Inhibition of FLT3-ITD Kinase in Acute Myeloid Leukemia by New Imidazo[1,2-b]pyridazine Derivatives Identified by Scaffold Hopping

FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.


■ INTRODUCTION
Acute myeloid leukemia (AML) is a malignant clonal disorder of the hematopoietic system characterized by infiltration of the bone marrow, peripheral blood, and other tissues by abnormally differentiated blasts of myeloid lineage. 1,2Although AML can occur at any age, it is the most common acute type of leukemia in adults and increases in incidence with age.The five-year overall survival of patients diagnosed with AML is estimated to be less than 50%. 3Despite the growing number of drugs available for the treatment of AML, the need for efficient therapeutic strategies persists.Advances in molecular cancer biology have resulted in the identification of potential therapeutic targets for the treatment of AML.Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases.Patients with FLT3 mutations have higher relapse rates and worse outcomes for both overall survival and disease-free survival in comparison with patients without FLT3 mutations.
FLT3 is a membrane-bound cytokine receptor closely related to KIT, FMS, and PDGFR.Binding to an extracellular ligand results in receptor dimerization and autophosphorylation of tyrosine residues in the intracellular domain, which activates downstream signaling pathways, including RAS/ MAPK, JAK/STAT5, and PI3K/AKT/mTOR.These path-ways promote the growth, proliferation, survival, and differentiation of myeloid cells. 4,5nternal tandem duplication (ITD), which represents the most common group of FLT3 mutations, occurs in 20−25% of all AML patients.ITD promotes ligand-independent dimerization and downstream signaling. 6,7Point mutations in the tyrosine kinase domain (FLT3-TKD) are approximately twice less prevalent.TKD mutations stabilize the kinase in its active conformation.Both FLT3-ITD and FLT3-TKD mutations can cause ligand-independent FLT3 kinase activation and promote cell proliferation, resulting in a high leukemic burden.
−10 First-generation inhibitors comprise nonspecific receptor tyrosine kinase inhibitors, such as sunitinib, sorafenib, and midostaurin, originally developed for other indications.A second generation of more selective and efficient inhibitors, which exhibit lower toxicity and off-target effects, has also been developed.These inhibitors, which include quizartinib, crenolanib, and gilteritinib, produce significant responses in AML patients.
In our previous studies, we investigated trisubstituted purines as kinase inhibitors and carbocyclic nucleoside derivatives with CDK2 inhibitory activity, among other analogues. 11We revealed that some of these compounds display nanomolar inhibitory potency toward FLT3 kinase (unpublished observation, Supporting Information, Table S1).These findings are substantiated in another of our studies, which found that trisubstituted purine derivatives are potent FLT3 inhibitors that selectively block the proliferation of AML cell lines harboring FLT3-ITD mutations. 12In order to identify potent and selective FLT3 inhibitors, we focused on synthesizing heterocyclic mimics of the purine base bearing similar substitution patterns as the parent purine derivatives.We designed trisubstituted derivatives containing various heterocyclic cores (Figure 1B) and then evaluated their inhibitory effects on FLT3 kinase in vitro and in vivo.

■ RESULTS AND DISCUSSION
−15 In order to explore this understudied chemical space and generate new active compounds, we designed new isosteric trisubstituted derivatives of several heterocyclic cores, including thieno[3,2-  For standard deviation (SD) values, see Table S3 in the Supporting Information.NT = not tested.d]pyrimidine, 16−18 pyrazolo[1,5-a]pyrimidine, 19−21 imidazo-[4,5-b]pyridine, 22 pyrido [4,3-d]pyrimidine, 23 and imidazo-[1,2-b]pyridazine 24,25 (Figure 1B).All of the prepared compounds were tested for their inhibitory activity against recombinant FLT3-ITD and CDK2/E.The most active compounds were screened against the FLT3-D835Y mutant, which is the most common resistance initiator in AML patients treated with clinically approved FLT3 inhibitors.To evaluate the FLT3-dependent mechanism of action, compounds were further screened for antiproliferative activity in a panel of human leukemia cell lines.Two AML cell lines, MV4-11 and MOLM-13, characterized by the presence of FLT3-ITD (full FLT3-dependency) and SEM, an acute lymphoblastic leukemia (ALL) cell line overexpressing FLT3-wt (with partial dependency on FLT3 signaling), were supplemented with four FLT3independent cell lines.These included the AML-derived cell lines NOMO-1 and ML-2, the ALL-derived cell line CEM, and chronic myeloid leukemia (CML)-derived K562 cells.
Compounds containing the thieno [3,2-d]pyrimidine core did not display significant inhibitory activity against recombinant FLT3-ITD.Their antiproliferative activities against leukemic cell lines varied mainly within the micromolar range (see Table 1).
Synthesis and Activity of Pyrido[4,3-d]pyrimidines.Pyrido [4,3-d]pyrimidine derivatives (Scheme 4) were prepared according to a procedure described by Jansa et al. 33 Activation of bromonicotinate with triphenylphosphine and ring closure with isocyanate afforded derivative 21.Chlorination and subsequent substitution with an aniline derivative afforded compound 23, which reacted with cyclohexene-1-boronic acid to give derivative 24 in a high yield.Final reduction with H 2 (15 bar) on Pd/C for 2 days afforded amino derivative 25 with an unsaturated cyclohexene ring.However, modification of the pyrido [4,3-d]pyrimidine core proved counterproductive, given the prepared compounds failed to show any promising activity (Table 4).
Synthesis and Activity of Imidazo[1,2-b]pyridazines. Finally, we focused on imidazo [1,2-b]pyridazine derivatives (Scheme 5).We started with 3-bromo-6-chloro derivative 27.However, it showed very poor reactivity under Suzuki crosscoupling conditions and afforded only a small amount of 28 together with the starting material as an inseparable mixture.Next, we treated the mixture with 1,4-trans-cyclohexendiamine, separated the products by reverse phase chromatography, and isolated compounds 29a and 29b.Unsaturated derivative 29b was hydrogenated by H 2 on Pd/C to give 30 (Scheme 5).Attempts to prepare 2,5-diaminopyridine derivatives using the Buchwald−Hartwig reaction failed, and 6-chloro derivative 28 proved poorly reactive.For SD values, see Table S3 in the Supporting Information.NT = not tested.

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The  For SD values, see Table S3 in the Supporting Information.

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cyclohexyl (30), and cyclohexenyl (29b).These compounds displayed promising inhibitory activities against the tested recombinant kinases within the nanomolar range.Antiproliferative activities confirmed that these compounds employed an FLT3-dependent mechanism of action: FLT3-dependent MV4-11 and MOLM-13, as well as SEM cell lines, were several times more sensitive than FLT3-independent cell lines (Table 5).
In the first part of this study, we identified new compounds by scaffold hopping and evaluated central cores as suitable replacements for the purine scaffold.As the most promising inhibitory activities were observed for imidazo [1,2-b]pyridazine derivatives, we decided to extend the series and modify the substituent in position 3 of the core.We performed a docking study using the active site of FLT3 to predict the binding poses of imidazo [1,2-b]pyridazine derivatives bearing aliphatic and aromatic substituents.We based the structures of the proposed ligands on the most potent inhibitor identified up to this point in the study, compound 29a (Figure 2A).Various aliphatic and aromatic substituents were placed in position 3 of the heterocycle to induce interaction with a pocket lined by A642, K644, V675, F691, and L767.Proposed analogues of 29a were docked in silico, and the binding affinity of each compound was evaluated using Glide built-in scoring functions (Table S2, Supporting Information).In agreement with previously published docking studies, 12 our results confirmed that the most important residues participating in the interaction are K614, C694, N765, and D778.Another residue that proved important was F691 in the hydrophobic cavity, which presumably interacts with aromatic residues such as phenyl in 34f (Figure 2B), pyrazole, or other hydrophobic species.A docking study also suggests that the binding mode of our molecules is similar to that of type I FLT3 inhibitors.
Based on our preliminary biological results and in silico docking analysis, we extended the imidazo[1,2-b]pyridazine series and prepared derivatives substituted in position 3 with various aliphatic and aromatic substituents (Table 6, Schemes 6 −8).As the phenyl derivative 34f showed activity toward FLT3-ITD-positive kinase in the single-digit nanomolar range together with high selectivity in comparison with CDK2, we Table 6.Substituted Imidazo [1,2-b] extended our study to phenyl derivatives substituted at various positions in the phenyl ring (Table 6, entries g−p).The series was prepared from 3-iodo derivative 32 (Scheme 6), 34 which is more reactive than 3-bromo derivative 27 used in the previous synthesis (Scheme 5).
While the reaction of 32 with sodium azide did not proceed (data not shown), 3-amino derivative 39 was synthesized via 3nitro intermediate 36 by nitration 35 of imidazo[1,2-b]pyridazine 35 and further substitution of the heterocyclic core (Scheme 7).
We explored the structure−activity relationship using a diverse series of compounds bearing the conserved 4-(pyrrolidin-1-ylsulfonyl)aniline substituent in position 8 (Table 7).Although a number of compounds featuring chloro substitution in position 6 were tested against FLT3-ITD as well, our results confirmed (Table S4, Supporting Information) that the introduction of the trans-1,4-diaminocyclohexyl substituent into this position is crucial for the anti-FLT3 activity of imidazo [1,2-b]pyridazines.
Compounds lacking the substituent in position 3 (42b) or containing a small polar amino group (39) are among the less potent in the series displaying IC 50 values against FLT3-ITD within a high nanomolar range.The introduction of small aliphatic substituents (methyl in 34e, isopropyl in 34d, isobutyl in 34c), cyclic aliphatic substituents (34b, 34a, 30, 29b), or furanyl (34r) and thienyl (34q) resulted in low nanomolar activity against recombinant FLT3-ITD as well as FLT3-D835Y.These results correspond with the potent antiproliferative activities in MV4-11 and MOLM-13 cell lines within a nanomolar concentration range.In contrast,

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FLT3-independent cell lines were several orders of magnitude less sensitive.On the other hand, these compounds showed very strong potency against CDK2, where the inhibitory ratio of CDK2 and FLT3-ITD was between 1 and 82.These results indicate the lower selectivity of these molecules.
On the other hand, dimethylcarbamoyl and dimethylsulfamoyl substituents of compounds 34o and 34p, respectively, probably affected the binding of compounds into active sites of the tested kinases and resulted in reduced activity.For example, IC 50 values increased more than 100-fold in comparison with the values of other members of the group.From all of the prepared compounds, 34f was selected as the tool compound for further biochemical and mechanistic evaluation.This molecule showed single-digit nanomolar IC 50 values against recombinant FLT3-ITD and FLT3-D835Y (0.004 and 0.001 μM, respectively), whereas CDK2 was nearly 250 times less sensitive.FLT3-ITD-inhibitory activity of 34f was comparable to the standards quizartinib (0.010 ± 0.004 μM) and gilteritinib (0.012 ± 0.001 μM).Although 34f shows promising potency also against FLT3-D835Y (0.001 μM), comparable to the clinically approved gilteritinib (0.002 ± 0.0003 μM), quizartinib is more than 100 times less potent against FLT3-D835Y than 34f (0.136 ± 0.002 μM for quizartinib).The same trend was also observed for FLT3-ITD-F691L.While 34f and gilteritinib showed low nanomolar IC 50 values (0.004 ± 0.003 and 0.010 ± 0.005 μM, respectively), quizartinib lost its potency against this mutant variant of FLT3 (>5 μM).
Synthesis of 34f on a Larger Scale.Compound 34f was selected for in vivo experiments in mice, requiring the preparation of hundreds of milligrams of the material.Given For SD values, see Table S3 in the Supporting Information.

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that Suzuki cross-coupling of 3-iodo derivative 32 gave products in relatively low yields from 15 to 30% and upscaling of the reaction proved problematic, we developed an alternative synthetic procedure employing phenylacetaldehyde (Scheme 9).Bromination 36 and subsequent cyclization with 3aminopyridazine gave compound 43 in a 50% yield after two steps.Further substitution with aniline and amine gave 34f in a 20% overall yield after four steps.This synthetic strategy, starting from cheap substituted acetaldehyde, proved suitable for the synthesis of larger quantities of 3-substituted imidazo-[1,2-b]pyridazine derivatives.
Cellular Effects of 34f.Specific FLT3 inhibition induces G1 arrest of FLT3-dependent AML cells but does not affect other cell lines (FLT3-independent).This was validated by flow cytometry analysis of FLT3-ITD-positive MV4-11 cells treated for 24 h with nanomolar concentrations of 34f.The number of G1 cells increased in a dose-dependent manner (Figure 3), but the NOMO-1 cell line was not affected within the same 34f concentration range (Figure 3).These results were comparable to the effects seen in MV4-11 and NOMO-1 cells treated with both quizartinib and gilteritinib (Figures S1, S2).The primary cause of this phenomenon is the blocking of FLT3-subordinate   signaling pathways, which are of crucial importance in cell proliferation.Dose-dependent attenuation of phosphorylation of FLT3 as well as its downstream targets, Y694 of STAT5 and T202/Y204 of ERK1/2, was confirmed after 1 h of treatment with 34f in MV4-11 cells.This demonstrated the FLT3dependent mechanism of action (Figure 4) and efficacy comparable to quizartinib and gilteritinib (Figures S3A, S4A).
Expression analysis of the MYC gene, a key transcription factor and common oncogene whose deregulation often contributes to the development of hematological malignancies, further confirmed the FLT3-dependent mechanism of action of 34f.MYC transcript levels were significantly reduced by 34f in treated MV4-11 cells, whereas they remained more stable in FLT3-independent NOMO-1 cells.Comparable effects were also observed in cells treated with quizartinib (Figure S3B) as well as gilteritinib (Figure S4B), a finding consistent with previously reported studies. 37ne of the most common obstacles to FLT3-inhibitor therapy of AML is the development of drug resistance.Therefore, we used the MOLM-13 cell line and its resistant clone expressing the FLT3-ITD-D835Y mutant to evaluate the effect of the lead compound 34f on proliferation.FLT3 inhibitors sorafenib, gilteritinib, and quizartinib were used for comparison.The graphs of the relative proliferation of MOLM-13 cells (Figure 5) show that compound 34f, as well as quizartinib, gilteritinib, and sorafenib, blocked proliferation in a time-dependent manner at low nanomolar concentrations.The antiproliferative ability of compound 34f was also confirmed in MOLM-13-resistant cells with the D835Y mutation; the GI 50 value obtained after 72 h treatment did not change significantly (0.010 and 0.004 μM in MOLM-13  and its resistant variant, respectively).A similar outcome was also observed for clinically approved gilteritinib.In contrast, the efficacy of quizartinib and sorafenib dramatically decreased, and the GI 50 values increased significantly.
Kinase Selectivity of 34f.The preliminary kinase selectivity of 34f in the panel of 48 kinases selected across the human kinome demonstrated the outstanding inhibitory activity of this compound against FLT3 (Figure 6).The IC 50 values for the most important off-targets were determined (Table S5).Although 34f also inhibits other kinases, it notably does not target KIT kinase, which is one of the most common off-targets of the known FLT3 inhibitors.Simultaneous inhibition of FLT3 and KIT results in myelosuppression, 38 which complicates the clinical use of these compounds.Therefore, avoiding KIT inhibition is a crucial goal in the development of novel FLT3 inhibitors.Kinase selectivity profiling demonstrated that 34f at a concentration of 100 nM reduced KIT activity to 71% (in comparison with 1% obtained for FLT3).A subsequent concentration-dependent experiment showed that the IC 50 value of 34f for KIT kinase is 680 nM (Table S5), a hundred times higher than for FLT3.These results indicate a favorable inhibitory ratio among these kinases.Hence, we decided to verify this finding using the Kasumi-1 cell line, which is characterized by activating N822K point mutation in KIT.Based on an evaluation of the antiproliferative properties of 34f, the GI 50 value measured in Kasumi-1 was 0.188 ± 0.019 μM, which was more than 18 times higher than the GI 50 values obtained for quizartinib in Kasumi-1 (0.010 ± 0.004 μM) and for 34f in FLT3-ITD MV4-11 cells (0.007 ± 0.004 μM).The GI 50 value determined for gilteritinib in Kasumi-1 cells was 0.124 ± 0.013 μM.The limited ability of 34f to block KIT activity was also confirmed by immunoblotting.At a concentration of 125 nM, 34f only partially reduced the phosphorylation of two tyrosine residues (Y703 and Y719) of the KIT kinase and T202/Y204 in the KIT downstream ERK1/2 (Figure S5).
Plasma and Microsomal Stability of 34f.In vitro stability of 34f in blood plasma and liver microsomes (from human and mouse sources) was tested prior to in vivo experiments in order to predict the clearance of compounds in the whole organism.Propantheline bromide and verapamil were used as reference compounds for plasma and microsomal stability, respectively, demonstrating the usual stability profiles.
Compound 34f was stable in both human and mouse plasma for up to 120 min of incubation (Figure 7A).As for microsomal stability, a slow decay by approximately 25% at 45 min was observed (Figure 7B).The calculated intrinsic clearance (CL int ; Table S6) values were 18 μmol/min/mL for human microsomes and 13 μmol/min/mL for mouse microsomes, indicating that the compound falls within the low-tomoderate clearance category.Overall, the metabolic stability of 34f was considered acceptable for in vivo experiments.
In Vivo Efficacy of 34f.Encouraged by these results, we performed in vivo experiments on immunodeficient mice bearing subcutaneous MV4-11 xenografts, the widely accepted simple in vivo model.As shown in Figure 8A, tumor growth was blocked in groups of mice treated repeatedly with intraperitoneal injections of 34f at doses of 5 and 10 mg/kg.By the end of the drug administration (day 7), the tumor growth rate remained restricted.On the other hand, our vehicle-treated control group of mice exhibited a steep increase in tumor size.For this reason, the experiment in this cohort had to be terminated prematurely.In addition to displaying strong anticancer efficacy in vivo, 34f administration had no adverse effect on mouse weight during the experiment (Figure 8B).
Moreover, immunoblotting analysis of MV4-11 xenografts exposed for 6 or 24 h to 34f at a dose of 10 mg/kg revealed reduced phosphorylation of FLT3 at Y589/591 and of STAT5 at Y694 in most of the analyzed tumors in comparison with vehicle-treated mice, thus confirming the FLT3-dependent mechanism of action of 34f in vivo (Figure 9).
In addition, the pharmacokinetic properties of 34f were determined in mice following intraperitoneal administration at a dose of 10 mg/kg.The results showed that 34f has a half-life of 71.3 min, including the absorption and elimination phases.The compound reaches a maximal plasma concentration of 384 pg/mL (722 nmol/L) after approximately 49 min following administration.For the details, see the Supporting information.

■ CONCLUSIONS
In this study, we investigated the suitability of several series of heterocyclic derivatives as potential FLT3 kinase inhibitors.Compounds derived from the imidazo[1,2-b]pyridazine heterocyclic core proved to be potent inhibitors of FLT3 kinase, and modification of position 3 resulted in a pronounced effect on activity and selectivity in comparison with CDK2.In the extensive structure−activity relationship (SAR), the 3phenyl substituent and some of its derivatives (e.g., 3-or 4methoxyphenyl, 4-fluorophenyl, or 4-(trifluoromethyl)phenyl) displayed activity toward FLT3 within a single-digit nanomolar range, where the selectivity ratio for CDK2/FLT3-ITD was more than 200.Candidate compound 34f showed high antiproliferative efficacy in the FLT3-ITD-positive AML cell lines MV4-11 and MOLM-13 (7 and 9 nM, respectively) as well as in the MOLM-13 variant bearing the FLT3-ITD-D835Y mutation (4 nM) in comparison with low sensitivity of FLT3-independent cell lines, proving the FLT3-dependent mechanism of action.Immunoblotting and flow cytometry analysis confirmed the blocking of signaling pathways subordinate to FLT3 as well as induced G1 arrest of FLT3dependent MV4-11 AML cells.As the derivative 34f showed sufficient plasma and microsomal stability, we continued with in vivo experiments in immunodeficient mice bearing subcutaneous MV4-11 xenografts.We observed a strong effect of 34f on tumor growth without any side effects on mouse weight.Additionally, immunoblotting analysis of MV4-11 xenografts confirmed reduced phosphorylation of FLT3 at Y589/591 and of STAT5 at Y694 in the analyzed tumors, confirming the FLT3-dependent mode of action in vivo.In summary, we found a novel substitution pattern of imidazo-[1,2-b]pyridazine that shows excellent potency toward FLT3 kinase in vitro and in vivo without any pronounced side effects.The activity displayed by this series of compounds, mainly the derivatives 34f, 34g, 34h, 34i, 34j, 34l, 34n, and 34m, indicates their suitability for further development as potential AML drug candidates.

■ EXPERIMENTAL SECTION
Starting compounds and reagents were purchased from commercial suppliers (Sigma-Aldrich, Fluorochem, Acros Organics, Carbosynth, TCI) and used without further purification.Dry tetrahydrofuran was distilled with lithium aluminum hydride pellets under an argon atmosphere.Analytical thin-layer chromatography (TLC) was performed on silica gel pre-coated aluminum plates with a fluorescent indicator (Merck 60 F 254 ).Flash column chromatography was carried out using Teledyne ISCO CombiFlash Nextgen.Preparative HPLC purification was performed on the INGOS HPLC system (LCD5000 and LCP5020 modules, chromatography column: Luna 5 μm C18(2) 100 Å).Mass spectra, UV absorbency, and purity of compounds were measured on the Waters UPLC-MS system, consisting of the Waters UPLC H-Class Core System (Waters Acquity UPLC BEH C18 1.7 mm column, 2.1 mm × 100 mm), the Waters Acquity UPLC PDA detector, and the Waters SQD2 mass spectrometer.The universal LC method was used (eluent H 2 O/CH 3 CN, gradient 0−100%, run length 4 min or 7 min) in conjunction with the MS method (ESI+ and/or ESI−, cone voltage = 30 V, mass detector range 100−1000 Da for standard cases and 500−1600 Da for larger molecules).Highresolution mass spectra were measured on the LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific).NMR spectra were obtained using the Bruker Avance III HD 500 MHz spectrometer operating at 125.7 MHz for 13 C and 500 MHz for 1 H.The spectra were referenced to solvent residual signals (dimethyl sulfoxide (DMSO): 2.50 for 1 H and 39.70 for 13 C, CDCl 3 : 7.26 for 1 H and 77.16 for 13 C).The assignment of hydrogen and carbon spectra was based on a combination of one-dimensional (1D) and two-dimensional (2D) experiments ( 1 H− 13 C APT, 1 H− 1 H COSY, 1 H− 13 C HSQC, and 1 H− 13 C HMBC).The purity of the final compounds was determined by ultra-performance liquid chromatography−mass spectrometry (UPLC-MS) and was 95% or higher, with the exception of compounds 5b, 10, 25, 34n, and 39 due to the problematic separation of highly polar compounds; nevertheless, the purity was still higher than 90%.
Molecular Docking.A docking study was performed using Schrodinger built-in modules.The homology model of the active DFG-in conformation of FLT3 (based on the crystal structure of FLT3 kinase, 39 PDB 1RJB, resolution 2.10 Å) was used. 12The structure was optimized prior to docking using the Schrodinger Protein Preparation Wizard Maestro Suite (version 12.9.123,release 2021-3).Inconsistencies in the structure, such as missing hydrogens, incorrect bond orders, and poor orientation of amino-acid side chains, were rectified during the optimization process.The LigPrep module was used to convert two-dimensional structures to three-dimensional (3D), correct improper bond distances and bond orders, ionize compounds to correspond with pH 7 ± 1, and minimize ligand energy.Structures generated by LigPrep were then used for ligand docking.Ligand docking was performed using the Schrodinger Gridbased Ligand Docking with Energetics (Glide) Suite 2021 application.Receptor grid generation was based on the ligand from the original PDB structure.The default selection of 20 poses per ligand was set for Glide.Extra precision (XP) mode was selected for the Glide redocking stage.
General Procedure 2 (GP2): Reaction with Trans-1,4-diaminocyclohexane.The heterocyclic derivative (1 mmol) and trans-1,4diaminocyclohexane (10 mmol) in NMP (2.5 mL) were heated in a tightly sealed 4 mL vial at 210 °C overnight.The mixture was diluted with DMSO (2 mL) and directly applied to the RP FC (H 2 O/ACN + 0.1% of formic acid).Products containing fractions were evaporated and codistilled with water; the final compound was dried in vacuo or freeze-dried from dioxane.
For antiproliferative assays, cells were seeded into 96-well plates in appropriate densities and subsequently treated with test compounds.After the incubation period, resazurin (Merck) solution was added for 4 h.Fluorescence of resorufin corresponding to live cells was measured at 544/590 nm (excitation/emission) using a Fluoroskan Ascent microplate reader (Labsystems).
Cell Cycle Analysis.Leukemia cells were seeded and, after a preincubation period, treated with tested compounds for 24 h.After staining with propidium iodide, DNA content was analyzed by flow cytometry using a 488 nm laser (BD FACSVerse with BD FACSuite software, version 1.0.6).Cell cycle distribution was analyzed using ModFit LT (Verity Software House).
RNA Isolation and qPCR.Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN) according to the manufacturer's instructions.RNA concentration and purity were measured using the DS-11 Series Spectrophotometer (DeNovix).RNA was transcribed into first-strand cDNA using the SensiFAST cDNA Synthesis Kit (Bioline).Quantitative RT-PCR was carried out on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and the SensiFAST SYBR No-ROX Kit (Bioline).Suitable primers were designed using Primer-BLAST 44 and synthesized by Generi Biotech.Primary data were analyzed using CFX Maestro Software 2.2 (Bio-Rad).Relative gene expressions were determined using the delta− delta Ct method. 45Expression of the MYC gene was normalized per the GAPDH and RPL13 genes, which were determined to be the most stable according to CFX Maestro Software 2.2 (Bio-Rad).

C G A C A A G A A A A A G C G G A T G G ; R : TTCTCTTTCCTCTTCTCCTCC).
Plasma Stability Assay.To determine plasma stability, 5 μM of the given compound was incubated with human pooled plasma from 50 donors (Biowest) for 20, 60, and 120 min at 37 °C.The reactions were terminated by adding four volumes of ice-cold methanol.The samples were then mixed vigorously and left at −20 °C for 30 min before being centrifuged.The supernatants were diluted with four volumes of 30% methanol in water and then analyzed using the Echo MS system (SCIEX).Zero time points were prepared by adding icecold methanol to the compound prior to the addition of plasma.
Microsomal Stability Assay.A microsomal stability assay was performed using 0.5 mg/mL of pooled human liver microsomes (Thermo Fisher Scientific) and 5 μM compounds in 90 mM TRIS-Cl buffer (pH 7.4) containing 2 mM NADPH and 2 mM MgCl 2 for 10, 30, and 45 min at 37 °C.The reactions were terminated by the addition of four volumes of ice-cold methanol, mixed vigorously, and left at −20 °C for 30 min; the samples were then centrifuged.The supernatants were diluted with four volumes of 30% methanol in water and then analyzed using the Echo MS system (SCIEX).Zero time points were prepared by adding ice-cold methanol to the mixture of compounds and cofactors prior to the addition of microsomes.
In Vivo Efficacy.The experimental design was approved by the Institutional Animal Care and Use Committee (Charles University, MSMT-37334/2020-4).Immunodeficient adult female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (referred to as NSG mice) purchased from the Jackson Laboratory were preserved in a pathogen-free environment in individually ventilated cages and provided with sterilized food and water.NSG mice were subcutaneously inoculated with MV4-11 cells.After all mice developed palpable tumors, they were stratified into cohorts (6 mice per group) with comparable calculated tumor volumes.Therapy involving 34f (5 and 10 mg/kg dissolved in 5% DMSO in saline, intraperitoneal administration, final volume 500 μL per mouse) was then initiated.Three perpendicular dimensions (in millimeters) were measured with a digital caliper.Tumor volumes were calculated using the following formula: π/6 × length × width × height.The experiment was terminated when the tumors reached a maximum diameter of 20 mm.For the purpose of immunoblotting analysis, mice that had developed tumors were treated with a 10 mg/kg intraperitoneal dose of 34f for 6 or 24 h.The mice were then euthanized, and the tumors were processed for further analysis.
■ ASSOCIATED CONTENT * sı Supporting Information

Figure 1 .
Figure 1.Structural modifications of kinase inhibitors leading to (A) FLT3 inhibitors and (B) heterocyclic cores explored in this study.Scheme 1. Synthesis of Thieno[3,2-d]pyrimidine Derivatives a

Figure 2 .
Figure 2. Docked binding poses of (A) compound 29a and (B) its phenyl derivative 34f in the active FLT3 site.

Scheme 9 .
Scheme 9. Synthesis of 34f on a Larger Scale a

Figure 4 .
Figure 4. (A) Immunoblotting analysis of FLT3 and its downstream signaling pathways in MV4-11 treated with 34f for 1 h.(B) Relative normalized expression of the MYC gene in MV4-11 and NOMO-1 cells treated with 34f for 4 h.

Figure 5 .
Figure 5. Antiproliferative activity of 34f in the MOLM-13 cell line and its clone expressing FLT3-ITD-D835Y (MOLM-13 resistant).Quizartinib, gilteritinib, and sorafenib were used as standards.T72 GI 50 = 50% growth inhibition concentration determined at the final point of the experiment after 72 h of treatment.

Figure 6 .
Figure 6.Kinase selectivity profiling of 34f.The efficacy of 34f at 100 nM concentration was compared with 48 human kinases across the kinome (coverage shown in the phylogenetic tree).

Figure 7 .
Figure 7. (A) Plasma and (B) microsomal stability of 34f.Propantheline bromide and verapamil were used as standards to determine plasma stability and microsomal stability, respectively.

Figure 8 .
Figure 8.In vivo efficacy of 34f.(A) Growth of subcutaneous MV4-11 xenografts (mean volume ± SD) in groups of mice treated with 34f (5 and 10 mg/kg) or a vehicle only every other day until day 7 (4 doses) by intraperitoneal administration.(B) The weight of mice (mean ± SD) during the experiment.