Background

For the past several years, thiophene-containing compounds have gained popularity in the field of organic and medicinal chemistry, and have attracted tremendous interest among organic and medicinal chemists owing to their remarkable and wide range of biological activities, such as antidepressant [1], analgesic [2], anti-inflammatory [3], anticonvulsant [4,5,6,7], and other antimicrobial properties [8]. In addition, the thiophene moiety is central in the structure of different antiepileptic drugs (AEDs) such as brotizolam [9], etizolam [10], and tiagabine [11], structures of which are shown in Fig. 1. Very recently, we have reported on the synthesis, X-ray structure, and bioactivity of new thiophene-containing compounds [11, 12]. We have described the synthesis, X-ray structure, and calculations pertaining to the new compound, (2E,2′E)-1,1′-(3,4-diphenylthieno [2,3-b] thiophene-2,5-diyl) bis (3-(dimethylamino)prop-2-en-1-one) [11]. In addition, we have prepared and characterized a number of novel thieno [2,3-b] thiophene derivatives and have evaluated their bioactivity against fungi and gram-negative bacteria [12].

Fig. 1
figure 1

Structures of some bioactive compounds containing thiophene moiety

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As part of our ongoing research in the synthesis of new heterocyclic compounds containing a thiophene core (Scheme 1), we describe herein the synthesis, characterization, and X-ray structure determination of novel thiophene-containing compounds. In addition, we found that compound 2 was formed in two isomeric forms; 2a where the bromine atom is on the side chain, and 2b, where the bromine is attached to the benzene ring. We performed energy analysis and explored other thermodynamic parameters on the two structural isomers 2a and 2b to account for the stability of one over the other. Furthermore, we have employed DFT/B3LYP calculations to highlight the molecular structural characteristics along with the electronic and spectroscopic properties of the newly prepared isomers, 2a and 2b. Additionally, the bioactivities of the newly synthesized compounds against some fungi and bacteria were investigated in vitro.

Scheme 1
scheme 1

Synthesis of compounds 2, 3, and 4

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Results and discussion

Chemistry

Shown in Scheme 1 are reactions involved in the synthesis of compounds 2, 3, and 4. 5-Acetyl-4-methyl-2-phenylamino-thiophene-3-carboxylic acid ethyl ester (2), a synthone required in this work, was prepared and characterized according to a procedure outlined by Mabkhot et al. [13] that involved stirring a mixture of ethyl acetoacetate and anhydrous potassium carbonate followed by addition of phenyl isocyanate and then chloroacetone. Compound 2, on the other hand, was prepared in 90% yield (75% 2a and 15% 2b) from the reaction of compound 1 with bromine in glacial acetic acid as a solvent. Condensation of 2-aminobenzimidazole and compound 2 in ethanol containing triethylamine under reflux afforded compound 3 [14], whereas treatment of compound 2 with 3-amino-1,2,4-triazol in ethanol under reflux for 7 h yielded compound 4. Structures of compounds 2, 3, and 4 where confirmed with the aid of IR, 1H NMR and 13C NMR spectra and with mass spectrometry, where the NMR spectra were in total agreement with the assigned structures. Similarly, mass spectra displayed the molecular ions corresponding to the respective molecular formulas of prepared compounds.

When compound 2 was prepared, we noticed that part of it dissolves in ethanol. Therefore, when it was recrystallized from this solvent followed by slow evaporation of ethanol, compound 2b was obtained as crystals. This compound was characterized by NMR and x-ray crystallography. In the 1H NMR spectrum, the signal at δ 3.47 ppm has disappeared and a new signal due to a methyl group appeared instead at δ 2.45 ppm. Moreover, the aromatic region in the new compound was different from that of 2a. Compound 2a was obtained via a typical bromination of α-hydrogen of the methyl group next to the carbonyl group. However, bromination was also possible on the activated benzene ring; due to steric effect, substitution took place at the para rather than the ortho position, leading to the formation of compound 2b (formation of compound 2b was achieved via an electrophilic aromatic substitution reaction).

Crystal structure of compound 2

In the crystal structure of compound 2, the asymmetric unit consists of one independent molecule with disorder in the position of bromine atom which eventually leads to two different isomers, 2a (Br is on the side-chain) and 2b (Br is on the benzene ring). Crystal structure of compound 2 is shown in Fig. 2, whereas depicted in Fig. 3 are the two isomers 2a and 2b for comparison. In the crystal structure of 2, the phenyl ring (C14–C19) is nearly perpendicular to the central thiophene ring (C1–C4/S1) with a dihedral angle of 88.11°. On the other hand, the second phenyl ring (C5–C10) is coplanar with the central thiophene ring with a dihedral angle of 3.27°. All bond lengths and angles are in the normal range [15]. In addition, the two isomers contain strong intramolecular hydrogen bonds between H1N1 and O2 1.934 (9) and 2.650 (12) Å for N–H–O and N–O, respectively, Fig. 4. Crystallographic data and refinement information for compound 2 are summarized in Table 1.

Fig. 2
figure 2

The ORTEP diagram of compound 2. Displacement ellipsoids are plotted at the 50% probability level for non-H atoms showing the two different isomers

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Fig. 3
figure 3

ORTEP diagram of the titled compound showing the two isomers, 2a and 2b, separately for clarification

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Fig. 4
figure 4

A view along the b axis of the crystal packing of compound 2. Dashed lines indicate week hydrogen bonds

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Table 1 Crystal data and structure refinement for 2
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Energetic and thermodynamic parameters

The calculated total energy (Etot), zero point correction (ZPVE), and thermodynamic parameters such as enthalpy (H), entropy (S) and Gibbs free energy (G) for the two isomers 2a and 2b are listed in Table 2. The optimized structure of these isomers is given in Fig. 5. Both isomers are stabilized by intramolecular H-bonding interactions of the type N–H–O. To account for the extra stability of 2b compared to 2a, we employed the data presented in Table 1. Results of energy analysis show that 2b has lower energy than 2a by 3.51 kcal/mol, hence, 2b represents the stable isomer of compound 2. Using the equation K = e−(∆G/RT), where the gas constant (R) is 2 × 10−3 kcal/mol k, the temperature (T) is 298.15 k, and the quantity ∆G is the difference between the Gibbs free energies of 2a isomer relative to 2b, we calculated the mole fractions of the two isomers to be 99.6 and 0.4 for 2b and 2a, respectively. These values confirm the predominance of 2b.

Table 2 The calculated energies and thermodynamic parameters of the studied isomers of 2
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Fig. 5
figure 5

The optimized structures of studied compounds

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The calculated optimized structural parameters of the studied isomers are given in Table 3. Both calculated structures differ geometrically in the plane–plane dihedral angels, affording the three planes C14–C15–C16–C17–C18–C19, S1–C1–C2–C3–C4, and C5–C6–C7–C8–C9–C10. Both disorders (2a and 2b) have the same dihedral angles but differ in the X-ray structure. This can be explained by two factors: 1) the crystallographic structure is an averaged structure 2) Gas phase calculations omit the packing interactions, therefore we are comparing solid state with gas phase which has more degrees of freedom. Another feature is the intramolecular hydrogen bonding, both disorders are stabilized by these H-bonding interaction of the type N–H–O (calculated 1.798 and 1.796 Å; experimental 1.934 Å) and by non-classical interaction C–H–S (calculated 2.487 and 2.479; experimental 2.480).

Table 3 The geometric parameters of both disorders, 2a and 2b (calculated and experimental)
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Antibacterial and antifungal activity

We investigated the in vitro antibacterial and antifungal activity of the newly synthesized compounds against two Gram-positive (Streptococcus pneumoniae and Bacillis subtilis) and two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) which are known to cause infections in humans. On the other hand, the antifungal activity of these compounds was assessed against four fungal species; Aspergillus fumigates, Syncephalastrum racemosum, Geotricum candidum, and Candida albicans. Activity against those pathogens was expressed as diameter of the inhibition zone, in mm, using the well-diffusion agar method. In this investigation, we have employed ampicillin, gentamicin, and amphotericin B as standard antimicrobial agents to compare the potency of the tested compounds. Results from this study are shown in Table 4.

Table 4 Antibacterial and antifungal activity of compounds 2, 3, and 4 (diameter of inhibition zone is given in mm)
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Results in Table 4 reveal that compound 3 has remarkable activity against the tested fungi A. fumigates, S. racemosum, and G. candidum, whereas compounds 2 and 4 exhibited moderate activities against these fungi. On the other hand, compound 3 displayed significant activity against the gram positive bacterial strains S. pneumoniae and B. subtilis and showed excellent activity against the gram negative strain E. coli. Compounds 2 and 4 showed moderate activities against the aforementioned bacterial strains. In addition, results suggest that the new skeletons possessing benzimidazole and thiophene moieties may provide valuable leads for the synthesis and development of novel antimicrobial agents. Moreover, compound 3 could be a promising antifungal and antibacterial agent, however, more work is needed to evaluate the safety and efficacy of this compound.

Experimental

Reagents and instrumentation

Reagents used throughout this work were obtained from commercial sources and were used as received without further purification. Progress of reactions was monitored with TLC using Merck Silica Gel 60 F–254 thin layer plates (Billerica, MA, USA). Infrared Spectra were recorded, as KBr pellets, on a Nicolet 6700 FT-IR Nicolet spectrophotometer (Madison, WI, USA). Melting points were determined on a Gallenkamp apparatus in open glass capillaries and are uncorrected. We acquired 1H- and 13C-NMR spectra with a Varian Mercury Jeol-400 NMR spectrometer (Akishima, Japan) with CDCl3 as solvent. Chemical shifts are reported in ppm (δ) relative to tetramethylsilane as an internal reference and coupling constants, J, are given in Hz. Mass spectral data were obtained with the aid of a Jeol of JMS-600H mass spectrometer (Tokyo, Japan). Single-crystal X-ray diffraction measurements were performed using a Bruker SMART APEX II CCD diffractometer (Karlsruhe, Germany). Elemental analyses were performed on a Euro Vector Elemental Analyzer (EA 3000 A, Via Tortona, Milan, Italy).

Synthesis of 5-(2-bromo-acetyl)-4-phenyl-2-phenylamino-thiophene-3-carboxylic acid ethyl ester (2)

Compound 2a was synthesized according to the following general procedure: A solution of 5-acetyl-4-phenyl-2-phenylaminothiophene-3-carboxylic acid ester (1) (3.0 g, 10 mmol) in glacial acetic acid (100 mL) was heated to 90–100 °C with vigorous stirring. To this hot solution, bromine (1.1 ml) in glacial acetic acid (50 mL) was added dropwise over a period of 30 min. After complete addition of bromine, the reaction mixture was stirred vigorously at room temperature for further 2 h until evolution of hydrogen bromide gas ceased, then was poured onto ice. The solid product was collected by filtration, washed with water, dried, and recrystallized from ethanol to give 2 as white yellowish crystals. Yield 75%; m.p.: 120–122 °C; IR (KBr): 3452 (NH), 1655 (C=O), 1633 (C=O) cm−1. 1H NMR (400 MHz, CDCl3): δ 0.72 (t, J = 6.0 Hz, 3H, CH3–CH2), 3.47 (s, 2H, CH2–Br), 3.91 (q, J = 6.1 Hz, 2H, CH2–CH3), 7.21–7.51 (m, 10H, aromatic), 10.81 (s, 1H, NH–ph). 13C NMR (100 Hz, CDCl3): δ 28.7 (CH3), 33.0 (CH2Br), 60.1 (CH2O), 110.5, 117.8, 120.5, 121.8, 125.2, 128.3, 129.8, 132.7, 136.7, 138.3. 139.2, 147.8, 166.3 (C=O), 184.4 (C=O). Anal. calcd. For C21H18BrNO3S: C, 56.76; H, 4.08; N, 3.15; S, 7.22; Found: C, 56.66; H, 3.98; N, 3.18; S, 7.34.

Compound 2b. Yield 15%; 1H NMR (400 MHz, DMSO-d6): δ 0.88 (t, J = 6.0 Hz 3H, CH3–CH2), 2.45 (s, 3H, CH3), 3.98 (q, J = 6.2 Hz, 2H, CH2–CH3), 7.45-7.83 (m, 9H, aromatic), 10.48 (s, 1H, NH–amine), ppm. 13C NMR (100 Hz, DMSO-d6): δ 11.9 (CH3), 12.0 (CH3), 60.0 (CH2), 111.2, 113.2, 118.3, 119.2, 122.8, 123.0, 127.8, 132.3, 134.0, 137.8, 150.0, 165.2 (C=O), 180.0 (C=O).

Synthesis of 4-phenyl-2-phenylamino-5-(1H-1,3-a,8-triaza-cyclopenta[α]inden-2-yl)-thiophene-3-carboxylic acid ethyl ester (3)

The following procedure was employed to prepare the title compound: A mixture of compound 2 (0.44 g, 1 mmol) and 2-aminobenzimidazole (0.133 g, 1 mmol) was refluxed in ethanol (15 mL) for 8 h in the presence of 0.5 mL of triethylamine (TEA). After cooling, the solid product was collected by filtration to afford the title compound 3 as a yellow powder. Yield 82%; m.p.: 146–148 °C; IR (KBr): 3452 (NH), 1633 (C=O), 1586 (C=N) cm−1. 1H NMR (400 MHz, CDCl3): δ 0.95 (t, J = 6.0 Hz 3H, CH3–CH2), 3.25 (q, J = 6.1 Hz, 2H, CH2–CH3), 6.57–7.51 (m, 14 H, aromatic), 7.54 (s, 1H, CH-imidazo), 10.73 (s, 1H, NH–ph) 10.81 (s, 1H, NH) ppm. 13C NMR (100 Hz, CDCl3): δ 12.1 (CH3), 54.5 (CH2), 111.0, 119.4, 119.7, 120.0, 126.2, 127.3, 128.0, 131.0, 135.0, 153.0, 164.9 (C=O). MS m/z 478 [M+, 1.2%] calcd. for C28H22N4O2S; 442 (18.9%); 328 (22.6%), 112 (100%); Anal. calcd. For C28H22N4O2S: C, 70.27; H, 4.63; N, 11.71; S, 6.70; Found: C, 70.50; H, 4.53; N, 11.66; S, 6.84.

Synthesis of 5-(1H-Imidazo[1,2-b][1,2,4]triazol-5-yl)-4-phenyl-2-phenylamino-thiophene-3-carboxylic acid ethyl ester (4)

Compound 4 was prepared according to the procedure employed for the synthesis of compound 3 with some modifications: a mixture of compound 2 (0.44 g, 1 mmol) and 3-amino-1H-1,2,4-triazole (0.84 g, 1 mmol) was heated under reflux for 8 h in ethanol (10 mL) in the presence of 0.5 mL of trimethylamine (TEA). The solid product was collected by filtration to afford the desired product as a brown powder. Yield 49%; mp 150–152 °C; IR (KBr): 3409 (NH), 1658 (C=O), 1627 (C=N), 1586 cm−1 (C=C). 1H NMR (400 MHz, CDCl3): δ 0.69 (t, J = 6.0 Hz 3H, CH3–CH2), 3.52 (q, J = 6.0 Hz, 2H, CH2–CH3), 5.14 (s, 1H, NH–amine), 7.24–7.53 (m, 14 H, aromatic), 7.56 (s, 1H, CH–imidazol), 10.74 (s, 1H, CH–triazol) 10.85 (s, 1H, NH–triazol) ppm. 13C NMR (100 Hz, CDCl3): δ 12.1 (CH3), 54.8 (CH2), 119.1, 119.9, 120.0, 121.3, 125.0, 126.9, 127.2, 127.3, 127.5, 128.1, 128.7, 128.9, 131.6, 131.9, 148.5, 148.7, 164.8 (C=O). MS m/z 429 [M+, 81.3%] calcd. for C23H19N5O2S; 275 (53.8%); 211 (47.4%); 91 (100%); Anal. calcd. For C23H19N5O2S: C, 64.32; H, 4.46; N, 16.31; S, 7.47; Found: C, 64.55; H, 4.39; N, 16.50; S, 7.66.

X-ray measurements

Crystals of compound of 2 were obtained by slow evaporation from an ethanol solution at room temperature. Crystallographic data were collected on a Bruker Kappa APEXII Duo diffractometer, equipped with graphite monochromatic Mo Kα radiation, λ = 0.71073 Å at 100 (2) K. Cell refinement and data reduction were accomplished with the aid of a Bruker SAINT, whereas structure was solved by means of SHELXT [16, 17]. The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F2. CCDC 1450887 contains the supplementary crystallographic data for compound 2 and can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

Computational details

X-ray structure coordinates of the two isomers of 2 were employed as input files for comparing their relative stability. Structure optimizations were accomplished using the B3LYP method and 6‒311G(d,p) basis set with the aid of Gaussian 03 software [18]. The optimized geometries gave no imaginary vibrational modes. GaussView4.1 [19] and Chemcraft [20] programs have been employed to extract the calculation results and to visualize the optimized structures.

Antimicrobial activity

In vitro antibacterial screening tests of the newly synthesized compounds were performed against four bacterial strains: two Gram-positive (Streptococcus pneumonia and Bacillis subtilis) and two Gram-negative (P. aeruginosa and E. coli) in addition to four different fungi; A. fumigates, S. racemosum, G. candidum, and C. albicans. The disc diffusion method [21] was used in this assay and each experiment was performed in triplicate; experimental details of these techniques can be found elsewhere [22, 23]. Readings of the zone of inhibition, which are shown in Table 4, represent the mean value of three readings. Amphotericin B, ampicillin, and gentamicin were employed as standard drugs in this assay.

Conclusions

Three new thiophene derivatives were synthesized in good yield. These newly synthesized compounds were characterized by means of different spectroscopic methods and by elemental analysis. Furthermore, X-ray crystallography was performed on the two isomeric forms of compound 2 in addition to DFT and energy calculations to show the dominance of one of the isomers over the other. Additionally, the new compounds were screened for their antimicrobial activity against a number of bacterial and fungal strains. Results showed that compound 3 was a promising candidate as a potential antibacterial and antifungal agent; it exhibited remarkable activity against the studied bacterial strains, especially the gram negative bacteria E. coli in addition to some fungi. More work is needed to evaluate its safety and efficacy.