Synthesis of Phthalimide Imine Derivatives as a Potential Anticancer Agent

Department of Pharmaceutical Chemistry, Pharmacy College, Hawler Medical University, Erbil, Kurdistan Region, Iraq Department of Pharmaceutical Chemistry, Pharmacy College, University of Baghdad, Baghdad, Iraq Nanotechnology & Catalysis Research Centre (NANOCAT), Institute for Advanced Studies, Block 3a, University of Malaya, Kuala Lumpur 50603, Malaysia Department of Pharmaceutical Chemicals, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia Centre for Natural Products and Drug Discovery (CENAR), University of Malaya, Kuala Lumpur, Malaysia


Introduction
e current pursue of innovative, biological-active chemicals continues to occupy a major part in medicinal chemistry and drug development research, in hope for beneficial, safer, and more potent outcomes than available medications [1]. Antioxidant and anticancer potentials represent two of the most widely searched biological activities targeting various disease types including cancer [2][3][4]. It is now clear that conventional therapies of chemotherapy and radiotherapy are not the definite answer for a selective cancer cure, and accordingly, the high mortality rates were maintained during the recent decades (WHO, 2018). is, in turn, necessitates the development of novel therapies [5]. Antioxidants, on the other hand, have been evidence-based to aid in the treatment and/or prevention of numerous diseases, especially those with etiology related to redox-state imbalances [6,7]. Herein, phthalimide derivatives have been the focus in an increasing number of studies recently [8][9][10][11]. e-CO-N(R)-CO structural feature connection to the imide ring in phthalimide chemical had proven immense biological impacts as antimicrobial [8], anti-inflammatory, antiviral, and anticancer activities [12]. e hydrophobic characteristics, as one example to the point, enable membrane interaction and improve bioavailability, which explains phthalimide use as a starting material for the synthesis of various pharmacophores [9,13]. e rational hybridization of phthalimide core to different functionalities further widens possible application areas [14]. In this field, the derivatives of phthalimide-Schiff bases demonstrated multiple strengths in different biological processes [14]. e nitrogen atom of imine (-C�N-) Schiff base group is thought to involve in hydrogen bonding with several cellular constituents [15] which can modulate activities and processes. Published data have indicated the promising avenues explored to date for hybridized phthalimide-Schiff base structures [11,14,16,17]. However, the complex multistep synthesis procedures, along with the low yield and high cost, are recognized as challenging factors [18,19]. In addition, different biological effects were observed in response to varying structures [20]. In this work, we aim to exploit both of phthalimide ring and Schiff base biological potentials into designing various hybridized structures, while innovatively altering attached functional groups in terms of types and positions, in relation to commonly tested derivatives [11,12,17,21]. A total of 16 compounds were synthesized using one-step N-aminophthalimide hybridization with functionalized aldehydes. Resulted compounds displayed a wide variety of groups pertaining to halogens, methoxy groups, phenolics, among others, which are known for biological interaction possibilities [22,23]. e alteration in types and positions of these groups within compounds is anticipated to reveal new perspectives in exploring possible differential biological outcomes. Physicochemical properties are also investigated along with antioxidant activity and cancer cytotoxicity, as potential biological activities. Distinctly, safety profile and selectivity of the synthesized imines are also explored using respective normal cell lines, in a step forward to comply with the recent up-to-date toxicology research and recommendations [24][25][26].

Chemistry.
e synthetic route used in this study to obtain hybridized compounds labeled H1 through H16 is illustrated in Figure 1, while the final compounds' structures are presented in Table 1.
Generally, the imine compounds are formed using two types of chemical reactions, i.e., addition and elimination ( Figure 2). It must be noted that the acid concentration must be low since the amines were basic compounds [27]. e structures of final imines were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, and high-resolution mass spectra as described in the Methods section (full spectra data of FT-IR, 1 H-NMR, and 13 C-NMR can be found in Supplementary Figures through Section 1-Section 3). e IR spectra of compounds H5, H7, and H8, bearing the O-H group, showed absorption peaks at 3328, 3317, and 3319 cm −1 , respectively. Compounds H1-H16 showed absorption band(s) at regions between 3099 and 2918 cm −1 due to C-H of the ring and C-H of CH 3 for compounds H3, H7-H10, and H14-H16. Compound H9, the only compound having cyano-functional group (C≡N), showed an absorption peak at 2252 cm −1 . Each compound shows an absorption peak at 1719-1707 cm −1 , 1641-1598 cm −1 , 1596-1481 cm −1 , and 1314-1297 cm −1 due to (C�O), (C�N), (C�C), and (C-N), respectively. Compound H2, the single compound among the synthesized compounds with (C-O) bond in the furfural ring, showed an absorption peak at 1113 cm −1 . Compounds that present ether in their structures showed absorption peaks at 1115 and 1085 cm −1 , 1105 and 1083 cm −1 , 1116 and 1081 cm −1 , and 1119 and 1081 cm −1 for compounds H7, H10, H14, and H15, respectively. e 1 H-NMR spectra of compounds H1-H16 showed a multiplate peak between 7.69 and 7.95 ppm for proton on carbon 4, 7.68-7.95 ppm for proton on carbon 5 and 6, and 7.69-7.94 ppm for proton on carbon 7. All compounds showed a singlet peak in the region between 8.68 and 9.82 ppm, due to a single proton at benzylidene carbon (CH�N). Compounds H5 and H7 have hydroxyl functional group, and compound H5 1 H-NMR gives a singlet peak at 11.13 ppm, due to the phenolic OH bonded to carbon 4 \\ . In contrast to compound H5, the phenolic OH of compound H7, which is linked to carbon 4 \\ , which was also surrounded by two methoxy groups, is significantly shielded to 5.81 ppm due to greater steric effect between two bulky groups than that of compound H5. e 13 C-NMR spectra of compounds H1-H16 indicated the presence of one sp 2 methine shifts between 140.60 and 159.93 ppm.

Antioxidant Activity.
e antioxidant activity of the synthesized compounds was evaluated using DPPH free radical inhibition assay. Results reveal a concentration-dependent trend among all synthesized imines (Supplementary Figure S4), while Table 2 lists the respective DPPH scavenging activity in terms of IC 50 values (mean ± SEM). Compound H7 scored the highest antioxidant activity among other active compounds as shown in Figure 3, with an IC 50 value of 28.37 ± 0.89 µM, surpassing that of standard antioxidant BHT (IC 50 35.25 ± 1.08 µM).

In Vitro Anticancer Activity.
e chemotherapeutic potential of the synthesized imines was screened against four human in vitro cell lines, representing colon and breast cancer cells (HT-29 and MCF-7), along with the normal tissue counterparts (CCD-841 and MCF-10A) cells. e panel uses 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay that measures cellular mitochondrial activity as dictating factor for healthy, metabolically active cells [28], upon treatment with compound's various concentrations for 24-hour incubation time. Selectivity ratios were also calculated and expressed as selectivity index (SI) for each compound in colon and breast tissues. Results revealed a concentration-dependent anticancer activity for the majority of compounds (full data presented in Supplementary Figures S5-S6), while the respective IC 50 and SI values are presented in Table 3         Journal of Chemistry displayed in Figure 4, in comparison to the starting material (N-aminophthalimide) activities. In colon cancer, various imines displayed lower IC 50 values compared to the precursor chemical N-aminophthalimide. Compounds H6, H7, and H10 recorded the lowest IC 50 values along with concurrent acceptable SI threshold. On the other hand, H6 and H14 compounds were most active and safe against breast cancer tissue.

Discussion
e rational and concept of the design of this study are based on modifying phthalimide structure by molecular hybridization with various aldehydes. e phthalimide focal point was maintained during synthesis, while changing aldehydes functionalities in terms of types and position took place in the latter synthetic steps. It is proposed here that the integration of imine (-N�C-) functional group with its broad biological activities on the one hand [15] and the various cancer inhibition potentials of phenolic functional groups on the other hand [22,29,30] is the path to enhanced and possible differential biological activities. e synthesized compounds' structural properties were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, and high-resolution mass spectra, as explained earlier. In relation to previous studies proving the presence of biological or industrial activities to similar phthalimide-derivative structures [11,21], we hypothesize that the presence of the same functional group at alternating positions (compounds H11, H12, and H13) or the different functionalities (compounds H5, H6, and H7) designates vital target and additional merits in measuring the possible differential biological activities which will aid in dictating the suitable application path. e antioxidant potential measured by DPPH free radical inhibition as the commonly used assay for chemicals screening [31][32][33] revealed a relatively weak antioxidant activity of the precursor chemical N-aminophthalimide that was only enhanced after introducing imines hybridization, at compounds H7 > H5 > H1 > N-aminophthalimide ( Figure 3). In this regard, the relative higher antioxidant activity trends demonstrated especially in compounds H7 and H5 may reflect their structures containing particular antioxidant fragments, which are very important for antioxidant activity especially in a single structure; these include phenolic hydroxyl group, substituted phenyl to increase the free radical stabilization, electron-donating group, and imino group to extend free radical delocalization [34,35]. Additionally, the presence of methoxy substitutions is documented to further enhance the antioxidant efficiency of monophenols [23]. e second line of biological activities' measurement was dedicated to cancer treatment potentials. e high incidence and mortality rates imposed by this disease emphasize the vital role of chemicals' development and design, with special care to the safety parameters. Reporting of early biocompatibility and selectivity signs toward normal tissue is now essential in exploring anticancer activities, and the recent toxicology reviews and recommendations call for such implementation that aid in obtaining a clear overview of studied agent potentials and pitfalls, leading to effective future translation into clinical use [24][25][26]. In this work, normal colon and breast cell lines were tested in parallel with malignant counterparts, through measuring selectivity index (SI). Results generally displayed varied compounds' responses among colon and breast cancer cell lines, which may reflect the differential and heterogeneous cancer microenvironments affecting treatment outcomes, as documented previously [36,37]. N-aminophthalimide demonstrated a concentration-dependent decrease in the viability of both cancer types, which agrees with previous reports stating potential anticancer activity of phthalimide pharmacophore (Figure 4) [38]. Nevertheless, the effective and safe anticancer potency was only achievable after hybridization and the production of imine compounds (Table 3). Such remark confirms the enhanced anticancer action of the hybridized imines.
Among the tested compounds, the H6 Schiff base displayed the most potent anticancer activity in both colon and breast cancer cell lines. It is anticipated that its structure   Journal of Chemistry including two halogens, namely, bromine and fluorine, might be relevant to its anticancer activity [29,[39][40][41].
Herein, several studies have indicated the effective compounds' bromination as a strategy to enhance biological properties and increase cytotoxicity, by targeting cellular nucleic acid production as one possible pathway among others [50]. e underlying mechanisms of these effects are still unclear; however, higher rates of lipophilicity, cellular permeability, and intermolecular bonds with biomolecules constitute probable explanatory axes [42]. On the other hand, fluorine has been reported to own the strongest hydrogen binding among halogens, which enable selective binding to specific proteins or enzymes, hence providing good anticancer activity [30,[43][44][45]. e presence of a phenyl ring is also important to provide more lipophilic character, which is reported to own the potential to inhibit proteins for cell growth [46]. Variously, the presence of methoxy and hydroxyl groups in H7, H14, and H10 may enhance the anticancer activity [29,47]. Studies of cytotoxicity activity have demonstrated that electron releasing groups, especially at para position, will enhance cancer cytotoxicity [29,47]. In particular, H14 compound has displayed good anticancer activity against MCF-7 breast cancer cell line with an acceptable selectivity index, despite its high molecular weight and presence of two bulky groups, which might be thought to cause steric hindrance preventing interaction with cellular proteins, as reported previously [29,46]. However, the active functional group, namely, the methoxy group at the ortho position, is expected to increase the compound's activity against the cancer cell environment.
In summary, this work highlights the differential biological outcomes within the synthesized compounds. Herein, some compounds displayed weak activity against used cancer cell lines, while they have the highest antioxidant potency and vice versa. is is related to the presence of phenolic and methoxy substitutions at aromatic imines that improve the antioxidant activity of phthalimide, while the  presence of meta bromo and para fluoro substituted imines will improve the anticancer activity.

Materials.
All materials and solvents were purchased from commercial suppliers, including ermo Fisher, Merck, and Sigma Aldrich and used without additional purification. Melting points (MPs) were determined on a MEL-TEMP II melting point instrument. Fourier Transform Infra-Red spectroscopy (FT-IR) was recorded on a Perki-nElmer spectrum 400 FT-IR/FT-FIR spectrometer. e 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) spectra were recorded on BRUKER 600 MHz spectrometer using CDCl 3 or DMSO as a solvent. High-resolution electron ionization mass spectrometry (HREIMS) m/z was obtained with LC/ MS mass spectrometer.

General Synthesis of Imines.
e N-aminophthalimide chemical was dissolved in ethanol, and then target aldehyde was added along with glacial acetic acid. e mixture was then refluxed for 6 hours at 78°C, followed by using thinlayer chromatography TLC. After completion, the mixture was cooled down to room temperature (25°C) and then kept at 4°C for 1 hour, and the precipitate was collected, filtered, and recrystallized from ethanol to give a crystal product. Further specific details of synthesis can be found in Supplementary Material. e DPPH radical scavenging assay was done according to the procedure followed in the literature [34]. DPPH solution (0.1 mM DPPH solution in methanol) was added (1.0 ml) to a range of various samples' concentrations (5, 10, 50, 100, 150, 300, and 600 µM), while methanol served as negative control and BHT as a positive control. e mixture was then incubated in the dark for 60 minutes at room temperature. Absorbance at 517 nm for each sample was then measured. e free radical scavenging activity of the compounds was calculated as a percentage of radical inhibition using the following equation: percentage of inhibition � (Ac − As) Ac × 100, where As is the absorbance of the compounds/positive control and Ac is the absorbance of the negative control.
Each assay was carried out in triplicate. e concentration required to achieve 50% inhibition (IC 50 ) of the DPPH radical was determined by plotting each compound's percentage result and exact concentration.

MTT Cytotoxicity
Assay. Cell viability assay 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was conducted based on a previously modified protocol [48]. Briefly, the cells were plated into 96-well plates at the density of 5000 cells/well in the final volume of 100 μL culture medium per well. On the following day, the cells were treated with the respective samples' treatments (800, 400, 200, 100, 50, and 25 µM), while the standard cytotoxic drugs 5-fluorouracil (5-FU) and tamoxifen (Tmx) served as positive controls. Cells were incubated at 37°C with 5% CO 2 for 24 hours. Cells without treatment were used as a negative control. At the end of the incubation period, 10 μL of MTT reagent (5 mg/mL) was added to each well and incubated again for 4 hours at 37°C with 5% CO 2 , then the supernatant was removed and 100 μL of dimethylsulphoxide (DMSO) was added to each well, and the absorbance was determined at 570 nm using microplate reader (Infinite-M200Pro-TECAN). e experiment was carried out in triplicate, and cellular viability was calculated according to cell viability (%) � sample absorbance control absorbance × 100. (2)

Selectivity Index (SI).
e degree of selectivity of chemicals to each cancer cell line is explored as SI ratio as per previous reports [49], according to the following equation: SI � IC 50 of sample on normal cell lines IC 50 of sample on cancer cell line . (3)

Conclusions
A series of hybridized imines were synthesized combining the active phthalimide pharmacophore and various phenolic aldehydes with various functionalities. e facile one-step synthesis method substituted the conventional chemical two-step route. Among these compounds, H7 presented superior antioxidant activity against DPPH free radical which may reflect the phenolic group-bearing structure. As such, H7 and H6 were colon cancer cytotoxic active agents with concurrent acceptable safety, surpassing the starting material N-aminophthalimide. Breast cancer was also screened and was inhibited by compounds H6 and H14. ese findings suggest that hybridized imines and especially the halogenated H6 structure have successfully amplified anticancer activities, which create a promising window for a novel therapeutic strategy.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request. Disclosure e funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Conflicts of Interest
e authors declare no conflicts of interest.