Identification of Thiazolo[5,4-b]pyridine Derivatives as c-KIT Inhibitors for Overcoming Imatinib Resistance

Simple Summary c-KIT has been regarded as a promising therapeutic target against gastrointestinal stromal tumor (GIST). Overcoming drug resistance of c-KIT inhibitors including imatinib is required. We designed and synthesized novel thiazolo[5,4-b]pyridine derivatives and performed structure-activity relationship (SAR) studies to overcome imatinib resistance. The SAR studies led to the identification of the derivative 6r as a potent c-KIT inhibitor. The derivative 6r is capable of strongly inhibiting a c-KIT V560G/D816V double mutant that is resistant to imatinib and remarkably attenuates proliferation of GIST-T1 and HMC1.2 cancer cells. Moreover, 6r possesses differential cytotoxicity on c-KIT D816V Ba/F3 cells relative to parental Ba/F3 cells. Kinase panel profiling revealed that 6r has reasonable kinase selectivity. Furthermore, 6r not only blocks migration and invasion, but also suppresses anchorage-independent growth of GIST-T1 cells. Abstract c-KIT is a promising therapeutic target against gastrointestinal stromal tumor (GIST). In order to identify novel c-KIT inhibitors capable of overcoming imatinib resistance, we synthesized 31 novel thiazolo[5,4-b]pyridine derivatives and performed SAR studies. We observed that, among these substances, 6r is capable of inhibiting significantly c-KIT and suppressing substantially proliferation of GIST-T1 cancer cells. It is of note that 6r is potent against a c-KIT V560G/D816V double mutant resistant to imatinib. Compared with sunitinib, 6r possesses higher differential cytotoxicity on c-KIT D816V Ba/F3 cells relative to parental Ba/F3 cells. In addition, kinase panel profiling reveals that 6r has reasonable kinase selectivity. It was found that 6r remarkably attenuates proliferation of cancer cells via blockade of c-KIT downstream signaling, and induction of apoptosis and cell cycle arrest. Furthermore, 6r notably suppresses migration and invasion, as well as anchorage-independent growth of GIST-T1 cells. This study provides useful SAR information for the design of novel c-KIT inhibitors overcoming imatinib-resistance.


Introduction
c-KIT, a class III receptor tyrosine kinase, consists of five immunoglobulin-like domains, one transmembrane domain, one juxta-membrane domain, and two split kinase domains [1].Binding of a stem cell factor (SCF) ligand to c-KIT induces the c-KIT dimerization, leading to subsequent activation of its intracellular signaling cascades [2].Dysfunctions of c-KIT, such as gain of function caused by overexpression and point mutation, result in c-KIT activation and tumorigenesis [3,4].Activation of c-KIT is observed in various cancers including GIST, mast cell tumors, and malignant melanomas [4][5][6].In particular, c-KIT activation is deeply associated with GISTs which are the most common gastrointestinal tumor [7,8].More than 90% of 419 GIST cases are associated with c-KIT activation and 6−7% of the cases are implicated with PDGFRα mutations [9].
Mutations within exon 11 encoding the juxta-membrane domain of c-KIT, with a relative frequency of 67%, are most common in GIST [10].The mutations in exon 9 (IgGlike D5 domain of c-KIT) of which the majority are duplications of A502_Y503 occur at nearly 15% in GIST [11,12].Notably, secondary c-KIT mutations causing imatinib resistance have been known to occur most commonly in exon 13/14 (ATP-binding domain of c-KIT) and exon 17/18 (activation loop of c-KIT) [13][14][15].The c-KIT T670I gatekeeper mutant within exon 13 corresponds to the BCR-ABL T315I gatekeeper mutant and causes drug resistance through constitutive activation and steric hindrance [16,17].D816H/V mutants occurring in the activation loop of c-KIT are capable of accelerating auto-activation rather than wild-type c-KIT, which leads to resistant to imatinib [18].Moreover, the double mutant of c-KIT V560G/D816V has disruption of auto-inhibitory mechanism and SCF-independent constitutive activation of c-KIT [19].
Imatinib, an innovative BCR-ABL kinase inhibitor against chronic myelogenous leukemia, has been approved as a first-line treatment for advanced GIST (Figure 1) [20].However, 10-20% of patients taking imatinib show secondary mutations that decrease c-KIT sensitivity to imatinib [21].Sunitinib, a second-line therapy for the treatment of GIST, possesses inhibitory activity against c-KIT V654A and gatekeeper mutant c-KIT T670I [13].However, preclinical studies have revealed that both imatinib and sunitinib do not effectively inhibit exon 17 KIT mutations (activation loop mutants) [14].Regorafenib was approved for the third-line treatment against imatinib-and sunitinib-resistant GISTs but it has moderate activities against the secondary mutations [22,23].The potent c-KIT/PDGFRα inhibitor avapritinib has been approved for the GIST treatment [24].However, secondary PDGFRα mutations (Val658Ala, Asn659Lys, Tyr676Cys, and Gly680Arg) have been observed in patients with drug resistant recurrent GIST [25].Ripretinib, the fourth-line treatment approved for GIST, interacts reversibly to both the switch pocket and activation loop of c-KIT [26].Although ripretinib is capable of inhibiting a wide range of c-KIT and PDGFRα mutants, it shows an overall response rate of 9.4%, suggesting additional resistance and disease progression [27].
Cancers 2023, 15, 143 2 of 18 domains [1].Binding of a stem cell factor (SCF) ligand to c-KIT induces the c-KIT dimerization, leading to subsequent activation of its intracellular signaling cascades [2].Dysfunctions of c-KIT, such as gain of function caused by overexpression and point mutation, result in c-KIT activation and tumorigenesis [3,4].Activation of c-KIT is observed in various cancers including GIST, mast cell tumors, and malignant melanomas [4][5][6].In particular, c-KIT activation is deeply associated with GISTs which are the most common gastrointestinal tumor [7,8].More than 90% of 419 GIST cases are associated with c-KIT activation and 6−7% of the cases are implicated with PDGFRα mutations [9].Mutations within exon 11 encoding the juxta-membrane domain of c-KIT, with a relative frequency of 67%, are most common in GIST [10].The mutations in exon 9 (IgG-like D5 domain of c-KIT) of which the majority are duplications of A502_Y503 occur at nearly 15% in GIST [11,12].Notably, secondary c-KIT mutations causing imatinib resistance have been known to occur most commonly in exon 13/14 (ATP-binding domain of c-KIT) and exon 17/18 (activation loop of c-KIT) [13][14][15].The c-KIT T670I gatekeeper mutant within exon 13 corresponds to the BCR-ABL T315I gatekeeper mutant and causes drug resistance through constitutive activation and steric hindrance [16,17].D816H/V mutants occurring in the activation loop of c-KIT are capable of accelerating auto-activation rather than wildtype c-KIT, which leads to resistant to imatinib [18].Moreover, the double mutant of c-KIT V560G/D816V has disruption of auto-inhibitory mechanism and SCF-independent constitutive activation of c-KIT [19].
Imatinib, an innovative BCR-ABL kinase inhibitor against chronic myelogenous leukemia, has been approved as a first-line treatment for advanced GIST (Figure 1) [20].However, 10-20% of patients taking imatinib show secondary mutations that decrease c-KIT sensitivity to imatinib [21].Sunitinib, a second-line therapy for the treatment of GIST, possesses inhibitory activity against c-KIT V654A and gatekeeper mutant c-KIT T670I [13].However, preclinical studies have revealed that both imatinib and sunitinib do not effectively inhibit exon 17 KIT mutations (activation loop mutants) [14].Regorafenib was approved for the third-line treatment against imatinib-and sunitinib-resistant GISTs but it has moderate activities against the secondary mutations [22,23].The potent c-KIT/PDG-FRα inhibitor avapritinib has been approved for the GIST treatment [24].However, secondary PDGFRα mutations (Val658Ala, Asn659Lys, Tyr676Cys, and Gly680Arg) have been observed in patients with drug resistant recurrent GIST [25].Ripretinib, the fourthline treatment approved for GIST, interacts reversibly to both the switch pocket and activation loop of c-KIT [26].Although ripretinib is capable of inhibiting a wide range of c-KIT and PDGFRα mutants, it shows an overall response rate of 9.4%, suggesting additional resistance and disease progression [27].In addition to clinically approved drugs, various scaffolds have been used as c-KIT inhibitors.A potent dual c-KIT/PDGFRα 1H-pyrazolo [3,4-b]pyridine inhibitor for the In addition to clinically approved drugs, various scaffolds have been used as c-KIT inhibitors.A potent dual c-KIT/PDGFRα 1H-pyrazolo [3,4-b]pyridine inhibitor for the treatment of imatinib-resistant GISTs has been reported [28], but its anti-proliferative activity against TEL-c-KIT D816V Ba/F3 cells was not significant (GI 50 > 10 µM).The derivatives having the 2-aminothiazole scaffold switch off activated c-KIT to its inactivated state, and possess activities against constitutively activated mutants in the activation loop [29].Moreover, the thiazolo [5,4-b]pyridine scaffold has been used to discover various kinase inhibitors against PI3K [30], ITK [31], BCR-ABL [32], RAF [33], and VEGFR2 [34].Thiazolo [5,4-b]pyridine derivatives have distinct types of binding modes depending on target kinases.The 4-nitrogen of thiazolo [5,4-b]pyridine is hinge-binding motif of PI3K kinase inhibitors while the 1-nitrogen and 2-amino group of the scaffold form H-bonding contact with the ITK kinase hinge region.In addition, the 5-position of thiazolo [5,4-b]pyridine has been functionalized to target the ATP-binding site of BCR-ABL, RAF, and VEGFR2.It is worthwhile to note that this study has, for the first time, reports functionalization on the 6-position of thiazolo [5,4-b]pyridine scaffold to identify novel c-KIT inhibitors.
In an effort to overcome drug resistance, we synthesized 31 novel thiazolo [5,4-b] pyridine derivatives and carried out a structure-activity relationship study against c-KIT enzyme and c-KIT-activated cells.The results of the SAR study for thiazolo [5,4-b]pyridine derivatives revealed that 6r possesses higher enzymatic and anti-proliferative activities than imatinib and comparable activities relative to sunitinib.Notably, 6r has 8.0-fold higher enzymatic inhibitory activity (IC 50 = 4.77 µM) against c-KIT V560G/D816V double mutant and 23.6-fold higher anti-proliferative activity (GI 50 = 1.15 µM) on HMC1.2 cells harboring both V560G and D816V c-KIT mutations compared with imatinib.Moreover, 6r exhibits a reasonable selectivity in the biochemical kinase panel profiling.Furthermore, 6r suppresses proliferation of cancer cells (GIST-T1 and HMC1.2) and c-KIT D816V Ba/F3 cells via blockade of c-KIT downstream signaling, and induction of apoptosis and cell cycle arrest.Notably, 6r remarkably blocks migration and invasion, as well as anchorage-independent growth of GIST-T1 cells.

Chemistry
All commercial reagents and solvents were purchased from chemical suppliers and directly used for synthesis without purification.Reaction monitoring was performed by thin-layer chromatography (TLC) analysis using a UV lamp, ninhydrin, or p-anisaldehyde stain for the compound detection.Reaction products were purified by column chromatography on silica gel (230−400 mesh).Purities of all compounds were analyzed by Waters LC/MS system and shown to be over 95%. 1 H and 13 C NMR spectra were recorded either by a Bruker 300 MHz FT-NMR (300 MHz for 1 H, and 75 MHz for 13 C) or a 400 MHz FT-NMR (400 MHz for 1 H, and 100 MHz for 13 C) spectrometer.The synthetic procedures and chemical characterizations of all compounds are described in File S1.

In Vitro Kinase Assay
Biochemical kinase assays on c-KIT protein kinase were performed at Reaction Biology Corp. (San Diego, CA, USA).Compounds were tested with 10 µM ATP in a 10-dose IC 50 mode with 3-fold serial dilution.The bioluminescent-based kinase assay was tested using an ADP-Glo assay kit.c-KIT (V560G/D816V) kinase was purchased from Promega (# VA7063).Compounds were tested with 10 µM ATP in a 10-dose IC 50 mode with 3-fold serial dilution.The kinase assay followed the manufacturer's instruction.IC 50 values were calculated by GraphPad prism 8.0 software (GraphPad Software, San Diego, CA, USA).

Molecular Docking Study
The X-ray co-crystal structure of c-KIT complexed with imatinib (PDB code: 1T46) was retrieved from the Protein Data Bank and loaded into Maestro software (Schrödinger Release 2020-4, New York, NY, USA).The Protein Preparation Wizard was executed for replenishment of missing residue, addition of hydrogens, and assignment of bond orders.Restrained energy minimization was applicated in the OPLS3e force field.Ligand 6r was prepared using the LigPrep and the receptor c-KIT grid was generated considering the imatinib binding pocket.Ligand docking of 6r on c-KIT was performed using GLIDE.

Kinase Panel Profiling
Kinase panel profiling was conducted by Reaction Biology Corp. (San Diego, CA, USA).A single dose (1.0 µM) of 6r was tested against 371 recombinant human kinases in the presence of 10 µM ATP.

Cell Cycle Arrest and Apoptosis Analysis
After compound treatment in GIST-T1 and HMC1.2 cells for 24 h, cells were fixed with 70% cold ethanol.Then, cells were incubated with 50 µg propidium iodide/mL solution (PI, Sigma) containing 1 mg RNase A/mL for 30 min in the dark.Cell cycle arrest was analyzed (BD Accuri), and data were processed using BD Accuri TM C6 software.For apoptosis analysis, compound treatment for 24-48 h, cells were harvested and stained with annexinV and PI solution.1 × 10 5 cells were analyzed using flow cytometric analysis.

Migration Assay
GIST-T1 cells were seeded in 6-well plates at 2 × 10 6 cells.Next day, cells were washed with PBS and wounded in the center of each well.Then, GIST-T1 cells were washed with PBS and added to fresh DMEM.After 36 h, the scratch recovery of GIST-T1 cells in each compound-treated groups were observed and taken with a Nikon microscope at a magnification of 1000×.

Invasion Assay
The cell invasion assay was performed using Transwell membrane filter inserts (Corning Costar Corp., Corning, NY, USA).GIST-T1 cells were then suspended in serum-free DMEM at 2 × 10 6 cells and seeded in the upper chambers.In the lower chambers, the Cancers 2023, 15, 143 5 of 18 DMEM medium containing 10% FBS was added.After 48 h, the cells migrating down the membrane were stained with crystal violet.

Soft Agar Assay
Anchorage-independent growth was assessed by determining colony formation on soft agar.GIST-T1 cells in DMEM media containing 0.7% agar were plated at 5,000 cells per well.Cells were treated with each compound for 3 weeks, and the media were changed every 3 days.Colonies were stained using iodonitrotetrazolium chloride (Sigma Aldrich).The average number colonies were counted using ImageJ software (version 1.53e, National Institutes of Health, Bethesda, MD, USA)

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0.Graphpad software, San Diego, CA, USA.All data are reported as average ± standard deviation (S.D.).p < 0.05 was considered to be statistically significant.

Synthesis of Thiazolo[5,4-b]pyridine Derivatives
The synthetic route for the thiazolo [5,4-b]pyridine derivatives is described in Scheme 1.To synthesize thiazolo [5,4-b]pyridine scaffold, we carried out the aminothiazole formation using commercially available 3-amino-5-bromo-2-chloropyridine 1 and potassium thiocyanate in 75% yield.The amino group of 2 underwent Boc protection to afford compound 3 in 90% yield.Then, 3 was coupled with 2-methyl-5-nitrophenylboronic acid pinacol ester by Suzuki cross-coupling reaction using Pd(dppf)Cl 2 as catalyst to form 4 in 70% yield.Reduction of the nitro group of 4 afforded the key intermediate 5 in 80% yield.The amide formation of aniline 5 with various carboxylic acids, followed by subsequent Boc deprotection yielded 6a-i and 6k-w.Urea formation between aniline 5 and 3-(trifluoromethyl)phenyl isocyanate was followed by subsequent Boc deprotection gave 6j.The primary amino group of selected derivatives was transformed into the corresponding amides 7a-h in 21-38% yields.
DMEM medium containing 10% FBS was added.After 48 h, the cells migrating down t membrane were stained with crystal violet.

Soft Agar Assay
Anchorage-independent growth was assessed by determining colony formation soft agar.GIST-T1 cells in DMEM media containing 0.7% agar were plated at 5,000 ce per well.Cells were treated with each compound for 3 weeks, and the media w changed every 3 days.Colonies were stained using iodonitrotetrazolium chloride (Sigm Aldrich).The average number colonies were counted using ImageJ software (versi 1.53e, National Institutes of Health, Bethesda, MD, USA)

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0.Graphpad softwa San Diego, CA, USA.All data are reported as average ± standard deviation (S.D.).p < 0 was considered to be statistically significant.

Synthesis of Thiazolo[5,4-b]pyridine Derivatives
The synthetic route for the thiazolo [5,4-b]pyridine derivatives is described in Schem 1.To synthesize thiazolo [5,4-b]pyridine scaffold, we carried out the aminothiazole f mation using commercially available 3-amino-5-bromo-2-chloropyridine 1 and potassiu thiocyanate in 75% yield.The amino group of 2 underwent Boc protection to afford co pound 3 in 90% yield.Then, 3 was coupled with 2-methyl-5-nitrophenylboronic acid nacol ester by Suzuki cross-coupling reaction using Pd(dppf)Cl2 as catalyst to form 4 70% yield.Reduction of the nitro group of 4 afforded the key intermediate 5 in 80% yie The amide formation of aniline 5 with various carboxylic acids, followed by subseque Boc deprotection yielded 6a-i and 6k-w.Urea formation between aniline 5 and 3-(triflu romethyl)phenyl isocyanate was followed by subsequent Boc deprotection gave 6j.T primary amino group of selected derivatives was transformed into the corresponding a ides 7a-h in 21-38% yields.

Molecular Docking Studies of 6j, 6r, and 7c with c-KIT
To investigate the binding mode of thiazolo [5,4-b]pyridine derivatives on c-KIT, we performed molecular docking studies of 6j, 6r and 7c using the X-ray co-crystal structure of c-KIT complexed with imatinib (Figure 2).For comparison, the binding mode of imatinib on c-KIT is presented in Figure 2C (PDB code: 1T46) [40].The results shows that 6r and 7c form a pair of hydrogen bonding with a Cys673 backbone in the hinge region.In addition, 6r and 7c also makes hydrogen bonding with Glu640/Asp810 and Ile789/His790 backbone, and these hydrogen bondings are also observed in the co-crystal structure of imatinib and c-KIT.Moreover, the thiazolo [5,4-b]pyridine moiety of 6r and 7c participates in hydrophobic interactions with Leu799, Val603, Ala621, and Val654.Furthermore, the 3-trifluoromethyl group of 6r and 7c fits well into the hydrophobic binding pocket which is also occupied by ponatinib [35].However, 6j could not form a hydrogen bonding with Asp810 observed in imatinib and could not fit into the hydrophobic binding pocket occupied by ponatinib.The predicted binding model suggests that 6r and 7c possess inhibitory activity, and 6j is inactive against c-KIT.

Suppression of Anchorage-Independent Growth in GIST-T1 Cells
Pharmacological inhibition and knockdown of c-kit decreased th GIST cells [48].A soft agar assay in GIST-T1 cells was performed to e blocks anchorage-independent growth (Figure 7).Treatment with tw trations of 6r and imatinib for 3 weeks resulted in dramatic inhibition pendent growth of the GIST-T1 cells.Notably, incubation of 6r at 0 suppressed anchorage-independent growth compared to control, indic of c-KIT by 6r suppresses tumorigenesis of GIST-T1 cells.

Suppression of Anchorage-Independent Growth in GIST-T1 Cells
Pharmacological inhibition and knockdown of c-kit decreased the colony growth of GIST cells [48].A soft agar assay in GIST-T1 cells was performed to evaluate whether 6r blocks anchorage-independent growth (Figure 7).Treatment with two different concentrations of 6r and imatinib for 3 weeks resulted in dramatic inhibition of anchorageindependent growth of the GIST-T1 cells.Notably, incubation of 6r at 0.05 µM completely suppressed anchorage-independent growth compared to control, indicating that blockade of c-KIT by 6r suppresses tumorigenesis of GIST-T1 cells.

Figure 2 .
Figure 2. (A,B) Docking model prediction of 6rand 7c on c-KIT (PDB code: 1T46).(C) Binding mode of imatinib on c-KIT (PDB code: 1T46).(D) Superimposition with docking model of 6r and 6j.Protein residues of c-KIT are represented by white cartoon and violet stick.The 6j, 6r, 7c, and imatinib are depicted in yellow, green, cyan, and orange, respectively.

Figure 3 .
Figure 3. Kinase panel profiling of 6r.Kinases that are more than 90% inhibited are represented by red circles.Illustration is provided by Cell Signaling Technology, Inc. (www.cellsignal.com,accessed on 3 November 2022) [41].

Figure 2 .
Figure 2. (A,B) Docking model prediction of 6r and 7c on c-KIT (PDB code: 1T46).(C) Binding mode of imatinib on c-KIT (PDB code: 1T46).(D) Superimposition with docking model of 6r and 6j.Protein residues of c-KIT are represented by white cartoon and violet stick.The 6j, 6r, 7c, and imatinib are depicted in yellow, green, cyan, and orange, respectively.

Figure 2 .
Figure 2. (A,B) Docking model prediction of 6rand 7c on c-KIT (PDB code: 1T46).(C) Binding mode of imatinib on c-KIT (PDB code: 1T46).(D) Superimposition with docking model of 6r and 6j.Protein residues of c-KIT are represented by white cartoon and violet stick.The 6j, 6r, 7c, and imatinib are depicted in yellow, green, cyan, and orange, respectively.

Figure 3 .
Figure 3. Kinase panel profiling of 6r.Kinases that are more than 90% inhibited are represented by red circles.Illustration is provided by Cell Signaling Technology, Inc. (www.cellsignal.com,accessed on 3 November 2022) [41].

Figure 3 .
Figure 3. Kinase panel profiling of 6r.Kinases that are more than 90% inhibited are represented by red circles.Illustration is provided by Cell Signaling Technology, Inc. (www.cellsignal.com,accessed on 3 November 2022) [41].

Figure 7 .
Figure 7. Effects of 6r and imatinib on anchorage-independent growth.(A) GIST-T1 cells were tured in 0.35% agar for 3 weeks.(B) The number of colonies was counted using ImageJ.(averag S.D., n = 3 one-way ANOVA and Tukey's multiple comparisons test; * p < 0.05) 4. Conclusions Dysregulation of c-KIT is associated with GIST.In order to identify a unique class c-KIT inhibitors capable of inhibiting clinically relevant c-KIT mutations and overrid imatinib resistance, we designed and synthesized 31 novel thiazolo[5,4-b]pyridine der
a Radiometric biochemical kinase assay results.b less than 50% inhibition at a concentration of 10 μM.
a Radiometric biochemical kinase assay results.b less than 50% inhibition at a concentration of 10 μM.

GI 50 (µM) a Entry GI 50 (µM) a
GI 50 means the concentration at which a compound causes half-maximal growth inhibition.b less than 50% inhibition at a concentration of 50 µM. a
a ADP-Glo kinase assay.