Imidazo[1,2-b]pyrazole-7-carboxamides Induce Apoptosis in Human Leukemia Cells at Nanomolar Concentrations

Leukemia, the malignancy of the hematopoietic system accounts for 10% of cancer cases with poor overall survival rate in adults; therefore, there is a high unmet medical need for the development of novel therapeutics. Eight imidazo[1,2-b]pyrazole-7-carboxamides have been tested for cytotoxic activity against five leukemia cell lines: Acute promyelocytic leukemia (HL-60), acute monocytic leukemia (THP-1), acute T-lymphoblastic leukemia (MOLT-4), biphenotypic B myelomonocytic leukemia (MV-4-11), and erythroleukemia (K-562) cells in vitro. Imidazo[1,2-b]pyrazole-7-carboxamides hampered the viability of all five leukemia cell lines with different potential. Optimization through structure activity relationship resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Human primary fibroblasts were much less sensitive in the applied concentration range. Both monolayer or spheroid cultures of murine 4T1 and human MCF7 breast cancer cells were less sensitive to treatment with 1.5–10.8 μM IC50 values. Flow cytometry confirmed the absence of necrosis and revealed 60% late apoptotic population for MV-4-11, and 50% early apoptotic population for HL-60. MOLT-4 cells showed only about 30% of total apoptotic population. Toxicogenomic study of DU385 on the most sensitive MV-4-11 cells revealed altered expression of sixteen genes as early (6 h), midterm (12 h), and late response (24 h) genes upon treatment. Changes in ALOX5AP, TXN, and SOD1 expression suggested that DU385 causes oxidative stress, which was confirmed by depletion of cellular glutathione and mitochondrial membrane depolarization induction. Imidazo[1,2-b]pyrazole-7-carboxamides reported herein induced apoptosis in human leukemia cells at nanomolar concentrations.


Introduction
Leukemias and lymphomas accounts for almost 10% of cancer cases worldwide [1]. Leukemias are heterogeneous diseases due to differential cellular origin along with self-characteristic genetic abnormalities [2]. The discussion of current therapies is beyond the scope of the present article. Although bone marrow transplantation reached a break-through in the last decades for the treatment of childhood leukemias, chemotherapy remains the main option for adults, and especially for the elderly with much less therapeutic success nowadays. Based on the maturation status, differentiation level, and/or lineage commitment acute myeloid leukemia cells (AML) have been classified by the French-American-British classification (FAB) system [3][4][5]. To determine the specificity of imidazo[1,2-b]pyrazole-7-carboxamides as potential anti-leukemic agents we have focused on the following differentially matured AML cells: HL-60 acute promyelocytic leukemia, (FAB M2) [6,7], MV-4-11 biphenotypic B myelomonocytic leukemia (FAB M4) [8,9], THP-1 acute monocytic leukemia (FAB M5) [10,11], K-562 erythroleukemia, (FAB M6) [12,13]. One acute lymphoblastic leukemia cell line (ALL), the MOLT-4 acute T-lymphoblastic leukemia, was also involved in the study.
Pyrazole derivatives are pharmacologically relevant active scaffolds in diverse therapeutic fields, reviewed recently by Karrouchi et al. [14]. Moreover, the synthesis and the anticancer activity of N-fused pyrazoles, such as imidazo[1, 2-b]pyrazoles have also been reported [15,16]. We have recently published the synthesis of a library (67 compounds) of novel imidazo[1,2-b]pyrazole-7-carboxamides, where among different cell lines, HL-60 leukemia cells showed the highest sensitivity upon treatment [17]. Based on the previous structure-activity relationship (SAR) experience, hit molecules have been further optimized by changing certain substituents to increase the cytotoxic effect against different leukemia cells. These efforts resulted in more active compounds with anti-leukemic potential reported first in this study.

Imidazo[1,2-b]pyrazole-7-carboxamides Hampered the Viability of Leukemia Cells with Different Potential
The substitutions chosen were based on the previously established structure-activity relationship (SAR) results [17], with the goal to design compounds with improved anti-leukemic effect. Seven compounds out of eight hampered the viability of all five tested leukemia cell lines with different potential. Superior efficiency was obtained for DU325 and DU385 (Table 1). The presence of tBu as a bulky group in the R 2 position in combination with 4-NH 2 and 4-OH moieties in the R 1 determined the strength of anti-leukemic efficiency. In addition, the hydroxyl moiety located in para position on the aromatic ring (R 1 = 4-OH) provided the highest cytotoxicity (DU385). Optimization

Imidazo[1,2-b]pyrazole-7-carboxamides Hampered the Viability of Leukemia Cells with Different Potential
The substitutions chosen were based on the previously established structure-activity relationship (SAR) results [17], with the goal to design compounds with improved anti-leukemic effect. Seven compounds out of eight hampered the viability of all five tested leukemia cell lines with different potential. Superior efficiency was obtained for DU325 and DU385 (Table 1). The presence of tBu as a bulky group in the R 2 position in combination with 4-NH2 and 4-OH moieties in the R 1 determined the strength of anti-leukemic efficiency. In addition, the hydroxyl moiety located in para position on the aromatic ring (R 1 = 4-OH) provided the highest cytotoxicity (DU385). Optimization resulted in the following IC50 values for the most effective lead compound DU385: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11, respectively (Table 1).
In the case of DU441, incorporating a tOctyl function in R 2 position, demonstrated an excellent anti-leukemic activity (60.48 nM IC50 value for MOLT-4). However, the tested DU443 with the same R 2 substituent did not show a similar result (104.2 nM IC50 value for MOLT-4) (Table 1). Accordingly, the substitution of R 1 = OH to NH2 (if R 2 = tert-Octyl) decreased the measured biological effects (DU443). Incorporating an EWG group in the framework (R 1 = 4-F; DU283) led to less active antitumor behavior (146.2 nM IC50 value for MOLT-4) ( Table 1).
On the other hand, all 4-OH-phenyl substituted carboxamides proved to be very efficient against leukemia cell lines independent from the quality of R 2 group. Each aliphatic functionalized, tBu,   (Table 1).
Interestingly, the anti-leukemic behavior was diminished in those where a hydroxyl group was in ortho or meta position (DU455 and DU456). The effect on human primary fibroblasts used as control cells were much less cytotoxic reducing the viability maximum to 60%, suggesting that the compounds reported herein were not as much as cytotoxic to non-malignant primary cells compared to leukemia cells (Table 1, Figure S7).
Cytotoxicity was further investigated on 4T1 murine mammary carcinoma and MCF7 human breast adenocarcinoma cancer cells to determine whether the cytotoxic effect was specific against leukemia cells or the compounds possessed a general antitumor effect. The four selected compounds diminished the viability of both 4T1 and MCF7 cells in micromolar concentrations irrespective of the culture method (minimum IC 50 = 1.559 µM of DU442 on MCF7 2D, maximum IC 50 = 10.8 µM of DU325 on 4T1 3D ( Table 2). The micromolar effective dose is not pharmacologically feasible compared to the nanomolar activity against leukemias, so imidazo[1,2-b]pyrazole-7-carboxamides were considered as potential anti-leukemic drug candidates instead of general antitumoral compounds. Cells were treated with imidazo[1,2-b]pyrazole-7-carboxamides in different concentrations (0.625 µM, 1.125 µM, 2.50 µM, 5 µM, 10 µM, 20 µM) in duplicates for 72 h. Viability was examined by resazurin assay as described in Section 4.4. Materials and Methods. Dose response curves can be found in Figure S8.

Toxicogenomic Data upon Treatment by Imidazo[1,2-b]pyrazole-7-carboxamide DU385
To have a deep insight into the disturbance of cellular homeostasis of leukemic cells due to treatment by imidazo[1,2-b]pyrazole-7-carboxamides we have designed a toxicogenomic panel for a gene expression study (Table S1). The most sensitive MV-4-11 cells were treated by the most active lead compound DU385 as described in the Section 4.6 Materials and Methods. Functional categories of the investigated genes have been listed in Table S2. The toxicogenomic panel consists of genes

Toxicogenomic Data upon Treatment by Imidazo[1,2-b]pyrazole-7-carboxamide DU385
To have a deep insight into the disturbance of cellular homeostasis of leukemic cells due to treatment by imidazo[1,2-b]pyrazole-7-carboxamides we have designed a toxicogenomic panel for a gene expression study (Table S1). The most sensitive MV-4-11 cells were treated by the most active lead compound DU385 as described in the Section 4.6 Materials and Methods. Functional categories of the investigated genes have been listed in Table S2. The toxicogenomic panel consists of genes which have been previously reported by Puskas et al. [18] and Zhang et al. in connection with drug induced cytotoxicity: Among others EGR1, GDF15, ATF3, FGF21 [19]. The expression of genes, published by Wang et al. involved in the differentiation and cytotoxicity of HL-60 cells induced by retinoids was also monitored in our study: SLC21A3, LGALS1, LBR, ALOX5AP, TXN, UBC, BCL2A1, CALR [20]. We implemented genes from our previous toxicogenomic study: ANXA2, GADD153, HSPA1A, SOD1 [21]. G-protein coupled receptor 84 (GPR84), a key player of β-catenin mediated signaling maintaining leukemogenesis was also investigated [22]. Finally, the expression of interleukin-6 (IL6) and tumor necrosis factor alpha (TNF) inflammatory cytokines was investigated [23,24].
The determined temporal pattern of differential expression divided the tested genes into three groups as: Early (6 h) ( Figure 3A), midterm (12 h) ( Figure 3B), and late response (24 h) genes upon treatment ( Figure 3C).

Imidazo[1,2-b]pyrazole-7-carboxamide DU385 Exerted Oxidative Stress of MV-4-11 Cells
Changes in the gene expression of oxidative stress-related genes (ALOX5AP, TXN, SOD1) opened the way for the investigation of cellular glutathione (GSH) level upon treatment. The luminescence in the assay is proportional with the GSH level, which can be reduced by reactive oxygen species (ROS) via oxidation or reaction with the thiol group [25]. Hydrogen peroxide validating the assay as a positive control was effective to decrease the GSH level prominently in 1 mM after 6, 12, and 24 h ( Figure 4A). The imidazo[1,2-b]pyrazole-7-carboxamide, DU385, was effective to generate ROS and the loss of GSH after 24 h to the 75% or 50% of the untreated cells with 40 nM and 200 nM treatment, respectively.  Since oxidative stress may subsequently confound mitochondrial homeostasis, the mitochondrial membrane potential was measured by JC-1 assay. After 24 h, the percentage of cells with decreased mitochondrial membrane potential three and four times increased after the treatment with 40 nM or 200 nM DU385, respectively ( Figure 4B, Figure S12).

Discussion
We have shown the anti-leukemic effect of imidazo[1,2-b]pyrazole-7-carboxamides ( Figure 1 and Figure S1) using the resazurin viability assay (Table 1, Figures S2-S6). The dose-response curves of the lead compound DU385 determined the following IC 50 values: 16.54 nM, 27.24 nM, and 32.25 nM on HL-60, MOLT-4, MV-4-11 cells, respectively. Importantly, human primary fibroblasts were much less sensitive in the applied concentration range (12.3 nM-3 µM) suggesting selective cytotoxic effect, especially on leukemia cells (Table 1, Figure S7). Since carcinoma cells establish solid tumors in vivo, breast cancer cells were grown as three-dimensional (3D) spheroids compared to traditional tissue culture dishes (2D) (Table 2, Figure S8). Monolayer cultures ignore the role of microenvironmental factors, such as structural niche, with the plethora of molecular and cellular constituents [26]. Three dimensional cell cultures as drug discovery models better represent the in vivo situation like the partial oxygen tension, local pH, or extracellular matrix composition [27]. Although monolayer cultures were much sensitive to treatment, the effect was much lower (between 1.5-10.8 µM IC 50 values, Table 2) both in 2D and 3D models of murine 4T1 or human MCF7 breast carcinoma cells, compared to the nanomolar IC 50 values in leukemias (Table 1).
Incorporating R 1 hydroxyl group in para position with a tBu (DU385), tOctyl (DU441), or Cy (DU442) functions in R 2 position, demonstrated an excellent anti-leukemic activity. Moreover, R 1 para NH 2 group combined with R 2 tOctyl (DU325) had an anti-leukemic effect at nanomolar concentration. This potent anti-leukemic activity was diminished in those compounds where the R 1 hydroxyl group was in ortho or meta position (DU455 and DU456), or the R 2 tOctyl function was combined with the R 1 NH 2 group (DU443). Incorporating an EWG group in the framework (R 1 = 4-F; DU283) led to a less active analog. All four selected compounds, DU325, DU385, DU441, and DU442 induced apoptosis of biphenotypic B myelomonocytic leukemia MV-4-11, acute promyelocytic leukemia HL-60, and the T-lymphoblastic leukemia MOLT-4 cells with different potential (Figure 2 and Figures S9-S11). None of the compounds induced necrosis. Myeloid cells were more sensitive to imidazo[1,2-b]pyrazole-7-carboxamides than the lymphoid MOLT-4, regarding the apoptotic process detected by flow cytometry.
Changes in the gene expression of oxidative stress-related genes (ALOX5AP, TXN, SOD1) initiated the investigation of cellular GSH level. The amount of intracellular GSH dropped upon DU385 treatment only after 24 h. The consistency in the GSH level with DU385 treatment after 6 and 12 h may indicate that the generation of ROS is not a fast stress response (like to H 2 O 2 ), rather a well-organized process after 24 h. Since oxidative stress induces mitochondrial dysfunction, and disturbance in the mitochondrial membrane potential can lead to apoptosis [34], the MMP was measured upon treatment by JC-1 assay. We have shown that mitochondria of MV-4-11 leukemia cells were depolarized when treated with 40 nM or 200 nM DU385 after 24 h.
Since leukemia cells used in this study were immature cells, malignant cells of myeloid or lymphoid precursors, authors may speculate that cells differentiate after treatment and the mitochondrial pathway of apoptosis is followed after differentiation (Figure 4). Granulation (side scatter = SSC) of the treated MV-4-11 ( Figures S9 and S12), HL-60 ( Figure S10) and MOLT-4 ( Figure S11) cells increased after treatment, which proposes the differentiation concept. Apoptosis followed after differentiation to chemotherapy has already been published for immature leukemias [35][36][37], e.g., all-trans retinoic acid (ATRA) acts via that mechanism, which is applied in the clinical protocols [38]. Authors hypothesize that human fibroblasts and mouse 4T1 or human MCF7 breast carcinoma cells are less sensitive to DU385, probably because these are already differentiated cells with less apoptotic susceptibility or more complex anti-apoptotic mechanisms. Anyhow, further research is needed to reveal the cause of the sensitivity of immature leukemias to imidazo[1,2-b]pyrazole-7-carboxamide DU385.

Ethical Statement
Participant informed consent was obtained prior to surgical intervention for the isolation of human primary fibroblasts. All tissue collection complied with the Guidelines of the Helsinki Declaration and was approved by the Regional and Institutional Research Ethics Committee (2799, 3517).

Skin Biopsies and Cell Culture of Human Primary Fibroblasts
Healthy volunteers (age 18-60 years) were enrolled into the study. The punch biopsies were taken from healthy subjects from the breast area undergoing plastic surgery. Primary fibroblasts were obtained from the skin by enzymatic digestion according to a standard protocol. Briefly, skin specimens were first washed in Salsol A solution (Human Rt, Gödöllő, Hungary) supplemented with 2% antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA). Skin samples were then cut into narrow strips and incubated in Dispase solution (Roche Diagnostics, Mannheim, Germany) overnight at 4 • C. The epidermis was subsequently separated from the dermis. Fibroblasts were obtained by incubating the dermis in Digestion Mix solution (Collagenase, Hyaluronidase and Deoxyribonuclease) for 2 h at 37 • C. Cell suspensions were filtered through a 100 µm nylon mesh (BD Falcon, San Jose, CA, USA), and cells were pelleted by centrifugation (Thermo Fisher Scientific, Waltham, MA, USA, Megafuge 16). Fibroblasts were grown in low glucose DMEM/F12 medium containing 15% FCS, 1% antibiotic/antimycotic (PAA, Pasching, Austria) and 1% L-glutamine solution (PAA). Fibroblasts were cultured at 37 • C and 5% CO 2 in humidified conditions. Depending on the cell growth, the medium was changed every 2-4 days, and cells were passaged at 80% of confluence.

Cell Culturing, 3D Spheroid Formation and Treatments
The following cells were purchased from the American Type Culture Collection (ATCC, Manassas, WV, USA): HL-60 acute promyelocytic leukemia, THP-1 acute monocytic leukemia, MOLT-4 acute T-lymphoblastic leukemia, MV-4-11 biphenotypic B myelomonocytic leukemia, and K-562, erythroleukemia maintained in RPMI 10% FCS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) using tissue culture dishes (Corning Life Sciences, Corning, NY, USA). The human breast adenocarcinoma MCF-7 and mouse mammary carcinoma 4T1 cells were also purchased from the ATCC. The MCF-7 cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) 10% fetal calf serum (FCS, Gibco), and the 4T1 were maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640) with 10% FCS. The pH of the cell culture media was controlled to be between 7.2-7.4 prior use. All used media were supplemented with 2 mM GlutaMAX, and 100 U/mL penicillin, 100 µg/mL streptomycin (Life Technologies, Carlsbad, CA, USA) before use. Cells were passed every three days and placed in a humidified incubator at 37 • C 5% CO 2 (Sanyo, Osaka, Japan).
The 3D spheroid production using pellet culture system was previously described by Johnstone, B et al. [39]. Briefly, the MCF-7 and 4T1 cells were gently detached from the conventional tissue culture flasks using trypsin, washed with PBS, counted, and then suspended to have 6000 cells in 100 µL medium per well. The cellular suspensions were dispended into cell repellent, U shaped CellStar ® 96 microplates (Cellstar ® Cell-Repellent Microplate, Greiner Bio-One, Kermsmünster, Austria) and centrifuged at 1200 g for 10 min. After spheroid formation (one spheroid/well), the spherical aggregates were directly used for viability assay without any detaching procedures and transfer steps. The cell repellent plates were incubated and maintained together with conventional 2D cell culture plates at 37 • C in a humidified incubator in an atmosphere of 5% CO 2 (Sanyo).
Compounds were dissolved in dimethyl sulfoxide (DMSO) at 10 mM concentration freshly before being used. Since DMSO can be toxic for cellular systems above 1%, the stock solution was further diluted in serial dilutions in all cases in the appropriate cell culture media.

Detection of Phosphatidylserine Exposure
Apoptosis was measured by flow cytometry as described previously in References [40,41] Table S1. The PCR protocol was as follows: Enzyme activation at 95 • C for 2 min, 45 cycles of denaturation at 95 • C for 10 s, annealing at 60 • C, and extension at 60 • C for 10 s. All the PCRs were performed with three replicates. After amplification, the melting curve was checked to verify the specificity of the PCR reactions. The Ct values were normalized to GAPDH gene for each time point. The presented relative gene expression ratios were ∆∆CT values (log 2 ). All values were presented as mean ± standard deviation (SD).
Mitochondrial membrane potential was measured as described previously in Reference [44]. Briefly, MV-4-11 cells (2 × 10 5 ) were plated in 24-well tissue culture plates (Corning Life Sciences, Corning, NY, USA) in RPMI 10% FCS and were treated in 500 µL media containing 40 nM or 200 nM DU385 compound. Untreated controls cells were supplemented with 500 µL cell culture media.

Statistical Analysis
Statistical analysis was performed using two-tailed, heteroscedastic Student s t-test to evaluate the statistical significance (set at * p< 0.05, ** p < 0.01, *** p < 0.001) between two given experimental groups: Pairwise comparison of each sample to the untreated control.

Statistical Analysis
Statistical analysis was performed using two-tailed, heteroscedastic Student′s t-test to evaluate statistical significance (set at * p< 0.05, ** p < 0.01, *** p < 0.001) between two given experimental ps: Pairwise comparison of each sample to the untreated control.