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. 2020 Nov 12;5(46):29988–30000. doi: 10.1021/acsomega.0c04358

Synthesis of Novel Biocompatible Thienopyrimidine Chromophores with Aggregation-Induced Emission Sensitive to Molecular Aggregation

Mostafa Ahmed †,*, Osama Younis †,‡, Esam A Orabi §,∥, Ahmed M Sayed §, Adel M Kamal El-Dean §, Reda Hassanien †, Rebecca L Davis ∥, Osamu Tsutsumi ‡, Mahmoud S Tolba †,*
PMCID: PMC7689934  PMID: 33251435

Abstract

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Biocompatible luminogens with aggregation-induced emission (AIE) have several applications in the biology field, such as in detecting biomacromolecules bioprobes and in bio-imaging. Due to their bioactivities and light-emitting properties, many heterocyclic compounds are good candidates for such applications. However, heterocyclic π-conjugated systems with AIE behavior remain rare as strong intermolecular π–π interactions usually quench their emission. In this work, new thienopyrimidine heterocyclic compounds were synthesized and their structures were verified by elemental analysis and Fourier transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), and 13C NMR spectra. The photophysical properties of some compounds were investigated in the solution and solid states. Density functional theory calculations were also performed to confirm the observed photophysical properties of the compounds. The studied dyes displayed AIE properties with spectral shapes related to the aggregate structure and a quantum yield up to 10.8%. The emission efficiency of the powder is attributed to the incorporation of multiply rotatable and twisted aryl groups to the fused heterocyclic moieties. The dyes also showed high thermal stability and potent antimicrobial activities against numerous bacterial and fungal strains. Additionally, the cytotoxicity of the new compounds was evaluated against the Caco-2 cell line, and molecular docking was used to investigate the binding conformation of the most effective compound with the MNK2 enzyme. Therefore, the presented structures may potentially be used for bioapplications.

1. Introduction

Aggregation-caused quenching was considered a common problem for organic luminescent dyes because molecular aggregation is natural in the condensed phases.1 Tang et al. have, however, discovered an interesting phenomenon in which some dyes are luminescent in their solid states and named it “aggregation-induced emission (AIE)”.2 This discovery was followed by the design and synthesis of several organic compounds with AIE.3−12 The spectral properties of some AIE-luminogens depend on the structure of the molecular aggregate without changing the luminescent core.13−16 AIE-active chromophores have recently been used in numerous biological applications, such as detecting biomacromolecules and as bioprobes for DNA or protein.17−20 To be suitable for such applications, the chromophores should additionally be biocompatible.21 Heterocyclic compounds seem good candidates for this purpose as many of them are light-emitting and have various biological activities.22 Organic heterocyclic π-conjugated molecules have applications in bio-imaging, light-emitting diodes, and nonlinear optics.22 However, strong intermolecular π–π interactions usually open nonradiative pathways and quench the emission of these materials.23−27 Thus, heterocyclic π-conjugated systems with AIE behavior are still rare and highly needed. Incorporating multiply rotatable and twisted aryl moieties into fused heterocycles may block the nonradiative relaxations and give AIE behavior.10,28

Most cancer types exhibit dysregulation in the protein synthesis process. Cancer cell lines depend on cap-dependent translation more than normal cells. 7-Methylguanosine (m7GDP) (the Cap) is located on 5′ extremity of mRNA in the cytoplasm to protect mRNA from ribonuclease degradation. Eukaryotic initiation factor (eIF4E) binds specifically with the 5′ cap structure and interacts with other initiation factors to induce ribosome recruitment to mRNA. eIF4E is highly overexpressed in various types of human cancers and activated through the phosphorylation by MAP kinase signal-integrating kinases (MNKs).29 Several studies reported that thienopyrimidines bind ATP-binding pocket of MNKs and inhibit its activity30 that subsequently inhibit the phosphorylation of eIF4E protein and prevent cancer progression and tumorigenesis.31 Thienopyrimidines are fused heterocyclic compounds that have also been shown to display various biological activities like antimalarial,32 anti-inflammatory,33 antimicrobial,34 antiproliferative,35 antiprotozoal,36 antiviral,37 antidiabetic,38 antianxiolytic,39 antitumor,40 and antioxidant41 activities. Thienopyrimidines with AIE properties are thus expected to be good candidates as luminescent materials for bioapplications.

In this work, we synthesized novel thienopyrimidine-based heterocycles with multiply twisted and rotatable aryl moieties. The photophysical properties of the compounds were investigated experimentally both in solution and in the solid state. The dye’s emission was conducted at different temperatures and various solution concentrations. Theoretical calculations were also performed with the density functional theory (DFT) to understand the difference in the photophysical properties of the compounds. Moreover, the thermal, antimicrobial, antifungal, and cytotoxic properties of the compounds were studied to assess the thermal stability and the biological activity.

2. Results and Discussion

2.1. Synthesis

As described in Scheme 1, the reaction of the amino carboxamide (1)42 with diethyl malonate in acetic acid under reflux for 1.5 h results in ring closure and formation of ethyl thieno[2,3-d]pyrimidine acetate derivative (2) in good yield (63%). Spectral analyses confirmed the formation of 2. IR spectrum of 2 confirmed the vanishing of NH2 bands at 3290 and 3248 cm–1 and the appearance of new bands at 3423, 3379, and 3139 cm–1 for 2NH groups and another new band at 1734 cm–1 for ester C=O. Signals at δ = 1.36 and 4.08 ppm in the 1H nuclear magnetic resonance (NMR) spectrum and 170.39 ppm in the 13C NMR spectrum correspond to the ethyl and C=O ester groups, respectively. Refluxing hydrazine hydrate with compound 2 in ethanol produced its corresponding carbohydrazide 3. The IR spectrum of 3 displays bands at 3336, 3254, and 3179 cm–1 for NH2 and 3NH groups. The disappearance of the ethyl proton signals also indicates the formation of 3. The mass spectrum and the 1H NMR, 13C NMR, and IR spectral data of 1–3 are reported in Figures S1–S9.

Scheme 1. Synthesis of Compounds 2–6.

Scheme 1

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Reacting compound 3 with triethyl orthoformate in acetic acid gave the cyclized compound 4. The structure of 4 was affirmed by the new signal at δ = 8.72 ppm due to NH in the new diazepine ring. The spectral data of 4 are given in Figures S10–S13. Refluxing 3 with phenyl isothiocyanate in ethanol gave thiosemicarbazide 5, which underwent ring closure by reaction with aqueous sodium hydroxide to afford compound 6. Compound 5 showed bands at 3335, 3212, 3113, 1663, and 1636 cm–1 for 5NH and 2CO functions in its Fourier transform infrared (FT-IR) spectrum. Also, multiplet aromatic signals appeared in the region of 6.88–7.58 ppm due to 14H in the 1H NMR spectrum. The elucidation of triazole compound 6 was affirmed by the appearance of vibrational bands in the IR spectrum at 3385 cm–1 (2NH), 3055 cm–1 (C–H aromatic), and 1594 cm–1 (C=N) in the new triazole ring. Also, the 1H NMR spectrum gave signals at δ = 8.36 and 9.98 ppm due to 2NH groups. Moreover, two singles at 165.44 and 167.12 ppm in the 13C NMR confirm the C=S and C=O groups, respectively. The appearance of the C=S signal in the 13C NMR is strong evidence for the formation of the triazole thione structure, not the thiazole-imine structure. The spectral data of 5 and 6 are included in Figures S14–S20.

2.2. Thermal Properties

The thermal stability of compounds 2–6 was evaluated using thermogravimetry–differential thermal analysis (TG-DTA). The thermal decomposition temperature (Tdec, 5% weight loss) of the compounds is 101, 290, 281, 202, and 180 °C, respectively. The results were represented in the Supporting Information as Figure S21. No liquid crystal phase was detected for compounds 2–6 by polarizing optical microscopy (POM) because their molecular structures lack a suitable spacer.

2.3. Molecular Properties of Compounds 2–6

The highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO) of the most stable structures of compounds 2–6 calculated with M062X/6-31+G(d,p) in the ground gaseous state are presented in Figure 1. The dipole moment, electronic energy, energy gap between HOMO and LUMO (Egap = ELUMO – EHOMO), and the maximum absorption wavelength (λabs) calculated in the gas phase and dimethyl sulfoxide (DMSO) with M062X/6-31+G(d,p) and B3LYP/6-31+G(d,p) are summarized in the Supporting Information (Table S1). Calculations in DMSO showed 40–50% larger dipole moments as compared to calculations in the gas phase (Table S1). Egap between the two molecular orbitals in the gas phase is similar to that in DMSO, which explains the similarity in the calculated absorption spectra (Figure 2). Compared to M062X, B3LYP calculations yield ∼1.2 eV higher energy of the HOMOs, ∼0.9 eV lower energy of the LUMOs, and ∼2.1 eV smaller Egap. Interestingly, the HOMO and LUMO have the same characteristics (Figure 1) with essentially the same Egap, which explains the observed similar λabs of the compounds. The 2.1 eV decrease in Egap translates into ∼70 nm increase in λabs for B3LYP (λabs = 383-419 nm) than M062X (λabs = 314–331 nm) (Figure 2). In comparison, the experiment shows λabs = 314–325 nm (Figure 6), indicating that the experimental values are better reproduced by the M062X functional.

Figure 1.

Figure 1

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HOMO (lower) and LUMO (upper) of the most stable structures of 2 and 3 in the gas phase calculated with M062X/6-31+G(d,p). Atomic coordinates are provided in the Supporting Information.

Figure 2.

Figure 2

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UV–vis absorption spectra calculated in the gas phase (black) and in DMSO (red) for compounds 2 (a), 3 (b), 4 (c), 5 (d), and 6 (e) with TD-DFT using M062X/6-31+G(d,p) (dotted curves) and B3LYP/6-31+G(d,p) (solid curves).

Figure 6.

Figure 6

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Photophysical properties of solutions of 2 (a) and 3 (b) (blue: absorbance of 2 × 10–6 M, red: excitation of 2 × 10–6 M, gray: emission of 2 × 10–3 M, green: emission of 2 × 10–5 M, black: emission of 2 × 10–6 M, cyan: emission of 2 × 10–7 M); excitation spectra (λem = 407 nm for 2 and 401 nm for 3); emission spectra (λex = 340 nm for both). CIE diagrams of emission spectra of 2 (c) and 3 (d). (e) Photos under UV (λex = 365 nm) for solutions of 2 (top) and 3 (bottom). Emission spectra of solutions of 4 (f) and 5 (g) (black: 1 × 10–3 M, green: 2 × 10–6 M, red: 1 × 10–7 M); the insets are their photos under UV lamp.

The emission spectra of compounds 2 and 3 were calculated using time-dependent DFT (TD-DFT) following geometry optimization of the first excited state and are shown in Figure 3. Comparing the curves calculated in DMSO with those reported from the experimental measurements (see below) reveals again that M062X calculations better reproduce the experimental data.

Figure 3.

Figure 3

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Emission spectra in the gas phase (black) and DMSO (red) calculated with TD-DFT using M062X/6-31+G(d,p) (dotted curves) and B3LYP/6-31+G(d,p) (solid curves) for compounds 2 (a) and 3 (b).

Stable structures (no imaginary frequencies) for dimers of compounds 2 or 3 calculated using B3LYP/6-31+G(d,p) are shown in Figure 4. σ-type H-bonding (N–H···O, N–H···N, and C–H···O) stabilized all structures. Note that while none of the structures display π–π interactions, these are expected to persist in the solid state between molecular layers. Compared to B3LYP/6-31+G(d,p), M062X/6-31+G(d,p) calculations yield 2–7 kcal/mol more stable dimers and ∼2 eV larger Egap between HOMO and LUMO (Table S2). Interestingly, Egap of these dimers is either similar to or larger than that of the monomer (isolated molecules), showing that molecular aggregation affects the absorption and emission properties of the molecules (Table S2 vs Table S1).

Figure 4.

Figure 4

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Stable structures for dimers of 2 (d2a–d2e) and 3 (d3a–d3d) in the gas phase, calculated with B3LYP/6-31+G(d,p). Atomic coordinates are involved in the Supporting Information.

2.4. Photophysical Properties

The photophysical behaviors of the powders of dyes 1–6 (Figure 5) and the DMSO solutions of some selected dyes (Figure 6) were investigated, and the results are summarized in Tables S3 and S4. Powders of 1, 3, 5, and 6 gave emissions in the range of 400–560 nm with wavelength of maximum emission (λmax) at 469, 460, 448, and 480 nm to produce Commission Internationale de l’Eclairage (CIE) coordinates of (x, y) = (0.23, 0.27), (0.21, 0.23), (0.18, 0.19), and (0.19, 0.18), respectively, in the light-blue region as shown also from the photos of the samples under a UV lamp (Figure 5b,c). Compounds 2 showed a strong emission at >520 nm besides the emission at 407 nm to give a red-color emission with (0.41, 0.33) CIE coordinates. Dye 4 has an enhanced emission at the shorter wavelengths with λmax = 370 nm and CIE coordinates at (0.22, 0.21). The formation of a nonplanar seven-membered ring in dye 4 can decrease the conjugation length, explaining the diminished emission at the longer wavelengths. Changing the excitation wavelength of dye 3 (Figure 5d) changes the relative emission intensities at 460 and 487 nm, which suggests the presence of dissimilar excited states from different molecular aggregations. Dyes 1–6 showed a significant change in the shape of the excitation spectra, indicating the sensitivity of the excited state to substitution and aggregation, Figure 5e. The solid-state dyes 1–6 gave emission quantum yields (Φem) = 5.9, 6.0, 10.8, 7.6, 9.2, and 8.6%, respectively.

Figure 5.

Figure 5

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Photophysical properties of powders: (a) normalized luminescence spectra at λex = 340 nm (1: red, 2: green, 3: blue, 4: black, 5: cyan, 6: gray). (b) CIE plot of the spectra in (a). (c) Photographs of 1–6 (from top to bottom) under UV (λex = 365 nm). (d) Normalized luminescence spectra of 3 at different excitation wavelengths (λem = 290: red, 340: green, 373: blue, 410 nm: black). (e) Normalized excitation spectra (1: red (λem = 469 nm), 2: green (λem = 630 nm), 3: blue (λem = 460 nm), 4: black (λem = 460 nm), 5: cyan (λem = 460 nm), 6: gray (λem = 460 nm)).

Figure 6 displays the emission, excitation, and absorption spectra of solutions of dyes 2 and 3 in DMSO. Due to π–π* transitions, dyes 2 and 3 showed absorption maxima at λabs = 314 and 325 nm, respectively (Table S4). Both dyes are mostly transparent in the region of 400–700 nm, which is a useful feature for luminescent materials. The emission spectra of the DMSO solutions of both dyes were also measured at different concentrations (2 × 10–7–2 × 10–3 M). The 2 × 10–7 M solution of 2 has a strong band at the short wavelengths (λmax = 380 nm) with a weaker emission band at ∼400 nm and is transparent at λ > 550 nm. This spectral shape produced CIE coordinates (x, y = 0.15, 0.14) with blue emission under UV irradiation (Figure 6 and Table S4). The emission intensity at 400 nm was enhanced and became comparable to that at 380 nm at a higher concentration (2 × 10–6 M); CIE coordinates are (0.18, 0.13). Increasing the concentration to 2 × 10–5 M improved the emission in the 420–550 nm range and resulted in CIE coordinates of (0.20, 0.22). At 2 × 10–3 M concentration, compound 2 gave a spectrum similar to that of 2 × 10–5 M, with a slight increase in the emission at the long wavelengths of the visible range. The emission of 2 × 10–3 M solution has (0.25, 0.25) CIE coordinates and emitted light-blue color. The emission enhancement at longer wavelengths by increasing molecular aggregation (powder and concentrated solutions) is a normal behavior in organic luminescent chromophores due to intermolecular π–π interactions of the close molecules.43,44

On the other hand, different solution concentrations (2 × 10–7, 2 × 10–5, and 2 × 10–3 M) of dye 3 have similar spectral shapes with λmax = 403 nm (Figure 6b) with CIE (x, y) = (0.17, 0.08), (0.17, 0.08), and (0.17, 0.11), respectively, producing deep-blue photoluminescence as seen from the CIE diagram and the photos under UV irradiation (Figure 6d,e). The solution of 3 with a concentration of 2 × 10–3 M showed a negligible increase in the emission intensity at 480–600 nm, showing that, in contrast to 2, the emission of dye 3 is less sensitive to molecular aggregation. This can be attributed to a higher solubility of 3 in DMSO due to its polar NH2 group. Also, the excitation and absorption spectra of 3 are similar, which indicates that the excited and ground states are similar. In contrast, the excitation and absorption spectra of 2 are different. The difference in powder (Figure 5e) and solution (Figure 6a,b) excitation spectra indicate the effect of aggregation on the excited state. Note that while solutions of 3 gave a strong emission at 403 nm (Figure 6b), the solid-state 3 emits much weaker at this wavelength compared to its emission at 460 and 487 nm. The emission spectra of different solution concentrations of dyes 4 and 5 (Figure 6f,g) showed similar behavior to dye 2. Dilute solutions (1 × 10–7 M) of dyes 4 and 5 exhibited a major emission band at λmax = 379 nm with a very weak emission at long wavelengths. Increasing the concentration to 2 × 10–6 M slightly improved the emission at 440–600 nm. The highly concentrated solution (2 × 10–3 M) showed significant quenching for the emission at λmax = 379 nm with a broad emission at long wavelengths (>440 nm). The solid-state and solution emission spectra of dyes 2–5 clearly indicate that the emission at short wavelength (379–407 nm) is a characteristic emission of the monomer, whereas the emission at longer wavelengths >460 nm, emitted from the powders and concentrated solutions, is a result of molecular aggregations. This behavior is evidence for the AIE in compounds 2–5.

The luminescence spectra of 2 in mixtures of DMSO (good solvent) and benzene (poor solvent) with various benzene fractions were measured to confirm the AIE behavior (Figure 7). Dye 2 solution in DMSO (2 × 10–6 M) gave two bands at λmax = 380 and 400 nm. An additional band appeared at 430 nm when benzene was added, and the emission intensity of this band reached the maximum intensity at 60% benzene fraction (∼5.5 times of its intensity at 0% benzene). Active molecular motions of the monomers (isolated molecules) in solutions with 0–20% benzene fractions weaken the emission by consuming the energy of the excited state via nonradiative relaxations. Interestingly, increasing the concentration of the poor solvent from 20 to 60% enhances the long-wavelength emission intensity. This is attributed to molecular aggregations in the dye, which restrict intramolecular rotations and quench nonradiative relaxation pathways. Conversely, the emission intensities of all bands diminished drastically by increasing benzene fraction over 60%, which can be attributed to the reduced solubility of the dye.45 To compare the emission efficiency in the solution and solid states, Φem values were estimated for the solutions 2 and 3 (Φem = 0.01 and 1.7%) using 9,10-diphenylanthracene as a reference. In comparison, the solid-state dyes have Φem = 6.0 and 10.8%, respectively. The higher Φem of 3 than 2 is likely due to the presence of the NH–NH2 group in 3, which increases the degree of intramolecular H-bonding and H-bonding with the solvent (DMSO). Also, the electron conjugation and charge transfer may extend upon H-bonding between the NH2 and C=O groups in 3. Hence, the AIE feature of the studied dyes was confirmed from several sides: (1) the emission at longer wavelengths that appeared with solids could be obtained from concentrated solutions due to increasing the molecular aggregation; (2) enhancement of the emission intensity by adding more fractions of a poor solvent; and (3) quantitative comparison of Φem in both solutions and solids.

Figure 7.

Figure 7

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(a) Emission spectra of 2 in mixtures of DMSO and benzene with different benzene fractions (2 × 10–6 M, λex = 340 nm). (b) Normalization of the emission spectra shown in (a). (c) Relative luminescence intensity (I/I0) at 380 nm as a function of the fractions of benzene (vol %) in the solvent mixtures; I0 is the initial intensity of 2 in DMSO only (0% benzene fraction).

The stability of the luminescence under severe conditions was investigated by measuring the emission spectra of the powder of compound 3 at high temperatures (Figure 8). Raising the temperature increased the thermally activated molecular motions, causing nonradiative consumption of the excited-state energy and hence diminishing the emission intensity. Oppositely, recooling the sample improves the emission again. Although the emission intensity changes with temperature, the spectral shape did not show significant modification by repeating the heating and cooling for two cycles (Figure 8a–e). The dye is still emitting efficiently, even at 210 °C, as indicated by integrating the area under the emission curve.

Figure 8.

Figure 8

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Emission spectra of 3 (λex = 340 nm) at high temperatures: (a) first heating; (b) normalization of (a); (c) first cooling; (d) second heating; (e) second cooling; (f) maximum emission intensity vs temperature (red: second heating, blue: second cooling).

2.5. Cytotoxic Activity

The cytotoxic effect of compounds (2–6) was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)46 against the human colorectal adenocarcinoma Caco-2 cell line. Table S5 shows the IC50 of all compounds compared to Paclitaxel as a reference cytotoxic drug. Compound 6 showed the highest cytotoxic effect with IC50 = 35 μg/mL (paclitaxel = 7.5 μg/mL). Toward understanding the higher cytotoxicity of compound 6, we performed molecular docking experiments to investigate its interaction with MNK2 kinase. Results show the potential interaction of compound 6 with residue K207 and N210 in the vicinity of the ATP pocket of MNK2 kinase (L90, E92, F159, D226, F227, and D228)47 (Figure 9). Figure 9a illustrates the binding of compound 6 with K207 through two hydrogen bonds of N1 and N2 of the triazole ring with the positively charged side chains of K207. The docking pose additionally suggests n→π* interaction between the carbonyl group of the pyrimidinone ring and the carbonyl group of the side chain of N210. The surface map also shows that compound 6 is well fitted in the ATP-binding pocket of MNK2 (Figure 9b).

Figure 9.

Figure 9

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(a) Binding mode of compound 6 with MNK2 kinase. Compound 6 is located in the vicinity of the ATP-binding pocket (L90, E92, F159, D226, F227, and D228) and is engaged in two hydrogen bonds with K207 and potentially in n→π* interaction with N210. (b) Surface map of MNK2 kinase binding site, showing that compound 6 is well fitted in the ATP-binding pocket. ATP-binding pocket and the proposed binding sites of MNK2 with compound 6 (K207 and N210) are colored purple and green, respectively.

2.6. Antimicrobial Activity

In vitro antimicrobial activity of the synthesized heterocycles against some bacterial and fungal strains was screened by the agar well diffusion method.48 The inhibition zone diameter (mm) and minimum inhibition concentration (MIC) of the studied samples were recorded against clotrimazole and nitrofurantoin as antifungal and antibacterial references, respectively (Table S6). The prepared derivatives represented significant antibacterial and antifungal activity, as shown in Figure 10. Representative photographs of the colony formation are shown in Figure S22. Compounds 2–6 displayed moderate to high antimicrobial activity for most investigated bacteria and fungi compared with the standard nitrofurantoin and clotrimazole drugs. Compounds 2 and 4 have no antibacterial activity against Pseudomonas aeruginosa and Escherichia coli, respectively, and compound 4 has no antifungal activity against Candida albicans.

Figure 10.

Figure 10

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Inhibition zone in mm for (a) antibacterial and (b) antifungal activities of compounds 2–6 with standard microorganisms.

The synthesized tricyclic thienopyrimidines exhibited significant biological activities at the cytotoxic and antimicrobial assays. Sasikumar et al. reported high permeability of other tricyclic thienopyrimidine derivatives on the Caco-2 cell line and have a high binding affinity to the mGluR1 receptor.49 Also, Tolba et al. synthesized some thienopyrimidines as anti-inflammatory agents,33 and some dimorpholine substituted thienopyrimidines were found as potential class I PI3K/mTOR dual inhibitors.50 These results highlight the importance of these compounds in biology.

3. Experimental Section

3.1. Synthesis

Solvents and reagents used here were of analytical grade and used as purchased without additional purification. Elemental analyses were performed on the (GMBH-Vario El V2.3 micro-analyzer), central laboratory of the Faculty of Science, Assiut University, and the results agreed (±0.3%) with expected values. Melting points are measured on a Fisher-Johns apparatus. Infrared spectra are estimated on the (Pye-Unicam Sp-100 spectrophotometer) using the KBr disk technique. 1H NMR and 13C NMR spectra were performed on Bruker BioSpin GmbH spectrometers (1H: 400 MHz, 13C: 100 MHz) using CHCl3 and DMSO-d6 as solvents and Me4Si as reference. Electron impact (EI) mass spectra were obtained by a JEOL JMS-600 spectrometer, National Research Center, Cairo, Egypt. All chemical reactions were conducted in the atmosphere. Compound 1 was synthesized according to the reported procedure, EI-MS (m/z): 375.47 [M+].42

3.1.1. Ethyl 2-(4-oxo-9-phenyl-7-(p-tolylamino)-3,4-dihydropyrimido[4′,5′:4,5]thieno[2,3-d]pyrimidine-2-yl)acetate (2)

A mixture of amino carboxamide 1 (1.00 g, 2.66 mmol) and diethyl malonate (2 mL, 12.4 mmol) in acetic acid (5 mL) was heated with reflux for 1.5 h. The solid product was separated during reflux, filtered off, and then recrystallized from dioxane as orange crystals in 63% yield, m.p. 344–346 °C FT-IR: υ (cm–1): 3423, 3379, 3139 (2NH), 3057 (C–H aromatic), 2922, 2853 (CH aliphatic), 1734 (C=O ester), 1667 (C=O amide), and 1582 (C=N). 1H NMR (CDCl3) δ (ppm): 1.36 (t, 3H, CH3 ester), 2.31 (s, 3H, CH3p-tolylamino), 4.08 (s, 2H, CH2CO), 4.17 (q, 2H, CH2 ester), 6.93–7.67 (m, 9H, Ar-H), 8.92 (s, 1H, NH), and 10.13 (s, 1H, NH). 13C NMR (CDCl3) δ (ppm): 14.70, 21.13, 38.98, 61.18, 116.09, 118.86, 128.52, 128.86, 129.75, 132.24, 149.33, 155.05, 156.16, 158.34, 165.44, 170.01, and 174.38. EI-MS (m/z): 470.76 [M+]. Anal. Calcd. for C25H21N5O3S (471.54): C, 63.68; H, 4.49; N, 14.85; S, 6.80%. Found: C, 63.64; H, 4.46; N, 14.87; S, 6.85%.

3.1.2. 2-(4-Oxo-9-phenyl-7-(p-tolylamino)-3,4-dihydropyrimido[4′,5′:4,5]thieno[2,3-d] pyrimidine-2-yl)acetohydrazide (3)

Compound 2 (0.50 g, 1.06 mmol) was added to hydrazine hydrate (1 mL, 17 mmol) and heated under neat conditions for 5 min then refluxed in ethanol (20 mL) for 3 h. After filtration and drying the solid product, it was recrystallized from hot ethanol to produce yellow crystals (78.6% yield), m.p. >360 °C. IR: υ (cm–1): 3336, 3254, and 3179 (3NH and NH2), 3018 (CH aromatic), 2918, 2849 (CH aliphatic), 1663, 1636 (2CO). 1H NMR (DMSO-d6) δ (ppm): 2.31 (s, 3H, CH3), 3.65 (s, 2H, CH2CO), 5.88 (s, 2H, NH2), 6.93–7.70 (m, 9H, Ar-H), 8.43 (s, 1H, NH carbohydrazide), 8.44 (s, 1H, NH), and 10.12 (s, 1H, NH). 13C NMR (DMSO-d6) δ (ppm): 21.13, 56.58, 116.09, 118.86, 128.52, 128.86, 129.75, 148.98, 149.33, 155.05, 158.34, 165.44, 171.18, and 174.38. EI-MS (m/z): 458.13 [M + H]+. Anal. Calcd. for C23H19N7O2S (457.51): C, 60.38; H, 4.19; N, 21.43; S, 7.01%. Found: C, 60.41; H, 4.16; N, 21.47; S, 7.05%.

3.1.3. Synthesis of 4-phenyl-2-(p-tolylamino)pyrimido[5″,4″:4′,5′]thieno[3′,2′:4,5]pyrimido [1,2-d][1,2,4]triazepine-7,12(6H,8H)-dione (4)

Triethyl orthoformate (2.85 mL, 19.24 mmol) was added to a solution of 3 (4.00 g, 8.74 mmol) and boiled; then, a few drops of acetic acid were added. The reaction mixture was refluxed for 1.5 h. The solid product was filtered off, dried, and recrystallized from an ethanol/dioxane mixture (2:1) as yellow crystals in (2.89 g, 70%) yield, m.p. 290–292 °C. FT-IR (KBr), ν (cm–1): 3428, 3241 (2NH), 3089 (C–H aromatic), 2918, 2849 (CH aliphatic), 1671, 1618 (2CO) and 1601 (C=N) cm–1. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 2.31 (s, 3H, CH3), 4.03 (s, 2H, CH2 triazepine), 6.93–7.67 (m, 9H, Ar-H), 7.94 (s, 1H, CH triazepine) and 8.72, 10.01 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): 21.13, 59.27, 116.09, 118.86, 124.70, 128.52, 128.86, 129.75, 131.11, 132.24, 136.74, 139.02,157.94, 158.34, 159.15, 163.70, 165.44, 170.24, 174,38. EI-MS (m/z): 467.71 [M+]. Anal. Calcd. for: C24H17N7O2S (467.51): C, 61.66; H, 3.67; N, 20.97; S, 6.86%. Found: C, 61.63; H, 3.65; N, 20.99; S, 6.83%.

3.1.4. 2-(2-(4-Oxo-9-phenyl-7-(p-tolylamino)-3,4-dihydropyrimido[4′,5′:4,5]thieno[2,3-d] pyrimidine-2-yl)acetyl)-N-phenylhydrazine-1-carbothioamide (5)

A mixture of phenyl isothiocyanate (0.24 mL, 2 mmol) and 3 (0.5 g, 1.09 mmol) in ethanol (20 mL) was refluxed for 1 h. The hot solid product obtained was collected and recrystallized from benzene as pale-yellow crystals in (0.33 g, 65%) yield, m.p. 284–286 °C. FT-IR (KBr), ν (cm–1): 3335, 3212, 3113 (5NH), 3011 (C–H aromatic), 2933 (C–H aliphatic), 1663, 1636 (2CO), 1599 (C=N), 1224 (C=S). 1H NMR (400 MHz, CDCl3), δ (ppm): 2.30 (s, 3H, CH3), 3.60 (s, 2H, CH2), 6.88–7.58 (m, 14H, Ar-H), 7.94 (s, 2H, NH), 8.32 (s, 1H, NH), 8.52 (s, 1H, NH), 8.932 (s, 1H, NH), 9.84 (s, 1H, NH). EI-MS (m/z): 592.23 [M+]. Anal. Calcd. For: C30H24N8O2S2 (592.70): C, 60.80; H, 4.08; N, 18.91; S, 10.82% Found: C, 60.83; H, 4.05; N, 18.94; S, 10.86%.

3.1.5. 9-Phenyl-2-((4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-7-(p-tolylamino)pyrimido[4′,5′:4,5]thieno[2,3-d]pyrimidine--4(3H)-one (6)

Thiosemicarbazide 5 (0.5 g, 0.84 mmol) in sodium hydroxide solution (0.4 g, 10 mmol in 5 mL of H2O) was heated at 80 °C 6 h, then cooled and acidified using acetic acid. The solid product was collected and recrystallized from ethanol as pale-green crystals in (0.36 g, 75%) yield, m.p. 328–330 °C. FT-IR (KBr), ν (cm–1): 3385 (2NH), 3055 (CH aromatic), 2919, 2849 (C–H aliphatic), 1667 (CO), 1594 (C=N). 1H NMR (400 MHz, DMSO-d6), δ (ppm): 2.33 (s, 3H, CH3), 3.82 (s, 2H, CH2), 7.15–7.48 (m, 14H, Ar-H), 7.57, 8.36, 9.98, (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): 21.13, 29.46, 116.09, 118.86, 127.92, 128.52, 128.58, 128.86, 129.37, 129.67, 129.75, 131.11, 132.24, 136.38, 136.74, 139.02, 145.52, 149.33, 152.49, 155.05, 158.34, 165.44, 167.12. EI-MS (m/z): 573.92 [M+H]+. Anal. Calcd. For: C30H22N8OS2 (574.68) C, 62.70; H, 3.86; N, 19.50; S, 11.16%. Found: C, 62.74; H, 3.82; N, 19.53; S, 11.19%.

3.2. Thermal Properties

The thermal stability was assessed by TG-DTA with a Shimadzu DTG-60AH instrument under a nitrogen atmosphere at a heating rate of 10 °C/min. POM was used at a heating rate of 5 °C/min using an Olympus BX51 microscope equipped with an mk1000 controller and Instec HC302 hot stage to check the liquid crystal phase possibility.

3.3. Computational Details

Quantum mechanical calculations were achieved with Gaussian 0951 using DFT with the 6-31+G(d,p) basis set and both B3LYP52 and M062X53 functionals. The latter functional was considered to better account for dispersion forces.54 The ground-state geometries of compounds 2–6 were optimized in the gas phase, and the global minimum structures are reported. To get insight into their excitation spectra, the geometry of the first excited state of compounds 2 and 3 was also calculated. Calculations were not performed on the excited state of compounds 4–6 due to the large size of these molecules. The solvent effect on the properties of the ground and excited states was studied using DMSO as an implicit solvent by the polarizable continuum model (PCM). We also reported HOMO, LUMO, and Egap in the ground state of each structure. The UV–Vis absorption of 2–6 and the emission spectra of 2 and 3 were calculated using the TD-DFT.55,56 Toward understanding the effect of molecular aggregation on the photophysical properties of the compounds, the intermolecular interaction between two molecules of 2 or 3 were investigated with B3LYP/6-31+G(d,p). The geometry of each dimer was optimized considering different initial conformers that account for stacking, head-to-head, and head-to-tail arrangements of the two molecules. The energy difference between the complex and its two isolated monomers was considered the interaction energy, which was corrected for basis set superposition error using the counterpoise (CP) procedure of Boys and Bernardi.56 The interaction energies were also calculated with M062X/6-31+G(d,p) using the B3LYP/6-31+G(d,p) optimized geometries, and both uncorrected (E) and corrected (ECP) values are reported.

3.4. Optical Properties

A JASCO V-550 absorption spectrometer was used to study the UV–visible (UV–Vis) absorption spectra, while the steady-state photoluminescence spectra were measured using a Hitachi F-7000 fluorescence spectrometer with an R928 photomultiplier (Hamamatsu) detector. For measuring the emission spectra at different temperatures, a 1 mm quartz cell with the sample was placed on a homemade heating stage. The Φem values in the solid state were estimated by a Hitachi calibrated integrating sphere, while the Φem values for solutions were evaluated using 9,10-diphenylanthracene as a standard.

3.5. Molecular Docking

Molecular docking was done using an Autodock Vina 1.5.6 to examine the binding mode between compound M1 and MNK2 (PDB ID: 2HW7).57 The site of the bound ligand in the crystal structure of MNK2 was utilized to check the binding conformation of M1, and the best-scored conformation is selected then visually analyzed using the PyMoL 1.7.6 software.

3.6. Antimicrobial Activity

The antimicrobial potency was evaluated by agar diffusion decomposition. Various solution concentrations of the studied heterocycles in DMSO were located on filter paper disks (5 mm diameter). Fungi and bacteria were inoculated by Sabouraud’s dextrose and nutrient agar media, respectively. Also, blank and standard antibiotic disks were used as negative and positive controls, respectively. The plates were incubated at 37 °C for 24 h with bacteria, but the incubation was done for 6 days at 25 °C with fungi. The inhibition zones were measured and recorded in units of mm. Also, the MIC of the organism’s growth was presented.

3.7. In Vitro Antitumor Efficacy

3.7.1. Cell Culture

The human colorectal adenocarcinoma Caco-2 cell line was procured from VacSERA, Egypt. The cell line was cultured in RPMI medium, including sodium bicarbonate, 10% fetal bovine serum (FBS), 1% (v/v) penicillin–streptomycin solution, l-glutamine 1% (v/v), and 7.5% NaHCO3. The cells were kept in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.

3.7.2. MTT Protocol

The 96-well tissue culture plate was inoculated with 1 × 105 cells/mL (100 μL/well) of Caco-2 cell line and incubated under conditions of 5 and 95% of air at 37 °C for 24 h to develop a complete monolayer sheet. After the confluent sheet of cells was formed, the growth media were decanted from 96-well microtiter plates. Then, the cell monolayer was washed twice with wash media. The tested samples were dissolved in DMSO and then diluted in RPMI medium with 1% serum (maintenance medium). Three different concentrations of each compound were added to the cells for the next 24 h, and all of the treatments were performed in triplicate. The MTT solution (BIO BASIC CANADA INC) was prepared (5 mg/mL in PBS), and 20 μL of the MTT solution was added to each well in the plate. The plate was placed on a shaking table for 5 min at 150 rpm to mix the MTT thoroughly into the media. Then, the plate was incubated at 37 °C and 5% CO2 for 5 h to allow the MTT to be metabolized. The media was dumped off, and the insoluble formazan (MTT metabolic product) was resuspended in 200 μL DMSO. The absorbance of the plate was measured at 570 nm to assess the cell viability.

4. Conclusions

The synthesis and spectral properties of five novel thienopyrimidines are presented. The compounds exhibited significant antibacterial activity against Bacillus cereus, Staphylococcus aureus, P. aeruginosa, and E. coli and antifungal activity against Geotrichum candidum, C. albicans, Trichophyton rubrum, and Aspergillus flavus strains. In addition, compound 6 has potent cytotoxic activity against the Caco-2 cell line. The studied dyes exhibited good thermal stability and AIE effect with a spectral shape that depends on the aggregate structure.

Acknowledgments

O.T. and O.Y. acknowledge financial supports from Japan–Egypt Research Cooperative Program (JSPS and MOSR-STDF), JSPS KAKENSHI (18K05265 and 18H03764), and Cooperative Research Program of the Network Joint Research Center for Materials and Devices (Tokyo Institute of Technology). O.Y., M.S.T., and M.A. acknowledge financial supports from New Valley University. E.A.O. and R.L.D. acknowledge Compute Canada (www.computecanada.ca) for the computational resources. This work was financially supported also by the Academy of Scientific Research & Technology (ASRT), Egypt, Grant No. 6371, under the project science up.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c04358.

  • Spectral and photophysical properties of compounds 1–6, thermal and DFT-calculated properties of compounds 2–6, properties of the dimers of compounds 2 and 3, cytotoxic and antimicrobial properties of compounds 2–6, and the atomic coordinates of the structures presented in Figures 1 and 4 (PDF)

Author Contributions

⊥ M.A., O.Y., and E.A.O. contributed equally and each one of them should be considered the first author.

The authors declare no competing financial interest.

Supplementary Material

ao0c04358_si_001.pdf (3.3MB, pdf)

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