New Imatinib Derivatives with Antiproliferative Activity against A549 and K562 Cancer Cells

Tyrosine kinase enzymes are among the primary molecular targets for the treatment of some human neoplasms, such as those in lung cancer and chronic myeloid leukemia. Mutations in the enzyme domain can cause resistance and new inhibitors capable of circumventing these mutations are highly desired. The objective of this work was to synthesize and evaluate the antiproliferative ability of ten new analogs that contain isatins and the phenylamino-pyrimidine pyridine (PAPP) skeleton, the main pharmacophore group of imatinib. The 1,2,3-triazole core was used as a spacer in the derivatives through a click chemistry reaction and gave good yields. All the analogs were tested against A549 and K562 cells, lung cancer and chronic myeloid leukemia (CML) cell lines, respectively. In A549 cells, the 3,3-difluorinated compound (3a), the 5-chloro-3,3-difluorinated compound (3c) and the 5-bromo-3,3-difluorinated compound (3d) showed IC50 values of 7.2, 6.4, and 7.3 μM, respectively, and were all more potent than imatinib (IC50 of 65.4 μM). In K562 cells, the 3,3-difluoro-5-methylated compound (3b) decreased cell viability to 57.5% and, at 10 µM, showed an IC50 value of 35.8 μM (imatinib, IC50 = 0.08 μM). The results suggest that 3a, 3c, and 3d can be used as prototypes for the development of more potent and selective derivatives against lung cancer.


Introduction
Protein tyrosine kinases (PTKs) are a group of approximately 90 enzymes that play essential roles in cells, such as in the regulation of cell division, cell differentiation and morphogenesis [1]. PTKs can be divided into receptor tyrosine kinases (RTKs) and nonreceptor tyrosine kinases (NRTKs). Examples of RTKs and NRTKs are insulin receptors and growth factor receptors (GFRs), such as epidermal growth factor receptor (EGFR) and ABL1, respectively [2]. The deregulated action of these PTKs is directly related to the development of some types of cancer [3].
PTK BCR-ABL1 is not expressed in healthy organisms because it is a product of cellular deregulation, and this PTK has been described as an oncogene that is present in 95% of patients with chronic myeloid leukemia (CML) [4]. The discovery of the relationship between this TK and CML made the development of imatinib (1) possible, as the first drug to be used against the PTK BCR-ABL1, which revolutionized the treatment of CML [5]. This drug acts by competitively inhibiting ATP binding to the BCR-ABL1 enzyme, preventing substrate phosphorylation, blocking its activity, and avoiding transduction of the signals essential for cellular functions. However, the emergence of cases of resistance to this drug has shown the need to develop new second-and third-generation inhibitors. However, even these new tyrosine kinase inhibitors (TKIs) have shown many cases of resistance, highlighting the need for a continuous search for new compounds that can treat these resistant tumors [6][7][8].
EGFR is a PTK receptor, also known as ErbB1/HER1, that belongs to the ErbB family, which also includes ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER42. EGFR participates in cell proliferation and apoptosis and has been classified as a proto-oncogene because it is commonly seen in cancers, such as non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer and breast cancer [9]. EGFR is a key mediator that plays a crucial role in cell growth, proliferation, survival and migration. This protein belongs to the kinase family and, in recent years, the discovery of the relationship between the overexpression of EGFR and solid tumors has made EGFR a target in modern medicinal chemistry for the planning of new anticancer agents [10].
The demand imposed by frequent mutations occurring in the kinase domain is still among the greatest limitations to treatment by target therapy, especially in oncology [11]. Therefore, it is imperative to develop new TKIs that can reduce both known resistances and others that may arise.
By continuing the work that the group has been developing to obtain new imatinib analogs [12], in this work, we synthesized a new series of ten imatinib analogs (2a-e and 3a-e, Figure 1) and evaluated their cytotoxic activity in two human cancer cell lines: K562 (CML) and A549 (lung cancer). The compounds were designed as molecular hybrids containing phenylamino-pyrimidine pyridine (PAPP, in blue) and isatin (in red) scaffold units, connected by a 1H-1,2,3-triazole ring (in purple) (Figure 1). PAPP is an important pharmacophoric fragment of imatinib [13,14]. Isatins and some derivatives have relevance as TKIs [15], as shown for the cytotoxic activity of compound 4 against K562 cells, a cell lineage that has the constitutive activity of the TK BCR-ABL1. Compound 4 showed an IC 50 of 0.03 µM, which was comparable to that obtained with the standard drug, irinotecan (CPT-11, IC 50 = 0.07 µM) (Figure 1) [16]. Moreover, isatin scaffolds are present in sunitinib (5) and nintedanib (6), which are important TKIs. In derivatives 2a-e and 3a-e, the isatins have different substituents, which were chosen to verify their electronic contributions as electron attractor (Cl, Br, F) and electron donor (CH 3 ) groups. The 1H-1,2,3-triazole ring was used as a spacer between the two units, PAPP and isatin, since this nucleus has been described in compounds with potential TK inhibitory activity, as in compound 7 [17].
The cell lines used herein (K562 and A549) are sensitive to imatinib. Although there is a vast literature showing the cytotoxicity of imatinib analogs in K562 cells, little is known about their effects in the A549 cell line. Shijie and coworkers showed that imatinib and its derivatives presented good inhibitory results in A549 cells. An example is compound 8, which has a PAPP skeleton and decreased cell viability to 38.5% at a concentration of 150 µM (Figure 1) [18]. This result encouraged us to also test our compounds against this cell line.

Chemistry
Ten new hybrids presenting the PAPP group and isatin or derivatives spaced with a 1H-1,2,3-triazole ring were synthesized, as shown in Scheme 1.
The fluorination of compounds 11a-e with diethylaminosulfurtrifluoride (DAST) in CH 2 Cl 2 at 25 • C produced intermediates 13a-e after 4 h, with a 68-88% yield. The IR spectra of intermediates 13a-e showed absorption bands in the region from 3200 to 3271 cm −1 , corresponding to the axial strain of the NH bond, and stretches between 1268 and 1335 cm −1 , corresponding to the axial strain of the CF 2 bond [19]. Spectroscopic data of intermediates 13a-d have already been described in the literature, but that of the intermediate 13e is unpublished. The 1 H NMR spectrum of intermediate 13e showed a singlet at 11.20 ppm corresponding to a N-H hydrogen. In the 13 C NMR spectrum, a triplet at 165.7 ppm with J = 29 Hz, corresponding to a carbonyl carbon and a triplet at 110.8 ppm with J = 247. 9 Hz, corresponding to the C-3 carbon were observed, and this multiplicity is due to carbon-fluorine coupling with the difluoromethylene group. The 19 F NMR spectrum showed a chemical shift at −111.1 ppm, corresponding to CF 2 , and the peak at 119.0 ppm corresponded to the C-F connection.
Gem-difluorinated intermediates 14a-e were prepared at a 52-94% yield by the N-alkylation of compounds 13a-e. The IR spectra of intermediates 14a-e showed absorption bands in the region from 1276 to 1335 cm −1 , corresponding to the axial deformation of CF 2 . For compounds 14d-e, NMR analyses were performed, as they have not yet been described in the literature. In the 1 H NMR analysis of intermediates 14d and 14e, it is possible to observe a signal at 7.30 or 7.37 ppm, related to H-7, and a doublet at 4.60 or 4.61 ppm, with a coupling constant of 2.5 Hz for H-1 . In the 13 C NMR analyses of the intermediates 14d-e, signals corresponding to C-1', C-2' and C-3' were observed at 29.5 ppm, 76.5 ppm, and between 75.5 and 75.6 ppm, respectively. The 19 F NMR spectrum showed chemical shifts between −110.6 and −110.7 ppm corresponding to CF 2 .
The 1,3-dipolar cycloaddition reaction between the azide derivative (10) and the terminal alkynes 12a-e and 14a-e, was performed via click chemistry conditions, using sodium ascorbate and copper sulfate in THF/H 2 O (1:1) at room temperature for 3 h, to obtain only one 1,4-regioisomer of the final products 2a-e and 3a-e, respectively.
New hybrids 2a-e and 3a-e were obtained in good yields, although some unsatisfactory yields may be associated with the low solubility of the final compounds, making their purification process difficult. The IR spectra for compounds 2a-e showed absorption bands between 3383 cm −1 and 3440 cm −1 , corresponding to the axial deformation of the N-H bond; 1736 and 1739 cm −1 , corresponding to the axial deformation of the ketone carbonyl; and 1579 and 1618 cm −1 , corresponding to the deformation of an amylic carbonyl. Compounds 3a-e showed absorption bands between 1284 and 1298 cm −1 , corresponding to the axial deformation of the CF 2 bond. The 1 H NMR analysis of compounds 2a-e and 3a-e showed that methylene hydrogens were present and simple, with displacements between 2.33 and 2.43 ppm (see Supplementary Materials). The CH 2 hydrogens presented as simple with chemical shifts between 5.04 and 5.10 ppm. The hydrogens of the triazole ring characteristic of these compounds were observed as singlets in the region of 8.02 to 8.89 ppm (see Supplementary Materials).

In Vitro Biological Evaluation
Biological evaluations of compounds 2a-e and 3a-e were performed in K562, A549 and WSS-1 cells. As previously described, K562 is a CML cell line, A549 is a human pulmonary carcinoma epithelial cell line, and WSS-1 is a healthy human cell line. WSS-1 cells were used as a reference for the calculation of the selectivity index (SI).
In WSS-1 cells, 3a, 3c, and 3d showed IC50 values of 11.6, 13.5 and 18.6 µM (imatinib, IC50 = 9.6 µM) and SI values of 1.6, 2.1 and 2.5, respectively, and were up to 25-fold more selective than imatinib (SI = 0.1). Thus, in A549 cells, the new compounds 3a, 3c and 3d were more potent and selective than imatinib ( Figure 3 and Table 2). Imatinib was used as a standard, even though it is not a drug used to treat lung cancer. This was due to the good results obtained by Shijie and coworkers, which decreased A549 cells viability to 38.8% at 150 µM ( Figure 1) [18]. In addition, the literature states that imatinib can be used as a potential treatment for NSCLC, as it was able to inhibit the growth of A549 cells with an IC50 value in the range of 2-3 µM [20].  (Table 1). In WSS-1 cells, compound 3b showed an IC50 of 69.3 µM (imatinib, IC50 = 9.6 µM), resulting in an SI value of 1.9 (imatinib, SI = 120) ( Figure 2 and Table 1).  * CI-95% confidence interval; SI = IC50 (WSS-1)/IC50 (cancer cell).

Cytotoxic Effects in A549 and WSS-1 Cells
The evaluations performed with A549 cells showed that compounds 3a, 3c, and 3d reduced cell viability by 24.6%, 34.0%, and 49.3% at 10µM, respectively ( Figure 3). Subsequently, the concentration-response curves were constructed, and derivatives 3a, 3c, and 3d exhibited IC 50 values of 7.2 µM, 6.4 µM, and 7.3 µM, respectively, proving to be approximately 10-fold more potent than imatinib (IC 50 = 65.4 µM) ( Table 2).  Table 2 shows the IC50 and SI values of imatinib and its most cytotoxic analogs (3a, 3c and 3d) in A549 cells. Table 2. Cytotoxic activity and selectivity index of imatinib and derivatives 3a, 3c and 3d in the   Figure 3. Screening of imatinib and its derivatives 2a-e and 3a-e at a concentration of 10 µM against human cell lines A549 (purple) and WSS-1 (green). Bars represent the mean ± standard deviation. Table 2 shows the IC50 and SI values of imatinib and its most cytotoxic analogs (3a, 3c and 3d) in A549 cells. The study of the relationship between the structure and the biological activity of the synthesized compounds showed the importance of the CF2 group since, in compounds 2a-e, the absence of this group showed a loss of activity. In addition, according to the biological tests performed, the intrinsic characteristics of the substituents on the C-5 carbon of the isatin-derived ring do not seem to influence the level of activity.  Figure 3. Screening of imatinib and its derivatives 2a-e and 3a-e at a concentration of 10 µM against human cell lines A549 (purple) and WSS-1 (green). Bars represent the mean ± standard deviation. Table 2 shows the IC50 and SI values of imatinib and its most cytotoxic analogs (3a, 3c and 3d) in A549 cells. The study of the relationship between the structure and the biological activity of the synthesized compounds showed the importance of the CF2 group since, in compounds 2a-e, the absence of this group showed a loss of activity. In addition, according to the biological tests performed, the intrinsic characteristics of the substituents on the C-5 carbon of the isatin-derived ring do not seem to influence the level of activity.  Figure 3. Screening of imatinib and its derivatives 2a-e and 3a-e at a concentration of 10 µM against human cell lines A549 (purple) and WSS-1 (green). Bars represent the mean ± standard deviation. Table 2 shows the IC50 and SI values of imatinib and its most cytotoxic analogs (3a, 3c and 3d) in A549 cells. The study of the relationship between the structure and the biological activity of the synthesized compounds showed the importance of the CF2 group since, in compounds 2a-e, the absence of this group showed a loss of activity. In addition, according to the biological tests performed, the intrinsic characteristics of the substituents on the C-5 carbon of the isatin-derived ring do not seem to influence the level of activity. In WSS-1 cells, 3a, 3c, and 3d showed IC 50 values of 11.6, 13.5 and 18.6 µM (imatinib, IC 50 = 9.6 µM) and SI values of 1.6, 2.1 and 2.5, respectively, and were up to 25-fold more selective than imatinib (SI = 0.1). Thus, in A549 cells, the new compounds 3a, 3c and 3d were more potent and selective than imatinib ( Figure 3 and Table 2). Imatinib was used as a standard, even though it is not a drug used to treat lung cancer. This was due to the good results obtained by Shijie and coworkers, which decreased A549 cells viability to 38.8% at 150 µM (Figure 1) [18]. In addition, the literature states that imatinib can be used as a potential treatment for NSCLC, as it was able to inhibit the growth of A549 cells with an IC 50 value in the range of 2-3 µM [20]. Table 2 shows the IC 50 and SI values of imatinib and its most cytotoxic analogs (3a, 3c and 3d) in A549 cells.
The study of the relationship between the structure and the biological activity of the synthesized compounds showed the importance of the CF 2 group since, in compounds 2a-e, the absence of this group showed a loss of activity. In addition, according to the biological tests performed, the intrinsic characteristics of the substituents on the C-5 carbon of the isatin-derived ring do not seem to influence the level of activity.

Kinase Inhibition Assay
The compounds 2a-e and 3a-e did not show ABL1-inhibitory activity at 0.5 or 10 µM under the given assay conditions. A possible explanation for this result is that the compounds may have a different mechanism of action from imatinib, which showed a subse-Molecules 2022, 27, 750 7 of 14 quent percentage inhibition at 0.5 and 10 µM in the same assay. Another hypothesis could be a probable higher affinity of the substrate to the enzyme than the analogs, which may have interfered with the interaction of the latter with this kinase. Thus, more experiments are needed to characterize the mechanism of these compounds, by varying the enzyme inhibition assay conditions.

Chemistry
All reagents and solvents used were of analytical grade. Briefly, 1 H, 13 C and 19 F nuclear magnetic resonance (NMR) spectra were generated at 400.00, 100.00 and 376.00 MHz, respectively, at 25 • C using a Bruker Avance III HD instrument (Bruker AG, Fällanden, Switzerland) equipped with a prodigy BBO 400 S1 probe (Bruker AG, Fällanden, Switzerland). Tetramethylsilane was used as an internal standard. The chemical shifts (δ) are reported in ppm, and the coupling constants (J) are reported in Hertz. Fourier transform infrared (FTIR) absorption spectra were recorded on a Thermo Scientific spectrophotometer (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA). The melting point (m.p.) values were determined using a Büchi model B-545 apparatus (Büchi Corporation, Flawil, Switzerland). Thin-layer chromatography (TLC) was performed using Merck TLC silica gel 60 F254 aluminum sheets (Merck KGaA, Darmstadt, Germany), 20 × 20 cm (eluent hexane/ethyl acetate 2:8). A mass spectrometry system was coupled to obtain electron impact gas chromatography (MS-GC) spectra at 70 eV on an Agilent 6890 apparatus, with an Agilent 5973 mass spectrometer (Agilent, Santa Clara, CA, USA). Low-resolution mass spectra were obtained by electrospray ionization (MS-ESI) on a Micromass ZQ4000 apparatus (Waters, Milford, MA, USA). High-resolution mass spectra (HR-MS) were registered using electron ionization mass spectrometry (EI-MS, digitalizing ES + capillary) on a QTOF Compact (Brucker AG, Fällanden, Switzerland). (1:1) were added to a monotubulated flask. The reaction was kept under magnetic stirring at room temperature for 3 h. The progress of the reaction was monitored using TLC (hexane/ethylacetate 3:7). On completion, 30 mL of water was added and the mixture was extracted with CHCl 3 (3 × 50 mL). The organic phase was dried with anhydrous Na 2 SO 4, and the solvent was removed by evaporation. The residual crude product was purified via silica gel column chromatography, using the gradient mixture of chloroform: methanol (9.5:0.5).
The attributes can be summarized as follows:  . On completion, 30 mL of water was added, and the mixture was extracted with CHCl 3 (3 × 50 mL). The organic phase was dried with anhydrous Na 2 SO 4 , and the solvent was removed by evaporation. The residual crude product was purified via silica gel column chromatography using the gradient mixture chloroform: methanol (9.5:0.5).
The attributes can be summarized as follows: