Dual Inhibitors of P-gp and Carbonic Anhydrase XII (hCA XII) against Tumor Multidrug Resistance with Piperazine Scaffold

A new series of piperazine derivatives were synthesized and studied with the aim of obtaining dual inhibitors of P-glycoprotein (P-gp) and carbonic anhydrase XII (hCA XII) to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing the two proteins, P-gp and hCA XII. Indeed, these hybrid compounds contain both P-gp and hCA XII binding groups on the two nitrogen atoms of the heterocyclic ring. All compounds showed good inhibitory activity on each protein (P-gp and hCA XII) studied individually, and many of them showed a synergistic effect in the resistant HT29/DOX and A549/DOX cell lines which overexpress both the target proteins. In particular, compound 33 displayed the best activity by enhancing the cytotoxicity and intracellular accumulation of doxorubicin in HT29/DOX and A549/DOX cells, thus resulting as promising P-gp-mediated MDR reverser with a synergistic mechanism. Furthermore, compounds 13, 27 and 32 induced collateral sensitivity (CS) in MDR cells, as they were more cytotoxic in resistant cells than in the sensitive ones; their CS mechanisms were extensively investigated.


Introduction
Multidrug resistance (MDR) is a type of acquired resistance displayed by cancer cells that show reduced sensitivity to structurally and mechanistically unrelated anticancer drugs [1].This phenomenon is one of the main problems in the chemotherapy of malignant tumors.MDR is mainly due to the overexpression on the cell membrane of some ATP binding cassette (ABC) proteins such as P-glycoprotein (P-gp), Multidrug-Resistance-associated Protein-1 (MRP1), and Breast Cancer Resistance Protein (BCRP).These transporter proteins work as efflux pumps, reducing the intracellular concentration of anticancer drugs below their active dose lowering their therapeutic efficacy [2].
P-glycoprotein (P-gp) is the most studied ABC transporter in clinical research; it is overexpressed in many blood and solid tumors causing reduced chemotherapeutic responses [3].Due to the extensive involvement of P-gp in MDR, the co-administration of efficient P-gp inhibitors with chemotherapeutic agents has been proposed as a powerful therapeutic strategy to overcome MDR [4].Over the years, many P-gp inhibitors have been identified which have been classified into three generations according to their chronology and characteristics [5][6][7].Some of these compounds have reached clinical evaluation; however, in most cases, the results obtained were disappointing due to unexpected adverse effects and toxicity, which led to the early conclusion of many trials [8,9].As a result, currently, no compounds have been approved for clinical use.Therefore, there is still an urgent need to find potent and effective MDR reversal agents with minimal adverse effects.
One of the causes of the toxicity of P-gp inhibitors is the presence of this transporter in many healthy tissues where it is responsible for several physiological and pharmacological effects [10,11], and the ability of P-gp inhibitors to affect the pharmacokinetics of coadministered chemotherapeutic agents [12].A strategy to improve the therapeutic efficacy of drugs may involve the development of molecules that simultaneously inhibit two drug targets, thus exploiting synergistic therapeutic effects.
It has recently been reported that P-gp is co-localized and physically associated with the XII isoform of human carbonic anhydrase (hCA XII) on the membrane of several resistant tumor cells.The hCA XII isoform belongs to the family of carbonic anhydrases (CAs, EC 4.1.1.1)which are metalloenzymes that catalyze the conversion of carbon dioxide to bicarbonate and a proton, playing crucial functions related to pH regulation, homeostasis, and metabolism, and their inhibition leads to pharmacological responses [13].
hCA XII is a tumor-associated enzyme overexpressed in many solid and hypoxic tumors that is associated with their progression and metastasis formation [14][15][16].It maintains an alkaline intracellular pH and an extracellular acidosis that favors the growth of tumor cells, compromising that of normal cells [17,18].The intracellular alkalinization maintained by hCA XII is optimal for P-gp efflux activity; thus, the high expression of hCA XII in some chemoresistant P-gp-positive tumor cells contributes to MDR [19].Therefore, inhibition of hCA XII causes a decrease in intracellular pH which reduces the ATPase activity of P-gp and consequently the efflux activity of the transporter [13,19].
Based on these observations, it is possible to develop a strategy that allows selective targeting of the efflux effect of P-gp in resistant tumor cells overexpressing both P-gp and hCA XII.Therefore, dual inhibitors of P-gp and hCA XII may serve as useful chemosensitizers to overcome P-gp-mediated MDR in tumor cells.
In previous papers [19,20], we reported the design and synthesis of a new series of N,N-bis(alkanol)amine aryl diesters able to modulate the activity of P-gp and hCA XII in cancer cells that overexpress both proteins (Figure 1, structures A and B of the most potent compounds of the series).These derivatives are characterized by the presence of both P-gp and hCA XII-binding groups to achieve a synergistic action to overcome the resistance.All compounds showed a multitarget effect being able to modulate the activity of both P-gp and hCA XII taken individually.Moreover, many of these hybrid derivatives showed a synergistic effect in cell lines that overexpress both proteins (LoVo/DOX, HT29/DOX, and A549/DOX cell lines), enhancing the cytotoxicity effect of doxorubicin in these resistant cells.These compounds can be considered promising chemosensitizer agents for selective inhibition in MDR cancer cells overexpressing both P-gp and hCA XII proteins.
Figure 1.Structures of the leads (A,B) and general structure of piperazine derivatives 1-36, synthesized in this study (C).For the structure of final compounds 1-36 see Table 1.
The synthesized compounds 1-36, as dihydrochlorides, were first tested to evaluate their inhibitory effects on P-gp and hCA XII proteins, taken individually.The P-gp modulating activity was measured by evaluating the doxorubicin cytotoxicity enhancement on K562/DOX cells that overexpress only P-gp [24].The hCA inhibition activity was measured on four different hCA isoforms: the cytosolic hCA I and hCA II, and the tumor-associated transmembrane hCA IX and hCA XII isoforms, to evaluate their hCA selectivity profiles.Eight selected compounds were then tested in doxorubicin-resistant human adenocarcinoma colon cells (HT29/DOX) and in doxorubicin-resistant non-small cell lung cancer cells (A549/DOX), that overexpress both P-gp and hCA XII [19], to verify the synergistic effect on the MDR reversal activity due to the dual P-gp/hCA XII inhibition.
Moreover, three compounds were further investigated as collateral sensitivity (CS)promoting agents since they were more cytotoxic in resistant HT29/DOX and A549/DOX cells than in the parental ones.Therefore, we evaluated their effects on the intracellular amount of ROS and peroxidized lipids, P-gp catalytic activity, and membrane fluidity in HT29/DOX and A549/DOX cells.
The synthesized compounds 1-36, as dihydrochlorides, were first tested to evaluate their inhibitory effects on P-gp and hCA XII proteins, taken individually.The P-gp modulating activity was measured by evaluating the doxorubicin cytotoxicity enhancement on K562/DOX cells that overexpress only P-gp [24].The hCA inhibition activity was measured on four different hCA isoforms: the cytosolic hCA I and hCA II, and the tumor-associated transmembrane hCA IX and hCA XII isoforms, to evaluate their hCA selectivity profiles.Eight selected compounds were then tested in doxorubicin-resistant human adenocarcinoma colon cells (HT29/DOX) and in doxorubicin-resistant non-small cell lung cancer cells (A549/DOX), that overexpress both P-gp and hCA XII [19], to verify the synergistic effect on the MDR reversal activity due to the dual P-gp/hCA XII inhibition.
Moreover, three compounds were further investigated as collateral sensitivity (CS)promoting agents since they were more cytotoxic in resistant HT29/DOX and A549/DOX cells than in the parental ones.Therefore, we evaluated their effects on the intracellular amount of ROS and peroxidized lipids, P-gp catalytic activity, and membrane fluidity in HT29/DOX and A549/DOX cells.

CA Inhibitory Activity
The CA inhibitory activity of the new compounds 1-36, evaluated by a stopped-flow CO 2 hydrase assay [32], is reported in Table 1.Four hCA isoforms were used in this assay: the cytosolic hCA I and II and the tumor-associated transmembrane hCA IX and XII isoforms.Acetazolamide (AAZ) was used as a standard inhibitor.The results confirm that the coumarin group directs activity only towards hCA IX and XII [33].Indeed, all these derivatives inhibited both hCA IX and XII at nanomolar concentrations, while they were inactive against off-target hCA I and II isoforms.

Doxorubicin Cytotoxicity Enhancement Assay in K562/DOX Cells
The P-gp modulating activity of compounds 1-36 was measured by evaluating the cytotoxicity enhancement of the co-administered doxorubicin in K562/DOX cells.K562 is a highly undifferentiated erythroleukemia cell line [34].The doxorubicin-resistant K562/DOX cells overexpress almost exclusively the transporter membrane protein Pgp [24].Since doxorubicin is a P-gp substrate and is expelled out of the cell by the pump, it is generally inactive in tumor cells that express P-gp.
Compounds were first studied at 1 and 3 µM concentrations to evaluate their intrinsic cytotoxicity in both the parental K562 and the resistant K562/DOX cell lines, using the MTT assay [35].All compounds had no intrinsic cytotoxicity in the parental line.Most of them showed a toxicity of approximately 20% in the resistant cells at the two concentrations tested, while compounds 7, 9, 25, and 27 reduced the cellular viability by 30-50% at 3 µM (Supplementary Materials, Figure S1).
Compounds 1-36 were studied at 1 and 3 µM concentrations to evaluate their ability to decrease the IC 50 value of co-administered doxorubicin in K562/DOX cells.The results are reported in Table 1 as RF (Reversal Fold) values that are the ratio between the IC 50 value of doxorubicin alone and in the presence of the studied compounds: the higher the RF value, the higher the MDR reversal activity.Verapamil (Ver) was used as a reference compound.
All these derivatives enhanced the cytotoxicity of doxorubicin to different extents, except compound 7, which shows RF = 1 for both concentrations, and most of them showed higher RF values than those of verapamil.
A thorough analysis of the obtained results seems to indicate the absence of a correlation between the structural requirements necessary to modulate the efflux activity of P-gp and those to inhibit hCA XII.

Doxorubicin Cytotoxicity Enhancement Assay in HT29/DOX and A549/DOX Cells
Compounds 1-36 were also tested in doxorubicin-resistant human adenocarcinoma colon cells (HT29/DOX) and in doxorubicin-resistant non-small cell lung cancer cells (A549/DOX), that overexpress both P-gp and hCA XII [19], to study the effect of these dual P-gp/hCA XII inhibitors in a specific environment where the two target proteins coexist.
The expression levels of P-gp and hCA XII in sensitive HT29 and A549 cells and their resistant counterparts (HT29/DOX and A549/DOX cells) were checked by immunoblotting analysis, as previously reported [20].The resistant sublines also showed increased expression of MRP1, another transporter involved in doxorubicin resistance, which, however, was not associated with hCA XII nor was affected in its efflux activity by hCA XII [19,20].
All compounds were preliminarily studied to evaluate their intrinsic cytotoxicity at different concentrations (from 10 nM to 50 µM) in the parental HT29 and A549 and the resistant HT29/DOX and A549/DOX cells using the MTT assay [35].
Based on the results obtained in the intrinsic cytotoxicity assays, the compounds with the best profiles in terms of potency on P-gp in the K562/DOX cells test and selectivities towards the hCA XII isoform, 1, 2, 4-6, 14, 19 and 33, were selected for further evaluation in HT29/DOX and A549/DOX cell lines in co-administration with doxorubicin.
These compounds were then assayed at 1 and 3 µM concentrations in co-administration with doxorubicin at a concentration of 5 µM, which is known to discriminate well between sensitive and resistant cells [36].In HT29 and A549 cells, which express low levels of P-gp [20], the tested compounds did not significantly increase the cytotoxicity of doxorubicin used alone (Supplementary Materials, Figure S6).Otherwise, in the resistant counterparts (HT29/DOX and A549/DOX), which displayed high levels of P-gp and hCA XII, the selected compounds at both 1 and 3 µM concentrations enhanced the cytotoxicity of doxorubicin, measured as the percentage of cell growth, in a dose-dependent way (Figure 2).In general, compounds were more active in HT29/DOX than in A549/DOX cells at both concentrations.In particular, compounds 4, 5, 14, and 19 at 3 µM concentration were the most potent in doxorubicin-treated HT29/DOX cells causing a reduction in cell viability by 65.0%.Moreover, in the A549/DOX cell line, the co-administration of 1, 2, 4-6, 14 and 19, tested at 3 µM, led to the highest enhancement of doxorubicin toxicity reaching a reduction of nearly 55.0% in cell viability.In both tested cell lines, the best compound was derivative 33, which, at the highest concentration, in combination with the anticancer drug, caused a reduction in cell viability of 67.6% in HT29/DOX cells and 62.6% in A549/DOX cells.Interestingly, most compounds at 3 µM achieved the same potency as verapamil tested at 1 mM concentration.

Doxorubicin Accumulation Assay in HT29/DOX and A549/DOX Cells
Furthermore, we evaluated the intracellular accumulation of 5 µM doxorubicin in HT29/DOX and A549/DOX cells alone and in the presence of derivatives 1, 2, 4-6, 14, 19 and 33, studied at 1 and 3 µM.This test allowed us to investigate if the reduced viability measured in the co-administration assay was due to different retention of the anticancer drug within the cells.In the parental HT29 and A549 cell lines, we measured a greater amount of intracellular doxorubicin compared to their resistant counterparts, which was not enhanced by our selected compounds (Supplementary Materials, Figure S7).However, in HT29/DOX and A549/DOX cells, derivatives 1, 2, 4-6, 14, 19 and 33 were able to increase the intracellular retention of the anticancer drug, in a dose-dependent way.Intracellular doxorubicin accumulation was lower in A549/DOX than in HT29/DOX cells probably due to a slightly greater basal expression of the transporter protein MRP1 [20], which can contribute to doxorubicin efflux [2].
The most potent molecules were 5, 19, and 33 in both cell lines (Figure 3).In particular, at the highest concentration, compound 33 allowed a 3-or 4-fold greater accumulation of doxorubicin in HT29/DOX or A549/DOX cells, respectively, showing a potency comparable to that of verapamil tested at 1 mM in HT29/DOX (Figure 3).increase the intracellular retention of the anticancer drug, in a dose-dependent way.Intracellular doxorubicin accumulation was lower in A549/DOX than in HT29/DOX cells probably due to a slightly greater basal expression of the transporter protein MRP1 [20], which can contribute to doxorubicin efflux [2].
The most potent molecules were 5, 19, and 33 in both cell lines (Figure 3).In particular, at the highest concentration, compound 33 allowed a 3-or 4-fold greater accumulation of doxorubicin in HT29/DOX or A549/DOX cells, respectively, showing a potency comparable to that of verapamil tested at 1 mM in HT29/DOX (Figure 3).

Collateral Sensitivity Studies
Recently, it emerged that P-gp-expressing cells are paradoxically more sensitive to specific compounds than their counterpart with no level of P-gp [37][38][39].This phenomenon, called "collateral sensitivity" (CS), relies on different mechanisms, among which the increased generation of reactive oxygen species (ROS) that damage the structural cell components, including the plasma membrane lipid where P-gp is embedded, and the increased membrane fluidity [40][41][42].
Interestingly we noticed that compounds 13, 27, and 32 administered alone were 15%-20% more cytotoxic in the resistant HT29/DOX and A549/DOX cells than in the parental sensitive cells (Figure 4).We thus hypothesized that these compounds could be potential CS inducers.
Molecules 2024, 29, x FOR PEER REVIEW

Collateral Sensitivity Studies
Recently, it emerged that P-gp-expressing cells are paradoxically more se specific compounds than their counterpart with no level of P-gp [37][38][39].This p non, called "collateral sensitivity" (CS), relies on different mechanisms, among w increased generation of reactive oxygen species (ROS) that damage the structural ponents, including the plasma membrane lipid where P-gp is embedded, an creased membrane fluidity [40][41][42].
Interestingly we noticed that compounds 13, 27, and 32 administered al 15%-20% more cytotoxic in the resistant HT29/DOX and A549/DOX cells than i rental sensitive cells (Figure 4).We thus hypothesized that these compounds cou tential CS inducers.For this purpose, we tested the selected compounds in HT29/DOX and A cells to investigate possible mechanisms through which they could work as pot inducers.Resistant cells often display a peculiar sensitivity to compounds able to For this purpose, we tested the selected compounds in HT29/DOX and A549/DOX cells to investigate possible mechanisms through which they could work as potential CS inducers.Resistant cells often display a peculiar sensitivity to compounds able to increase ROS [43].Interestingly, in both HT29/DOX and A549/DOX cells, compounds 13, 27, and 32 significantly enhanced the intracellular amount of ROS and peroxidized lipids, thiobarbituric reactive substances (TBARS), considered indexes of oxidative stress [44] (Figure 5).In particular, in HT29/DOX, they were able to increase the levels of ROS and TBARS by 3-4 and 2.6 times, respectively, compared to the control, while in A549/DOX cells, these compounds caused a major increase in both oxidative stress indexes (7-8 times for ROS, and 4 times for TBARS).

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11 of 32 5).In particular, in HT29/DOX, they were able to increase the levels of ROS and TBARS by 3-4 and 2.6 times, respectively, compared to the control, while in A549/DOX cells, these compounds caused a major increase in both oxidative stress indexes (7-8 times for ROS, and 4 times for TBARS).
It is noteworthy that some coumarin-derivatives may act as anticancer agents thanks to their ability to inhibit thioredoxin reductase 1, eliciting oxidative stress [45,46].This mechanism can explain the increase in ROS and TBARS as well as the cytotoxic potential of the compounds against chemoresistant cells that are even more susceptible to ROS than chemosensitive cells [42].However, for the moment, this aspect has not yet been explored in depth for these new compounds.Oxidized lipids, besides triggering cell death, e.g., via ferroptosis [47], are important to modulate the activity of P-gp.Resistant cells usually have a lower amount of oxidizable fatty acid incorporated in the plasma membrane and this lipid composition creates an optimal environment for P-gp catalytic activity [48].However, every factor increasing the amount of oxidized lipids alters the chemical-physical properties of the membrane impairing P-gp conformation, drug binding, and release [48].A similar scenario also occurred in HT29/DOX and A549/DOX cell lines treated with compounds 13, 27, and 32, which lowered the catalytic efficacy of P-gp in resistant cells to levels comparable to drugsensitive HT29 and A549 cells (Figure 6).It is noteworthy that some coumarin-derivatives may act as anticancer agents thanks to their ability to inhibit thioredoxin reductase 1, eliciting oxidative stress [45,46].This mechanism can explain the increase in ROS and TBARS as well as the cytotoxic potential of the compounds against chemoresistant cells that are even more susceptible to ROS than chemosensitive cells [42].However, for the moment, this aspect has not yet been explored in depth for these new compounds.
Oxidized lipids, besides triggering cell death, e.g., via ferroptosis [47], are important to modulate the activity of P-gp.Resistant cells usually have a lower amount of oxidizable fatty acid incorporated in the plasma membrane and this lipid composition creates an optimal environment for P-gp catalytic activity [48].However, every factor increasing the amount of oxidized lipids alters the chemical-physical properties of the membrane impairing P-gp conformation, drug binding, and release [48].A similar scenario also occurred in HT29/DOX and A549/DOX cell lines treated with compounds 13, 27, and 32, which lowered the catalytic efficacy of P-gp in resistant cells to levels comparable to drug-sensitive HT29 and A549 cells (Figure 6).
Similarly, P-gp activity is high in plasma membrane rich in saturated fatty acid that allows a proper conformation of the transporter [48].By contrast, a more fluid membrane reduces the catalytic activity of the transporter [49,50].This happens, for instance, when the ratio between polyunsaturated and saturated fatty acid is increased [49] or when specific compounds act as membrane fluidifiers [42].This seems also to be the case of compounds 13, 27 and 32, which increased membrane fluidity of HT29/DOX and A549/DOX cells (Figure 7), displaying an additional mechanism by which they can act as CS inducers and by which they reduce the activity of P-gp.
amount of oxidized lipids alters the chemical-physical properties of the membrane impairing P-gp conformation, drug binding, and release [48].A similar scenario also occurred in HT29/DOX and A549/DOX cell lines treated with compounds 13, 27, and 32, which lowered the catalytic efficacy of P-gp in resistant cells to levels comparable to drugsensitive HT29 and A549 cells (Figure 6).

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Similarly, P-gp activity is high in plasma membrane rich in saturated fatty acid that allows a proper conformation of the transporter [48].By contrast, a more fluid membrane reduces the catalytic activity of the transporter [49,50].This happens, for instance, when the ratio between polyunsaturated and saturated fatty acid is increased [49] or when specific compounds act as membrane fluidifiers [42].This seems also to be the case of compounds 13, 27 and 32, which increased membrane fluidity of HT29/DOX and A549/DOX cells (Figure 7), displaying an additional mechanism by which they can act as CS inducers and by which they reduce the activity of P-gp.

Transport Inhibition of Fluorescent Probes in MDCK Transfected Cells
The derivatives studied as collateral sensitizers, 13, 27 and 32, together with compound 33, which displayed the highest potency in doxorubicin co-administration assays, were further tested in three Madin-Darby Canine Kidney (MDCK) transfected cell lines that overexpress P-gp, MRP1 or BCRP.
The MDR interaction potency of these compounds was studied by detecting the inhibition of the P-gp-, MRP1-or BCRP-mediated efflux of a substrate in cells overexpressing the studied pumps.In detail, we measured the following: (i) the transport inhibition of the profluorescent probe calcein-AM (P-gp and MRP1 substrate) in MDCK-MDR1 and MDCK-MRP1 cells (P-gp-and MRP1-overexpressing cells, respectively); (ii) the transport inhibition of the fluorescent probe Hoechst 33342 (BCRP substrate) in MDCK-BCRP cells (BCRP-overexpressing cells) [51].
The results, reported in Table 2, showed that CS inducers 13, 27, and 32 modulated the P-gp-mediated transport of calcein-AM, with EC50 values in the micromolar range, demonstrating their specific influence on the activity of this efflux pump in an MDCK-MDR1 model, in which only P-gp is present.Also, compound 33 confirmed its effect as a P-gp modulator, displaying an EC50 value of 3.02 µM.
Regarding MRP1 inhibition, only compound 13 displayed an EC50 value in the micromolar range, while all these derivatives were not active on BCRP.

Transport Inhibition of Fluorescent Probes in MDCK Transfected Cells
The derivatives studied as collateral sensitizers, 13, 27 and 32, together with compound 33, which displayed the highest potency in doxorubicin co-administration assays, were further tested in three Madin-Darby Canine Kidney (MDCK) transfected cell lines that overexpress P-gp, MRP1 or BCRP.
The MDR interaction potency of these compounds was studied by detecting the inhibition of the P-gp-, MRP1-or BCRP-mediated efflux of a substrate in cells overexpressing the studied pumps.In detail, we measured the following: (i) the transport inhibition of the profluorescent probe calcein-AM (P-gp and MRP1 substrate) in MDCK-MDR1 and MDCK-MRP1 cells (P-gp-and MRP1-overexpressing cells, respectively); (ii) the transport inhibition of the fluorescent probe Hoechst 33342 (BCRP substrate) in MDCK-BCRP cells (BCRP-overexpressing cells) [51].
The results, reported in Table 2, showed that CS inducers 13, 27, and 32 modulated the P-gp-mediated transport of calcein-AM, with EC 50 values in the micromolar range, demonstrating their specific influence on the activity of this efflux pump in an MDCK-MDR1 model, in which only P-gp is present.Also, compound 33 confirmed its effect as a P-gp modulator, displaying an EC 50 value of 3.02 µM.Regarding MRP1 inhibition, only compound 13 displayed an EC 50 value in the micromolar range, while all these derivatives were not active on BCRP.

Discussion and Conclusions
In this work, we described the design, synthesis, and biological activity of novel N 1 ,N 4 disubstituted piperazine derivatives able to reverse the P-gp-mediated MDR in cancer cells overexpressing both P-gp and hCA XII transmembrane proteins.In several MDR cancer cells P-gp is co-localized with hCA XII isoform and the catalytic activity of hCA XII modulates the efflux activity of P-gp.Indeed, the structure of these dual inhibitors contains both P-gp and hCA XII binding moieties to synergistically overcome MDR.Therefore, these compounds had a piperazine ring carrying on one nitrogen atom three different methoxy-substituted arylalkyl moieties, which conferred good inhibitory activity on P-gp, and on the second nitrogen atom four different coumarin groups, to maintain a selectivity towards hCA XII, linked to the heterocycle by an alkoxy chain.
All compounds were able to inhibit the hCA XII isoform at nanomolar concentrations, to different extents: indeed, derivatives carrying the coumarin group IV were the least active, while the (E)-3-(3,4,5-trimethoxyphenyl)vinyl (a) and 3,4,5-trimethoxyphenyl (b) residues conferred higher potency on hCA XII, regardless of linker length.Moreover, most of our new derivatives displayed good inhibitory effects on P-gp, by increasing the cytotoxicity of doxorubicin in resistant K562/DOX cells, which overexpress only the transporter.All compounds with the 4,4-bis(4-methoxyphenyl)butyl (c) residue were the most potent ones, also showing RF values higher than 10.0 or 20.0, when used at 1 or 3 µM, respectively.
Selected compounds (1, 2, 4-6, 14, 19 and 33) were also studied in doxorubicinresistant human adenocarcinoma colon cells (HT29/DOX) and in doxorubicin-resistant non-small cell lung cancer cells (A549/DOX), that overexpress both P-gp and hCA XII.The results showed that, in a specific environment where the two target proteins coexist, these compounds enhanced the cytotoxicity of the co-administered doxorubicin in a dosedependent way, demonstrating a synergic effect on the MDR reversal activity due to the dual P-gp/hCA XII inhibition.In particular, compounds 4, 5, 14, 19 and 33 were the most potent in both the doxorubicin-treated HT29/DOX and A549/DOX cell lines, when tested at a concentration of 3 µM.As regards SARs, it is not possible to find a certain correlation between the nature of the substituents on the two nitrogen atoms and the activity of these compounds, since their structures are quite different.
Moreover, we demonstrated that the reduced viability measured in the doxorubicin co-administration assay in the presence of our compounds was due to different retention of the anticancer drug within the cells.Indeed, all tested compounds were able to increase intracellular doxorubicin in HT29/DOX or A549/DOX cells, and the most potent derivative was 33, which allowed the greatest accumulation of the drug in both resistant cell lines.
Evidence that these compounds (4, 5, 14, 19, 33) had higher activity in HT29/DOX and A549/DOX cells, that overexpress both P-gp and hCA XII, than in K562/DOX cells, overexpressing only P-gp, demonstrates that they were endowed with a synergistic mechanism on the two target proteins.
Together, these results confirm that most of our new N 1 ,N 4 -disubstituted piperazine derivatives act as dual P-gp/hCA XII inhibitors.Among them, compound 33 resulted as a promising P-gp-mediated MDR reverser with a synergistic mechanism.Indeed, although it did not prove to be the most potent compound on the two target proteins taken individually (Ki = 46.8nM on hCA XII isoform and RF = 9.9 and 31.7, at 1 and 3 µM concentrations, respectively, in K562/DOX cells, devoid of hCA XII), compound 33 displayed the greatest potency in enhancing the cytotoxicity and accumulation of doxorubicin in assays performed in cancer cells expressing both P-gp and hCA XII.
Interestingly, compounds 13, 27 and 32 showed an unexpectedly higher cytotoxicity in resistant cells than in the sensitive ones, appearing as possible CS inducers.The mechanistic investigations revealed that they acted as multitarget agents: indeed, they induced oxidative stress and increased the amount of peroxidized plasma membrane lipids; they altered the chemical-physical properties of the membrane by increasing its fluidity and creating an unfavorable lipid environment for the activity of P-gp.Notwithstanding, compounds 13, 27 and 32 proved to be more potent cytotoxic agents against P-gp-expressing cells than their sensitive counterparts due to increased generation of ROS and lipoperoxides, and an increased membrane fluidity, three events that are sufficient to induce CS in drug-resistant cells [41,42].
In conclusion, these results confirm our starting hypothesis that compounds containing both P-gp and hCA XII binding groups on the two nitrogen atoms of the piperazine ring were able to synergistically overcome MDR by acting as dual P-gp/hCA XII inhibitors.However, these piperazine derivatives, bearing arylalkyl and aryloxyalkyl substituents on the two nitrogen atoms, were generally less potent on the two proteins, P-gp and hCA XII, taken individually, than the previously synthesized N,N-bis(alkanol)amine aryl derivatives containing ester functions.Therefore, a possible development of this project could be the synthesis of new N 1 ,N 4 -disubstituted piperazine derivatives containing aryl ester moieties to improve the potency of this series of molecules.
Furthermore, three collateral-sensitizing compounds were identified that could be considered interesting leads for the development of selective cytotoxic agents for MDR cells.

Chemistry
All melting points were taken on a Büchi apparatus and are uncorrected.NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz for 1 H-NMR, 100 MHz for 13 C-NMR). 1 H and 13 C NMR spectra were measured at room temperature (25 • C) in an appropriate solvent. 1 H and 13 C chemical shifts are expressed in ppm (δ) referenced to TMS.Spectral data are reported using the following abbreviations: s = singlet, d = doublet, dd = doublet of doublets, dt = doublet of triplets, t = triplet, bs = broad singlet, m = multiplet, and coupling constants are reported in Hz, followed by integration.Assignments of the 13 C signals were performed using the attached proton test (APT) technique.
Chromatographic separations were performed on a silica gel column by flash chromatography (Kieselgel 40, 0.040-0.063mm; Merck, Darmstadt, Germany).Yields are given after purification unless otherwise stated.The high-resolution mass spectrometry (HRMS) analysis was performed with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with an electrospray ionization source (ESI).The accurate mass/charge ratio measure was carried out by introducing, via a syringe pump at 10 µL min −1 , the sample solution (1.0 µg mL −1 in mQ water/acetonitrile 50:50), and the signal of the positive ions was acquired.The proposed experimental conditions allowed to monitoring the protonated molecules of studied compounds ([M+H] + species), that were measured with a proper dwell time to achieve 60,000 units of resolution at Full Width at Half Maximum (FWHM).The elemental composition of each compound was calculated based on its measured accurate mass/charge ratio, accepting only results with an attribution error less than 2.5 ppm and a not integer double bond/ring equivalents (RDB) value, to consider only the protonated species [52].The final compounds were checked by HPLC/diode array detection (DAD) analysis and were found a purity ≥ 95%.HPLC/DAD conditions of the analytical method, the chromatograms and UV spectra of final compounds 1-36 are included in the Supplementary Materials (Figures S8-S47).
Compounds were named following IUPAC rules as applied by ChemBioDraw Ultra 14.0 software.When reactions were performed in anhydrous conditions, the mixtures were maintained under nitrogen.Free bases 1-36 were transformed into the corresponding dihydrochlorides by treatment with a solution of acetyl chloride (2-3 equiv.) in anhydrous CH 3 OH.The salts were crystallized from abs. ethanol/petroleum ether.

General Procedures for the Synthesis of Piperazine Derivatives 1-36
To a solution of the proper methoxy-substituted aryl piperazine (37-39) [21] (1 equiv.) in dry acetonitrile, K 2 CO 3 (1.2equiv.)and the adequate bromoalkoxy-2H-chromen-2-one (42-53) (1.2 equiv.)were added.The mixture was stirred at 60 • C overnight, then the solvent was removed under reduced pressure and the residue was treated with CH 2 Cl 2 .The organic layer was washed twice with 10% NaOH solution, dried over Na 2 SO 4 and concentrated under vacuum.Finally, the residue was purified by flash chromatography using the proper eluting system, yielding the desired compound as an oil.
7-(4-Bromobutoxy)-4-methyl-2H-chromen-2-one 47 [26].Following the general procedure, compound 47 (0.16 g, yield: 76.3%) was synthesized as a white solid, from 40 (0.12 g, 0.68 mmol) and 1,4-dibromobutane (0.  C were used as substrate.Stock solutions of inhibitors were prepared at a concentration of 10 µM (in DMSO-water 1:1, v/v) and dilutions up to 0.01 nM were carried out with the assay buffer mentioned above.At least 7 different inhibitor concentrations have been used for measuring the inhibition constant.Inhibitor and enzyme solutions were preincubated together for 6 h at room temperature prior to assay in order to allow for the formation of the E-I complex.Triplicate experiments were conducted for each inhibitor concentration, and the values reported throughout the paper are the mean of such results.The inhibition constants were obtained by non-linear least-squares methods using the Cheng-Prusoff equation, as reported earlier [53], and represent the mean from at least three different determinations.All CA isozymes used here were recombinant proteins obtained as reported earlier by our group and their concentrations in the assay system were 5.6-12 nM [54].

Cell Lines and Cultures
The K562 leukemia cells were derived from a patient with chronic myelogenous leukemia [34] and the P-gp over-expressing K562/DOX cells were obtained from Prof. J. P. Marie (Hospital Hotel-Dieu, Paris, France).These cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; GIBCO, Grand Island, NY, USA) at 37 • C in a humidified incubator with 5% CO 2 .To maintain the resistance, every month, resistant cells were cultured for three days with 400 nM doxorubicin.
Human chemosensitive colon cancer HT29 cells and lung cancer A549 cells were purchased from ATCC (Manassas, VA, USA).Human HT29/DOX and A549/DOX were generated by stepwise selection in a medium with increasing concentration of doxorubicin, as reported in the literature [20], and maintained in a culture medium with a final concentration of 200 nM and 100 nM doxorubicin, respectively.All cell lines were authenticated by microsatellite analysis, using the PowerPlex kit (Promega Corporation, Madison, WI, USA; last authentication: January 2022).Cells were maintained in media supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin-streptomycin, and 1% v/v L-glutamine.

Drugs and Chemicals
Doxorubicin hydrochloride, verapamil hydrochloride, dimethylsulphoxide (DMSO) and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Milan, Italy).Stock solutions of the tested compounds as hydrochloride salts were prepared in DMSO at 10 −2 M. Stock solutions of doxorubicin hydrochloride and verapamil hydrochloride were prepared in water at 10 −2 M. All the stock solutions were then diluted with complete medium to obtain the 10× desired final maximum test concentrations.

Intrinsic Cytotoxicity
The intrinsic toxicity of the studied compounds was determined by an MTT assay [35].K562 and K562/DOX were exposed for 72 h with compounds at 1 and 3 µM concentrations while HT29, A549, HT29/DOX and A549/DOX cells were incubated for 48 h with compounds used at different concentrations (from 10 nM to 50 µM).The percentage of growth compared to the untreated control was transformed into histograms with the GraphPad Prism 5 program.4.2.5.Co-Administration Assays in K562/DOX, HT29, A549, HT29/DOX and A549/DOX Cells K562/DOX cells were incubated for 72 h with different concentrations of doxorubicin in combination with 1 or 3 µM of all compounds.IC 50 was the concentration killing 50% of cells and was determined graphically from relative survival curves obtained by GraphPad Prism 5 software (GraphPad, San Diego, CA, USA).HT29, A549, HT29/DOX and A549/DOX cells were incubated for 48 h with 5 µM doxorubicin, alone or in combination with 1 or 3 µM of selected compounds.Cell viability of the cell lines was measured by the MTT assay, using a Synergy HT microplate spectrofluorometer (Bio-Tek Instruments, Winooski, VT, USA).The absorbance of untreated cells was considered 100%; results were expressed as a percentage of viable treated cells versus the control untreated cells [51].4.2.6.Intracellular Doxorubicin Accumulation in HT29, A549, HT29/DOX and A549/DOX Cells Intracellular doxorubicin was measured in 10,000 cells seeded into 96-well plates and incubated for 24 h with 5 µM doxorubicin with or without 1 µM and 3 µM of each compound.The intracellular drug content was measured fluorimetrically as detailed previously [51] using a Synergy HTX 96-well plate reader.The results were expressed as nmol doxorubicin/mg cell proteins based on the titration curve previously prepared.

Calcein-AM Experiment
Experiments were performed as previously described [51].MDCK-MDR1 and MDCK-MRP1 cells (30,000 cells per well) were seeded in a 96-well black CulturePlate in 100 µL of medium and allowed to grow overnight in a humidified atmosphere of 5% CO 2 at 37 • C. Tested compounds (100 µL) in a final concentration ranging between 0.01 nM and 100 µM, solubilized in culture medium, were added to the monolayers.After 30 min in a humidified atmosphere of 5% CO 2 at 37 • C, 100 µL of Calcein-AM in phosphate-buffered saline (PBS) was added to obtain a final concentration of 2.5 µM and the plate was incubated for 30 min.All wells were washed 3 times with 100 µL ice-cold PBS and read with Victor3 (PerkinElmer) after adding 100 µL of cold PBS.Excitation and emission wavelengths of 485 nm and 535 nm, respectively, were used.In these experimental conditions, cell Calcein-AM accumulation in the absence and in the presence of each tested compound was evaluated and compared to the basal level of fluorescence derived from untreated cells.The increase in fluorescence from the baseline level was measured in the treated wells.EC 50 values were determined by fitting the rate of fluorescence increase versus log[dose].

Hoechst 33342 Experiment
These experiments were conducted as previously described [51].MDCK-BCRP cells (30,000 cells per well) were seeded in a black CulturePlate 96/well in 100 µL of medium and allowed to grow overnight in a humidified atmosphere of 5% CO 2 at 37 • C. Tested compounds (100 µL) in a final concentration ranging between 0.01 nM and 100 µM, solubilized in culture medium, were added to the monolayers.After 30 min in a humidified atmosphere of 5% CO 2 at 37 • C, 100 µL of Hoechst 33342 at the final concentration of 8 µM in PBS was added and the plate was incubated for 30 min.The supernatants were drained, and the cells were fixed for 20 min under protection from light using 100 µL per well of a 4% PFA solution.All wells were washed 3 times with 100 µL ice-cold PBS and read with Victor3 (PerkinElmer) after adding 100 µL of cold PBS.Excitation and emission wavelengths of 340/35 nm and 485/20 nm, respectively, were used.In these experimental conditions, the baseline fluorescence level of Hoechst 33342 was estimated using untreated cells and compared with the fluorescence emissions of cells treated with the tested compounds.EC 50 values were determined by plotting the percent increase in fluorescence versus log[dose].

Oxidative Stress Measurement
Selected compounds have been tested for their cytotoxic activity at 24 h on HT29/DOX and A549/DOX cells and on the parental HT29 and A549 cells by MTT assay.To evaluate the mechanisms involved in CS, reactive oxygen species (ROS) were measured using

Figure 1 .
Figure 1.Structures of the leads (A,B) and general structure of piperazine derivatives 1-36, synthesized in this study (C).For the structure of final compounds 1-36 see Table1.

Figure 5 .
Figure 5. Effects on ROS or TBARS levels of 13, 27 and 32 at 1 µM in HT29/DOX (left) and A549/DOX (right) cell lines.Each bar represents the mean ± SD of three independent experiments, with technical duplicates.Control (CTR) is ROS and TBARS levels in both HT29/DOX and A549/DOX cells.One-way analysis of variance (ANOVA) analysis: *** p < 0.0001 vs. control.

Figure 6 .
Figure 6.P-gp activity in (A) HT29 or HT29/DOX cells and (B) A549 or A549/DOX cells, in the absence (CTR) or presence of 13, 27 and 32 at 1 µM.Each bar represents the mean ± SD of three inde-

Figure 5 .
Figure 5. Effects on ROS or TBARS levels of 13, 27 and 32 at 1 µM in HT29/DOX (left) and A549/DOX (right) cell lines.Each bar represents the mean ± SD of three independent experiments, with technical duplicates.Control (CTR) is ROS and TBARS levels in both HT29/DOX and A549/DOX cells.One-way analysis of variance (ANOVA) analysis: *** p < 0.0001 vs. control.

Table 1 .
Inhibitory activity on hCA I, II, IX and XII isoforms and doxorubicin cytotoxicity enhancement effect in K562/DOX cells of compounds 1-36 and of the two reference compounds acetazolamide (AAZ) and verapamil (Ver).

Table 1 .
Inhibitory activity on hCA I, II, IX and XII isoforms and doxorubicin cytotoxicity enhancement effect in K562/DOX cells of compounds 1-36 and of the two reference compounds acetazolamide (AAZ) and verapamil (Ver).KI (nM)