1 Introduction

Studies of donor–acceptor conjugates capable of undergoing light-induced electron transfer are current interest to develop optical and molecular electronic devices [1,2,3,4,5] and biomedical applications [6,7,8]. Toward constructing such conjugates, various covalently linked systems have been constructed based in the recent years [1,2,3,4,5]. Among the utilized materials, 1,4-dihydropyridine (DHP) parent nucleus, the 1,8-dioxodecahydroacridines, or acridine-1,8-diones, attracted much attention as an important class of nitrogen heterocyclic compounds [9,10,11]. Acridin-1,8-diones have been extensively used as photosensitizers [12, 13], laser dyes [12, 14, 15], and promoters [12, 13]. The optical, electrochemical, biologic, and biomedical uses are expected to be greatly impacted by the little changes made to the 1,4-dihydropyridine (DHP) molecule [16, 17]. The synthetic community has expressed increased interest in this fundamental structure [18]. Because of their wide range of biologic action, 1,8-acridinediones may be used as a medicinal drug to treat a variety of pathologic disorders. Acridinedione N-acetic acid so effectively inhibits the growth of cancer by binding to DNA [19]. N-aminoacridinedione has anticancer properties as well.

Taking these unique optical and biologic properties of acridinedione into consideration, we reported herein the optical characterization and singlet oxygen generation of acridinedione derivatives (Acr 1–5) and acridinedione-phthalimide conjugates (AcrPhth 1–5) considering that the phthalimide is a crucial starting material for a variety of biologically active compounds [20,21,22,23]. In addition, phthalimide derivatives are well known to act as electron accepting units for the optoelectronic applications [24,25,26]. The optical studies of the synthesized acridinediones-phthalimide conjugates (AcrPhth 1–5) have been well characterized utilizing various spectroscopic techniques, e.g., steady-state absorption and fluorescence, and time-correlated singlet photon counting (TCSPC). These compounds have been compared with the acridinediones derivatives (Acr 1–5). The electron-transfer reactions have been supported by computational studies using DFT at B3LYP/6-311G methods. To examine the ability of the examined compounds as photosensitizers for the photodynamic therapy (PDT), the singlet oxygen quantum yields were directly determined in the polar acetonitrile utilizing the powerful nanosecond laser flash photolysis technique.

2 Experimental section

2.1 Synthesis of Acr 1–5 and AcrPhth 1–5 conjugates

Acrididine-1,8-diones derivatives (Acr 1–5) and acridinediones-phthalimide conjugates (AcrPhth 1–5) have been synthesized according to the reported methods [27]. The synthetic procedure can be summarized in Schemes 1 and 2. The details are described in the supporting information.

Scheme 1.
scheme 1

Synthesis of some acridine-1,8-diones (Acr 1–5)

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Scheme 2.
scheme 2

Synthesis of acridinedione phthalimide derivatives AcrPhth 1–5

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2.2 Instruments

Optical absorption and fluorescence measurements were conducted on the UV–vis spectrophotometer (Shimadzu UV-2600) and spectrofluorophotometer (Shimadzu RF-6000), respectively. The rates and efficiencies of the electron-transfer reactions are evaluated using theTCSPC via fluoroHub (Horiba Scientific). The lifetimes of the examined compounds were determined using Fluofit software affixed to the apparatus. Geometry optimizations of the examined AcrPhth materials are conducted using the Becke3LYP functional and the 6-311G basis set, with the restricted Hartree–Fock (RHF) formalism and as implemented in the Gaussian 03 program [28]. Graphical outputs of the computational results generated with the Gauss View software program (ver. 16) developed by Semichem, Inc.

Singlet oxygen quantum yields are detected directly by measurement of singlet oxygen phosphorescence (λem ~ 1275 nm) following photoexcitation of the examined compounds at ambient temperature in acetonitrile. The compounds are excited using the third harmonic of a Q-S Nd:YAG laser (λ = 355 nm, ~ 8 ns pulse length, pulse energy ≤ 20 mJ). The specific laser model used is the LP980 from Edinburgh instruments laser flash photolysis system. The luminescent signal of singlet oxygen (1O2) at a wavelength of 1275 nm was measured using the Hamamatsu H10330-45 near-infrared (NIR) detector. The optical densities of the substance under study and the standard were equated at a wavelength of 355 nm [29,30,31].

2.3 Molecular Docking

The study employed Molecular Operating Environment (MOE 2019) software to simulate how promising compound (AcrPhth 2) interacted and bound with Mitogen-activated protein kinase (MEK1) protein. To begin, the 3D structure of the MEK1 protein was preprocessed using MOE, involving steps such as removing water molecules, eliminating repeating chains, adding protons, and performing energy minimization to refine the protein structures. Subsequently, the binding site was isolated and verified by accurately redocking the original ligands from their respective PDB IDs (e.g., 4LMN), resulting in a root mean square deviation (RMSD) of less than 2, ensuring structural validity. The promising compound (AcrPhth 2) was prepared for docking within MOE through the software’s chemical structure creation process. Protons were incorporated into the compounds’ 3D structures, followed by further energy minimization using the Force Field MMFF94x. These optimized structures were then integrated into the MOE database. The newly synthesized compounds underwent docking simulations within MOE, allowing the determination of their binding energies and elucidation of their binding mechanisms based on the outlined procedures [27].

3 Results and discussions

3.1 Optical absorption and fluorescence studies

The absorption spectrum of Acr 1 in DMF shown in Fig. 1 exhibited maxima absorption band at 374 nm, in addition other bands in the UV region (200–300 nm). The absorption band at the highest wavelength has been assigned to the charge transfer from nitrogen to oxygen center [9, 32]. Similar to Acr 1, the absorption band of the examined Acr samples were found to be 375 nm (for Acr 2), 374 nm (for Acr 3), 374 nm (for Acr 4), and 373 nm (for Acr 5). When turning to AcrPhth conjugates where the Acr and Phth entities are connected covalently, the absorption spectra of the AcrPhth 1–5 conjugates showed a considerable blue shift (~ 7–10 nm) compared to that of the Acr compounds; 365 nm (for AcrPhth 1), 365 nm (for AcrPhth 2), 366 nm (for AcrPhth 3), 366 nm (for AcrPhth 4), and 376 nm (for AcrPhth 4). From the listed values in Table 1, one can see that the absorption maxima of the AcrPhth conjugates tend to shift to shorter wavelength compared to that of Acr compounds suggest the existence of weak ground state interactions between the Acr and Phth entities of the conjugates [33].

Fig. 1
figure 1

Absorption spectra of Acr 1–5 (left figure) and AcrPhth 1–5 (right figure) in DMF

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Table 1 Photophysical properties of Acr 1–5 and AcrPhth 1–5
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The fluorescence spectrum of Acr 1 exhibited emission band at 434 nm. Based on the fluorescence maxima, the energy of the singlet-excited state of Acr was determined to be 2.90 eV. For Acr 2–5, it was observed that the emission band was slightly shifted (1–3 nm) with changing the substituents over the Acr entity (Fig. 2 and Table 1). For this, the emission bands were found to be 436 nm (for Acr 2), 432 nm (for Acr 3), 435 nm (for Acr 4), and 431 nm (for Acr 5). The fluorescence efficiency of Acr 1–5 compounds was determined to be 0.79 (for Acr 1), 0.58 (for Acr 2), 0.42 (for Acr 3), 0.635 (for Acr 4), and 0.50 (for Acr 5). These values suggest a moderate fluorescent property of the examined Acr derivatives.

Fig. 2
figure 2

Fluorescence spectra of the synthesized Acr 1–5 and AcrPhth 1–5 in DMF; λex = 338 nm

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When turning to connected AcrPhth 1–5, it was found that the fluorescence intensity of the singlet-excited state of Acr entity is significantly quenched compared to the control Acr 1–5 compounds accompanied with considerable blue shifts (nearly 4–6 nm). As seen, the emission maxima bands were found to be 429 nm (for AcrPhth 1), 430 nm (for AcrPhth 2), 428 nm (for AcrPhth 3), 427 nm (for AcrPhth 4), and 427 nm (for AcrPhth 5). The fluorescence quantum yields of AcrPhth 1–5 were determined to be 0.21 (for AcrPhth 1), 0.12 (for AcrPhth 2), 0.08 (for AcrPhth 3), 0.16 (for AcrPhth 4), and 0.13 (for AcrPhth 5). It is most likely that the fluorescence quenching of AcrPhth 1–5 compared to the control Acr 1–5 is due to the electron transfer from the electron donating Acr entity to the electron accepting Phth moiety in the polar medium.

3.2 Fluorescence-lifetime measurements

The time-resolved fluorescence spectral features are in track of steady-state fluorescence measurements (Fig. 3). Upon excitation with 370 nm laser light, the fluorescence-decay profile of the singlet Acr compounds at 440 nm showed a mono-exponential decay, from which the fluorescence lifetimes of the singlet state of Acr were determined to be 6.05 ns (for Acr 1), 4.56 ns (for Acr 2), 3.74 ns (for Acr 3), 4.20 ns (Acr 4), and 3.78 ns (Acr 5). When turning into AcrPhth conjugates, substantial quenching of the fluorescence lifetimes of the singlet states of Acr were recorded in the polar dimethylformamide. The decay profiles could be fitted to a bi-exponential decay. The recorded quick decay component may possibly be ascribed to the electron-transfer process from the electron donating Acr to the Phth where the lifetimes of the Acr entity were found to be 1.20 ns (for AcrPhth 1), 0.88 ns (for AcrPhth 2), 0.74 ns (for AcrPhth 3), 0.92 ns (for AcrPhth 4), and 0.83 ns (for AcrPhth 5). Based on the lifetimes of the singlet-excited states of Acr compounds (τ0)reference and the quick decay of AcrPhth conjugates (τf)sample, the rates of electron-transfer process (ket) were determined from Eq. 1 [34]:

$$k_{{{\text{et}}}} \, = \,({1}/\tau_{{\text{f}}} )_{{{\text{sample}}}} {-\!\!-}({1}/\tau_{{{\text{f}}0}} )_{{{\text{reference}}}}$$
(1)
Fig. 3
figure 3

Fluorescence decay time profiles of the examined compounds in DMF. The decay profiles monitored at the maximum emission wavelength each compoud; the excitation light was fixed at 370 nm

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Based on Eq. 1, the rates of electron transfer from the singlet-excited states of the electron donating Acr entities to the electron accepting Phth entities were found to be 6.68 × 108 s−1 (for AcrPhth 1), 9.17 × 108 s−1 (for AcrPhth 2), 1.08 × 109 s−1 (for AcrPhth 3), 8.48 × 108 s−1 (for AcrPhth 4), and 9.40 × 108 s−1 (for AcrPhth 5). These values indicate fast intramolecular electron-transfer processes of the examined AcrPhth conjugates.

3.3 Determination of the triplet quantum yield

The extinction coefficient for the triplet–triplet absorption for Acr2 and AcrPhth2 were determined using the singlet depletion method (Eq. 2):

$$\varepsilon_{{\text{T}}} \, = \,\frac{{\varepsilon_{S} \times \Delta {\text{OD}}_{{\text{T}}} }}{{\Delta {\text{OD}}_{{\text{S}}} }}$$
(2)

where both DODS and DODT are obtained from the triplet-singlet difference transient absorption spectra. The obtained εT for Acr2 and AcrPhth2 were found to be 3310– 4980 M−1 cm−1, respectively [35]. The triplet quantum yield, ϕT, values were obtained using benzophenone as a reference according to the following equation by comparing the ΔODT triplet absorbance of benzophenone at 530 nm and that of the unknown compound where both samples are optically matched at the 355 nm laser excitation wavelength (Eq. 3):

$$\Phi_{T}^{u} = (\varepsilon_{TT}^{Bz} )/(\varepsilon_{TT}^{u} ) \, \times (\Delta OD_{\max }^{u} )/(\Delta OD_{\max }^{Bz} ) \, \times \Phi_{T}^{Bz}$$
(3)

where \({\upvarepsilon }_{\text{TT}}^{\text{Bz}}\) and \({\upvarepsilon }_{\text{TT}}^{\text{u}}\) are the extinction coefficient of benzophenone and the unknown, respectively, while \(\Delta {\text{OD}}_{\max }^{u}\) is the optical density of the unknown at its max triplet absorption (as shown in Fig. 4) and\(\Delta {\text{OD}}_{\max }^{Bz}\) is the corresponding optical density of benzophenone. \({\Phi }_{\text{T}}^{\text{Bz}}\) is the triplet quantum yield of benzophenone [36]. The obtained values of ϕT for Acr3 and AcrPhth3 were found to be 0.47 and 0.35, respectively.

Fig. 4
figure 4

Triplet–triplet absorption spectra of Acr2 (left figure) and AcrPhth 2 (right figure) obtained using 355 nm excitation light in Ar-saturated DMF solutions

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The triplet decay at 660 nm of both Acr2 and AcrPhth2 in Argon purged DMF solution shows a triplet state lifetime of 24.8 and 22.7 μs, respectively. These values suggest no significant. On the other hand, the triplet decay of Acr2 and AcrPhth2 in air equilibrated DMF solution gives triplet lifetimes of 376– 438.3 ns, respectively. These values in turn result in a triplet state oxygen quenching rate constant of 9.5 × 108 M−1 s−1 and 8.2 × 108 M−1 s−1 for Acr2 and AcrPhth2, respectively. It is most likely that the significant quenching of the triplet Acr states in the air is attributed to the energy transfers from the triplet-excited states of Acr to oxygen generating the singlet oxygen (1O2). The details of the singlet oxygen generation will be discussed in the forthcoming section.

3.4 Computational calculations

To gain insights into the molecular geometry and the electronic structure, computational studies were performed using ab initio B3LYP/6-311G methods on the AcrPhth 1–5 conjugates. For this, in a first step, the starting moieties, Acr and Phth entities were fully optimized to a stationary point on the Born–Oppenheimer potential energy surface and allowed to interact. The geometric parameters of the conjugates AcrPhth 1–5 were obtained after complete energy minimization. In the optimized structures of AcrPhth 1–5 conjugates, the N–N distance between the nitrogen atom of Acr moiety and the nitrogen atom of the Phth moiety was found to be 6.79 Å. In the optimized structures shown in Fig. 5, the electron distribution of the highest occupied molecular orbitals (HOMOs) was found to be entirely located on the Acr entity, which suggests no charge transfer interaction between Acr and Phth entities in the ground state. Similarly, while the lowest unoccupied molecular orbitals (LUMOs) were found to be entirely on the Phth entity. This observation predicts the formation of Acr.+-Phth.− as electron-transfer states [37]. For conjugate AcrPhth 1, the energies of the LUMO and HOMO were found to be 4.48 and 2.97 eV, respectively. From these values, the HOMO–LUMO gaps (gas phase) of the conjugate AcrPhth 1 was found to be 1.32 eV. Similarly, the HOMO–LUMO gaps for the other examined derivatives were found to be 1.38 eV (for AcrPhth 2), 0.95 eV (for AcrPhth 3), 0.9416 eV (for AcrPhth 4), and 1.33 eV (for AcrPhth 5). The small HOMO–LUMO gap values agreed fairly well with that determined by the electrochemical measurements.

Fig. 5
figure 5

Frontier HOMO and LUMO orbitals of AcrPhth 3 conjugate obtained by the ab initio B3LYP/6-311G method

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3.5 Generation of singlet oxygen (1O2) generation using nanosecond laser photolysis technique

The amount of the singlet oxygen quantum yield (1O2) is considered as a sign of the capability of the examined materials to function as photosensitizers in PDT of cancer treatment. Singlet oxygen 1O2 can be produced in a practical and controlled manner using the photosensitized generation process. As is well known, the generation of singlet oxygen requires oxygen, light with the appropriate wavelength, and a photosensitizer that can absorb the light. The process of photoexcitation of a sensitizer primarily involves the excitation of the photosensitizer to its singlet-excited state, which decayed to its lowest energy triplet state via the intersystem crossing state. Considering that the excited triplet state (T1) exhibits a longer lifetime (typically in microseconds time scale), the generation of singlet oxygen arises from the energy transfer from the triplet-excited state of photosensitizer (3Acr*) and ground state triplet oxygen. The produced singlet oxygen quantum yield (ΦΔ) is detected directly by recording the weak signal of the singlet oxygen phosphorescence in the NIR region at ~ 1275 nm in acetonitrile, according to the modified reported method by McKenzie et al. [38,39,40,41,42]. The ΦΔ values provided in this study were acquired under conditions of low energy when the decay of singlet oxygen emission follows a mono-exponential pattern. The kinetic trace obtained was analyzed by a mono-exponential decay using OriginPro ExpDecay1 fitting function (Eq. 4):

$${\text{y}} = {\text{A}}_{1} {\text{e}}^{{{{ - \left( { - {\text{X}} - {\text{X}}_{0} } \right)} \mathord{\left/ {\vphantom {{ - \left( { - {\text{X}} - {\text{X}}_{0} } \right)} {t_{1} }}} \right. \kern-0pt} {t_{1} }}}}$$
(4)

The determination of the quantum yield of singlet oxygen generation (ΦΔ) involves comparing the extrapolated emission intensity at 0 time for the compounds with that of the standard, which is tris(bipyridine)ruthenium(II) chloride. The quantum yield value for singlet oxygen production of the standard tris(bipyridine)ruthenium(II) chloride is reported as ΦΔ = 57% in acetonitrile [43,44,45]. The values of the photosensitized singlet oxygen quantum yield of the prepared Acr 1–5 and AcrPhth 1–5 derivatives have been compiled in Table 1. From Fig. 6 (left), the singlet oxygen quantum yields of Acr 1–5 were determined to be 0.12 (for Acr 1), 0.16 (for Acr 2), 0.27 (for Acr 3), 0.25 (for Acr 4), and 0.22 (for Acr 5). Among the examined Acr 1–5 derivatives, Acr 3 with the substituted two methoxy groups showed the highest value of singlet oxygen quantum yield.

Fig. 6
figure 6

Decay traces of singlet oxygen phosphorescence at 1275 nm produced by Acr 1–5 (left) and AcrPhth 1–5 (right) in acetonitrile; λex = 355 mm

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When turning to AcrPhth conjugates shown in Fig. 6 (right), it was found that the singlet oxygen quantum yields were found to be 0.07 (for AcrPhth 1), 0.07 (for AcrPhth 2), 0.19 (for AcrPhth 3), 0.18 (for AcrPhth 4), and 0.14 (for AcrPhth 5). From these values, the singlet oxygen quantum yields of AcrPhth 1–5 derivatives are found to be lower than that of the Acr 1–5 conjugates. Such a decrease of the singlet oxygen quantum yields can be rationalized by the additional intramolecular electron-transfer process from the electron donating Acr moiety to the electron accepting Phth moiety generating the radical ion pairs (Acr.+Phth.−). The finding that the energy levels of the radical ion pairs Acr.+Phth.− (~ 1.50 eV) [46] were found to be lower than that of the triplet Acr (2.29 eV) [9, 32] suggests that the energy levels of Acr.+Phth.− recombine to populate the ground states, but not the triplet state (3Acr*). This may rationalize the obtained lower singlet oxygen generation of the AcrPhth conjugates compared to Acr derivatives. In other words, the decrease of the singlet oxygen quantum yields of the AcrPhth conjugates compared to the Acr derivatives may arise from the additional electron transfers pathway of AcrPhth 1–5 that results in decreasing the population of the triplet state of Acr moiety, which is an important factor for generating the singlet oxygen in the presence of oxygen and the appropriate light source (Scheme 3).

Scheme 3
scheme 3

illustrates the fluorescence (hνf), intersystem crossing (kisc), intramolecular electron transfer (ket), back electron transfer (kbet), and singlet oxygen generation (ϕΔ) pathways of Acr compounds (left scheme) and AcrPhth conjugates (right scheme)

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3.6 Molecular docking

For biomedical applications, compound (AcrPhth 2) has demonstrated significant efficacy against melanoma skin cancer in vitro, surpassing its analogs with the highest effectiveness and a notable selectivity index [27]. This is supported by its IC50 value of 11.9 ± 3.3 μg/mL and a selectivity index (SI) of 15.9 [27]. Moreover, the in silico-docking analysis revealed that the compound AcrPhth 2 exhibited a high binding affinity with the Mitogen-activated protein kinase (MEK1) protein, with a binding energy of −9.6195 kcal/mol. This binding energy is notably similar to that of the reference drug Cobimetinib, as shown in Table 2. The observed high binding affinity of AcrPhth 2 toward MEK1 protein further underscores its potential as a therapeutic candidate for melanoma treatment. This computational finding suggests that AcrPhth 2 could effectively interact with MEK1, a protein known to be overexpressed in melanoma, potentially inhibiting its activity, and thus contributing to anti-melanoma effects. Further experimental validation and characterization of this interaction could substantiate AcrPhth 2’s candidacy for melanoma therapy. The results of the docking analysis involving compound (AcrPhth 2) with the MEK1 protein are depicted in graphical representations (Fig. 7).

Table 2 Binding energy (BE) (kcal/mol), RMSD Value and Binding amino acids in compound (AcrPhth 2) Targeting (MEK1) protein compared to reference drug (Cobimetinib) to the MEK1
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Fig. 7
figure 7

(Left) 3D visualization showcases the binding interactions between compound (AcrPhth 2) and the MEK 1 protein, emphasizing Pi–H bonding interactions highlighted in black. Conversely. (Right) 2D depiction illustrating detailed insights into the molecular interactions between compound and the protein

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To confirm the accuracy of the MOE program, a validation process involved comparing co-crystallized ligands with the respective protein target. This was achieved by visually overlaying the native co-crystallized ligand (shown in green) with the redocked co-crystallized ligand (depicted in purple) using 3D diagrams. Root mean square deviation (RMSD) values were calculated for these overlays, and the results were graphically presented, as depicted in Fig. 8.

Fig. 8
figure 8

The provided 3D diagram displays the overlay of the native co-crystallized ligand, Cobimetinib (in green), with the redocked co-crystallized ligand (in purple) within the MEK1 protein target. The calculated RMSD value of 1.6 Å quantifies the disparity between these two structures, indicating their level of deviation from each other

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4 Conclusion

Spectroscopic and computational studies of series of N-substituted acrididine-1,8-dione derivatives were reported in this manuscript. The examined compounds were designed as hybrids of phthalimide and acridine-1,8-diones, with the aim of developing light harvesting, and singlet oxygen photosensitizers for PDT. Based on the steady-state and time-resolved emission studies, the optical studies of AcrPhth 1–5 showed fast electron-transfer character from the singlet-excited states of the electron donating Acr entiy to the electron accepting Phth entity to generate the charge-separated states (Acr.+-Phth.−) in polar dimethylformamide. This finding suggests the potential of the examined materials for optical applications. Such intramolecular electron-transfer character was confirmed by computational calculations using DFT methods at the B3LYP/6-311G level of theory.

By utilizing the powerful nanosecond laser flash photolysis, we have examined the ability of the examined materials to directly detect the weak emission signals of the singlet oxygen species at the NIR region (1270 nm). The singlet oxygen quantum yields (ΦΔ) were determined to be in the range of 0.12–0.27 (for Acr 1–5) and 0.07–0.19 (for the conjugates AcrPhth 1–5) render the examined materials as potential photosensitizers for PDT. The finding that Acr 1–5 exhibited higher values of singlet oxygen quantum yields compared to the AcrPhth 1–5 conjugates may rationalized by the extra quenching pathway of the conjugates due to the intramolecular electron transfer from the singlet-excited state of Acr to the covalently linked Phth entities, which consequently might decrease the population of the triplet states. The molecular docking studies revealed that compound AcrPhth 2 exhibited high binding affinity with for key genes (p53, TOP2B, p38, and EGFR) suggesting its potential as a targeted anticancer therapy. Such finding indicates that N-substituted acrididone-1,8-dione derivatives may allow us to explore their potentials in biomedical treatments in the near future.