Design and Biological Evaluation of Mannich-Modified 8-Hydroxyquinoline–Phthalimide Hybrids Against Drug-Resistant Cancer Cells

TL;DR

This study designs and tests new chemical compounds that show strong activity against drug-resistant cancer cells, particularly colorectal cancer.

Contribution

The paper introduces Mannich-modified 8-hydroxyquinoline–phthalimide hybrids with enhanced selectivity for multidrug-resistant cancer cells.

Findings

Compounds 2 and 4 showed the strongest cytotoxic activity with IC50 values as low as 4.88 µM.

Compounds 9c, 9a, and 8a exhibited the highest selectivity for MDR cells with RR values of 0.29, 0.33, and 0.35.

Mannich modification improved selectivity and some compounds inhibited P-glycoprotein ATPase activity.

Abstract

Background: 8-Hydroxyquinoline and phthalimide are two significant heterocyclic scaffolds in medicinal chemistry due to their pharmacological profiles. Hybridizing these pharmacophores and further modifying them via modified Mannich reactions provides a strategy to improve their physicochemical parameters and selectivity toward multidrug-resistant (MDR) cancer cells. Objectives: To synthesize a series of 8-hydroxyquinoline–phthalimide hybrids and their Mannich base derivatives and evaluate their cytotoxic activity and resistance-selective properties against sensitive Colo205 and resistant Colo320 cancer cell lines. Methods: Four hybrid compounds were synthesized by reacting 5-amino-8-hydroxyquinoline with different phthalic anhydride derivatives. Twelve fine-tuned derivatives were prepared by using the modified Mannich reaction. Cytotoxic activity was measured using the MTT assay, and relative resistance (RR) was calculated to determine selectivity toward the resistant cell line. P-glycoprotein (Pgp) ATPase activity was evaluated for the most active compounds. Results: All derivatives displayed cytotoxic activity, with higher potency toward the resistant Colo320 cell line. Compounds 2 and 4 showed the strongest activity against both cell lines (IC50 down to 4.88 µM). Compounds 5, 8a, 9a, and 9c retained potent activity against Colo320 (IC50 = 9.89–22.79 µM). Incorporating a CH2–N group at position C7 substantially enhanced the selectivity for MDR cells. Compounds 9c, 9a, and 8a exhibited the highest selectivity, with RR values of 0.29, 0.33, and 0.35, respectively. Compounds 2, 4, 5, 8a, and 9a showed inhibitory effects on Pgp ATPase activity. Conclusions: The newly synthesized HQ–phthalimide hybrids represent promising candidates for targeting MDR in colorectal cancer, with Mannich modification enhancing the selectivity toward resistant cells.

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Full text

1. Introduction

8-Hydroxyquinolines (8-HQs) have been considered to be valuable and privileged scaffolds in medicinal chemistry due to their versatile binding affinities, chelating abilities for various transition metal ions, multitargeted molecules, and drug-like properties [1,2]. In addition, an 8-HQ core is present in numerous biologically active compounds and several marketed drugs, such as clioquinol, broxyquinoline, nitroxoline, and iodoquinol [3]. Additionally, hydroxyquinoline derivatives exhibit a wide range of pharmacological and biological effects, including anticancer [4,5], anti-inflammatory [6], antioxidant [7], antibacterial [8], antimalarial [9,10], antiviral [11], and antifungal [12]. Furthermore, the anticancer activity of 8-HQ derivatives is related to their ability to chelate endogenous transition metals, particularly copper and iron. Under normal physiological conditions, these metal ions are regulated and maintained at low intracellular concentrations. However, in cancer cells—especially multidrug-resistant (MDR) phenotypes—metal homeostasis is dysregulated due to enhanced proliferation and increased metabolic demands, leading to elevated levels of iron and copper. As a result, 8-HQ derivatives can interfere with intracellular metal homeostasis, either through the deprivation of essential metal ions or by shuttling the excess of metal ions into cellular organelles such as mitochondria [13,14,15,16]. In addition, the interaction of 8-HQ derivatives with copper or iron leads to the formation of redox-active metal complexes capable of undergoing redox cycling, thereby generating cytotoxic reactive oxygen species (ROS). These ROS can induce oxidative damage to cellular lipids, proteins, DNA, and other essential biomolecules, ultimately promoting cancer cell death [5,17]. Similarly, the MDR-selective anticancer activity observed for 8-HQ derivatives is likely associated with these combined effects on iron homeostasis and copper-mediated oxidative stress [13,18]. Moreover, numerous studies have proposed alternative mechanisms whereby 8-HQ derivatives inhibit key metalloenzymes, including iron-dependent ribonucleotide reductases involved in DNA synthesis and matrix metalloproteinases associated with cancer invasion and metastasis. Additional potential metalloenzyme targets include cytosolic and nuclear oxygenases, as well as histone deacetylases, further contributing to the anticancer activity of these compounds [1,19,20].

The Mannich reaction is one of the most applied chemical reactions in medicinal chemistry, facilitating the synthesis of novel chemical compounds or the optimization of the physicochemical parameters of drug candidates [21]. 8-HQ has been reported as a promising substrate for the Mannich reaction, with reports indicating that Mannich bases derived from 8-HQ exhibit significant potency against human cancer cells [1,22]. The literature indicates that the biological activity and physicochemical properties of 8-HQ derivatives can be fine-tuned through the incorporation of Mannich bases [14,23]. A major limitation of many HQ derivatives is their poor water solubility, an unfavorable pharmaceutical property [24]. In contrast, 8-HQ-derived Mannich bases have been reported to exhibit significantly improved water solubility due to their zwitterionic nature, and introducing a CH_2_–N moiety at the C7 position enhances their cytotoxicity and selectivity against multidrug-resistant cells [13,19]. For example, compound I showed promising anticancer activity against a broad range of cancer cell lines (e.g., U251, MCF-7) [25]. Moreover, our research group has reported different HQ–Mannich derivatives with potent antiproliferative activity, improved physicochemical parameters, and selectivity toward MDR cells. Notably, compound II showed moderate activity against the sensitive Colo205 cell line and was more potent against the resistant cell line Colo320 [24]. Additionally, compound III displayed potent cytotoxicity against both the Colo205 and Colo320 cell lines, and its hydrolyzed product, compound IV, which contained an amino acid moiety, also exhibited promising cytotoxic activity against both cell lines (Figure 1) [26,27].

Phthalimide, a significant heterocyclic compound, exhibits diverse biological activity, including antioxidant [28], anticonvulsant [29,30], anti-inflammatory [31], antimycobacterial [32,33], analgesic [34,35], hypoglycemic [36,37], and anticancer properties [38,39,40]. Additionally, three phthalimide derivatives—thalidomide, lenalidomide, and pomalidomide—have been approved by the FDA for cancer treatment, highlighting the significance of the phthalimide scaffold in cancer therapy [41]. Moreover, the anticancer activity of phthalimide derivatives is associated with multiple mechanisms of action, such as the induction of tumor necrosis factor-alpha (TNF-α) synthesis; the inhibition of angiogenesis, tryptase, and HDACs; and proteolysis-targeting chimera (PROTAC) modulation [41,42,43,44]. For example, compound V exhibited potent antiproliferative activity against a broad range of cancer cell lines (e.g., HepG-2, HCT-116) [39]. Similarly, compound VI demonstrated significant anticancer activity against the MV4-11, A549, and MCF-7 cell lines [45]. Furthermore, compound VII showed promising activity against colon cancer cell line HCT-116 (Figure 1) [46].

Recently, hybridization has emerged as a prominent strategy in cancer therapy, involving the covalent fusion of two or more pharmacophoric moieties into a single chemical structure to achieve synergistically enhanced anticancer efficacy [47,48]. Such hybrid molecules are designed to exhibit broader biological activity and improved potency compared to their parent compounds, overcome drug resistance, and minimize toxicity [49,50,51]. Therefore, this study aimed to develop hybrid molecules by combining phthalimide and 8-HQ pharmacophores and incorporating Mannich bases to enhance the biological activity and fine-tune the pharmacological and physicochemical properties of the targeted compounds (Figure 1). The synthesized hybrids were characterized using spectroscopic techniques such as ^1^H NMR, ^13^C NMR, and HRMS. Finally, their anticancer activity was evaluated against the sensitive Colo205 and resistant Colo320 colon adenocarcinoma cell lines, along with an assessment of P-glycoprotein (Pgp) ATPase activity.

2. Results and Discussion

2.1. Synthesis

The synthesis of HQ–phthalimide hybrids 2–5 started with the reaction of 5-amino-8-hydroxy quinoline (1) with different phthalic anhydride derivatives in an acetic acid solvent under reflux conditions (Scheme 1). Additionally, to fine-tune the physicochemical and pharmacological parameters of these hybrids, the modified Mannich reaction was subsequently performed using paraformaldehyde and different secondary amines, applied either as free bases or acetate salts, in 1,4-dioxane at 80 °C (Scheme 2 and Scheme 3). Specifically, the reaction of compound 1 with the unsubstituted, nitro-, and methoxy- substituted phthalic anhydrides was carried out in acetic acid at 120 °C, and the progress of the reaction was monitored by TLC. After 4–6 h, the products were obtained in good yields of 83–90% by crystallization from diethyl ether, and these intermediates were then used as substrates in the modified Mannich reaction (Scheme 1).

Regarding the regular unsubstituted imide 2, the modified Mannich reaction with morpholine 6c was conducted using both the free base and its acetate salt (Scheme 2). When morpholine acetate was employed, the reaction proceeded faster (2 h) compared to using the free base (4 h), although the latter afforded only a slightly higher yield (Table 1). On the other hand, when stronger bases such as piperidine and N-methyl piperazine were used as free bases, the reaction yielded complex reaction mixtures and the targeted products 6a and 6b could not be isolated because the components displayed substantially close Rf values in TLC (Scheme 2, Table 1). We suggest that the strong basicity of piperidine and N-methyl piperazine facilitates nucleophilic attack on the carbonyl groups of the phthalimide ring, causing ring opening simultaneously with the formation of the Mannich product. This assumption was supported by crude MS and crude ^1^H-NMR analyses, which revealed traces of amidic N-H proton and carboxylic proton signals. However, this aspect requires further detailed investigation in future studies. Notably, when using the acetate form of piperidine and N-methyl piperazine, the reactions were progressive and yielded our targeted products 6a and 6b in short reaction times with good yields (2 h, 73% for 6a; 3 h, 88% for 6b). Based on the previous data, using secondary amines as acetate salts proved to be more effective than using free bases, as the mild acidity catalyzes iminium ion formation, stabilizes intermediates, and suppresses side reactions in the modified Mannich reaction [26].

Furthermore, the targeted compounds 7a–c, 8a–c, and 9a–c were prepared via the modified Mannich reactions of nitro- and methoxy-substituted imides 3–5, respectively (Scheme 3). For the morpholine derivatives (7c, 8c, and 9c), the reactions were performed using the free base or the acetate salt. In both cases, the isolated yields were comparable; however, the reactions proceeded faster when the acetate salts were employed. Regarding the piperidine and N-methyl piperazine derivatives, when using the free base forms of the amine, the reactions produced complex reaction mixtures that subdued the isolation of our desired products (Table 1). On the other hand, when using the acetate salts of the secondary amines, the reactions were completed within significantly shorter times, affording the target compounds with moderate to good yields, ranging from 55 to 75% (Table 1). All of the above confirmed the acetate form of the base to be more effective than the free base, in terms of both reaction progress and completion time. Finally, all synthesized compounds were identified by ^1^H-NMR and ^13^C-NMR and further confirmed by HRMS.

The aforementioned observations were supported by preliminary DFT calculations with the B3LYP-D3 ma-def2-SVP level of theory in the gas phase. For compounds 2–5, the visualization of the LUMO lobes (Figure 2) and calculation of the corresponding Fukui indices and electrophilicity indices were conducted (Table 2). It could be deduced that, due to the significantly high f(+) values of the phthalimide carbonyls and the spatial arrangement of the LUMO lobes, these sites are substantially prone to nucleophilic attack. By calculating the local electrophilicity (ω_k_) (via both the ΔSCF method and Koopmans’ theorem as a point of view), theoretical data aligning with our experiments were acquired, as compound 3 could be considered the most susceptible compound to nucleophilic attack under the reaction conditions [52,53,54,55,56,57,58].

2.2. Biological Evaluations

2.2.1. In Vitro Antiproliferative Activity

The cytotoxic activity of the newly synthesized derivatives was evaluated in vitro against the sensitive Colo205 and resistant Colo320 cell lines using the MTT assay. Doxorubicin (DOXO) was applied as a positive control, while DMSO was used as the solvent control (Table 3).

Based on the obtained results, the compounds exhibited potent to moderate cytotoxic activity against the sensitive Colo205 and resistant Colo320 cell lines, with IC_50_ values ranging from 7.22 (±1.22) µM to 64.79 (±1.92) µM for the Colo205 cell line and from 4.88 (±1.27) µM to 60.86 (±3.17) µM for the Colo320 cell line. Interestingly, compounds 2 and 4 showed potent cytotoxic activity against both cell lines that was comparable to that of doxorubicin, with IC_50_ values of 7.22 (±1.22) and 4.88 (±1.27) µM for compound 2 and 12.58 (±1.52) and 10.28 (±0.18) µM for compound 4 in Colo205 and Colo320, respectively. Additionally, compounds 5, 8a, 9a, and 9c demonstrated fair to moderate activity against the sensitive Colo205 cell line, with IC_50_ values of 22.03 (±0.96), 64.79 (±1.92), 49.93 (±4.85), and 39.52 (±2.76) µM, respectively. However, these compounds showed significantly higher potency against the resistant Colo320 cell line, with IC_50_ values of 9.89 (±1.68), 22.79 (±3.35), 16.39 (±3.31), and 11.5 (±1.42) µM, respectively. Compounds 6b, 6c, 7a, 8c, and 9b showed no detectable cytotoxicity in Colo205 cells but exerted fair to moderate activity against the resistant cell line. Conversely, compounds 7b, 7c, and 8b showed no anticancer activity against any of the measured cell lines. Representative dose–response curves in Colo320 cells for compounds 4, 5, 8a, and 9a are shown in the Supplementary Data (Figure S59). Data are presented as the mean ± SD (n = 3).

Relative resistance (RR) values were calculated as the ratio of the IC_50_ of each compound in resistant cells to that in the corresponding sensitive cells. Compounds with RR < 1 are considered selective toward resistant cells, whereas RR ≤ 0.5 means that compounds are highly selective toward resistant cells [59]. The results showed that all synthesized derivatives were selective toward the resistant Colo320 tumor cells, exhibiting RR values between 0.29 and 0.82, which were markedly superior to those of the reference drug, doxorubicin (RR = 2.43). Although the 8-hydroxyquinoline–phthalimide hybrids displayed notable activity, with RR values ranging from 0.45 to 0.82, the aminoalkylation of these hybrids (formation of Mannich bases) significantly enhanced their selectivity to the resistant cancer cells. For example, compound 4 exhibited an RR value of 0.82, while its Mannich base analog 8a showed substantially improved selectivity with an RR value of 0.35. Similarly, the Mannich base derivatives 9a and 9c of hybrid 5 exhibited significantly decreased RR values, from 0.45 (compound 5) to 0.33 and 0.29, respectively. Overall, the tested compounds demonstrated higher efficacy and selectivity against the resistant Colo320 cell line. Moreover, the data confirm that introducing a CH_2_–N moiety at the seventh position of the hybrid scaffold enhances the selectivity toward multidrug-resistant cells. Among the tested derivatives, Mannich bases 9c, 9a, and 8a displayed the highest relative potency against the MDR Colo320 cell line, with RR values of 0.29, 0.33, and 0.35, respectively. The main difference between the sensitive Colo205 and the resistant Colo320 cell lines is the expression of the ABCB1 (P-glycoprotein) multidrug efflux pump. It can be hypothesized that these potent derivatives may act directly or indirectly on the ABCB1 transporter, rendering the cancer cells more vulnerable to chemotherapeutic agents; however, further experiments are required to investigate the modes of action of the derivatives on ABCB1.

2.2.2. Pgp ATPase Activity

Compounds 2, 4, 5, 8a, 9a, and 9c, which showed pronounced antiproliferative activity and selectivity toward the MDR adenocarcinoma cell line Colo320, with favorable relative resistance (RR) values, were further evaluated for their interactions with P-glycoprotein (Pgp) by assessing the ATPase activity. The Pgp ATPase activity was measured using the Pgp-Glo^TM^ assay to determine whether the selected compounds acted as inhibitors or stimulators of Pgp. The basal Pgp ATPase activity (ΔRLU_basal_) was calculated as the difference between the luminescent signals of sodium orthovanadate-treated samples (RLU_Na3VO4_) and untreated samples (RLU_NT_), where sodium vanadate serves as a selective and potent Pgp inhibitor (Figure 3). The tested compounds could be classified as Pgp inhibitors or stimulators by comparing their ΔRLU values (ΔRLU_TC_) with the basal Pgp ATPase activity (ΔRLU_basal_). Verapamil (200 µM), a known Pgp substrate and ATPase stimulator, was used as a stimulator/substrate control in this assay. As shown in Figure 3, at the tested concentration, compounds 2, 4, 5, 8a, and 9a exhibited ΔRLU values lower than the basal level, demonstrating that these compounds are potential inhibitors of Pgp ATPase activity. In contrast, compound 9c displayed a ΔRLU value comparable to the basal level, suggesting that it has no effect on Pgp ATPase activity. Overall, the Pgp interaction profiles of the tested compounds were largely comparable, with no marked differences in activity among the most active compounds, and correlated well with their IC_50_ and RR values.

2.2.3. Structure–Activity Relationship (SAR)

Based on the aforementioned results regarding the antiproliferative activity of the targeted compounds, it was found that the molecular hybridization of the 8-HQ scaffold with the phthalimide fragment afforded a library of compounds with notable anticancer activity. Among the HQ–phthalimide hybrids, the unsubstituted phthalimide derivative (compound 2) exhibited the highest cytotoxicity against both the Colo205 and multidrug-resistant Colo320 cell lines.

The introduction of substituents at the phthalimide ring substantially influenced the antiproliferative activity. Substitution at position C4 with the representative electron-withdrawing nitro group (compound 3) resulted in an approximately 10-fold decrease in cytotoxic activity, while the relative resistance (RR) value remained unchanged. On the other hand, substitution with the electron-donating methoxy group at either the C4 or C5 position (compounds 4 and 5, respectively) caused only a 2–3-fold reduction in potency, with the compounds still retaining good antiproliferative activity (IC_50_ = 9.89–22.03 µM) on both cell lines. Interestingly, the substitution pattern exerted a pronounced effect on MDR selectivity. While the methoxy substitution at C4 (compound 4) slightly increased the RR value (1.2-fold), methoxy substitution at C5 (compound 5) showed a 1.5-fold decrease in the RR value, indicating enhanced selectivity toward the resistant Colo320 cell line (RR = 0.45).

Consistent with the previous literature on 8-hydroxyquinoline derivatives, unsubstituted or small halogen substituents such as chloro or nitro groups at the 5-position are generally considered optimal for anticancer activity [14,19,26,27]. Notably, our results demonstrate that the replacement of these small substituents with a bulkier phthalimide moiety is well tolerated and does not abolish the antiproliferative activity. This finding suggests that the 5-position of the 8-HQ scaffold can accommodate larger substituents and benefit from pharmacophore hybridization, potentially leading to synergistic biological effects.

The transformation of the HQ–phthalimide hybrids to their corresponding Mannich bases generally showed a reduction in antiproliferative activity. For instance, Mannich derivatives of the nitro-substituted hybrid (7a–c) were inactive on both cell lines. For hybrids 2, 4, and 5, the incorporation of the N-methylpiperazine fragment, containing two basic nitrogen atoms, led to a marked loss of activity against both cell lines. However, the incorporation of piperidine or morpholine moieties caused moderate increases in the IC_50_ values on both cell lines, reaching 5–7-fold (6a,c), 2.5–5-fold (8a,c), and 1.2–2-fold (9a,c) compared to their parent hybrids.

In spite of the reduced cytotoxic activity, the incorporation of the aminoalkyl moiety (Mannich modification) is a valuable tool to further fine-tune the MDR selectivity. Compounds 8a, 9a, and 9c exhibited significantly decreased RR values relative to their corresponding hybrids, with 8a showing a 2.5-fold reduction and 9a and 9c showing approximately 1.5-fold reductions in their RR values, highlighting their improved selectivity toward the resistant Colo320 cell line.

2.3. Prediction of Physicochemical and Pharmacokinetic Properties

The physicochemical properties and ADME profiles of the most active HQ–phthalimide hybrids (2, 4, and 5) and their Mannich base derivatives (8a, 9a, and 9c) were evaluated using the SwissADME web tool, pkCSM platform, and Chemicalize (ChemAxon, Budapest, Hungary). All tested compounds had a molecular weight (MW) below 500 g/mol and complied with Lipinski’s rule of five, with fewer than 10 hydrogen bond acceptors (HBAs), fewer than five hydrogen bond donors (HBDs), and fewer than 10 rotatable bonds. The determined topological polar surface area (TPSA), reflecting the contribution of polar atoms (N and O), ranged from 70.50 Å^2^ to 92.20 Å^2^, which is compatible with favorable oral bioavailability. Moreover, all derivatives showed predicted lipophilicity (iLogP) values lower than 5. In addition, the estimated water solubility was moderate for HQ–phthalimide hybrids 2, 4, 5, while the Mannich derivatives 8a, 9a, and 9c had high solubility, indicating that Mannich modification largely improved the aqueous solubility. Furthermore, all compounds displayed high predicted bioavailability scores (F > zero) (Table 4). Moreover, all evaluated derivatives exhibited high predicted intestinal absorption, with absorbed fractions ranging from 94.13% to 97.27%. Caco-2 permeability, used to model paracellular transport, indicated high permeability for compounds 2, 4, 5, 8a, and 9a (values > 0.90), suggesting efficient transcellular passage, whereas compound 9c showed moderate permeability (0.638) (Table 4). Importantly, all tested derivatives satisfied multiple drug-likeness criteria, including Lipinski, Ghose, Veber, Egan, and Muegge filters. In conclusion, these results suggest that the newly synthesized HQ–phthalimide hybrids possess favorable pharmacokinetic properties and promising drug-likeness characteristics.

The predicted pK_a_ values of the investigated compounds showed multiple ionizable sites, as presented in Table 4. For the HQ–phthalimide hybrids 2, 4, and 5, two pK_a_ values were observed, corresponding to the phenolic OH group (pK_a_ = 8.91) and the quinoline nitrogen (pK_a_ = 3.83–3.84). In contrast, the Mannich base derivatives 8a, 9a, and 9c exhibited three ionization constants, including the phenolic OH group (pK_a_ = 7.52–8.18), the quinoline nitrogen (pK_a_ = 2.24–2.25), and the tertiary amine of the Mannich moiety (pK_a_ = 6–80–9.62), reflecting their increased basicity and cationic character. In addition, the BOILED-Egg model (WLOGP vs. TPSA) for compounds 2, 4, 5, 8a, 9a, and 9c predicted high gastrointestinal (GI) absorption, with all compounds located within the human intestinal absorption (HIA) zone [60]. None of the evaluated compounds were predicted to penetrate the blood–brain barrier (BBB), except compound 2, which showed BBB permeability (Figure 4). Additionally, the model showed that compounds 8a, 9a, and 9c are predicted P-glycoprotein substrates (PGP+), whereas compounds 2, 4, and 5 are predicted non-P-glycoprotein substrates (PGP−). The latter feature suggests reduced susceptibility to active efflux mechanisms, which may have contributed to the enhanced antiproliferative activity observed for these compounds against the resistant Colo320 cell line.

Moreover, compounds 2, 4, 5, 8a, 9a, and 9c were predicted to possess high GI absorption, consistent with their desirable physicochemical properties, which fall within the optimal range for oral bioavailability. The bioavailability radar maps of the evaluated compounds are shown in Figure 5 and further support these findings. The radar maps comprise six axes, representing six crucial features for oral bioavailability: saturation (INSATU), flexibility (FLEX), lipophilicity (LIPO), size (SIZE), polarity (POLAR), and solubility (INSOLU). The pink region represents the range of ideal property values, and a red line indicates the compound’s expected attributes. While the parent HQ–phthalimide hybrids (2, 4, and 5) showed partial deviation from the optimal pink region, the Mannich-modified derivatives (8a, 9a, and 9c) fully fit within the ideal physicochemical space, indicating improved predicted oral bioavailability.

In order to gain further insights into the electronic descriptors in regard to biological activity, preliminary DFT calculations were conducted. Visualization of the electrostatic potential maps (EPM) and HOMO, HOMO-1, and LUMO lobes of compounds 2–5, 6a, 8a, 9a, and 9c was conducted. A comparison of the electrostatic potential maps shed light on the crucial electronic differences in regard to the phthalimide moieties of compounds 2–5 (Figure 6).

It can be observed that, regarding compound 3, the phthalimide ring has the lowest electron density among compounds 2–5; it can be also observed that the methoxy substituent at position 5 (compound 5) has a lower electron density than that of compound 4 at position 4. Additionally, regarding Mannich bases, it is apparent that the spatial extent of the HOMO lobes is localized around the aminoalkyl moiety, and the distribution of the HOMO-1 lobes is localized around the hydroxyquinoline scaffold (Figure 7). However, in the case of 9a and 9c, the HOMO encompasses the core as well. In correspondence to the Pgp ATPase activity assessments, it can be hypothesized that the strong negative charge distribution of the morpholine ring in compound 9c decreased the Pgp ATPase activity compared to 9a, bearing a more neutral aminoalkyl moiety.

3. Materials and Methods

3.1. Synthesis

Melting points were determined on a Hinotek X-4 melting point apparatus (Ningbo, China). Merck Kieselgel 60 F254 plates (Merck KGaA, Darmstadt, Germany) were applied for TLC.

^1^H and ^13^C NMR spectra were recorded in DMSO-d_6_ or CDCl_3_ solutions in 5 mm tubes at room temperature (RT) on a Bruker DRX-500 spectrometer (Bruker Biospin, Karlsruhe, Germany) at 500 (^1^H) and 125 (^13^C) MHz, with the deuterium signal of the solvent as the lock and TMS as an internal standard (^1^H, ^13^C).

The HRMS flow injection analysis was performed with a Thermo Scientific Orbitrap Q-Exactive Plus Hybrid Quadrupole–Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer coupled to a Waters Acquity I-Class UPLC™ (Waters, Manchester, UK).

3.1.1. General Procedure for the Synthesis of Compounds 2–5

Equimolar amounts of 5-amino-8-hydroxyquinoline 1 (1 mmol, 160 mg) and the appropriate phthalic anhydride derivative (1 mmol) were dissolved in 10 mL glacial acetic acid in a round-bottom flask. The reaction mixture was refluxed (120 °C), stirred in an oil bath for 4–6 h, and monitored by TLC. After the reaction was completed, the solvent was removed under reduced pressure and the product was crystallized from diethyl ether.

2-(8-Hydroxyquinolin-5-yl)isoindoline-1,3-dione (2)

Brown powder (245 mg, 85% yield); m.p. 265–268 °C; ^1^H NMR (DMSO-d6) δ 10.29 (s, 1H), 8.97 (d, J = 3.9 Hz, 1H), 8.27 (d, J = 8.6 Hz, 1H), 8.06–8.03 (m, 2H), 8.01–7.97 (m, 2H), 7.69–7.51 (m, 2H), 7.25 (d, J = 8.0 Hz, 1H); ^13^C NMR (DMSO-d6) δ 168.35, 154.91, 149.07, 138.85, 135.13, 132.52, 132.39, 129.47, 126.86, 123.97, 123.01, 118.86, 111.14; HRMS calcd for [M + H]^+^ m/z = 291.07642, found m/z = 291.07629.

2-(8-Hydroxyquinolin-5-yl)-4-nitroisoindoline-1,3-dione (3)

Dark brown powder (278 mg, 83% yield); m.p. 281–284 °C; ^1^H NMR (DMSO-d6) δ 10.29 (s, 1H), 8.92 (d, J = 3.4 Hz, 1H), 8.45–8.33 (m, 2H), 8.28 (d, J = 7.4 Hz, 1H), 8.15 (t, J = 7.8 Hz, 1H), 7.64–7.53 (m, 2H), 7.21 (d, J = 8.2 Hz, 1H). ^13^C NMR (DMSO-d6) δ 166.50, 163.87, 155.15, 149.13, 145.02, 138.79, 136.68, 134.43, 132.67, 129.51, 128.78, 127.55, 126.88, 123.94, 122.98, 118.36, 111.16; HRMS calcd for [M + H]^+^ m/z = 336.06150, found m/z = 336.06113.

2-(8-Hydroxyquinolin-5-yl)-4-methoxyisoindoline-1,3-dione (4)

Brown powder (288 mg, 90% yield); m.p. 250–253 °C; ^1^H NMR (DMSO-d6) δ 10.26 (s, 1H), 8.92 (d, J = 3.1 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.90 (t, J = 7.9 Hz, 1H), 7.60–7.52 (m, 4H), 7.20 (d, J = 8.1 Hz, 1H), 4.00 (s, 3H). ^13^C NMR (DMSO-d6) δ 167.96, 166.49, 157.08, 154.83, 149.04, 138.86, 137.38, 134.29, 132.46, 129.51, 126.91, 122.99, 119.23, 118.95, 117.30, 115.86, 111.13, 56.77; HRMS calcd for [M + H]^+^ m/z = 321.08698, found m/z = 321.08676.

2-(8-Hydroxyquinolin-5-yl)-5-methoxyisoindoline-1,3-dione (5)

Black powder (288 mg, 90% yield); m.p. 242–247 °C; ^1^H NMR (DMSO-d6) δ 10.29 (s, 1H), 8.92 (d, J = 3.8 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.62–7.47 (m, 3H), 7.42 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 3.98 (s, 3H). ^13^C NMR (DMSO-d6) δ 167.99, 167.96, 165.11, 154.86, 149.05, 138.86, 135.00, 132.42, 129.46, 126.85, 125.90, 124.14, 123.01, 120.67, 118.97, 111.13, 109.01, 56.89; HRMS calcd for [M + H]^+^ m/z = 321.08698, found m/z = 321.08701.

3.1.2. General Procedure for the Synthesis of Compounds 6a–c, 7a–c, 8a–c, and 9a–c

A mixture of HQ–phthalimide hybrids 2–5 (0.1 mmol), paraformaldehyde (0.12 mmol, 4 mg), and the appropriate amine [piperidine acetate (0.12 mmol, 17.4 mg) for 6a–9a, N-methylpiperazine acetate (0.12 mmol, 19.5 mg) for 6b–9b, or morpholine (0.12 mmol, 10.3 μL)/morpholine acetate (0.12 mmol, 18 mg) for 6c–9c] was dissolved in 2 mL of 1,4-dioxane in a sealed microwave tube. The reaction mixture was stirred in an oil bath at 80 °C for 2–3 h (for acetate salts) or 4–7 h (for free morpholine) and monitored by TLC. After the reaction was complete, the solvent was removed under reduced pressure, and the product was crystallized from diethyl ether.

2-(8-Hydroxy-7-(piperidin-1-ylmethyl)quinolin-5-yl)isoindoline-1,3-dione (6a)

Dark brown powder (28 mg, 73% yield); m.p. 209–213 °C; ^1^H NMR (DMSO-d6) δ 8.89 (d, J = 3.9 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.03–7.97 (m, 2H), 7.97–7.92 (m, 2H), 7.56 (s, 1H), 7.51 (dd, J = 8.5, 4.0 Hz, 1H), 3.77 (s, 2H), 2.5 (m, 4H, under solvent signal), 1.57–1.51 (m, 4H), 1.45–1.40 (m, 2H). ^13^C NMR (DMSO-d6) δ 168.35, 153.38, 149.10, 138.75, 135.11, 132.39, 132.30, 130.17, 125.72, 123.94, 122.53, 119.80, 118.17, 57.94, 54.20, 25.92, 24.20; HRMS calcd for [M + H]^+^ m/z = 388.16557, found m/z = 388.16544.

2-(8-Hydroxy-7-((4-methylpiperazin-1-yl)methyl)quinolin-5-yl)isoindoline-1,3-dione (6b)

Brown powder (36 mg, 88% yield); m.p. 255–259 °C; ^1^H NMR (CDCl_3_) δ 8.94–8.89 (m, 1H), 8.02–7.96 (m, 2H), 7.87–7.82 (m, 3H), 7.39 (d, J = 4.4 Hz, 1H), 7.26 (s, 1H, under solvent signal), 3.93 (s, 2H), 2.93–2.43 (m, 8H), 2.33 (s, 3H); ^13^C NMR (CDCl_3_) δ 168.00, 154.57, 149.22, 139.51, 134.60, 131.88, 130.91, 128.56, 125.67, 123.95, 122.01, 117.72, 117.35, 59.90, 54.87, 52.69, 45.84; HRMS calcd for [M + H]^+^ m/z = 403.17647, found m/z = 403.17615.

2-(8-Hydroxy-7-(morpholinomethyl)quinolin-5-yl)isoindoline-1,3-dione (6c)

Brown powder (33 mg, 85% yield); m.p. 226–229 °C; ^1^H NMR (DMSO-d6) δ 8.91 (d, J = 3.4 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.03–7.96 (m, 2H), 7.98–7.90 (m, 2H), 7.63 (s, 1H), 7.54 (dd, J = 8.3, 3.9 Hz, 1H), 3.76 (s, 2H), 3.63–3.57 (m, 4H), 2.5 (m, 4H, under solvent signal); ^13^C NMR (DMSO-d6) δ 168.34, 152.75, 149.07, 138.61, 135.10, 132.49, 132.41, 130.56, 125.69, 123.94, 122.66, 119.65, 118.33, 66.63, 56.71, 53.64; HRMS calcd for [M + H]^+^ m/z = 390.14483, found m/z = 390.14471.

2-(8-Hydroxy-7-(piperidin-1-ylmethyl)quinolin-5-yl)-4-nitroisoindoline-1,3-dione (7a)

Brown powder (25 mg, 55% yield); m.p. 238–240 °C (decomp.); ^1^H NMR (DMSO-d6) δ 8.90 (d, J = 3.5 Hz, 1H), 8.36 (d, J = 8.2 Hz, 2H), 8.27 (d, J = 7.4 Hz, 1H), 8.14 (t, J = 7.8 Hz, 1H), 7.62 (s, 1H), 7.54 (dd, J = 8.5, 4.1 Hz, 1H), 3.78 (s, 2H), 2.5 (m, 4H, under solvent signal), 1.58–1.52 (m, 4H), 1.45–1.40 (m, 2H); ^13^C NMR (DMSO-d6) δ 166.53, 163.90, 153.59, 149.18, 145.00, 138.65, 136.64, 134.45, 132.52, 130.31, 128.75, 127.51, 125.78, 123.98, 122.56, 119.67, 117.72, 57.73, 54.19, 25.82, 24.12; HRMS calcd for [M + H]^+^ m/z = 433.15065, found m/z = 433.14487.

2-(8-Hydroxy-7-((4-methylpiperazin-1-yl)methyl)quinolin-5-yl)-4-nitroisoindoline-1,3-dione (7b)

Brown powder (33 mg, 75% yield); m.p. 267–270 °C (decomp.); ^1^H NMR (CDCl_3_) δ 8.93 (d, J = 3.5 Hz, 1H), 8.26 (d, J = 7.4 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 8.03 (t, J = 7.7 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.42 (dd, J = 8.4, 3.9 Hz, 1H), 7.26 (s, 1H, under solvent signal), 3.92 (s, 2H), 2.81–2.46 (m, 8H), 2.33 (s, 3H); ^13^C NMR (CDCl_3_) δ 165.48, 162.52, 155.01, 149.35, 145.52, 139.45, 135.92, 133.82, 130.58, 129.08, 128.62, 127.56, 125.36, 123.54, 122.21, 117.39, 116.81, 59.82, 54.85, 52.67, 45.83; HRMS calcd for [M + H]^+^ m/z = 448.16155, found m/z = 448.16162.

2-(8-Hydroxy-7-(morpholinomethyl)quinolin-5-yl)-4-nitroisoindoline-1,3-dione (7c)

Brown powder (30 mg, 70% yield); m.p. 219–222 °C; ^1^H NMR (DMSO-d6) δ 8.92 (d, J = 3.6 Hz, 1H), 8.37 (t, J = 9.0 Hz, 2H), 8.27 (d, J = 7.4 Hz, 1H), 8.14 (t, J = 7.7 Hz, 1H), 7.68 (s, 1H), 7.56 (dd, J = 8.4, 3.8 Hz, 1H), 3.75 (s, 2H), 3.62–3.58 (m, 4H), 2.5 (m, 4H, under solvent signal); ^13^C NMR (DMSO-d6) δ 166.52, 163.89, 152.98, 149.14, 145.00, 138.52, 136.63, 134.47, 132.66, 130.65, 128.74, 127.50, 125.73, 124.00 (s), 122.67, 119.62, 117.86, 66.59, 56.62, 53.64; HRMS calcd for [M + H]^+^ m/z = 435.12991, found m/z = 435.12964.

2-(8-Hydroxy-7-(piperidin-1-ylmethyl)quinolin-5-yl)-4-methoxyisoindoline-1,3-dione (8a)

Brown powder (24 mg, 57% yield); m.p. 209–213 °C; ^1^H NMR (DMSO-d6) δ 8.89 (d, J = 3.6 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.54–7.49 (m, 3H), 4.00 (s, 3H), 3.75 (s, 2H), 2.5 (m, 4H, under solvent signal), 1.56–1.52 (m, 4H), 1.45–1.39 (m, 2H); ^13^C NMR (DMSO-d6) δ 167.98, 166.52, 157.06, 153.27, 149.07, 138.74, 137.37, 134.29, 132.25, 130.20, 125.75, 122.50, 119.87, 119.22, 118.22, 117.29, 115.83, 57.96, 56.77, 54.21, 25.96, 24.22; HRMS calcd for [M + H]^+^ m/z = 418.17613, found m/z = 418.17604.

2-(8-Hydroxy-7-((4-methylpiperazin-1-yl)methyl)quinolin-5-yl)-4-methoxyisoindoline-1,3-dione (8b)

Brown powder (25 mg, 60% yield); m.p. 221–225 °C; ^1^H NMR (CDCl_3_) δ 8.90 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.37 (dd, J = 8.2, 3.7 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 4.07 (s, 3H), 3.91 (s, 2H), 2.89–2.37 (m, 8H), 2.32 (s, 3H); ^13^C NMR (CDCl_3_) δ 167.73, 166.51, 157.19, 154.40, 149.13, 139.49, 136.70, 134.00, 131.09, 128.63, 125.77, 121.90, 117.96, 117.85, 117.37, 117.19, 116.02, 59.92, 56.46, 54.90, 52.71, 45.86; HRMS calcd for [M + H]^+^ m/z = 433.18703, found m/z = 433.18719.

2-(8-Hydroxy-7-(morpholinomethyl)quinolin-5-yl)-4-methoxyisoindoline-1,3-dione (8c)

Brown powder (30 mg, 72% yield); m.p. 222–225 °C; ^1^H NMR (DMSO-d6) δ 8.90 (d, J = 3.5 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.59–7.55 (m, 2H), 7.55–7.52 (m, 2H), 4.00 (s, 3H), 3.75 (s, 2H), 3.62–3.57 (m, 4H), 2.49–2.46 (m, 4H); ^13^C NMR (DMSO-d6) δ 167.96, 166.51, 157.06, 152.66, 149.04, 138.60, 137.36, 134.31, 132.45, 130.61, 125.73, 122.65, 119.70, 119.22, 118.38, 117.33, 115.85, 66.65, 56.77, 56.70, 53.64; HRMS calcd for [M + H]^+^ m/z = 420.15540, found m/z = 420.15546.

2-(8-Hydroxy-7-(piperidin-1-ylmethyl)quinolin-5-yl)-5-methoxyisoindoline-1,3-dione (9a)

Dark brown powder (25 mg, 60% yield); m.p. 184–188 °C; ^1^H NMR (500 MHz, CDCl_3_) δ 8.91 (d, J = 2.8 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 7.38 (dd, J = 8.4, 4.0 Hz, 1H), 7.28 (d, J = 2.0 Hz, 1H), 7.21 (s, 1H), 3.98 (s, 3H), 3.94 (s, 2H), 2.82–2.52 (m, 4H), 1.75–1.68 (m, 4H), 1.57–1.49 (m, 2H); ^13^C NMR (CDCl_3_) δ 167.92, 167.81, 165.15, 155.32, 149.21, 139.59, 134.49, 130.93, 128.50, 125.81, 125.67, 123.68, 121.97, 120.56, 117.61, 116.67, 108.44, 60.84, 56.24, 54.00, 25.57, 23.75; HRMS calcd for [M + H]^+^ m/z = 418.17613, found m/z = 418.17587.

2-(8-Hydroxy-7-((4-methylpiperazin-1-yl)methyl)quinolin-5-yl)-5-methoxyisoindoline-1,3-dione (9b)

Brown powder (30 mg, 70% yield); m.p. 228–232 °C; ^1^H NMR (CDCl_3_) δ 8.94–8.88 (m, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.46 (s, 1H), 7.39 (dd, J = 8.0, 3.7 Hz, 1H), 7.31–7.27 (m, 1H), 7.26 (s, 1H, under solvent signal), 3.98 (s, 3H), 3.93 (s, 2H), 2.83–2.48 (m, 8H), 2.34 (s, 3H); ^13^C NMR (CDCl_3_) δ 167.89, 167.77, 165.19, 154.42, 149.18, 139.48, 134.51, 131.00, 128.61, 125.74, 125.68, 123.71, 121.99, 120.57, 117.94, 117.31, 108.47, 59.80, 56.23, 54.84, 52.61, 45.78; HRMS calcd for [M + H]^+^ m/z = 433.18703, found m/z = 433.18796.

2-(8-Hydroxy-7-(morpholinomethyl)quinolin-5-yl)-5-methoxyisoindoline-1,3-dione (9c)

Dark brown powder (30 mg, 70% yield); m.p. 197–200 °C; ^1^H NMR (DMSO-d6) δ 8.91 (d, J = 4.0 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.60 (s, 1H), 7.54 (dd, J = 8.5, 4.1 Hz, 1H), 7.51 (s, 1H), 7.42 (d, J = 8.3 Hz, 1H), 3.98 (s, 3H), 3.76 (s, 2H), 3.61–3.58 (m, 4H), 2.5 (m, 4H, under solvent signal); ^13^C NMR (DMSO-d6) δ 167.99, 167.96, 165.09, 152.74, 149.07, 138.61, 135.01, 132.41, 130.56, 125.88, 125.70, 124.15, 122.68, 120.62, 119.60, 118.41, 109.01, 66.61, 56.88, 56.68, 53.61; HRMS calcd for [M + H]^+^ m/z = 420.15540, found m/z = 420.15521.

3.2. Biology

3.2.1. Cell Lines and Their Maintenance

The doxorubicin-sensitive Colo205 (ATCC-CCL-222) and the doxorubicin-resistant ABCB1- and LRP-expressing Colo320/MDR-LRP (ATCC-CCL-220.1) human colon adenocarcinoma cell lines were obtained from LGC Promochem in Teddington, UK. These cell lines were cultivated in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM Na-pyruvate, and 10 mM HEPES, along with nystatin and gentamicin. The cell lines were incubated in a humidified atmosphere (5% CO_2_, 95% air) at 37 °C. The cells were detached with a Trypsin–Versene (EDTA) solution for 5 min at 37 °C.

3.2.2. MTT Assay

The effects of increasing concentrations of the compounds on cell growth were tested in 96-well flat-bottomed microtiter plates. Namely, 1 × 10^4^ of human colonic adenocarcinoma cells in 100 μL of the medium (RPMI 1640) were added to each well, except for the medium control wells. Two-fold serial dilutions of the compounds were created in a separate plate (100–0.19 μM) and then transferred to the plates containing the cells. Plates were incubated at 37 °C for 24 h. At the end of incubation, 20 μL of MTT (thiazolyl blue tetrazolium bromide) solution (from a 5 mg/mL stock solution) was added to each well. After incubation at 37 °C for 4 h, 100 μL of sodium dodecyl sulfate (SDS) solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540 nm (ref. 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of cell growth was expressed as IC_50_ values, defined as the inhibitory dose that reduced the growth of the cells exposed to the tested compounds by 50%. The IC_50_ values and the SDs of triplicate experiments were calculated using the GraphPad Prism software version 5.00 for Windows, with a non-linear regression curve fit (GraphPad Software, San Diego, CA, USA; https://www.graphpad.com/, GPS-2615994-LAV1-F3C99). Doxorubicin (from a 2 mg/mL stock solution, Teva Pharmaceuticals Ltd. (Budapest, Hungary) was used as a positive control. The solvent (DMSO) did not have any effect on cell growth at the tested concentrations. The relative resistance (RR) was calculated as the ratio of the IC_50_ value in the resistant cancer cells to the IC_50_ in the sensitive cancer cell line [59].

3.2.3. Pgp ATPase Activity Assay

P-glycoprotein ATPase activity was determined using the Pgp-Glo^TM^ Assay System (Promega, WI, USA) [61]. The assay was performed according to the manufacturer’s instructions. Briefly, 20 µL of 1.25 mg/mL recombinant human Pgp membrane was incubated for 5 min in 20 µL of sample at 37 °C. The concentrations of the compounds were adjusted to approximately match the IC_50_ values obtained for the Colo320 cell line (5 µM for compound 2; 10 µM for 4, 5, and 9c; 20 µM for 8a and 9a). The treated samples contained less than 1% (v/v) DMSO. Sodium orthovanadate (Na_3_VO_4_, 100 µM) was applied as a selective inhibitor of Pgp, and verapamil (200 µM) was used as a stimulator/substrate control. The reaction was initiated by adding 10 µL of 25 mM MgATP, and samples were incubated at 37 °C for 40 min. The reaction was stopped after adding 50 µL of ATP detection reagent. Then, the tested compounds and controls were incubated at room temperature for 20 min. The emitted luciferase-generated luminescent signal was measured in a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany) at 580 nm. The basal Pgp ATPase activity was determined by calculating the difference in the average luminescent signal between Na_3_VO_4_-treated samples (RLU_Na3VO4_) and untreated (NT) samples (RLU_NT_). The Pgp ATPase activity in the presence of a test compound was determined by calculating the difference in the average luminescent signal between Na_3_VO_4_-treated and coumpound-treated (RLU_TC_) samples. Possible outcomes were as follows:

If ΔRLU_TC_ > ΔRLU_basal_, then the tested compound was a stimulator of Pgp ATPase activity;

If ΔRLU_TC_ = ΔRLU_basal_, then the test compound had no effect on Pgp ATPase activity;

If ΔRLU_TC_ < ΔRLU_basal_, then the test compound was an inhibitor of Pgp ATPase activity.

3.3. Prediction of Physicochemical and Pharmacokinetic Properties

The ADME profiles, pharmacokinetic parameters, and drug-likeness of the most active compounds 2, 4, 5, 8a, 9a, and 9c were predicted using the SwissADME online tool and pKCSM platform., and Chemicalize was used for pK_a_ and logS calculations (https://chemicalize.com), developed by ChemAxon [62,63]. The molecular structures were converted into smiles format and submitted to the online servers. All 3D molecular structures were built and preoptimized with the software Avogadro 2 version 1.102.1 (https://www.openchemistry.org) [64]. The preliminary DFT calculations were conducted and exported with the software ORCA 6.1.1. using the B3LYP-D3 ma-def2-SVP level of theory in the gas phase [65,66,67]. For the visualization and rendering of the results, Chemcraft Version 1.8, Build 682 (https://www.chemcraftprog.com) was used, with the HOMO, HOMO-1, and LUMO lobe contour value = 0.03; for the electrostatic potential maps, the contour value = 0.006, value range [−0.07, +0.07].

4. Conclusions

In this study, a series of novel 8-hydroxyquinoline–phthalimide hybrids was synthesized and transformed to their corresponding Mannich bases to fine-tune their pharmacological and physicochemical properties. Starting with 5-amino-8-hydroxyquinoline and phthalic anhydride derivatives, four new hybrid cores were formed, which were further subjected to a modified Mannich reaction using various secondary amines to develop twelve additional derivatives. Optimization of the Mannich reaction revealed that employing the acetate salt of the base was more effective than using the free base, improving the preparative efficiency and completion time. Based on the biological evaluations, the synthesized compounds showed cytotoxic activity against both the sensitive Colo205 and the doxorubicin-resistant Colo320 cell line, with consistently higher activity toward the resistant cell line. Among the tested compounds, compounds 2 and 4 exhibited the highest cytotoxic activity across both cell lines, with IC_50_ values ranging from 4.88 to 12.58 µM. Additionally, compounds 5, 8a, 9a, and 9c retained potent antiproliferative activity against the resistant cell line, with IC_50_ values of 9.89, 22.79, 16.39, and 11.5 µM, respectively, while showing lower activity against the sensitive cell line. Notably, the introduction of a CH_2_–N substituent at the seventh position of the hybrid scaffold markedly improved the selectivity toward multidrug-resistant cells. Compounds 9c, 9a, and 8a were the most selective, showing the lowest RR values (0.29, 0.33, and 0.35, respectively), indicating their strong potential for overcoming resistance mechanisms. Furthermore, compounds 2, 4, 5, 8a, and 9a showed potential inhibitory effects on Pgp ATPase activity. Overall, these findings highlight HQ–phthalimide hybrids as promising anticancer scaffolds, with Mannich modification providing a valuable strategy to improve their selectivity toward multidrug-resistant colorectal cancer cells.