant, and anticancer properties.³⁹ However, it is known that the magnetic NPs are unstable, especially under physiological conditions and in the presence of air. Therefore, protection of these NPs with suitable species is a very important challenge between chemists. Moreover, their physical and chemical properties are determined by their size, shape, composition, crystallinity, and structure. Consequently, the design of magnetic nanocrystals for biomedical applications needs to be well controlled in order to get magnetic NPs bearing ligands capable of bonding to specific receptors and having a specific narrow range of sizes and a high degree of biocompatibility. Therefore, to investigate the process of the formation of the nanosystem to examine its effect on the various biological activities, we have designed one compound as a representative example for future work. In this regard, Schiff bases have been acknowledged as excellent capping agents capable of inhibiting the overgrowth of NPs and preventing their aggregation/coagulation in colloidal synthesis.⁴⁰ In addition, Schiff bases have gained importance in medicinal and pharmaceutical fields due to their broad spectrum of biological activities like antimicrobial, anticancer, and antioxidant.⁴¹

Thus, in this study, the Schiff base 2-((3-hydroxy-4-methoxybenzylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile 11 was designed and prepared to be used as a coating shell for the new NPs, which will be evaluated for their efficacy as preventive or therapeutic agents against CRC. Initially, condensation of 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carbonitrile 1c with isovanillin 10 yielded the new Schiff base derivative 11 whose structure was confirmed based on its spectroscopic data. Thus, the IR spectrum revealed the disappearance of the characteristic stretching absorption band due to the primary amino group of 1c associated with the emergence of the characteristic stretching absorption bands at ν_max/cm^–1 = 3403 (broad) and 1595 cm^–1, attributable to the phenolic OH and C=N groups, respectively. Similarly, ¹H NMR (300 MHz, DMSO-d₆) analysis provided a rigid support for the assigned structure based on the disappearance of the signal at δ = 6.94 ppm of the NH₂ group of the precursor 1c. Moreover, this spectrum displayed three new singlet signals at δ_H = 9.52, 8.36, and 3.83 ppm due to the phenolic OH, azomethine (CH=N−), and methoxy (OCH₃) groups, respectively. Additionally, the ¹³C NMR (75 MHz, DMSO-d₆) spectrum indicated the presence of the methoxy group at δ_C = 55.65 ppm. Also, the sp²-carbons due to the azomethine group and phenyl ring were displayed in the region of 160.43– 104.87 ppm. Last, the mass spectrum of compound 11 showed the molecular ion peak [M⁺ + 3] at m/z = 315.74 (4%), [M⁺ + 2] at m/z = 314.82 (10%), and the base peak was observed at m/z = 77.13.

Afterward, the magnetite was prepared by coprecipitation method, and it was allowed to react with 4,5,6,7-tetrahydrobenzo[b]thiophene-Schiff base 11 to give aldimine-Fe₃O₄ (NPs12a). The latter product was coated with silica to generate aldimine-Fe₃O₄/SiO₂ nanoparticles (NPs12b) through stirring with tetraethoxysilane (TEOS), as shown in Scheme 2.

Scheme 2.

Reagent and conditions: (i) ethanol, glacial acetic acid catalytic amount, reflux 16 h; (ii) EtOH/ H₂O (70:30) mixture, Fe₃O₄ NPs, compound 11, reflux with stirring 4 h at 70 °C; (iii) EtOH/ H₂O/NH₃ (30:10:1) mixture, Schiff base-Fe₂O₃ NPs 12a, US stirring 30 min, 2.5 mL of TEOS, stirring 22 h.

A series of characterizations were applied to investigate the structures of the aldimine-NPs 12a,b. First, the morphologies of Fe₃O₄ and Fe₃O₄/SiO₂ capped with the organic compound were analyzed by transmission electron microscopy (TEM, JEOL JEM-2100 F). A drop of the colloidal solutions of the prepared NPs in the ethanol solvent was placed onto copper grids and was allowed to dry for TEM testing. The TEM micrograph of the Fe₃O₄-NPs 12a showed a cubic shape with good monodispersity and a mean diameter of 15 ± 2 nm (Figure 2a). Some slight aggregations were observed from the magnified image, which can be attributed to the aggregation of individual particles due to an incomplete coating with the organic molecules. An increase in the size of magnetite NPs 12b was observed as shown in Figure 2b due to the formation of a thin layer of SiO₂, which acted as a shell to the Fe₃O₄ core.

Figure 2.

TEM images of aldimine NPs: (a) Fe₃O₄ and (b) Fe₃O₄–SiO₂.

Second, a Malvern Instruments Zetasizer (model 2000) was used to determine the particle sizes of Fe₃O₄ and Fe₃O₄/SiO₂ colloids in ethanol by the dynamic light scattering technique. The measurements indicated the lowered particle size and polydispersity index for Fe₃O₄-NPs than for Fe₃O₄/SiO₂. These results are in agreement with the data obtained from TEM micrographs. The increase in particle size diameter for both Fe₃O₄ and Fe₃O₄/SiO₂ NPs compared with that obtained from TEM micrographs is due to the interaction between NPs in the ethanol solvent, which lead to the formation of some agglomerations (Figure 3a,b).

Figure 3.

Particle size distribution of Fe-NPs capped with 2-((3-hydroxy-4-methoxybenzylidene)amino)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carbonitrile: (a) Fe₃O₄-NPs and (b) Fe₃O₄/SiO₂-NPs.

Last, the crystal lattice structure and diffraction pattern of Fe₃O₄-NPs capped with aldimine and with SiO₂ were analyzed by X-ray powder diffraction (XRD; BDX-3300 diffractometer using Cu Kα radiation of a wavelength of λ = 1.5406 Å). The XRD diffractograms of aldimine-Fe₃O₄ and aldimine-SiO₂/Fe₃O₄ (Figure 4a,b) confirmed the formation of magnetite NPs without any other iron oxides. It was found that there are five characteristic peaks at 2-theta values of 30.5° (2 2 0), 35.9° (3 1 1), 43.5° (4 0 0), 57.3° (5 1 1), and 63.1° (4 4 0) to verify the crystalline structure of Fe₃O₄ particles, which is matched with the standard diffractions of Fe₃O₄ (JCPDS 89-4319). Also, the broad XRD peak at a low diffraction angle of 20–30° relates to the amorphous state of SiO₂ shells surrounding the Fe₃O₄-NPs.

Figure 4.

XRD patterns of 2-((3-hydroxy-4-methoxybenzylidene)amino)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carbonitrile loaded on (a) Fe₃O₄ and (b) Fe₃O₄/SiO₂.

Biological Investigation

The enzymatic inhibitory potencies of the synthesized derivatives were determined at a concentration of 100 μg/mL against both PDK1 and LDHA and compared to the inhibition efficiencies of the reference inhibitors sodium dichloroacetate and sodium oxamate at a concentration of 1 mM (1000 μM), respectively (Table 1 and Figure 5).

Table 1. Pyruvate Dehydrogenase Kinase-1 (PDK-1) and Lactate Dehydrogenase A (LDHA) Inhibitory Efficiencies of the Synthesized 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives Determined at a Concentration of 100 μg/mL and Expressed in %^a.

	mean inhibitory % at a concentration of 100 μg/mL ± SD
comp. #	PDK-1	LDHA
1a	12.35 ± 1.91	19.25 ± 3.18
1b	87.00 ± 2.83	74.10 ± 2.97
1c	11.25 ± 3.18	23.15 ± 3.047
3a	21.50 ± 2.40	28.75 ± 2.057
3b	51.50 ± 3.54	19.30 ± 0.85
3c	37.95 ± 2.48	46.80 ± 2.55
3d	11.25 ± 1.77	24.85 ± 2.67
3e	18.40 ± 1.98	18.40 ± 1.98
3f	29.00 ± 2.83	27.50 ± 3.54
5a	41.00 ± 2.83	30.80 ± 1.70
5b	16.75 ± 1.63	34.70 ± 3.25
5c	21.40 ± 1.98	25.65 ± 2.33
7	35.90 ± 4.10	48.75 ± 5.30
9	36.50 ± 2.12	32.65 ± 2.33
11	23.70 ± 2.40	29.60 ± 1.98
12a	29.00 ± 2.83	40.15 ± 4.45
12b	23.75 ± 2.48	36.30 ± 3.25
sodium dichloroacetate	100.00 ± 0.00 at150.92 μg/mL
sodium oxamate		100.00 ± 0.00 at111.03 μg/mL

^aSodium dichloroacetate (1 mM = 1000 μM = 150.92 μg/mL) and sodium oxamate (1 mM = 1000 μM = 111.03 μg/mL) were used as the standard inhibitors. Values of inhibitory percentage represent the mean ± SD of two different replicates.

Figure 5.

Enzymatic inhibitory assays of 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives.

The results of PDK1-assays revealed that compounds 1b and 3b were the most active with inhibition percentages of 87.0 and 51.50% relative to 100% produced by sodium dichloroacetate, respectively.

Similarly, compounds 1b, and 7 demonstrated the highest inhibitory effciencies of 74.10 and 48.75% against LDHA, respectively, compared with 100% by sodium oxamate.

The rest of the compounds exhibited poor inhibitory efficiencies ranging from 41.0 to 11.25% and from 46.80 to 18.40% against PDK1 and LDHA, respectively.

In the view of these results, the mean IC₅₀ values of compounds 1b, 3b, and 7 against both enzymes were determined in terms of μg/mL and μM and they were consistent with the observed percentages of inhibitory efficiencies as summarized in Table 2.

Table 2. Summary of Inhibitory Efficiencies (%) and the Half-Maximal Inhibitory Concentration (IC₅₀) Expressed in μg/mL and μM of Compounds 1b, 3b, and 7 against PDK1 and LDHA^a.

	PDK1			LDHA
comp. # (MWt)	IC50 (μg/mL) ± SD	IC50 (μM)	mean % inhibition ± SD at 100 μg/mL	IC50 (μg/mL) ± SD	IC50 (μM)	mean % inhibition ± SD at 100 μg/mL
1b (254.35)	57.10 ± 2.97	224.49	87.00 ± 2.83	64.1 ± 4.10	252.01	74.10 ± 2.97
3b (326.41)	98.50 ± 4.95	301.76	51.50 ± 3.54	291.0 ± 5.66	891.52	19.30 ± 0.85
7 (336.41)	144.00 ± 5.66	428.05	35.90 ± 4.10	93.0 ± 2.83	276.45	48.75 ± 5.30
sodium dichloro-acetate (150.92)	25.75±1.06	170.62	100.00% at 150.92 μg/mL = 1 mM = 1000 μM
sodium oxamate (111.03)				15.6 ± 0.85	140.50	100.00% at 111.03 μg/mL = 1 mM = 1000 μM

^aResults are the mean values of two separate determinations ± SD.

With regard to the antioxidant activities, they were assessed using DPPH radicals via determination of the percentages of scavenging activities of the studied compounds and butylated hydroxy toluene (BHT), which was used as the reference antioxidant drug at a 1000 μg/mL concentration. Additionally, the IC₅₀ values were deduced in μg/mL and μM as presented in Table 3 and Figures 6 and 7.

Table 3. Evaluation of the Antioxidant Activities of the Synthesized 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives by Determination of DPPH Free-Radical Scavenging Activity (%) at a Concentration of 1000 μg/mL and IC₅₀ Values Expressed as μg/mL and μM^a.

comp. # (MWt)	mean scavenging activity (%) at 1000 μg/mL ± SD	mean IC50 (μg/mL) ± SD	mean IC50 (μM)
1a (301.40)	63.35 ± 1.90	390.5 ± 13.44	1295.62
1b (254.35)	95.10 ± 1.27	110.0 ± 14.14	432.47
1c (178.25)	72.25 ± 1.77	332.5 ± 17.68	1865.35
3a (373.47)	57.35 ± 1.91	665.0 ± 21.21	1780.60
3b (326.41)	79.95 ± 2.76	130.0 ± 14.14	398.27
3c (377.88)	63.15 ± 1.63	365.0 ± 21.21	965.92
3d (330.83)	86.15 ± 1.63	127.0 ± 9.90	383.88
3e (411.53)	52.60 ± 2.26	595.0 ± 21.21	1445.82
3f (392.90)	58.25 ± 3.18	400.0 ± 14.14	1018.07
5a (428.55)	61.05 ± 2.90	440.0 ± 28.28	1026.72
5b (504.65)	73.95 ± 2.76	285.0 ± 21.21	564.74
5c (381.49)	59.50 ± 2.12	525.0 ± 35.36	1376.18
7 (336.41)	73.20 ± 2.55	222.5 ± 17.68	661.40
9 (309.43)	69.20 ± 1.13	210.0 ± 28.28	678.67
11 (312.39)	58.20 ± 1.13	332.5 ± 17.68	1064.37
12a	87.25 ± 1.77	80.0 ± 14.14
12b	84.40 ± 2.40	95.0 ± 7.07
BHT (220.35)	95.30 ± 0.42	54.0 ± 5.66	245.06

^aResults are the mean values of two separate determinations ± SD.

Figure 6.

DPPH radical scavenging activity expressed as % for the synthesized 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives and the reference antioxidant BHT at a concentration of 1000 μg/mL.

Figure 7.

Antioxidant activities of the synthesized 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives and the reference antioxidant BHT determined through calculations of the half-maximal inhibitory concentrations (IC₅₀ values) expressed as μg/mL.

These experiments revealed that compounds 1b, 12a, 3d, 12b, and 3b can be considered as promising antioxidant candidates as they exerted the strongest free-radical scavenging activities with efficiencies of 95.10 ± 1.27, 87.25 ± 1.77, 86.15 ± 1.63, 84.40 ± 2.40, and 79.95% ± 2.76 and the smallest IC₅₀ (μg/mL) values of 110.0 ± 14.14, 80.0 ± 14.14, 127.0 ± 9.90, 95.0 ± 7.07, and 130.0 ± 14.14, respectively, as compared with BHT, which displayed a radical scavenging potency of 95.30% ± 0.42 and an IC₅₀ value of 54.0 ± 5.66 μg/mL. The remaining compounds demonstrated good to moderate antioxidant properties with scavenging potencies (%) ranging from 73.95 ± 2.76 to 52.60 ± 2.26 and IC₅₀ (μg/mL) values ranging from 665.0 ± 21.21 to 210.0 ± 28.28.

The antibacterial evaluations were performed by determination of IC₅₀ (μg/mL and μM) against E. faecalis (ATCC 29122) and E. coli (ATCC 25922) using ampicillin as the reference antibiotic, as shown in Table 4 and Figure 8. The results indicated that the tested bacterial strains demonstrated varied sensitivities to the studied compounds, but none of them displayed stronger potency than ampicillin.

Table 4. Evaluation of the Antibacterial Activity of the Synthesized 4,5,6,7-Tetrahydro-benzo[b]thiophene Derivatives by Determination of IC₅₀ Values Expressed as μg/mL and μM^a.

	Enterococcus faecalis (ATCC 29122)		E. coli (ATCC 25922)
comp. # (Mwt)	mean IC50 (μg/mL) ± SD	mean IC50 (μM)	mean IC50 (μg/mL) ± SD	mean IC50 (μM)
1a (301.40)	44.10 ± 1.28	146.32	24.90 ± 0.99	82.61
1b (254.35)	21.20 ± 1.13	83.35	NS	NS
1c (178.25)	21.25 ± 1.06	119.21	26.25 ± 1.06	147.27
3a (373.47)	38.10 ± 1.27	102.01	27.05 ± 0.35	72.43
3b (326.41)	27.25 ± 1.06	83.48	NS	NS
3c (377.88)	26.15 ± 1.20	69.20	33.25 ± 1.06	87.99
3d (330.83)	40.60 ± 1.98	122.72	30.90 ± 1.56	93.40
3e (411.53)	37.30 ± 0.99	90.64	36.80 ± 1.70	89.42
3f (392.90)	42.00 ± 0.71	106.90	28.30 ± 1.70	72.03
5a (428.55)	26.65 ± 0.50	62.19	26.90 ± 0.57	62.77
5b (504.65)	24.75 ± 1.77	49.04	NS	NS
5c (381.49)	17.00 ± 0.28	44.56	NS	NS
7 (336.41)	28.10 ± 1.56	83.53	27.90 ± 2.26	82.93
9 (309.43)	34.20 ± 0.99	110.53	29.35 ± 1.20	94.85
11 (312.39)	39.35 ± 0.92	125.96	56.60 ± 1.98	181.18
12a	17.60 ± 0.57		26.20 ± 0.85
12b	18.60 ± 0.85		22.25 ± 1.06
Amp. (349.406)	12.75 ± 1.06	36.49	20.25 ± 1.77	57.96

^aResults are the mean values of two separate determinations ± SD. NS: not specified.

Figure 8.

Antibacterial activities of the synthesized 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives determined through the calculation of the half-maximal inhibitory concentrations, IC₅₀ values, expressed as μg/mL.

Considering the calculated IC₅₀ values (μg/mL) against E. faecalis, the compounds demonstrated higher values ranging from 44.10 ± 1.28 to 17.00 ± 0.28 with compounds 5c, 12a, and 12b exhibiting the smallest values of 17.00 ± 0.28, 17.60 ± 0.57, and 18.60 ± 0.85, respectively, in comparison to 12.75 ± 1.06 by ampicillin.

Similarly, the tested compounds inhibited E. coli with IC₅₀ (μg/mL) values ranging from 22.25 ± 1.06 to 36.80 ± 1.70 with 12b and 1a being the most active candidates by exerting the lowest concentrations of 22.25 ± 1.06 and 24.90 ± 0.99, respectively, as compared with ampicillin, which exhibited 20.25 ± 1.77 μg/mL.

In the view of all performed biological experiments, the most active candidates 1b, 3d, 5c, 12a, and 12b in addition to Schiff base 11 along with compounds 3b, 7, and 9 as representative examples for the newly introduced chemical functionalities on the starting amino thiophenes were further evaluated for their in vitro cytotoxic activities against HCT-116 and LoVo cell lines of CRC by determination of the percentages of residual viable cells after being exposed to each compound at a concentration of 100 μg/mL associated with utilization of 0.1% Triton X-100 in the assay medium and the assay medium as the positive and negative controls, respectively. The obtained results (Table 5 and Figure 9) indicated that the NPs 12a,b and carbamate derivative 3b were the most active by demonstrating the lowest cell viability percentages of 11.75 ± 1.06, 33.25 ± 1.77, and 35.50 ± 2.12 against LoVo cells, respectively.

Table 5. Mean % of Viable LoVo and HCT-116 Cancerous Cells after Being Treated with Selected 4,5,6,7-Tetrahydrobenzo[b]thiophene Derivatives^a.

comp. #	mean % of viable LoVo cells ± SD	mean % of viable HCT-116 cell ± SD
positive control Triton X-100 (0.1%)	0.00	0.00
negative control	99.50 ± 0.71	100.00 ± 0.00
1b	56.50 ± 0.71	53.50 ± 2.12
3b	35.50 ± 2.12	28.00 ± 1.41
3d	77.00 ± 2.83	65.50 ± 0.717
5c	79.50 ± 2.12	90.50 ± 2.12
7	51.50 ± 2.12	47.25 ± 1.06
9	71.80 ± 2.55	69.00 ± 1.41
11	63.75 ± 1.77	60.50 ± 2.12
12a	11.75 ± 1.06	15.75 ± 1.06
12b	33.25 ± 1.77	30.50 ± 2.12

^aResults are the mean values of two separate determinations ± SD.

Figure 9.

In vitro viability % of the LoVo and HCT-116 cancerous cells after being treated with selected 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives. The results are the mean values of two separate determinations ± SD.

Furthermore, the viability of HCT-116 cells was reduced to 15.75 ± 1.06, 28.00 ± 1.41, and 30.50 ± 2.12 by compounds 12a, 3b, and 12b, respectively.

Although compound 1b was recognized as the most active candidate against the PDK1 and LDHA enzymes (Table 2), it did not demonstrate significant cytotoxicity against the examined cell lines (% cell viabilities of 56.50 ± 0.71 and 53.50 ± 2.12); thus, it is worthwhile to investigate its antitumor potential on different cancer types and cell lines in the future.

Contrarily, NPs 12a,b and carbamate 3b, which showed moderate to poor PDK1 and LDHA inhibitory efficiencies were found to be the most active cytotoxic candidates, implying that the observed antitumor properties would be resulted from the interaction of these compounds with other molecular targets.

Next, the IC₅₀ values expressed in μg/mL and μM of compounds 3b, 7, 9, 11, and 12a against the tested HCT-116 and LoVo cell lines were calculated (Table 6). The NPs12a and carbamate 3b, respectively, exerted the smallest values of 60.35 ± 2.76 and 71.00 ± 2.83 μg/mL (against HCT-116 cells) and 57.15 ± 2.48 and 81.5 ± 3.54 μg/mL (against LoVo cells).

Table 6. Half-Maximal Inhibitory Concentrations (IC₅₀) Expressed as μg/mL and μM of the Active Cytotoxic Compounds against HCT-116 and LoVo Cells of CRC^a.

	LoVo cells		HCT-116
comp. # (MWt)	mean IC50 (μg/mL) ± SD	mean IC50 (μM)	mean IC50 (μg/mL) ± SD	mean IC50 (μM)
3b (326.41)	81.50 ± 3.54	249.68	71.00 ± 2.83	217.52
7 (336.41)	108.50 ± 3.54	322.52	97.00 ± 2.83	288.34
9 (309.43)	198.50 ± 4.95	641.50	162.00 ± 4.24	523.54
11 (312.39)	148.00 ± 4.24	473.77	126.00 ± 4.25	403.34
12a	57.15 ± 2.48		60.35 ± 2.76

^aResults are the mean values of two separate determinations ± SD.

Structure–Activity Relationship Studies

The structure–activity relationships of the newly synthesized 4,5,6,7-tetrahydrobenzo[b]thiophene derivatives can be derived on the basis of the results obtained from enzyme inhibition, antioxidant, antibacterial, and anticancer experiments as follows:

• 1.The enzymatic inhibitory activities against PDK-1 and LDHA were enhanced because of the presence of the primary amino group coupled with the nitrile group on the thiophene ring (compound 1b). The transformation of the amino group to other functionalities reduced the efficiency by at least 1.5-fold as indicated by comparing the IC₅₀ values of compound 1b with carbamate derivative 3 and cyclic imide analogue 7.
• 2.The presence of the electron-donating amino group capable of H-bond formation (compound 1b)⁴² and the formation of Fe₃O₄-NPs (compound 12a)⁴³ and the Fe₃O₄-NPs coated by SiO₂ (compound 12b) in addition to the insertion of chloroacetamide (in compound 3d) and the carbamate (in compound 3b)⁴⁴ moieties along with the presence of the nitrile group in the 4,5,6,7-tetrahydrobenzo[b]thiophene core is crucial for the potent free-radical scavenging capacities.
• 3.The antibacterial activity of the synthesized compounds was improved as a result of the formation of Fe₃O₄-NPs⁴⁵ (compound 12a) and the transformation of the amino group to the morphlino-acetamide moiety (in compound 5c) against E. faecalis and the formation of Fe₃O₄-NPs coated by SiO₂⁴⁶ (compound 12b) against E. faecalis and E. coli.
• 4.The anticancer potency of the synthesized compounds was improved against LoVo and HCT-116 cells of CRC as a result of the formation of Fe₃O₄-NPs (compound 12a) and Fe₃O₄-NPs coated by SiO₂⁴⁷ (compound 12b) and by the insertion of the carbamate moiety⁴⁸ (in compound 3b).

Thus, our results are in accordance with the previously published data, which showed that the carbamate group is a structural motif in several anticancer drugs and prodrugs that are approved by the Food and Drug Administration (FDA) in USA and the European Medicines Agency (EMA), as shown in Figure 10.⁴⁸

Figure 10.

Examples of carbamate containing antineoplastic drugs.

For example, docetaxel is a carbamate containing an antineoplastic drug against breast, ovarian, and non-small cell lung cancers. It produces its chemotherapeutic action through interfering with several tumorigenic pathways. Thus, it induces programmed cell death (apoptosis) in cancer cells by binding to the antiapoptotic Bcl-2 (B-cell leukemia 2) protein and thus arresting its function. Also, it is considered as an antimicrotubule agent capable of destroying the cell’s ability to use its cytoskeleton in a flexible manner through promoting microtubulin assembly and stabilizing the polymers against depolymerization. Further research indicated that docetaxel has been shown to inhibit angiogenesis.⁴⁹

Irinotecan is another key antineoplastic drug for metastatic CRC, which contains a carbamate moiety. The mechanism underlying its antitumor effect primarily depends on binding to topoisomerase I and inhibiting its action in addition to causing double-strand DNA breakage, which leads to irreversible inhibition of DNA synthesis, thus inducing arrest of the cell cycle in S-G2 leading to cell death.⁵⁰

Moreover, capecitabine (Xeloda) is an oral fluoropyrimidine carbamate prodrug against metastatic breast and colon cancers. It is selectively activated at the tumor site under the action of thymidine phosphorylase, which converts it to 5-fluorouracil (5-FU), which is further metabolized to the active metabolites to cause cell injury. This produrg is absorbed intact through the gastrointestinal tract; thus, it provides protection against gut toxicity induced by 5-FU itself.⁵¹

Collectively, the carbamate group is considered as an important motif that increases the biological activity of different pharmacophoric scaffolds when it is introduced on them. This functional group allows modification of the pharmacokinetic properties of the compounds containing them through varying the substituents on its amino and carboxylate termini. Additionally, it improves their stability, potency, duration of action, and target specificity.⁴⁸

In Silico Molecular Docking Studies to Unveil the Underlying Molecular Mechanism of Cytotoxic Effects

Molecular docking of a chemical inhibitor in the active pocket of the proper molecular target is widely used as an approach to investigate the possible underlying antitumor mechanism of action. Microtubules are cellular polymers, which play diverse roles within the cell, including chromosomal segregation, cytokine and chemokine secretion, maintenance of the cell structure, protein trafficking, cell migration, and cell division. Thus, targeting tubulin dynamics is a promising approach for new chemotherapeutic agents. Compounds capable of disturbing microtubules by either stabilizing or destabilizing them would produce antiproliferative activity via enhancing mitotic arrest and cell apoptosis. At least three main binding sites in the tubulin protein, including colchicine, taxane, and vinca alkaloid, have been identified as targets for the anticancer drugs.⁵²

Due to the fact that the promising cytotoxic candidate 3b is a benzo[b]thiophene derivative with carbamate functionality, where both these structural units were found to inhibit tubulin polymerization, which in turn leads to irreversible damage to the tumor vasculature and eventually causes mitotic arrest and tumor necrosis,^49,53 in the current study, the inhibitory potential of this compound against the tubulin (TUB) domain was studied using in silico molecular docking analyses to explore its underlying antitumor mechanism of action.

Initially, the docking setup was first validated by self-docking of the co-crystallized ligand (COL) in the vicinity of the binding site of the protein (Figure 11), the docking score (S) was −13.0770 kcal/mol, and the root-mean-square deviation (RMSD) value was 0.8996 Å, which is less than the cutoff value (2 Å) for the correct docking procedure.⁵⁴

Figure 11.

3D representation of the superimposition of the co-crystallized ligand (red) and the docking pose (green) of COL in the active site of TUB.

Through examination of the binding interactions of colchicine (COL) to the active site of the protein, it showed strong hydrogen bond interactions with Ala-A180, Val-A181, Leu-B248, and Lys-B352 (Figure 12).

Figure 12.

2D interactions of COL within the TUB active site.

With regard to compound 3b, the docking results, which are summarized in Table7 and Figure 13, showed that it exhibited a good binding score (−11.3097 kcal/mol), and it was capable of fitting inside the pocket of the target tubulin active site. The compound exhibited good binding interactions using the carbonyl group as a hydrogen bond acceptor with Ala-A180, Val-A181, and Lys-B352 residues. Moreover, the sulfur atom and the nitrile group were involved in the binding interactions as H-bond acceptors with Lys-B352 and Lys-B254 amino acids, respectively.

Table 7. Docking Results of 3b in the Colchicine Binding Site of the Tubulin (TUB) Domain.

compound	S (kcal/mol)	amino acids	interacting groups	type of interaction	length
3b	–11.3097	Ala-A180	O (C=O)	H-bond acceptor	3.26
		Val-A181	O (C=O)	H-bond acceptor	3.75
		Lys-B352	O (C=O)	H-bond acceptor	3.23
		Lys-B352	S	H-bond acceptor	3.51
		Lys-B254	N (CN)	H-bond acceptor	4.04

Figure 13.

2D and 3D interactions of compound 3b within the TUB active site.

ADME Predictions

The absorption, distribution, metabolism, and excretion (ADME) properties of compound 3b were predicted by the SwissADME free web tool (http://www.swissadme.ch/index.php). The studied compound showed a good oral bioavailability profile, which is clear through the bioavailability radar chart (Figure 14).

Figure 14.

Bioavailability radar chart for compound 3b.

Moreover, it exhibited suitable physicochemical properties (Table 8), which was tested through the following six parameters: lipophilicity (LIPO), size, polarity (POLAR), insolubility (INSOL), unsaturation (UNSAT), and flexibility (FLEX). Concerning the absorption property, compound 3b showed high gastrointestinal tract (GIT) absorption as it is located in the BOILED-EGG chart white area, indicating that it can be passively absorbed through the intestinal tract.

Table 8. Measured Physicochemical Parameters for Compound 3b.

no. of heavy atoms	23
C-sp3 fraction	0.33
no. of rotatable bonds	5
molar refractivity	91.55
topological polar surface area (TPSA)	90.36 Å2
consensus log Po/w	3.90

On the other hand, it is not expected to penetrate the blood brain barrier (located outside the chart yellow area). Moreover, it is not a potential substrate for permeability glycoprotein efflux protein (PGP), which is indicated by red, given for the tested compound (Figure 15).

Figure 15.

BOILED-EGG chart for compound 3b.

Finally, compound 3b showed no violation to Lipinski’s rule of five⁵⁵ as shown from data in Table 9.

Table 9. Compliance of Compound 3b to Lipinski’s Rule of Five.

log P = 3.90	no violation as it is <5
molecular weight = 326.41 g/mol	no violation as it is <500
no. of H-bond donor groups (OHs + NHs) = 1	no violation as it is <5
no. of H-bond acceptor atoms (Os + Ns) = 4	no violation as it is <10

Collectively, 3b possesses desirable drug-likeness and oral bioavailability properties.

Conclusions

A series of 4,5,6,7-tetrahydrobenzo[b]thiophene-conjugated amides, acetamides, carbamates, a cyclic imide and a Schiff base in addition to two Fe₃O₄-NPs were synthesized and characterized. These compounds were evaluated in vitro as inhibitors to the protumorigenic enzymes PDK-1 and LDHA, antioxidants, antibacterial, and anti-CRC agents on HCT-116 and LoVo cells. Although compound 1b showed promising enzymatic inhibitions and antioxidant potential, it failed to produce significant cytotoxic effects on the tested cell lines; probably, it may be selective toward other cell lines. The Schiff base derivative 11 did not display significant bioactivity. Contrarily, its modified Fe₃O₄ (12a) and Fe₃O₄/SiO₂ (12b) nanoparticles exhibited enhanced antioxidant, antibacterial, and cytotoxic activities with NPs 12a being the most active agent. Thus, the obtained data support further development of Fe₃O₄-NPs capped with Schiff bases derived from 3-substituted-2-amino-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene as potential CRC agents. Moreover, carbamate 3b significantly inhibited the growth of both types of the examined colorectal cancerous cells. Thus, its capability to inhibit tubulin polymerization was investigated using molecular modeling analyses in addition to its ADME chacteristics were predicted. The results of these analyses showed that 3b exhibited reasonable affinity to the colchicine site of the tubulin (TUB) domain. It is involved in hydrogen bond interactions through sulfur atom, as well as nitrile and carbonyl groups with the amino acid residues in the binding site, implying that carbamate 3b may induce antitumor effects on HCT-116 and LoVo cells via targeting tubulin polymerization. Furthermore, this 4,5,6,7-tetrahydrobenzo[b]thiophene derivative complied with Lipinski’s rule of five; thus, it is expected to show good in vivo oral absorption. In addition, it possesses a desirable pharmacokinetic profile, which makes it an interesting structural template for the development of new antimitotic agents against CRC.

Experimental Section

Chemistry

General Information

The reactions were monitored, and the purity of the final products was checked using TLC analysis on pre-coated silica gel sheets (60 F254, Merck, and Kenilworth, NJ). The developed plates were examined by exposure to ultraviolet light using a VL-6.LC (254, 365 nm, 50/60 Hz). All the melting points were determined in open capillary tubes with a Gallenkamp melting point apparatus (°C). The IR spectra were recorded on a PerkinElmer Fourier transform infra-red (FTIR) spectrophotometer (Spectrum BX 1000) in wave numbers (cm^–1) with potassium bromide (KBr) discs. Nuclear magnetic resonance (NMR) spectra were recorded on an Eclipse 300 FT NMR spectrometer operating at 300 MHz for ¹H and at 75 MHz for ¹³C at 25 °C (KSU, Riyadh, KSA). The chemical shifts (δ) were expressed in ppm using tetramethylsilane as the internal standard; coupling constants (J) were expressed in Hz. Mass spectra were recorded on a Shimadzu Qp-2010 Plus mass spectrometer in the ionization mode: EI (Regional Center for Mycology and Biotechnology, Al-Azhar University, Egypt).

The physical and spectroscopic data of compounds 1a–c(56,57) and 3c(58) are consistent with those previously reported.

Synthesis of Carbamate Derivatives (3a,b)

A mixture of aminothiophene 1a or 1b (0.0028 mol) and ethyl chloroformate 2a (0.038 mol) was refluxed for 10 h. The excess reagent was evaporated under reduced pressure; the remaining residue was treated with diethyl ether, and the precipitated solid was filtered off, washed with water, air-dried, and purified by recrystallization from benzene to give carbamates 3a,b, respectively.

Ethyl 2-((Ethoxycarbonyl)amino)-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (3a)

Buff powder; R_f: 0.88 (petroleum ether/ethyl acetate, 1:2); yield (86%); mp 107–109 °C; IR (KBr) ν_max/cm^–1: 3251 (NH), 3020 (CH-Ar), 2983, 2929 (CH-aliphatic), 1725, 1652 (2 × C=O), 1434, 1236, 1058, 846, 763, 697, 542; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.22 (t, 6H, J = 6.9 Hz, 2 × CH₃), 1.75–1.95 (m, 2H, CH₂), 2.58–2.88 (m, 5H, 2 × CH₂, CH), 4.20 (q, 4H, J = 7.2 Hz, 2 × CH₂O), 7.17–7.26 (m, 5H, 5 × Ar-H), 10.33 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 13.92, 14.12 (CH₃), 26.27, 29.54, 30.57, 31.34 (3 × CH₂, CH), 60.29, 61.99 (2 × OCH₂), 110.12, 124.72, 126.14, 126.67, 128.27, 130.44, 145.55, 148.46, 152.13, 165.21 (5 × CH-Ar, 5 × C_q-Ar & 2 × C=O); MS (EI, 70 eV) m/z (%): 376.27 [M⁺ + 3, 23] and 375.41 [M⁺ + 2, 32] for C₂₀H₂₃NO₄S, 366.64 (61), 343.07 (31), 329.98 (65), 328.97 (61), 321.08 (56), 296.61 (45), 277.34 (35), 230.48 (35), 190.70 (37), 177.89 (55), 173.89 (100), 155.22 (65), 134.01 (61), 125.66 (81), 89.03 (50), 52.57 (50).

Ethyl (3-Cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)carbamate (3b)

Buff powder; R_f: 0.89 (petroleum ether/ethyl acetate, 1:1); yield (80%); mp 177–178 °C; IR (KBr) ν_max/cm^–1: 3221 (NH), 3098 (CH-Ar), 2988, 2926 (CH-aliphatic), 2219 (CN), 1725 (C=O), 1465, 1236, 1069, 882, 761, 699, 547; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.22 (t, 3H, J = 7.1 Hz, CH₃), 1.87–1.97 (m, 2H, CH₂), 2.61–2.97 (m, 5H, CH, 2 × CH₂), 4.21 (q, 2H, J = 7.1 Hz, CH₂O), 7.22–7.30 (m, 5H, 5 × Ar-H), 11.18 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 14.31 (CH₃), 24.13, 29.00, 31.25 (3 × CH₂, CH), 61.78 (OCH₂), 93.90, 113.98, 126.34, 126.86, 128.41, 128.73, 131.18, 133.07, 145.38, 148.55, 153.57 (5 × CH-Ar, 5 × C_q-Ar, CN & C=O); MS (EI, 70 eV) m/z (%): 328.41 [M⁺ + 2, 26] for C₁₈H₁₈N₂O₂S, 320.29 (47), 316.67 (97), 285.67 (50), 284.40 (41), 272.55 (31), 253.21 (60), 241.01 (52), 229.56 (36), 192.02 (66), 186.39 (54), 170.74 (32), 140.77 (30), 123.84 (100), 85.28 (32), 77.22 (76), 63.22 (50).

Synthesis of Amide Derivatives (3c–f)

To a solution of compound 1a or 1b (0.02 mol) in CHCl₃ (30 mL) containing triethylamine (0.024 mol), the appropriate acid chloride (0.024 mol): chloroacetyl chloride 2b, thiophene-2-carbonyl chloride 2c, or 4-chlorobenzoyl chloride 2d, was added drop wise at 0 °C and the resulting reaction mixture in each case was stirred at r.t for 1 h then refluxed for 16 h. Evaporation of the solvent and treatment of the remaining residue with soln. of NaHCO₃ gave the crude product, which was collected by filtration, and air dried. Recrystallization from ethanol gave the pure amides, the previously reported 3c(41) and the new derivatives 3d–g, respectively.

2-Chloro-N-(3-cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) Acetamide (3d)

Brown crystal; R_f: 0.82 (petroleum ether/ethyl acetate, 1:1); yield (70%); mp 170–172 °C; IR (KBr) ν_max/cm^–1: 3239 (NH), 3089 (CH), 2980, 2940 (CH₂), 2208 (CN), 1708 (C=O), 1479, 1176, 1034, 806, 694, 536; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.90–2.00 (m, 2H, CH₂), 2.65–3.06 (m, 5H, 2 × CH₂, CH), 4.48 (s, 2H, CH₂–CO), 7.21–7.39 (m, 5H, 5 × Ar-H), 10.59 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 23.98, 28.98, 31.12, 42.28, 45.34 (3 × CH₂, CH, CH₂–CO), 93.38, 113.99, 126.33, 126.84, 127.64, 128.41, 130.83, 145.30, 146.17 (5 × CH-Ar, 5 × C_q-Ar & CN), 164.64 (C=O); MS (EI, 70 eV) m/z (%): 332.82 [M⁺ + 2, 4] for C₁₇H₁₅ClN₂OS, 328.17 (8), 315.13 (30), 253.17 (30), 181.26 (100), 163.09 (23), 153.07 (50), 119.24 (23), 101.37 (28), 91.05 (46), 89.26 (27).

Ethyl 6-Phenyl-2-(thiophene-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylate (3e)

Pale yellow powder; R_f: 0.76 (petroleum ether/ethyl acetate, 1:2); yield (60%); mp 185–187 °C; IR (KBr) ν_max/cm^–1: 3233 (NH), 3095 (CH-Ar), 2928 (CH-aliphatic), 1718, 1646 (2 × C=O), 1412, 1219, 1025, 841, 729, 694, 538.; ¹H NMR (300 MHz, CDCl₃): δ_H 1.36 (t, 3H, J = 6.9 Hz, CH₃), 1.80–2.12 (m, 2H, CH₂), 2.70–2.99 (m, 5H, 2 × CH₂, CH), 4.34 (q, 2H, J = 7.2 Hz, CH₂O), 7.08–7.11 (m, 1H, Ar-H), 7.19–7.31 (m, 5H, 5 × Ar-H), 7.54 (dd, 1H, J = 3.6, 1.2 Hz, Ar-H), 7.71 (dd, 1H, J = 2.4, 1.2 Hz, Ar-H), 12.13 (s, 1H, NH–C=O); ¹³C NMR (75 MHz, CDCl₃): δ_C 11.21 (CH₃), 23.59, 26.96, 29.03, 37.38 (3 × CH₂, CH), 57.54 (CH₂O), 108.42, 123.22, 123.44, 123.70, 124.90, 125.35, 126.12, 127.64, 128.66, 134.50, 142.60, 144.83, 155.08, 163.63 (8 × CH-Ar, 6 × C_q-Ar & 2 × C=O); MS (EI, 70 eV) m/z (%): 415.68 [M⁺ + 4, 8] for C₂₂H₂₁NO₃S₂, 414.27 (19), 373.12 (40), 357.50 (48), 350.06 (46), 330.06 (33), 296.42 (79), 292.80 (36), 269.46 (84), 268.30 (100), 253.11 (69), 239.41 (30), 224.29 (47), 176.90 (38), 163.16 (56), 149.29 (79), 135.21 (33), 119.15 (32), 106.85 (49), 93.55 (60), 90.36 (50), 77.09 (94), 65.46 (83), 41.13 (59).

4-Chloro-N-(3-cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl) Benzamide (3f)

Brown powder; R_f: 0.91 (petroleum ether/ethyl acetate, 1:1); yield (60%); mp 205–207 °C; IR (KBr) ν_max/cm^–1: 3246 (NH), 3079 (CH-Ar), 2978, 2930 (CH-aliphatic), 2218 (CN), 1663 (C=O), 1481, 1276, 1089, 846, 750, 695, 534; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.87–1.99 (m, 2H, CH₂), 2.65–2.75 (m, 2H, CH₂), 2.87–3.05 (m, 3H, CH₂, CH), 7.20–7.30 (m, 5H, 5 × Ar-H), 7.61 (d, 2H, J = 8.4 Hz, 2 × Ar-H), 8.00 (d, 2H, J = 8.4 Hz, 2 × Ar-H), 10.90 (s, 1H, NH–C=O); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 24.09, 29.03, 31.23, 45.31 (3 × CH₂, CH), 95.85, 114.16, 126.32, 126.83, 128.40, 128.51, 128.67, 128.85, 130.27, 131.11, 137.20, 145.31, 146.44, 164.15 (9 × CH-Ar, 7 × C_q-Ar, CN & C=O); MS (EI, 70 eV) m/z (%): 396.05 [M⁺ + 4, 17] for C₂₂H₁₇ClN₂OS, 395.34 (6), 392.00 (11), 390.79 (7), 389.51 (6), 388.86 (5), 384.50 (100), 372.20 (40), 357.81 (44), 341.13 (40), 304.04 (36), 254.82 (36), 240.84 (39), 178.47 (41), 157.43 (33), 116.94 (36), 102.50 (32), 91.56 (45), 78.24 (47), 65.22 (79), 51.70 (51).

General Procedures for Condensation of Chloroacetamides 3c,d with 2° Amines

A mixture of chloroacetamide derivative 3c or 3d (0.025 mol) and 0.03 mol of morpholine 4a or 1-(pyridin-2-yl)piperazine 4b in CH₃OH (30 mL) was refluxed for 10 h. The excess solvent was evaporated, and the remaining residue was treated with cold water. The obtained solid product in each case was collected by filtration, air-dried, and recrystallized from ethanol to give compounds 5a–c, respectively.

Ethyl 2-(2-Morpholinoacetamido)-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5a)

Beige powder; R_f: 0.32 (petroleum ether/ethyl acetate, 1:2); yield (55%); mp 90–93 °C; IR (KBr) ν_max/cm^–1: 3233 (NH), 2926 (CH₂), 1728, 1671 (2 × C=O), 1440, 1232, 1035, 866, 754, 699, 542; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.31 (t, 3H, J = 6.9 Hz, CH₃), 1.84–2.08 (m, 2H, CH₂), 2.51 (apparent. s, 4H, 2 × CH₂N), 2.64–2.92 (m, 5H, 2 × CH₂, CH), 3.24 (s, 2H, CH₂–CO), 3.69 (apparent. s, 4H, 2 × CH₂O), 4.32 (q, 2H, J = 7.2 Hz, CH₂O), 7.20–7.28 (m, 5H, 5 × Ar-H), 12.13 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 14.14 (CH₃), 26.30, 29.69, 31.43 (3 × CH₂, CH), 53.21, 60.24, 60.58, 66.15 (4 × CH₂-morphline ring, OCH₂-ester, CH₂–N), 111.22, 125.51, 126.18, 126.75, 128.32, 130.13, 145.62, 146.05, 164.83, 167.78 (5 × CH-Ar, 5 × C_q-Ar, 2 × C=O); MS (EI, 70 eV) m/z (%): 431.13 [M⁺ + 3, 25] and 430.23 [M⁺ + 2, 11] for C₂₃H₂₈N₂O₄S, 424.00 (10), 361.40 (26), 340.69 (31), 308.89 (27), 273.95 (31), 263.09 (37), 231.37 (29), 224.28 (45), 211.28 (39), 185.48 (70), 151.55 (100), 147.35 (55), 143.11 (63), 134.15 (31), 116.12 (70), 114.83 (79), 101.95 (93), 78.48 (52), 64.66 (57), 44.12 (95).

Ethyl-6-phenyl-2-(2-(4-(pyridin-2-yl)piperazin-1-yl)acetamido)-4,5,6,7-tetrahydro Benzo[b]thiophene-3-carboxylate (5b)

Beige powder; R_f: 0.32 (petroleum ether/ethyl acetate, 1:2); yield (70%); mp 189–191 °C; IR (KBr) ν_max/cm^–1: 3222 (NH), 3042 (CH-Ar), 2983, 2941, 2910 (CH-aliphatic), 1724, 1662 (2 × C=O), 1597 (C=N), 1522 (C=C), 1436, 1237, 1014, 772, 696, 541; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.29 (t, 3H, J = 6.9 Hz, CH₃), 1.82–2.08 (m, 2H, CH₂), 2.46–3.04 (m, 9H, 2 × CH₂ and CH-cyclohexene, 2 × CH₂N), 3.43 (s, 2H, CH₂–CO), 3.60 (apparent s, 4H, 2 × CH₂N), 4.27 (q, 2H, J = 7.2 Hz, CH₂O), 6.60–6.70 (m, 1H, CH-pyridine), 6.84 (d, 1H, J = 8.1 Hz, CH-pyridine), 7.18–7.31 (m, 5H, 5 × CH-Ph), 7.56 (apparent td, 1H, J = 8.1, 2.4 Hz, CH-pyridine), 8.10–8.20 (m, 1H, CH-pyridine), 12.16 (s, 1H, NH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 14.14 (CH₃), 26.32, 29.72, 31.46 (3 × CH₂ and CH of the cyclohexene ring), 45.00, 52.62 (CH₂C=O and 4 × CH₂-piprazine ring), 60.29 (CH₂-ester), 107.16, 111.30, 113.11, 125.61, 126.24, 126.83, 128.37, 130.22, 137.56, 145.67, 146.06, 147.57, 158.92, 164.86, 167.95 (9 × CH-Ar, 6 × C_q-Ar, 2 × C=O); MS (EI, 70 eV) m/z (%): 506.41 [M⁺ + 2, 24] for C₂₈H₃₂N₄O₃S, 505.86 (24), 453.56 (50), 451.55 (48), 422.36 (40), 369.47 (100), 338.04 (59), 325.11 (50), 300.70 (47), 293.04 (76), 280.27 (76), 174.82 (56), 165.63 (45), 71.68 (46), 54.45 (82).

N-(3-Cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-morpholino Acetamide (5c)

Dark brown powder; yield (58%); mp 182–185 °C; IR (KBr) ν_max/cm^–1: 3428 (NH), 2924, 2852 (CH-aliphatic), 2205 (CN), 1679 (C=O), 1446, 1229, 1039, 870, 754, 698, 515; MS (EI, 70 eV) m/z (%): 383.60 [M⁺ + 2, 11] for C₂₁H₂₃N₃O₂S, 382.81 (30), 381.45 (10), 379.07 (24), 371.66 (56), 341.76 (81), 317.82 (62), 283.91 (39), 281.04 (76), 279.77 (88), 244.76 (45), 218.42 (77), 205.23 (63), 197.82 (59), 173.35 (100), 133.29 (63), 97.00 (73), 91.16 (93), 81.14 (75), 74.10 (78), 63.35 (77), 44.12 (70).

Synthesis of 2-(2,5-Dioxopyrrolidin-1-yl)-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (7)

Equimolar amounts (5 mmol) of aminothiophene 1b and succinic anhydride 6 in the presence of freshly fused sodium acetate (5 mmol) were fused in an oil bath for 2 h. After cooling to r.t., the reaction mixture was treated with conc. HCl (37.5%, 2 mL). The separated solid was filtered off, washed with water, air-dried, and recrystallized from ethanol to yield the title compound as a yellow powder; R_f: 0.49 (petroleum ether/ethyl acetate, 1:1); yield (60%); mp 157–160 °C; IR (KBr) ν_max/cm^–1: 3027 (CH-Ar), 2923 (CH-aliphatic), 2216 (CN), 1728, 1662 (2 × C=O), 1495 (C=C), 1439, 1283,1164, 912, 757, 700, 540; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.98–2.08 (m, 2H, CH₂), 2.75–3.08 (m, 9H, 4 × CH₂, CH), 7.21–7.32 (m, 5H, 5 × Ar-H); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 24.40, 25.76, 28.67, 28.90, 29.33, 31.90 (5 × CH₂, CH), 107.55, 112.96, 126.44, 126.59, 127.00, 128.55, 128.62, 133.70, 136.70, 145.22, 145.70 (5 × CH-Ar, 5 × C_q-Ar and CN), 175.44 (2 × CO); MS (EI, 70 eV) m/z (%): 338.32 [M⁺ + 2, 26] for C₁₉H₁₆N₂O₂S, 336.64 (51), 315.16 (34), 281.54 (45), 258.21 (71), 243.27 (50), 231.75 (100), 220.56 (42), 158.80 (31), 119.22 (48), 45.82 (39).

Synthesis of N′-(3-Cyano-6-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-N,N-dimethyl Formamidine (9)

To a solution of aminothiophene 1b (4 mmol) in dry xylene (15 mL), DMF–DMA 8 (4 mmol) was added. The reaction mixture was heated under reflux for 10 h. The separated solid was filtered off and recrystallized from EtOH to give the title compound as a buff powder; R_f: 0.67 (petroleum ether/ethyl acetate, 1:1); yield (70%); mp 120–122 °C; IR (KBr) ν_max/cm^–1: 3027 (CH-Ar), 2920 (CH-aliphatic), 2205 (CN), 1628 (C=N), 1572, 1401, 1262, 972, 871, 758, 700, 546; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.74–1.97 (m, 2H, CH₂), 2.50–2.80 (m, 5H, 2 × CH₂, CH), 2.98 (s, 3H, CH₃–N), 3.08 (s, 3H, CH₃–N), 7.21–7.31 (m, 5H, 5 × Ar-H), 7.96 (s, 1H, N=CH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 24.46, 29.14, 32.12 (3 × CH₂, CH), 34.62, 38.81 (2 × CH₃–N), 95.48, 116.05, 122.97, 126.38, 126.93, 128.48, 131.93, 145.58, 155.28, 164.88 (5 × CH-Ar, 5 × C_q-Ar, CN & CH=N); MS (EI, 70 eV) m/z (%): 311.49 [M⁺ + 2, 57] for C₁₈H₁₉N₃S, 308.87 (14), 306.47 (64), 296.95 (39), 284.36 (32), 272.55 (74), 261.15 (42), 249.12 (45), 238.22 (55), 236.25 (64), 216.32 (60), 200.89 (68), 185.82 (39), 176.87 (47), 161.77 (53), 157.32 (73), 143.69 (81), 132.65 (61), 124.55 (82), 108.84 (59), 93.18 (100), 90.59 (92), 71.96 (44), 65.33 (59), 53.36 (78).

Synthesis of 2-((3-Hydroxy-4-methoxybenzylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (11)

To an equimolar (0.01 mol) solution of aminothiophene 1c and isovanillin 10 in ethanol (30 mL), acetic acid (3 mL) was added. The resulting reaction mixture was refluxed for 12 h. Evaporation of the excess solvent gave the crude product, which was recrystallized from ethanol to give the title compound as a yellowish green powder; R_f: 0.79 (petroleum ether/ethyl acetate, 4:1); yield (80%); mp 190–192 °C; IR (KBr) ν_max/cm^–1: 3403 (OH), 3027 (CH-Ar), 2931 (CH-aliphatic), 2220 (CN), 1595 (C=N); 1521 (C=C), 1440, 1281, 1018, 895, 795, 699, 540; ¹H NMR (300 MHz, DMSO-d₆): δ_H 1.73 (apparent s, 4H, 2 × CH₂), 2.06 (apparent s, 2H, CH₂), 2.60 (apparent s, 2H, CH₂), 3.83 (s, 3H, OCH₃), 7.02 (d, 1H, J = 8 Hz, Ar-H), 7.31 (dd, 1H, J = 6.4, 1.6 Hz, Ar-H), 7.47 (s, 1H, Ar-H), 8.36 (s, 1H, CH=N), 9.52 (s, 1H, OH); ¹³C NMR (75 MHz, DMSO-d₆): δ_C 21.61, 22.40, 23.72, 24.49 (4 × CH₂), 55.65 (OCH₃), 104.87, 111.43, 113.43, 114.43, 124.31, 127.79, 131.59, 134.00 (3 × CH-Ar, 4 × C_q-Ar & CN), 147.06, 152.22, 159.90, 160.43 (2 × C_q-O, S-C_q-N & HC=N); MS (EI, 70 eV) m/z (%): 315.74 [M⁺ + 3, 4] and 314.82 [M⁺ + 2, 10] for C₁₇H₁₆N₂O₂S, 313.55 (16), 312.34 (10), 213.66 (56), 201.35 (42), 105.26 (59), 77.13 (100), 74.39 (53), 69.91 (58), 64.21 (77), 50.30 (39).

Synthesis of Magnetite and Magnetic Silicate NPs Supported with the 4,5,6,7-Tetrahydrobenzo[b]thiophene-3-carbonitrile Schiff Base Derivative (11)

Synthesis of Fe₃O₄ NPs by the Co-precipitation Method

500 mL of 1.5 M NaOH was added dropwise with continuous stirring for 4 h at 80 °C to a mixture of (0.04 mol, 10.8 g) FeCl₃·6H₂O and (0.02 mol, 5.5 g) FeSO₄·7H₂O dissolved in 50 mL of 0.5 M HCl. The result Fe₃O₄ precipitant was separated with a magnet and washed a few times with deionized water and dried at 50 °C for 7 h.

Modification of the Surface of Fe₃O₄ NPs by 2-((3-Hydroxy-4-methoxy benzylidene)amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (12a)

A solution of Fe₃O₄ NPs (0.8 g) and compound 11 (0.4 g) in a mixture of EtOH/H₂O (70:30) were stirred for 4 h at 70 °C. The resulting Fe₃O₄ was collected with an external magnet, washed several times with ethanol, and dried at room temperature.

Synthesis of Silica-Coated Fe₃O₄ Nanomolecules (12b)

The coated Fe₃O₄ NPs (12a, 0.5 g) were dispersed under sonication for 30 min in a mixture of deionized H₂O/EtOH/NH₃ (10:30:10) v/v. TEOS (2.5 mL) was added dropwise to the magnetite colloid under mechanical stirring for 22 h. The prepared Fe₃O₄/SiO₂ nanocomposite was separated using an external magnet, washed several times with distilled H₂O and dried at room temperature.

Biological Screening

Enzymatic Inhibitory Assays

Pyruvate Dehydrogenase Kinase (PDK1) Activity Assay

A Kinase-Glo Luminescent Kinase kit was used to determine the PDK-1 activities. Briefly, 5 μL of the assay buffer (10 × 250 mM Tris–HCl, 50 mM MgCl₂, 5 mM EGTA, 10 mM EDTA, and 10 mM DTT) and 25 μL of distilled water were added to a 96-well plate. After that, different concentrations (25, 50, 75, 100, 200, and 300 μg) of each tested compound (5 μL) were added to each well, except for the two control wells containing 5 μL of PBS buffer or 1% DMSO. Then, 5 μL of PDH E1 protein (10 μM) and 5 μL of ATP (10 μM) were added to all the wells. Subsequently, 5 μL of PDK-1 (20 μM) in protein buffer (1 mM MgCl₂, 2000 mM KCl, 40 mM K₃PO₄) was added to all the wells, while 5 μL of protein buffer was added to the control wells. Thereafter, the plate was well mixed and incubated for 30 min at 37 °C. Then, 50 μL of the appropriate kinase-Glo reagent was added to each well. Finally, the plate was incubated at room temperature for 10 min  under gentle shaking, and then, the luminescence was recorded on a Microplate Reader. Sodium dichloroacetate (1000 μM) was used as the standard drug for inhibition of PDK1.

Lactate Dehydrogenase (LDH) Inhibition Assay

LDHA inhibition activity was investigated by measuring the amounts of consumed NADH.⁵⁹ Briefly, different concentrations of each compound (25, 50, 75, 100, 200, and 300 μg) were incubated in a buffer containing 20 mM of HEPES-K+ (pH 7.2), 20 μM of NADH, 2 mM of pyruvate, and 10 ng of purified recombinant human LDHA protein for 10 min. The fluorescence of NADH, which has an excitation wavelength of 340 nm and an emission wavelength of 460 nm, was detected using a spectrofluorometer. Sodium oxamate (1000 μM) was used as a standard inhibitor for LDHA.

DPPH Radical Scavenging Effect

The 1,1 diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method was used to investigate the antioxidant potency of the all studied compounds.⁶⁰ Briefly, a 0.5 mL volume of DPPH ethanolic solution was mixed with an equal volume of each sample concentration, shaken strongly, and incubated at room temperature for 1 h in darkness. The absorbance of the residual DPPH radicals was measured at 519 nm and compared to the control (without the tested compound), while BHT was used as a positive control. The DPPH radical scavenging potency was calculated using the following formula:

where A_compound and A_control are the absorbances of the tested compound and the control, respectively. The plot of the scavenging effect (%) versus the compound concentration was also performed to determine the compound concentration providing 50% inhibition (IC₅₀).

Antibacterial Assay

Pure standard microbial isolates collected from King Khaled University Hospital were tested in this study, including E. faecalis (ATCC: 29122) as Gram-positive bacteria and E. coli (ATCC: 25922) as Gram-negative bacteria. Fresh cultures of each microorganism were grown on nutrient agar plates (Oxoid, UK), of which small inoculums were suspended in 5 mL nutrient broth for bacterial suspension preparation of 0.5 MacFarland. Bacterial viability was investigated by determining the colony-forming ability (CFU) of bacteria incubated at different time intervals without or with appropriate amounts of the compound that were mixed with 2 × 10⁷ CFU/mL in sterile BHI and were incubated under shaking for 60 min at 37 °C. Samples were serially diluted into sterile BHI, streaked onto media agar plates, and incubated for 24 h at 37 °C. The antibacterial potency of each tested compound was expressed as the residual number of CFU with reference to the initial inoculums. The results presented as the half-maximal (50%) inhibitory concentration (IC₅₀) are means of two different measurements. Ampicillin was used as a positive standard reference.

Cytotoxicity Assays on HCT-116 and LoVo Human CRC Cells

Cytotoxic potency was examined on human CRC cell lines HCT-116 and LoVo (American Type Culture Collection; USA) using various amounts of tested compounds (25, 50, 75, 100, 200, and 400 μg). Samples were diluted in Dulbecco’s modified Eagle’s medium, consisting of 10% fetal bovine serum, added to cells grown and cultured for 24 h in a 5% CO₂-humidified incubator at 37 °C. Then, the activity of lactate dehydrogenase released from damaged cells was determined in the collected supernatant aliquots using an ELISA end-point assay (Benchmark Plus, Bio-Rad, CA, USA). 0.1% Triton X-100 in the assay medium and the assay medium only were used as positive and negative controls, respectively. Cell viability, expressed as a relative percentage of the OD (optical density) values (at 550 nm) for compound-treated cells (a final concentration of 100 μg) and the control, is shown as the mean ± SD (n = 2). The plot of the cell viability (%) versus the compound’s concentration was also performed to determine the compound’s concentration providing 50% inhibition (IC₅₀).

Molecular Docking

All the molecular modeling studies were carried out using Molecular Operating Environment (MOE, 2019.0102) software. All minimizations were performed with MOE until an RMSD gradient of 0.1 kcal·mol^–1 Å^–1 with the MMFF94x force field, and the partial charges were automatically calculated. The X-ray crystallographic structure of the tubulin (TUB) domain complexed with colchicine (COL) (PDB ID: 5NM5) was downloaded from the protein data bank (https://www.rcsb.org/structure/5NM5). For the co-crystallized enzyme, water molecules and ligands, which are not involved in the binding, were removed, and the protein was prepared for the docking study using the Protonate 3D protocol in MOE with default options. The co-crystallized ligand (COL) was used to define the binding site for docking. The Triangle Matcher placement method and London dG scoring function were used for docking.

ADME Profiling

The ADME properties of compound 3b were studied by the SwissADME free web tool (http://www.swissadme.ch/index.php), accessed on 26 July 2021.

Author Present Address

^¶ Medicinal Chemistry Department, Faculty of Pharmacy, King Salman International University, Ras-Sedr, South Sinai, Egypt

Author Contributions

S.K. performed the chemistry experiments, interpreted chemistry data, and wrote the original draft of the manuscript. N.N.E.E.-S. designed the work, interpreted chemistry data with S.K. and all the biological results, supervised the student, and rewrote the manuscript as is in the final form. H.A.D. reviewed and edited the manuscript. S.S.A. secured a place for the student at KSU, designed the Nano experiments, helped with morphological characterization of NPs, and provided with N.N.E.E.-S. all chemicals. A.B.B. and M.A. performed biological activity and fund acquisition. M.K.E.-A. performed and interpreted the docking and ADME analyses.

The authors declare no competing financial interest.