Research Article | Open Access
Hayman Sardar Abdulrahman, Mohamed Hassan Mohammed, Lina A. Al-Ani, Mohd Hafiz Ahmad, Najihah M. Hashim, Wageeh A. Yehye, "Synthesis of Phthalimide Imine Derivatives as a Potential Anticancer Agent", Journal of Chemistry, vol. 2020, Article ID 3928204, 13 pages, 2020. https://doi.org/10.1155/2020/3928204
Synthesis of Phthalimide Imine Derivatives as a Potential Anticancer Agent
The outstanding evidence of phthalimide pharmacophore in securing enhanced biological activities had encouraged further research and development into phthalimide-based derivatives as potential new drugs. In this study, phthalimide core was hybridized with aldehydes giving integrated imines displaying different types of functionalities and at alternating positions. The resulting compounds, therefore, provide an innovative window to explore possible differential biological effects as antioxidants and anticancer agents. A total of sixteen compounds were synthesized, and each was verified by FT-IR, H NMR, C NMR, and MS characterization. Herein, a facile single-step synthesis method was employed substituting the conventional two-step chemical production routes. Among the sixteen tested compounds, the H7 compound with hydroxyl phenolic group has shown an eminent antioxidant activity with a 19.52% decrease to the IC50 value compared to that of the control standard BHT antioxidant. On the other hand, the halogenated H6 Schiff base structure was successful in securing effective cancer inhibition to both colon and breast cancer cell lines, while maintaining selective action toward normal tissues. Results have collectively indicated the importance and impactful effects of functional groups position and types within similar basic structures, in directing different biological outcomes.
The 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 . Antioxidant and anticancer potentials represent two of the most widely searched biological activities targeting various disease types including cancer [2–4]. 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). This, in turn, necessitates the development of novel therapies . 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 [8–11]. The–CO-N(R)-CO structural feature connection to the imide ring in phthalimide chemical had proven immense biological impacts as antimicrobial , anti-inflammatory, antiviral, and anticancer activities . The 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].
The rational hybridization of phthalimide core to different functionalities further widens possible application areas . In this field, the derivatives of phthalimide-Schiff bases demonstrated multiple strengths in different biological processes . The nitrogen atom of imine (-C=N-) Schiff base group is thought to involve in hydrogen bonding with several cellular constituents  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 . 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]. The 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 [24–26].
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 .
The structures of final imines were confirmed by FT-IR, 1H-NMR, 13C-NMR, and high-resolution mass spectra as described in the Methods section (full spectra data of FT-IR, 1H-NMR, and 13C-NMR can be found in Supplementary Figures through Section 1–Section 3). The 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 CH3 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.
The 1H-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 1H-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. The 13C-NMR spectra of compounds H1–H16 indicated the presence of one sp2 methine shifts between 140.60 and 159.93 ppm.
2.2. Biological Activities
2.2.1. Antioxidant Activity
The 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 IC50 values (mean ± SEM). Compound H7 scored the highest antioxidant activity among other active compounds as shown in Figure 3, with an IC50 value of 28.37 ± 0.89 µM, surpassing that of standard antioxidant BHT (IC50 35.25 ± 1.08 µM).
NA: not applicable (above testing range).
2.2.2. In Vitro Anticancer Activity
The 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. The 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 , 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 IC50 and SI values are presented in Table 3, demonstrating variable effects among colon and breast cancer types. The highest cancer-active compound trends are displayed in Figure 4, in comparison to the starting material (N-aminophthalimide) activities. In colon cancer, various imines displayed lower IC50 values compared to the precursor chemical N-aminophthalimide. Compounds H6, H7, and H10 recorded the lowest IC50 values along with concurrent acceptable SI threshold. On the other hand, H6 and H14 compounds were most active and safe against breast cancer tissue.
NA: not applicable (above testing range).
The rational and concept of the design of this study are based on modifying phthalimide structure by molecular hybridization with various aldehydes. The 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  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.
The synthesized compounds’ structural properties were confirmed by FT-IR, 1H-NMR, 13C-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. The antioxidant potential measured by DPPH free radical inhibition as the commonly used assay for chemicals screening [31–33] 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 .
The second line of biological activities’ measurement was dedicated to cancer treatment potentials. The 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 [24–26]. 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) . 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 including two halogens, namely, bromine and fluorine, might be relevant to its anticancer activity [29, 39–41]. 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 . The underlying mechanisms of these effects are still unclear; however, higher rates of lipophilicity, cellular permeability, and intermolecular bonds with biomolecules constitute probable explanatory axes . 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, 43–45]. The 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 . 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. This 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.
4. Materials and Methods
All materials and solvents were purchased from commercial suppliers, including Thermo 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 PerkinElmer spectrum 400 FT-IR/FT-FIR spectrometer. The 1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were recorded on BRUKER 600 MHz spectrometer using CDCl3 or DMSO as a solvent. High-resolution electron ionization mass spectrometry (HREIMS) m/z was obtained with LC/MS mass spectrometer.
5.1. General Synthesis of Imines
The N-aminophthalimide chemical was dissolved in ethanol, and then target aldehyde was added along with glacial acetic acid. The mixture was then refluxed for 6 hours at 78°C, followed by using thin-layer 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.(1)(E)-2-(Benzylideneamino)isoindole-1,3-dione (H1): off-white crystal product of 0.57 gram (Yield 75%); MP: 132–135°C. IR, cm−1: = 3048 (C–H of ring), 1708 (C=O), 1599 (C=N), 1572 (C=C), 1314 (C–N). 1H NMR (600 MHz, CDCl3) ppm: 7.94 (m, 1 H, H-4), 7.94 (m, 1 H, H-5), 7.94 (m, 1H, H-6), 7.94 (m, 1H, H-7), 9.42 (s, 1H, H-of benzylidene carbon), 7.80 (m, 1H, H-2\\), 7.49 (m, 1H, H-3\\), 7.49 (m, 1H, H-4\\), 7.49 (m, 1H, H-5\\), 7.80 (m, 1H, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.11 (2C, C-1, C-3), 131.69 (2C, C-1\, C-3\), 123.78 (2C, C-4, C-7), 134.64 (2C, C-5, C-6), 158.65 (1C, C-benzylidene), 133.66 (1C, C-1\\), 128.78 (2C, C-2\\, C-6\\), 128.44 (2C, C-3\\, C-5\\), 130.36 (1C, C-4\\). HREIMS m/z: 251.0742 [M + H+] (Calcd for C15H10N2O2 250.2570).(2)(E)-2-((Furan-2-ylmethylene)amino)isoindoline-1,3-dione (H2): A yellow powder product of 0.53 gram (Yield 72%); MP: 139–141°C. IR, cm−1: = 3099 (C–H of ring), 1715 (C=O), 1607 (C=N), 1481 (C=C), 1299 (C–N), 1113 (C–O). 1H-NMR (600 MHz, CDCl3) ppm: 7.85 (m, 1H, H-4), 7.71 (m, 1H, H-5), 7.71 (m, 1H, H-6), 7.85 (m, 1H, H-7), 9.25 (s, 1H, H-of benzylidene carbon), 6.92 (d, 1H, J = 6 Hz, H-2\\), 6.49 (m, 1H, H-3\\), 7.56 (d, 1H, J = 6 Hz, H-4\\). 13C-NMR (150 MHz, CDCl3) ppm: 164.99 (2C, C-1, C-3), 146.01 (2C, C-1\, C-3\), 123.82 (2C, C-4,C-7), 134.69 (2C, C-5, C-6), 148.95 (1C, C- benzylidene), 146.50 (1C, C-1\\), 116.82 (1C, C-2\\), 112.27 (1C, C-3\\), 130.28 (1C, C-4\\). HREIMS m/z: 241.0536 [M + H+] (Calcd for C13H8N2O3 240.2180).(3)2-(((1E, 2E)-2-Methylbut-2-en-1-ylidene)amino)isoindoline-1,3-dione (H3): a milky woolly product of 0.26 gram (Yield 37%); MP: 86–88°C. IR, cm−1: = 2923 and 2857 (C–H of ring and of CH3), 1707 (C=O), 1634 (C=N), 1573 (C=C), 1309 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.82 (m, 1H, H-4), 7.68 (m, 1H, H-5), 7.68 (m, 1H, H-6), 7.82 (m, 1H, H-7), 8.68 (s, 1H, H-of benzylidene carbon), 6.14 (m, 1H, H-2\\), 1.85 (d, 3H, J = 6 Hz, H-3\\), 1.92 (s, 3H, H-4\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.24 (2C, C-1, C-3), 134.82 (2C, C-1\, C-3\), 123.58 (2C, C-4, C-7), 134.39 (2C, C-5, C-6), 165.12 (1C, C- benzylidene), 130.49 (1C, C-1\\), 140.60 (1C, C-2\\), 10.51 (1C, C-3\\), 14.63 (1C, C-4\\). HREIMS m/z: 229.0989 [M + H+] (Calcd for C13H12N2O2 228.2510).(4)(E)-2-((3-Bromobenzylidene)amino)isoindoline-1,3-dione (H4): a white crystal product of 1.69 gram (Yield 83%); MP: 151–153°C. IR, cm−1: = 2970 (C–H of ring), 1719 (C=O), 1606 (C=N), 1556 (C=C), 1305 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.87 (m, 1H, H-4), 7.73 (m, 1H, H-5), 7.73 (m, 1H, H-6), 7.87 (m, 1H, H-7), 9.35 (s, 1H, H-of benzylidene carbon), 8.02 (s, 1H, H-2\\), 7.53 (d, 1H, J = 12 Hz, H-4\\), 7.27 (t, 1H, J = 6, 12 Hz, H-5\\), 7.72 (d, 1H, J = 6 Hz, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.02 (2C, C-1, C-3), 134.46 (2C, C-1\, C-3\), 123.92 (2C, C-4, C-7), 134.80 (2C, C-5, C6), 156.18 (1C, C- benzylidene), 135.79 (1C, C-1\\), 130.78 (1C, C-2\\), 123.06 (1C, C-3\\), 130.28 (1C, C-4\\), 130.25 (1C, C-5\\), 127.19 (1C, C-6\\). HREIMS m/z: 330.9901 [M + H+] (Calcd for C15H9BrN2O2 329.1530).(5)(E)-2-((3-Bromo-4-hydroxybenzylidene)amino)isoindoline-1,3-dione (H5): a pale-yellow powder product of 1.92 gram (Yield 90%); MP: 209–211°C. IR, cm−1: = 3328 (O–H), 2930 (C–H of ring), 1709 (C=O), 1601 (C=N), 1562 (C=C), 1310 (C–N). 1H-NMR (600 MHz, DMSO) ppm: 7.93 (m, 1H, H-4), 7.89 (m, 1H, H-5), 7.89 (m, 1H, H-6), 7.93 (m, 1H, H-7), 9.07 (s, 1H, H-of benzylidene carbon), 8.03 (d, 1H, H-2\\), 7.08 (d, 1H, J = 6 Hz, H-5\\) 7.73 (dd, 1H, J = Hz, H-6\\), 11.13 (s, 1H, -OH). 13C-NMR (150 MHz, DMSO) ppm: 164.97 (2C, C-1, C-3), 133.08 (2C, C-1\, C-3\), 123.89 (2C, C-4, C-7), 135.36 (2C, C-5,C-6), 159.25 (1C, C- benzylidene), 126.35 (1C, C-1\\), 130.55 (1C, C-2\\), 110.46 (1C, C-3\\), 157.89 (1C, C-4\\), 117.14 (1C, C-5\\), 129.58 (1C, C-6\\). HREIMS m/z: 346.9845 [M + H+] (Calcd for C15H9BrN2O3 345.1520).(6)(E)-2-((3-Bromo-4-fluorobenzylidene)amino)isoindoline-1,3-dione (H6): white powder product of 1.64 gram (Yield 77%); MP: 177–179°C. IR, cm−1: = 3045 and 2935 (C–H of ring), 1715 (C=O), 1598 (C=N), 1508 (C=C), 1302 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.95 (m, 1H, H-4), 7.83 (m, 1H, H-5), 7.83 (m, 1H, H-6), 7.95 (m, 1H, H-7), 9.43 (s, 1H, H- of benzylidene carbon), 8.17 (dd, 1H, H-2\\), 7.23 (t, 1H, J = 6, 12 Hz, H-5\\), 7.83 (dd, 1H, J = Hz, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 164.99 (2C, C-1, C-3), 133.17 (2C, C-1\, C-3\), 123.92 (2C, C-4, C-7), 134.82 (2C, C-5, C-6), 154.99 (1C, C- benzylidene), 130.20 (1C, C-1\\), 131.44 (1C, C-2\\), 109.95 (1C, C-3\\), 160.15 (1C, C-4\\), 116.81 (1C, C-5\\), 129.26 (1C, C-6\\). HREIMS m/z: 348.9795 [M + H+] (Calcd for C15H8BrFN2O2 347.1434).(7)(E)-2-((4-Hydroxy-3,5-dimethoxybenzylidene)amino)isoindoline-1,3-dione (H7): a dark yellow powder product of 1.38 gram (Yield 69%); MP: 159–161°C. IR, cm−1: = 3317 (O–H), 2994-2937 (C–H of ring and of CH3), 1713 (C=O), 1609 (C=N), 1579 (C=C), 1303 (C–N), 1115 and 1085 (ether). 1H-NMR (600 MHz, CDCl3) ppm: 7.85 (m, 1H, H-4), 7.71 (m, 1H, H-5), 7.71 (m,1H, H-6), 7.85 (m, 1H, H-7), 9.13 (s, 1H, H- of benzylidene carbon), 7.08 (s, 1H, H-2\\), 5.81 (s,1H, OH), 7.08 (s, 1H, H-6\\), 3.91 (s, 6H, 2 X -OCH3). 13C-NMR (150 MHz, CDCl3) ppm: 165.20 (2C, C-1, C-3), 130.40 (2C, C-1\, C-3\), 123.72 (2C, C-4, C-7), 134.58 (2C, C-5, C-6), 159.48 (1C, C- benzylidene), 124.82 (1C, C-1\\), 105.47 (2C, C-2\\, C-6\\), 147.26 (2C, C-3\\, C-5\\), 138.37 (1C, C-4\\), 56.54 (2C, 2 X -OCH3). HREIMS m/z: 327.0966 [M + H+] (Calcd for C17H14N2O5 326.3080).(8)(E)-2-((3,5-Di-tert-butyl-2-hydroxybenzylidene)amino)isoindoline-1,3-dione (H8): a milky powder product of 1.89 gram (Yield 81%); MP: 164-166°C. IR, cm−1: = 3319 (O–H), 3033-2874 (C–H of ring and of CH3), 1718 (C=O), 1610 (C=N), 1582 (C=C), 1313 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.82 (m, 1H, H-4), 7.95 (m, 1H, H-5), 7.95 (m, 1H, H-6), 7.82 (m, 1H, H-7), 9.46 (s, 1H, H- of benzylidene carbon), 7.19 (d, 1H, J = 6 Hz, H-4\\), 7.76 (d, 1H, J = 6 Hz, H-6\\), 4.18 (s, J = 6 Hz, H-2\\ (OH)), 1.35 (s, 9H, t-butyl (8\)), 1.49 (s, 9H, t-butyl (7\\)). 13C-NMR (150 MHz, CDCl3) ppm: 164.36 (2C, C-1, C-3), 128.42 (2C, C-1\, C-3\), 123.89 (2C, C-4, C-7), 134.75 (2C, C-5, C-6), 156.71 (1C, C- benzylidene), 116.36 (1C, C-1\\), 123.50 (1C, C-6\\), 130.25 (1C, C-5\\), 127.21 (1C, C-4\\), 137.28 (1C, C-3\\), 162.31 (1C, C-2\\), 35.19 (1C, C-8\\), 34.21 (1C, C-7\\), 31.46 (3C, t-butyl C-7\\), 29.45 (3C, t-butyl C-8\\). HREIMS m/z: 379.2027 [M + H+] (Calcd for C23H26N2O3 378.4720).(9)(E)-3-((4(((1,3-Dioxoisoindolin-2-yl)imino)methyl)phenyl) (methyl)amino) propanenitrile (H9): a yellow crystal product of 1.74 gram (Yield 85%); MP: 155–157°C. IR, cm−1: = 3034 and 2907 (C–H of ring and of CH3), 2252 (C≡N) 1709 (C=O), 1610 (C=N), 1589 (C=C), 1306 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.69 (m, 1H, H-4), 7.82 (m, 1H, H-5), 7.82 (m, 1H, H-6), 7.69 (m, 1H, H-7), 9.05 (s, 1H, H- of benzylidene carbon), 6.64 (d, 1H, J = 12 Hz, H-2\\), 7.72 (d, 1H, J = 6 Hz, H-3\\), 7.72 (d, 1H, J = 6 Hz, H-5\\), 6.64 (d, 1H, J = 12 Hz, H-6\\), 3.07 (s, 3H, H-7\\), 3.73 (t, 2H, J = 6 Hz, H-8\\), 2.57 (t, 2H, J = 6 Hz, H-9\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.31 (2C, C-1, C-3), 130.50 (2C, C-1\, C-3\), 123.57 (2C, C-4, C-7), 134.42 (2C, C-5, C-6), 159.93 (1C, C- benzylidene), 123.57 (1C, C-1\\), 122.50 (2C, C-2\\, C-6\\), 111.72 (2C, C-3\\, C-5\\), 150.18 (1C, C-4\\), 38.93 (1C, C-7\\), 48.56 (1C, C-8\\), 15.59 (1C, C-9\\), 118.01 (1C, C-10\\). HREIMS m/z: 333.1280 [M + H+] (Calcd for C19H16N4O2 332.3630).(10)(E)-2-((3-(p-Tolyloxy)benzylidene)amino)isoindoline-1,3-dione (H10): a white crystal product of 1.92 gram (Yield 88%); MP: 103–105°C. IR, cm−1: = 2918 (C–H of ring and of CH3), 1716 (C=O), 1601 (C=N), 1571 (C=C), 1302 (C–N), 1105 and 1083 (ether). 1H-NMR (600 MHz, CDCl3) ppm: 7.71 (m, 1H, H-4), 7.84 (m, 1H, H-5), 7.84 (m, 1H, H-6), 7.71 (m, 1H, H-7), 9.27 (s, 1H, H- of benzylidene carbon), 7.41 (s, 1H, H-2\\), 7.02 (m, 1H, H-4\\), 7.32 (t, 1H, J = 6, 12 Hz, H-5\\), 7.53 (d, 1H, J = 12 Hz, H-6\\), 7.08 (d, 1H, J = 12 Hz, H-2\\\), 6.86 (d, 1H, J = 12 Hz, H-3\\\), 6.86 (d, 1H, J = 12 Hz, H-5\\\) 7.08 (d, 1H, J = 12 Hz, H-6\\\), 2.28 (s, 3H, H-7\\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.04 (2C, C-1, C-3), 130.41 (2C, C-1\, C-3\), 123.81 (2C, C-4,C-7), 134.68 (2C, C-5, C-6), 158.03 (1C, C- benzylidene), 135.38 (1C, C-1\\), 117.82 (1C, C-2\\), 158.33 (1C, C-3\\), 122.87 (1C, C-4\\), 121.57 (1C, C-5\\), 133.38 (1C, C-6\\), 154.30 (1C, C-1\\\), 119.29 (2C, C-2\\\, C-6\\\), 130.05 (2C, C-3, C-5\\\), 130.31 (1C, C-4\\\), 20.75 (1C, C-7\\\). HREIMS m/z: 357.1158 [M + H+] (Calcd for C22H16N2O3 356.3810).(11)(E)-2-((2-Fluorobenzylidene)amino)isoindoline-1,3-dione (H11): a white crystal product of 0.65 gram (Yield 79%); MP: 156–158°C. IR, cm−1: = 2934 (C–H of ring), 1717 (C=O), 1612 (C=N), 1510 (C=C), 1313 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.81 (m, 1H, H-4), 7.95 (m, 1H, H-5), 7.95 (m, 1H, H-6), 7.81 (m, 1H, H-7), 9.70 (s, 1H, H- of benzylidene carbon), 7.26 (t, 1H, J = 6, 6 Hz, H-3\\), 7.48 (m, 1H, H-4\\), 7.14 (t, 1H, J = 6, 6 Hz, H-5), 8.22 (m, 1H, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 164.95 (2C, C-1, C-3), 130.29 (2C, C-1\, C-3\), 123.87 (2C, C-4, C-7), 134.74 (2C, C-5, C-6), 151.85 (1C, C- benzylidene), 121.61 (1C, C-1\\), 161.45 (1C, C-2\\), 115.83 (1C, C-3\\), 127.28 (1C, C-4\\), 124.51 (1C, C-5\\), 133.31 (1C, C-6\\). HREIMS m/z: 269.0725 [M + H+] (Calcd for C15H9FN2O2 268.2474).(12)(E)-2-((3-Fluorobenzylidene)amino)isoindoline-1,3-dione (H12): a white crystal product of 0.58 gram (Yield 71%); MP: 147–149°C. IR, cm−1: = 3035 (C–H of ring), 1718 (C=O), 1609 (C=N), 1574 (C=C), 1305 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.73 (m, 1H, H-4), 7.86 (m, 1H, H-5), 7.86 (m, 1H, H-6), 7.73 (m, 1H, H-7), 9.37 (s, 1H, H- of benzylidene carbon), 7.59 (d, 1H, J = Hz, H-2\\), 7.11 (m, 1H, H-4\\), 7.35 (m, 1H, H-5\\), 7.54 (d, 1H, J = 6 Hz, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.01 (2C, C-1, C-3), 130.26 (2C, C-1\, C-3\), 123.88 (2C, C-4, C-7), 134.77 (2C, C-5, C-6), 156.49 (1C, C- benzylidene), 136.01 (1C, C-1\\), 114.24 (1C, C-2\\), 162.20 (1C, C-3\\), 118.52 (1C, C-4\\), 130.32 (1C, C-5\\), 124.64 (1C, C-6\\). HREIMS m/z: 269.0714 [M + H+] (Calcd for C15H9FN2O2 268.2474).(13)(E)-2-((4-Fluorobenzylidene)amino)isoindoline-1,3-dione (H13): a milky crystal product of 0.6 gram (Yield 73%); MP: 163–165°C. IR, cm−1: = 3037 (C–H of ring), 1716 (C=O), 1608 (C=N), 1507 (C=C), 1308 (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.94 (m, 1H, H-4), 7.94 (m, 1H, H-5), 7.94 (m, 1H, H-6), 7.94 (m, 1H, H-7), 9.40 (s, 1H, H- of benzylidene carbon), 7.80 (m,1H, H-2\\), 7.17 (m, 1H, H-3\\), 7.17 (m, 1H, H-5\\), 7.80 (m, 1H, H-6\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.74 (2C, C-1, C-3), 130.50 (2C, C-1\, C-3\), 123.81 (2C, C-4, C-7), 134.69 (2C, C-5, C-6), 157.15 (1C, C- benzylidene), 130.31 (1C, C-1\\), 130.44 (2C, C-2\\, C-6\\), 115.95 (2C, C-3\\, C-5\\), 164.07 (1C, C-4\\). HREIMS m/z: 269.0716 [M + H+] (Calcd for C15H9FN2O2 268.2474).(14)(E)-2-((3,5-Di-tert-butyl-2-methoxybenzylidene)amino)isoindole 1,3-dione (H14): a milky crystal product of 2.27 gram (Yield 94%); MP: 135–137°C. IR, cm−1: = 2952-2868 (C–H of ring, t-Bu and CH3), 1720 (C=O), 1608 (C=N), 1587 (C=C), 1307 (C–N), 1116 and 1083 (ether). 1H-NMR (600 MHz, CDCl3) ppm: 7.94 (m, 1H, H-4), 7.80 (m, 1H, H-5), 7.80 (m, 1H, H-6), 7.94 (m, 1H, H-7), 9.52 (s, 1H, H- of benzylidene carbon), 7.52 (d, 1H, H-4\\), 8.01 (d, 1H, H-6\\), 1.37 (s, 9H, t-butyl (C-8\\)), 1.45 (s, 9H, t-butyl (C-7\\)), 3.93 (s, 3H, -OCH3). 13C-NMR (150 MHz, CDCl3) ppm: 165.11 (2C, C-1, C-3), 130.50 (2C, C-1\, C-3\), 126.28 (2C, C-4, C-7), 134.54 (2C, C-5, C-6), 158.59 (1C, C- benzylidene), 122.36 (1C, C-1\\), 158.83 (1C, C-2\\), 142.26 (1C, C-3\\), 128.27 (1C, C-4\\), 146.23 (1C, C-5\\), 123.69 (1C, C-6\\), 35.25 (1C, C-7\\), 34.75 (1C, C-8\\), 64.66 (1C, -OCH), 31.44 (3C, t-butyl (C-7\\)), 30.92 (3C, t-butyl (C-8\\)). HREIMS m/z: 393.2089 [M + H+] (Calcd for C24H28N2O3 392.4990).(15)(E)-2-((4-Butoxybenzylidene)amino)isoindoline-1,3-dione (H15): a white powder product of 0.91 gram (Yield 89%); MP: 101–104°C. IR, cm−1: = 2953 and 2873 (C–H of ring and CH3), 1715 (C=O), 1641 (C=N), 1589 (C=C), 1313 (C – N), 1121 and 1083 (ether). 1H-NMR (600 MHz, CDCl3) ppm: 7.83 (m, 1H, H-4), 7.69 (m, 1H, H-5), 7.69 (m, 1H, H-6), 7.83 (m, 1H, H-7), 9.16 (s, 1H, H- of benzylidene carbon), 7.75 (d, 1H, J = 6 Hz, H-2\\), 6.87 (d, 1H, J = 12 Hz, H-3\\), 6.87 (d, 1H, J = 12 Hz, H-5\\), 7.75 (d, 1H, J = 6 Hz, H-6\\), 3.95 (t, 2H, J = 6, 6 Hz, H-7\\), 1.72 (m, 2H, H-8\\), 1.43 (m, 2H, H-9\\), 0.92 (t, 3H, J = 6, 12 Hz, H-10\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.22 (2C, C-1, C-3), 130.45 (2C, C-1\, C-3\), 123.65 (2C, C-4, C-7), 134.49 (2C, C-5, C-6), 159.21 (1C, C- benzylidene), 125.97 (1C, C-1\\), 130.26 (2C, C-2\\, C-6\\), 114.73 (2C, C-3\\, C-5\\), 162.24 (1C, C-4\\), 67.91 (1C, C-7\\), 31.20 (1C, C-8\\), 19.22 (1C, C-9\\), 13.84 (1C, C-10\\). HREIMS m/z: 323.1389 [M + H+] (Calcd for C19H18N2O3 322.3640).(16)(E)-2-((4-Isopropylbenzylidene)amino)isoindoline-1,3-dione (H16): a milky powder product of 0.38 gram (Yield 43%); MP: 99–101°C. IR, cm−1: = 2960-2876 (C–H of ring and CH3), 1717 (C=O), 1609 (C=N), 1598 (C=C), 1302. (C–N). 1H-NMR (600 MHz, CDCl3) ppm: 7.84 (m, 1H, H-4), 7.70 (m, 1H, H-5), 7.70 (m, 1H, H-6), 7.84 (m, 1H, H-7), 9.25 (s, 1H, H- of benzylidene carbon), 7.74 (d, 1H, J = 12 Hz, H-2\\), 7.24 (d, 1H, J = 6 Hz, H-3\\), 7.24 (d, 1H, J = 6 Hz, H-5\\), 7.74 (d,1H, J = 12 Hz, H-6\\), 2.89 (m, 1H, H-7\\), 1.20 (d, 3H, J = 6 Hz, H-8\\), 1.21 (d, 2H, J = 6 Hz, H-9\\). 13C-NMR (150 MHz, CDCl3) ppm: 165.15 (2C, C-1, C-3), 131.26 (2C, C-1\, C-3\), 123.71 (2C, C-4, C-7), 134.55 (2C, C-5, C-6), 159.12 (1C, C- benzylidene), 130.42 (1C, C-1\\), 128.59 (2C, C-2\\, C-6\\), 126.91 (2C, C-3\\, C-5\\), 153.17 (1C, C-4\\), 34.26 (1C, C-7\\), 23.76 (2C, C-8\\, C-9\\). HREIMS m/z: 293.1277 [M + H+] (Calcd for C18H16N2O2 292.3380).
5.2. Biological Activities
5.2.1. Antioxidant DPPH Free Radical Scavenging Assay
The DPPH radical scavenging assay was done according to the procedure followed in the literature . 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. The mixture was then incubated in the dark for 60 minutes at room temperature. Absorbance at 517 nm for each sample was then measured. The free radical scavenging activity of the compounds was calculated as a percentage of radical inhibition using the following equation: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. The concentration required to achieve 50% inhibition (IC50) of the DPPH radical was determined by plotting each compound’s percentage result and exact concentration.
5.2.2. Cell Culture
Human in vitro cell lines of colon cancer HT-29 (colon adenocarcinoma), breast cancer MCF-7 (breast adenocarcinoma), normal colon CCD841-Con, and normal breast cells MCF-10A were all obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cells were cultured in RPMI-1640 media (Sigma Aldrich, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% Pen-Strip antibiotic (10,000 units penicillin-10 mg streptomycin/mL, Sigma Aldrich, USA) in a 37°C, 5% CO2 incubator (Thermo Fisher Scientific, USA).
5.2.3. MTT Cytotoxicity Assay
Cell viability assay 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was conducted based on a previously modified protocol . 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% CO2 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% CO2, 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). The experiment was carried out in triplicate, and cellular viability was calculated according to
5.2.4. Selectivity Index (SI)
The degree of selectivity of chemicals to each cancer cell line is explored as SI ratio as per previous reports , according to the following equation:
A series of hybridized imines were synthesized combining the active phthalimide pharmacophore and various phenolic aldehydes with various functionalities. The 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. These 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.
The data used to support the findings of this study are available from the corresponding author upon request.
The 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
The authors declare no conflicts of interest.
Hayman Sardar Abdulrahman was responsible for data curation; Hayman Sardar Abdulrahman, Lina A. Al-Ani, and Mohd Hafiz Ahmad performed formal analysis; Wageeh A. Yehye was responsible for funding acquisition; Hayman Sardar Abdulrahman and Najihah M. Hashim investigated the study; Hayman Sardar Abdulrahman and Mohamed Hassan Mohammed were responsible for the methodology of the study; Mohd Hafiz Ahmad was involved in project administration; Mohamed Hassan Mohammed and Wageeh A. Yehye supervised the study; Hayman Sardar Abdulrahman and Mohamed Hassan Mohammed wrote the original draft; Lina A. Al-Ani, Wageeh A. Yehye, and Hayman Sardar Abdulrahman reviewed and edited the manuscript.
The authors would like to thank the University of Malaya, Malaysia, and Hawler Medical University, Erbil, Kurdistan Region, Iraq, for their cordial support in completing this work. This work was financially supported by the grant RP044C-17AET (UMRG) by the University of Malaya, Malaysia.
The following are available online at http://www.mdpi.com/xxx/s1. Figures 1.1–1.16: the full FT-IR spectra of the synthesized imines. Figures 2.1–2.16: the full 1H-NMR spectra of the synthesized imines. Figures 3.1–3.16: the full 13C-NMR spectra of the synthesized imines. Figure 4.1: DPPH free radical inhibition of the synthesized imines. Figure 5.1: colon cancer HT-29 cell line viability upon treatment with the synthesized imines. Figure 6.1: breast cancer MCF-7 cell line viability upon treatment with the synthesized imines. (Supplementary Materials)
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