Discovery of novel heteroaryl alkynes for highly potent KITD816V cells inhibition to treat gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GIST) is the most popular mesenchymal tumor in the gastrointestinal tract with approximately 80% of GIST harboring gain-of-function mutations at either the extracellular region (exon 9) or the juxtamembrane domain (JMD, exon 11) of KIT, resulting in uncontrolled proliferation and resistance to apotosis. Beyond surgical removal, targeted tyrosine kinase inhibitors (TKI) such as imatinib (1), sunitinib (2) and regorafenib (3) have been approved for the treatment of GIST by inhibiting malignant proliferation driven by KIT. However, these therapies are limited due to the acquisition of polyclonal secondary resistance mutations in ATP pocket (exons 13/14) and activation loop (A-loop, exons 17/18) of KIT. D816V, one of the major mutant forms in A-loop, shifts the KIT activation equilibrium from an inactive to active state and renders it insensitive to the approved treatments. Targeting KIT proved a formidable challenge for drug development and only a few multikinase inhibitors (4e6, Supporting Information Fig. S1) and newly approved drugs (7e8, Fig. S1) for drug resistant GISTs, show the potential to overcome KIT mutants. However, the clinical benefit of these medications is still limited due to their insufficient activity toward the KIT mutant as well as unwanted side effects from low selectivity


To the Editor:
Gastrointestinal stromal tumors (GIST) is the most popular mesenchymal tumor in the gastrointestinal tract with approximately 80% of GIST harboring gain-of-function mutations at either the extracellular region (exon 9) or the juxtamembrane domain (JMD, exon 11) of KIT, resulting in uncontrolled proliferation and resistance to apotosis 1 . Beyond surgical removal, targeted tyrosine kinase inhibitors (TKI) such as imatinib (1), sunitinib (2) and regorafenib (3) have been approved for the treatment of GIST by inhibiting malignant proliferation driven by KIT. However, these therapies are limited due to the acquisition of polyclonal secondary resistance mutations in ATP pocket (exons 13/14) and activation loop (A-loop, exons 17/18) of KIT.
D816V, one of the major mutant forms in A-loop, shifts the KIT activation equilibrium from an inactive to active state and renders it insensitive to the approved treatments 2 . Targeting KIT D816V proved a formidable challenge for drug development and only a few multikinase inhibitors (4e6, Supporting Information Fig. S1) and newly approved drugs (7e8, Fig. S1) for drug resistant GISTs, show the potential to overcome KIT D816V mutants. However, the clinical benefit of these medications is still limited due to their insufficient activity toward the KIT D816V mutant as well as unwanted side effects from low selectivity profiles over the wild-type KIT and other kinases. Beyond these approved medications for GIST, there is still an unmet clinical need for highly potent therapeutic agents to overcome drug resistant KIT D816V mutant, while minimizing the risk of potential toxicity by reducing the off-target effects. Many efforts (9e12, Fig. S1) have been made to achieve these goals, but so far none of them show highly potent and specific to KIT D816V cells and sufficient in vivo anti-tumor efficacy in KIT D816V xenograft models.
In this study, we report novel heteroaryl alkynyl compounds that are highly potent and specific against KIT D816V cells. The most promising compound, 54 displayed high potency against transformed 32D cell lines bearing KIT D816V mutant (GI 50 Z 0.7 nmol/ L) as well as varies of KIT mutations. Moreover, 54 demonstrated excellent selectivity profiles between KIT D816V mutant cells and a panel of cell lines, with no obvious toxicity for 32D normal cells (GI 50 > 1000 nmol/L) and high therapeutic windows of >491.7-fold change related to the KIT WT cells (NCI-H526, Mo7e and HMC-1 cells). Also, it showed much lower activities against EGFR wt amplified A431 cells and PDGFR-a wt driven U118MG cells (GI 50 > 1000 nmol/L). In addition, 54 displayed acceptable PK properties and significant in vivo anti-tumor efficacy in 32D KIT D816V xenograft mice models with low toxicity. The high potency and specificity, together with favorable druggabilities of 54 indicates that this compound can serve as a promising candidate for treatment of GIST.

Structural optimizations and biological evaluation for potent KIT D816V inhibitors
In our previous work 3 , a series of o-aminopyridyl alkyne CSF-1R inhibitors (such as 13, Fig. 1A) with type II binding modes were identified. On the other hand, according to the switch control concepts 4 , type II inhibitors can also inhibit a variety of KIT mutants, even secondary mutations located in A-loop, such as D816V, KIT D816V xenografts models. Nude mice bearing 32D KIT D816V xenografts were randomized into vehicle (n Z 10) or treatment (n Z 6) groups, and given treatment as indicated. Tumor volume and body weight were measured on the indicated days. *P < 0.05, ***P < 0.001 versus vehicle group. by switching back to its inactive state inducing by the binding of switch inhibition type II inhibitor. Thus, we hypothesized that our type II CSF-1R inhibitors might display inhibition effects against some KIT mutants due to the structural homology with CSF-1R and the precedent established by compounds like 1.
Given the unusual activation profile of KIT that the A-loop is independent on the presence of phosphate 5 and the type II inhibition in general is difficult to reproduce in biochemical assays, the screening was carried out on 32D cell lines transformed with KIT D816V mutant. The result showed that compound 13 displayed potential to (GI 50 Z 1010 nmol/L) inhibit the proliferation of these drug-resistant GIST cells. Subsequently, an iterative medicinal chemistry optimization focused on the "linker", "tail", "central ring" and "hinge binder" was carried out to yield heteroaryl alkynes (represented by 54 and 57) as highly potent inhibitors against KIT D816V cells (Fig. 1A).

Further biological assays of compounds 54 and 57 in vitro
The blockage effects of 54 and 57 on the KIT signaling pathways was confirmed in 32D KIT D816V cell lines (Fig. 1B). It was found that these compounds showed potent inhibition effects against the phosphorylation of KIT D816V and the downstream STAT3 and ERK phosphorylation in KIT D816V cells, which were more potent than that of ripretinib.
Compounds 54 and 57 was selected for further investigation in a panel of 32D cell lines with broad-spectrum mutants, which are transformed by a variety of clinically important KIT mutants as shown in Fig. 1C. The results showed that these compounds displayed strong potency against these KIT/mutants. Compound 54 showed a low nomolar and even picomolar GI 50 value against mutants of V559D (GI 50 Z 0.09 nmol/L), V559D/V654A (GI 50 Z 1.1 nmol/L), and V559D/N822K (GI 50 Z 0.8 nmol/L), which was 16.7, 18.8 and 2.2 times more potent than that of ripretinib, respectively. Moreover, the therapeutic windows for wild-type KIT related to these KIT/mutants were determined in those cells with amplified wild-type KIT, including NCI-H526, Mo7e and HMC-1 cells. Interestingly, compared to the KIT/mutants cell lines, 54 and 57 displayed much lower potency to these cell lines, indicating the enhanced therapeutic outcome for GIST treatment driven by KIT/mutants and minimized the potential risk of myelosuppression, and other toxicities related to the physiological function of KIT signaling pathways. Also, further investigation of selectivity profiles showed that these compounds displayed relatively low activities against a panel of cell lines dependent on other kinases, including EGFR wt , PDGFRa wt with favorable therapeutic windows of more than 1428.6-fold comparing to the KIT D81V mutant cells. Whereas, ripretinib exhibited considerable inhibitory effects toward the KIT wt amplified cells (Mo7e cells, GI 50 Z 260.6 nmol/L; HMC-1 cells, GI 50 Z 260.1 nmol/L) and the PDGFRa wt amplified cells (U118MG cells, GI 50 Z 268.9 nmol/L), showing relatively low therapeutic windows to the KIT/mutants.
Given the high cellular potency and therapeutic windows of 54, it was selected for further evaluation of kinome selectivity against a panel of 253 kinases (Supporting Information Table S6). Unfortunately, the molecular data did not match the cellular data. Compound 54 showed low inhibitory potency against KIT D816V while displayed strong potency against KIT wt and other KIT mutations (including D816H, V560G and V654A) at 1 mmol/L. We reasoned that 54 serving as a switch inhibitor, the inhibitory potency against KIT D816V was closely dependent on the kinase conformation change, which was restricted in this molecular assay utilizing the non-natural KIT D816V protein 5 . Further cellular thermal shift assay (CETSA) was carried out to confirm the on-target binding of compounds 54 and 57 (Supporting Information Fig. S2).

The in vivo PK and anti-tumor efficacy evaluations of compounds 54 and 57
The preliminary pharmacokinetic properties of 54 and 57 were evaluated in mice (Fig. 1D). After a single dose administration of these compounds (i.v., 2 mg/kg; p.o., 10 mg/kg), the key PK parameters of these compounds were determined. The results displayed that 54 exhibited the moderate bioavailability (29.9%) and drug exposure (AUC last Z 1278 h$ng/mL) as well as the relatively short t 1/2 (2.29 h) using 10 mpk oral administration. Whereas compound 57 displayed poor oral PK properties with low drug exposure (AUC last Z 233 h$ng/mL) and short t 1/2 (1.34 h). Based on these PK properties, both compounds were administrated twice a day (BID) via oral administration to maintain consistent plasma concentrations over a longer period of time and suppress the growth of cancer cells in vivo. Meanwhile, the dosage of 57 was increased appropriately given its relatively lower drug exposure as compared to the 54.
The in vivo antitumor activity of compounds 54 and 57 was further investigated in 32D KIT D816V xenograft mice models. Each group of mice were repeatedly administrated vehicle or compounds 54, 57 and ripretinib twice a day via intragastric administration for 12 continuous days. As shown in Fig. 1E, compound 54 (20, 40 mg/kg, i.g., BID Â 12) dose-dependently inhibited the growth of 32D KIT D816V xenografts with a TGI (tumor growth inhibition) value of 45% and 71%, respectively. Compound 57 (30, 100 mg/kg, i.g., BID Â 12) suppressed the growth of 32D KIT D816V xenografts by 50% at high dose group (100 mg/kg). However, ripretinib (30, 100 mg/kg, i.g., BID Â 12) showed no apparent efficacy in the 32D KIT D816V xenograft mice models. In addition, no mortality or obvious body weight loss was observed during the whole period of treatment (Fig. 1F). Collectively, these results indicated that compounds 54 and 57 displayed significant in vivo anti-tumor efficacy in 32D KIT D816V xenografts, which was much better than that of ripretinib.
In conclusion, starting from our previously reported type II inhibitors of CSF-1R with o-aminopyridinyl alkyne scaffold, an iterative medicinal chemistry optimization was carried out to improve the anti-KIT D816V activity, selectivity, and drug-like characteristics, leading to the discovery of the most promising compound 54, with a novel heteroaryl alkynyl scaffold. In vitro, 54 potently inhibited the KIT D816V mutant cells (GI 50 Z 0.7 nmol/L) and a variety of clinically important KIT mutants including both the primary mutations and secondary mutations. Also, this compound showed superior selectivity profiles over the 32D parental cells, and those cell lines driven by KIT wt , EGFR wt and PDGFR wt which could increase its therapeutic benefits for the GIST treatment driven by KIT/mutants and minimize potential off-target toxicity related to the physiological function of these kinases signaling pathways. In vivo, 54 exhibited acceptable druggabilities and excellent inhibitory efficacy in 32D KIT D816V xenograft mice model, which is resistant to ripretinib. To conclude, this study identifies compound 54 as a promising therapeutic agent for GIST with the potential to overcome clinical resistance induced by KIT D816V .