Novel insights on [1,2]oxazolo[5,4‐e]isoindoles on multidrug resistant acute myeloid leukemia cell line

Abstract A series of [1,2]oxazolo[5,4‐e]isoindole derivatives was evaluated against HL‐60 cell line and its multidrug resistance (MDR) variant, HL‐60R, resistant to doxorubicin and to other P‐gp substrates by overexpressing the efflux pump. They displayed antiproliferative activities, with IC50 values ranging from 0.02 to 5.5 µM. In particular, the newly synthesized compound 4k produced synergistic effects in terms of cell growth inhibition and cell death induction either in combination with a Vinca alkaloid, Vinblastine, and a Taxane, Paclitaxel in HL‐60R cells. The study of the mechanism of action indicated that all compounds showed antimitotic activity through inhibition of tubulin polymerization. Thus, [1,2]oxazoles could represent a valuable tool to overcome MDR mechanism, confirming the potential use of this class of compounds.

Even in leukemic patients, the phenomenon of MDR is reputed to be responsible for a strong limitation of therapeutic choice, and for this reason it has been studied for years and continues to receive great attention (Du & Chen, 2017;Leith, 1998;Oliai & Schiller, 2020).
For example, in acute myeloid leukemia (AML), drug resistance toward standard chemotherapeutic compounds, due to overexpression of P-gp, leads to failure of the treatment or relapse. This P-gp overexpression can result in highly aggressive AML clone, which needs more intensive treatment and invasive procedures (Kunadt et al., 2020;J. Zhang et al., 2019). MDR mechanisms produce resistance to microtubule-targeting agents (Van Vuuren et al., 2015), such as Vinca alkaloids and Taxanes. The latter, although approved for some types of solid tumors, are also investigated for hematological neoplastic diseases (Chao et al., 2017;Hijiya et al., 2009;Philchenkov et al., 2015;Schnerch et al., 2013;Trendowski et al., 2015;Barreca, Stathis et al., 2020).
In the course of our studies on small heterocyclic molecules with anticancer properties Barreca, Ingarra et al., 2022), we discovered the class of the [1,2]oxazolo [5,4-e]isoindole system of type 4 (Scheme 1), which showed antiproliferative activity against multiple tumor cell lines at micromolar-nanomolar level Spanò et al., 2020). [1,2]Oxazolo [5,4-e]isoindoles were further investigated in two additional cell lines derived from human diffuse malignant peritoneal mesothelioma (DMPM), showing a dosedependent inhibition of cell proliferation in both cellular models, with IC 50 values ranging from micromolar to nanomolar. The study of mechanism of action demonstrated the ability of [1,2]oxazole derivatives to impair cell cycle progression and induce apoptosis, as a consequence of the inhibition of tubulin polymerization. Selected derivatives, at well-tolerated doses, were able to significantly reduce tumor volume in a DMPM xenograft model (Spanò, Pennati, Parrino, Carbone, Montalbano, Cilibrasi, et al., 2016;.
On the basis of these observations, we wanted to examine the biological activity of these same compounds on AML cell line, HL-60, and its MDR variant HL-60R to assess their capacity to affect the MDR phenomenon. HL-60R was obtained by us through exposure of the sensitive cell line HL-60 with increasing doses of doxorubicin (Notarbartolo et al., 2002) and for this reason characterized by overexpression of P-gp, constitutive activation of the transcription factor NF-κB and as a consequence overexpression of inhibitor of apoptosis proteins (IAPs).  -1c, 2a-2j, 2l, 3a-3j, 3l, 4a-4j, 4l were prepared according to our previously published procedures (Barraja et al., 2009;Spanò, Pennati, Parrino, Carbone, Montalbano, Cilibrasi, et al., 2016;.
Vinblastine and Paclitaxel were purchased from Sigma (Sigma-Aldrich Srl), and all derivatives were dissolved in DMSO. 2.2.6 | P-gp ATPase activity determination P-gp ATPase activity was performed with Pgp-Glo™ Assay Systems (Promega) following manufacturer's instructions. The compounds 4e

| Cell growth assays
and 4k, at their IC 50s (test compound [TC]) were added to a 96-well white plate in duplicate and incubated with recombinant human P-gp membranes. The compound Na 3 VO 4 (0.25 mM) was used as the selective inhibitor of P-gp ATPase activity while negative control (no treatment [NT]) with only Pgp-GLO assay buffer was used to provide a measure of unregulated ATPase activity. Verapamil (0.5 mM) is a Pgp substrate that stimulates P-gp ATPase activity and represents the positive control for drug stimulation of P-gp ATPase activity. In fact, it is not a pure inhibitor of P-gp function as well as Na 3 VO 4 , but in association with another P-gp substrate like doxorubicin, it behaves as a competitive antagonist, that is, an efflux inhibitor. The ATP Standards curve serves to verify the correct execution of the assay.
MgATP (5 mM) was added to initiate the ATPase activity; after 40 min incubation at 37°C, the reaction was stopped with 50 µl ATPase Detection Reagent and then incubated for 20 min at room temperature.
Luminescence was measured using a GLOMAX Multidetection System (Promega). The data were presented as change in luminescence (ΔRLU), calculated as follows: ΔRLU basal is the difference between the average luminescent signals from Na 3 VO 4 -treated samples (RLU Na3VO4 ) and untreated (NT) samples (RLU NT ), ΔRLU TC that reflects P-gp ATPase activity in the presence of the test compounds, is the difference between the average luminescent signals from Na 3 VO 4 -treated samples (RLU Na3VO4 ) and test compound-treated samples (RLU TC ).

| Statistical analysis
Results are given as means ± SE. Statistical analysis was carried out by analysis of variance (one-way analysis of variance) followed by Tukey's test. Statistica ver. 12 (StatSoft Inc. 1984-2014 was used as software for the analyses.
Both key intermediates 3 are highly reactive toward dinucleophiles. Thus, ring closure leading to [1,2]oxazoles was achieved by reaction of intermediates 3 with hydroxylamine hydrochloride in refluxing ethanol to give the desired tricyclic derivatives 4 in good yield (57%−83%) (Scheme 1, Table 2).  (Table 3). Interestingly, it was observed that differently from the reference anti-mitotic drugs, all derivatives induced cell growth inhibition at similar concentration in both tumor cell lines.

| Anticancer activity
Notably, nontumorigenic cells are not affected by [1,2]oxazoles at their maximum concentration (μM) used in the MTS assay. The best results were obtained for derivative 4e, belonging to the 1,3-H substituted series and bearing a 3,4,5-trimethoxybenzyl group at the pyrrole nitrogen, which maintained nanomolar antiproliferative activity against both HL-60 and HL-60R cell lines.
Moreover, 4c and 4d belonging to the same series, bearing a 3methoxybenzyl and a 3,4-dimethoxybenzyl group on the pyrrole nitrogen respectively, were the second best showing antiproliferative activity at submicromolar level. Moving the methoxy group from position 3 to position 2 in the benzyl moiety (4b) or its removal (4a) generated a decrease of the activity but still at low micromolar level. Among [1,2]oxazoles bearing differently substituted phenyl substituents at position 6 of the tricyclic core system, the best activity was obtained for derivatives 4f, 4i, and T A B L E 1 Overview of α-substituted ketones 3a-3l 4j, bearing a 4-methoxybenzyl group at the pyrrole nitrogen, which showed comparable activity at submicromolar level. The introduction of a nitro group in position 3 of the benzyl moiety for the 6-phenyl derivative led to compound 4h, which maintains the activity in the submicromolar range (compare with 4f).
Compound 4g of the same series showed micromolar activity.
Decoration of the tricyclic system with a 3,4,5-trimethoxyphenyl group at position 6 led to a decrease of the activity at low micromolar level when the R group is a 3-nitro,4-methoxybenzyl (4l) and a 3,4-dimethoxybenzyl (4k). The only exception is for 4j probably have a tumor-specific action; myeloid leukemia cells are in fact much more sensitive and responsive to their antiproliferative action.
Finally, we analyzed the effects of all derivatives and VIN or PTX alone or in combination in the inhibition of cell growth by viable cell count with Trypan blue exclusion test, at sub-cytotoxic concentrations. Table 4 shows the percentages of cell growth inhibition obtained by co-treatment versus percentages expected.
IAPs are able to block apoptosis induced by different triggers including antitumor agents. For this reason, the overexpression of antiapoptotic proteins such as Bcl-2, or IAPs, for example, survivin, XIAP (X-Linked IAPs), IAP-1, is responsible for multi-drug resistance. The last result was corroborated by a substantial protein expression decrease of some antiapoptotic factors such as, XIAP, IAP-1, and Bcl-2, enhanced by the co-treatment of 4k and PTX ( Figure 2). In addition, we observed in some cases a significant increase of protein expression when HL-60R cells were treated with PTX or 4k alone. As reported by other authors, PTX, in fact, can cause an activation of NF-κB signaling and consequently an increase of its target such as XIAP (Aggarwal et al., 2005). For this reason, the results obtained highlight the importance of the synergic effects of cotreatments.

| Effects of compounds on P-gp activity
Since different behaviors were observed when [1,2]oxazolo isoindoles 4a-4l were tested in combination with PTX and VIN, we decided to further investigate if these differences could be due to specific alterations of P-gp function. Thus, it was To have a deeper insight into the mechanism of action of our compounds, effects of compounds 4e and 4k on P-gp ATPase activity were also evaluated by the P-gp-Glo™ assay, which detects a luminescent signal inversely proportional to the ATP consumption. Verapamil was used as positive control, being a substrate for transport by P-gp and stimulator of ATP-dependent drug efflux transporter. Only compound 4k produced a significative increase of verapamil-stimulated P-gp ATPase activity ( Figure 4).

| Antimitotic activity
On the basis of previous evidence ( Molinari et al., 1998).  (Notarbartolo et al., 2002(Notarbartolo et al., , 2004Sikic et al., 1997;Vanhoefer et al., 1996;Wang et al., 2016). Thus, the strong synergism of action of 4k in combination both with VIN and PTX in the induction of cell death caused by the hyperactivation of IAPs, appears to be a significant result.

T A B L E 5 Effects of compounds on intracellular accumulation of doxorubicin in HL-60R cell lines
Insight on the mechanism of action confirmed the antimitotic activity of 4k through inhibition of tubulin polymerization.
The potent antiproliferative effect confirmed against HL-60R cell line demonstrates that derivatives 4a-4l could further be explored as valuable tool to overcome MDR mechanism thus confirming the potentialities of the class of compounds and encouraging to conduct further studies.

Determination of doxorubicin accumulation was provided by ATeN
Center (University of Palermo). Open Access Funding provided by Universita degli Studi di Palermo within the CRUI-CARE Agreement.
F I G U R E 4 Effects of compounds 4e and 4k (at their IC 50s ) on verapamil-stimulated P-gp ATPase activity. The data are expressed as fold changes of P-gp ATPase activity compared to basal one (ΔRLU TC /ΔRLU basal ) and are presented as mean ± SE of three experiments, each in duplicate. Differences when treatments are compared to the basal activity, *p ˂ .05, **p < .01 (one-way analysis of variance followed by Tukey's test).
F I G U R E 5 Representative western blot showing the soluble (S) or polymerized (P) tubulin fraction in HL-60 and HL-60R cells. Cells were treated for 24 h with vinblastine (VIN), PTX, and 4k at the concentrations corresponding to the IC 50 at 72 h. Vinblastine and Paclitaxel were used as reference drugs due to their opposite mechanism of action on tubulin polymerization. The experiment repeated three times gave the same result.