Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine

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Discovery of asciminib (ABL001), an allosteric inhibitor of the tyrosine kinase activity of BCR-ABL1 Joseph Schoepfer, Wolfgang Jahnke, Giuliano Berellini, Silvia Buonamici, Simona Cotesta, Sandra W Cowan-Jacob, Stephanie Dodd, Peter Drueckes, Doriano Fabbro, Tobias Gabriel, JeanMarc Groell, Robert M. Grotzfeld, Asm Quamrul Hassan, Christelle Henry, Varsha Iyer, Darryl Jones, Franco Lombardo, Alice Loo, Paul William Manley, Xavier Pelle, Gabriele Rummel, Bahaa Salem, Markus Warmuth, Andrew Wylie, Thomas Zoller, Andreas L. Marzinzik, and Pascal Furet J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01040 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018

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Journal of Medicinal Chemistry

Discovery of asciminib (ABL001), an allosteric inhibitor of the tyrosine kinase activity of BCR-ABL1 Joseph Schoepfer#,+, Wolfgang Jahnke#,+, Giuliano Berelliniǂ, Silvia Buonamiciǂ, Simona Cotesta#, Sandra W. Cowan-Jacob#, Stephanie Dodd##, Peter Drueckes#, Doriano Fabbroǂ, Tobias Gabriel#, Jean-Marc Groell#, Robert M. Grotzfeld#, A. Quamrul Hassanǂ, Chrystèle Henry#, Varsha Iyerǂ, Darryl Jones#, Franco Lombardoǂ, Alice Loo##, Paul W. Manley#, Xavier Pellé#, Gabriele Rummel#, Bahaa Salem#, Markus Warmuthǂ, Andrew A. Wylieǂ, Thomas Zoller#, Andreas L. Marzinzik*,#,+, Pascal Furet#,+ # Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland ## Novartis Institutes for BioMedical Research, 250 Mass Ave, Cambridge, MA 02139 USA ǂ New address + These authors contributed equally

ACS Paragon Plus Environment

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ABSTRACT Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) which target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATPsite mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors, so that combinations of asciminib with ATPcompetitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.


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Figure 1: (A) Structures of FDA-approved BCR-ABL1 inhibitors, type I and type II ATP competitive inhibitors1,

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(B) Structures of compounds employed for X-ray crystallographic

studies of assembled inactive state of ABL13 (C) GNF-24 and GNF-5,5 activities on the short


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(kinase domain) and long (kinase and myristate binding domain) ABL1 constructs (radiometric filter binding assays) and cell proliferation data in Ba/F3 BCR-ABL1 (p210) dependent cells (ATPLite™).6


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Introduction Chronic myelogenous leukemia (CML) and some cases of adult acute lymphoblastic leukemia (ALL) arise from a reciprocal translocation between chromosomes 9 and 22 in hematological progenitor cells, leading to the fusion of the BCR and ABL1 genes on the Philadelphia chromosome (Ph).7 Transcription and the resulting expression of the BCR-ABL1 fusion protein, which has a constitutively active ABL1 kinase domain, leads to the aberrant activation of numerous signaling pathways which result in the dysregulated differentiation, growth and survival of leukemic cells.8 Inhibiting the kinase activity of BCR-ABL1 by targeting its ATPbinding site with drugs such as imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib (Tasigna®) and bosutinib (Bosulif®) greatly reduces leukemic tumor burden and increases the overall survival rate of CML patients, thereby validating ABL1 as a target for therapeutic intervention for this disease.9-12 With such targeted tyrosine kinase inhibitors (TKIs), the majority of chronic phase CML patients continuously treated according to current guidelines have a normal life expectancy.13 However, despite the success of these drugs in transforming the prognosis of a fatal malignancy, some patients suffer from either poor initial response, loss of response or tolerability issues.14, 15 Loss of response is frequently associated with drug resistance, which can emerge when clones harboring mutations in the BCR-ABL1 kinase (BCR-ABL1mut) that hinder TKI binding, expand to further drive the disease.16,

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Of these mutations the one harboring a

substitution of the threonine T315 to isoleucine (BCR-ABL1T315I) is only inhibited by ponatinib (Iclusig®), the dosing of which is limited by adverse events.18 Many of the adverse events recorded for TKIs which lead to tolerability issues in CML patients result from off-target activities and, consequently a drug that specifically targeted ABL kinase activity would probably have fewer side effects.


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All of the TKIs currently approved for the treatment of CML target the catalytic ATP binding site of ABL1, either in a type-I or type-II binding mode where they bind to either the catalytically active or catalytically inactive conformation of the SH1 domain.1,

2

Although

imatinib and nilotinib are more selective than ponatinib and the type-I TKIs, bosutinib and dasatinib, all of the drugs have distinct profiles leading to the inhibition of many other kinases.19, 20

Moreover, they tend to be susceptible to resistance mutations, a notorious one being the T315I

mutation involving the gatekeeper residue in the ATP site, which is only inhibited by ponatinib.21 In 2003, the research groups of Kuriyan and Superti-Furga 3, 22 reported that a myristoyl group is involved in the autoregulation of ABL1 and the closely related ABL2 kinase:23 By binding to an internal “myristate binding site / pocket” in the C-lobe of the ABL1 kinase domain, the myristoyl group that is covalently attached to the N-terminus of ABL1 induces an assembled inactive state in which the SH3 and SH2 domains dock against the kinase domain to reduce its conformational flexibility (Figure 2). This mechanism of natural autoinhibition seen in ABL1 is lost upon fusion with the fragment of BCR which replaces the N-terminal cap region of ABL1 that contains the myristoylation site, thus leading to the constitutive activation of BCR-ABL1.3 This assembled inactive state differs from the typical kinase inactive DFG-out conformation where ATP site directed type-II inhibitors stabilize the inactive conformation of the kinase domain by directly binding to the DFG-out loop conformation.24 Conceptually, a drug that inhibited ABL1 kinase by interacting with this myristoyl site would be more selective than ATPcompetitive TKIs, would maintain activity against resistance mutations and would not be subject to adverse events resulting from off-target kinase inhibition, and hence could be well tolerated. As an approach to identify novel BCR-ABL1 inhibitors, Adrian and colleagues employed a differential cytotoxicity screen using BCR-ABL1 transformed and parental mouse hematopoietic


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32D cells.4 Whereas parental 32D cells require interleukin 3 (IL3) as a growth factor for proliferation, the expression of BCR-ABL1 allows the cells to grow in the absence of IL3 and instead depend upon BCR-ABL1 for proliferation.25 From a library of heterocyclic compounds, an N-phenylpyrimidin-4-amine derivative showed remarkable selectivity and subsequent optimization to improve cellular activity afforded GNF-2, which exhibited potent and selective antiproliferative activity on BCR-ABL1-dependent cells (Figure 1). SAR around GNF-2 indicated that a para-CF3O- group and the free-NH of the CF3OC6H4NHpharmacophore were important for activity. Based on mutagenesis studies where amino acid substitutions within the myristoyl binding pocket modulated the activity of GNF-2, it was hypothesized that GNF-2 bound to myristoyl binding pocket of ABL1. Subsequently this hypothesis was validated using NMR and X-ray crystallography,5 which demonstrated that GNF2 bound to the myristate binding site and therefore inhibited ABL1 kinase activity allosterically. In the latter study an analogue with improved pharmacokinetic properties, GNF-5 (Figure 1), demonstrated efficacy in vivo. Although attempts to exploit this chemotype as a lead expanded the diversity of allosteric BCR-ABL1 inhibitors, no clinical development candidates were identified.26 Following a strategy to elaborate myristate binding site inhibitors as potential drugs for the treatment of CML, we identified asciminib (ABL001, compound 1), a potent inhibitor of BCRABL1-driven cell proliferation, which is currently undergoing clinical evaluation.27 Compound 1 has been shown to mimic the role of the myristoylated N-terminus of ABL1 by occupying its vacant binding site, thereby restoring the negative regulation of BCR-ABL1 kinase activity. We report here the main aspects of the discovery of compound 1, which include identification of a weak fragment screening hit employing an NMR-based conformational assay, and subsequent


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optimization for potency and drug-like properties, guided by X-ray crystallography and molecular modeling (Figure 3). The in vivo evaluation of compound 1 and analogs in BCRABL1-dependent tumor xenograft models, the full in vitro profile of compound 1, and its pharmacokinetics in preclinical species are discussed.

Figure 2. (A) The assembled inactive state of ABL1 kinase as seen in the minimal autoinhibitory construct, comprising the SH3, SH2 and SH1 domains, bound to PD166326 (Figure 1; a type-I tyrosine kinase inhibitor) and myristic acid (1opl).3 (B) The structure of the ABL1 SH1 domain in complex with imatinib (2hyy).28 Note the two different conformations of helix I, which is bent in the assembled inactive state (panel A, orange), but linear and partially disordered in the absence of autoinhibition (panel B, light blue).


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Figure 3. From fragment to clinical candidate: Medicinal chemistry progression from fragments to fragment-derived hit 6, to lead 7 and finally clinical candidate, asciminib (ABL001, 1),27 showing some NMR-derived Kd and GI50 values from the Ba/F3 cellular proliferation assay (ATPLite™).6

Fragment-based lead discovery Attempts to discover novel ligands for the myristate pocket of ABL1 kinase started with a fragment screen using NMR spectroscopy. Since only allosteric inhibitors of ABL1 were of interest, the ATP-site was blocked with imatinib and the ABL1-imatinib complex was screened using NMR T1ρ and water LOGSY ligand experiments. By screening approximately 500


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diverse, quality-controlled and soluble fragments29, 30 hits were identified. The dissociation constants of the hits were determined by NMR by either direct titration, measuring chemical shift changes in a one-dimensional 1H spectrum of the ABL1-imatinib complex, or by competition with an CF3O- containing tool compound derived from GNF-2, which had a Kd of (7.4 ± 1.0) µM, as determined by direct NMR titration. Both NMR-based methods for Kd determination were compared for a subset of hits, and the results were in full agreement. Figure 3 shows the progression from screening hits to follow-up compounds, with NMR-derived Kd values. Screening hit 2 was remarkable in terms of its affinity (Kd = 6 µM) and low molecular weight (185 Da), corresponding to a ligand efficiency of 0.6. SAR-by-archive analysis showed clear trends for 2, with replacement of Cl with Me (Kd = 12 µM), CF3 (Kd = 14 µM) or F (Kd = 31 µM) resulting in a loss in potency, whereas a replacement with Br gave an increase in potency (Kd = 1 µM). None of the fragment hits or initial follow-up compounds were active in a biochemical ABL1 kinase assay, which monitored substrate phosphorylation by capillary electrophoresis. This was not surprising since at the time, the assay employed only the kinase domain of ABL1, and lacked the minimal autoinhibitory construct (Figure 2A) capable of forming an assembled inactive state induced by allosteric inhibitors. More surprisingly, none of them were active at concentrations < 50 uM in cellular assays. The cellular assays comprised of proliferation or autophosphorylation assays in Ba/F3 BCR-ABL1 dependent cells (viability determined by luminescent ATP detection, ATPLite™ 6) which were established as being sensitive to ATP-competitive BCRABL1 inhibitors and with BCR-ABL1 inhibition being highly correlated with antiproliferative activity.19 Although fragment hits are generally not expected to be active in cellular assays, our


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hits and initial follow-up compounds exhibited low micromolar Kd values, and comprised of several chemotypes, such that cellular activity was expected at a concentration of 50 µM. A selection of the fragment hits were crystallized as ternary complexes with the ABL1imatinib complex. The X-ray crystal structures of these complexes confirmed that the hits bound to the myristate pocket and revealed why they were not functional in cellular assays, thus providing a path forward towards functional inhibitors: the structures, such as that structure involving fragment hit 2 (Figure 3) showed that the binding mode of the fragment was incompatible with bending of helix-I, which is a conformational change necessary for the formation of the assembled inactive state (Figure 2).30 In fact, 2 as well as other fragment hits would sterically clash with helix-I in the bent conformation. Therefore, hits such as 2 are not allosteric inhibitors of BCR-ABL1, but would actually activate ABL1 by interfering with its autoinhibition.30, 31

Figure 4. (A) Crystal structure of ABL1 kinase domain in complex with imatinib and fragment hit 2. (B) Structural detail, showing the position of 2 in the myristate pocket of ABL1 kinase. Superimposed from the structure of ABL1 kinase in complex with myristic acid (1opl) are the


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myristic acid ligand (orange), as well as helix-I (orange), in order to show the exit vector of 2, and the steric clash of helix-I (side chain of isoleucine I521 displayed) with the ester group of 2 (PDB code 3MS9, 1.80 Å). Based on the crystal structures, a strategy was developed to design analogs of 2 (Figure 4) which would bind without sterically clashing with the bent conformation of helix-I, and expected to be functional inhibitors. Removal of the methyl ester moiety would avoid the steric clash, and substituting the aromatic amine with an aliphatic carbon chain such as in the natural myristate ligand should restore the potency lost upon removal of the ester. The resulting compound 3 retained the affinity of the original fragment 2 (Kd = 4 µM) but was still inactive in cellular assays. In another approach to design allosteric inhibitors that bind solely to the bent conformation of helix-I, we performed similarity and pharmacophore searches based on the crystal structures of multiple crystallized fragments, and docked the resulting hits into the bent helix-I conformation of the myristate pocket. Compounds such as 4 were identified, which bound with moderate affinity (Kd = 6 µM), but still showed no cellular activity. We then questioned whether our assumption of the ability of follow-up fragments such as 3 and compound 4 to bend helix-I was correct. At this stage, the crystal structures only included the kinase domain of ABL1, and in this system helix-I was not visible. We therefore used NMR spectroscopy and developed an assay using a selectively 15N-valine-labeled ABL1 kinase domain to monitor the conformation of helix-I in solution.30 This was used to monitor valine V525 on helix-I, taking advantage of the fact that V525 is in a flexibly disordered region when helix-I is linear, but gets fully ordered when the helix is bent. Since the line-width and intensity of an NMR signal depends on the local correlation time and thus on the local flexibility, V525 gives


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rise to a strong and sharp signal in 15N, 1H-HSQC when helix-I is linear, while it gives rise to a peak with average intensity when helix-I is bent. This is illustrated in Figure 5, with the unliganded state as reference for the linear state of helix-I (Figure 5B), and the myristate-bound state as reference for the fully bent helix (Figure 5C). Similar to the myristoylated peptide, GNF2 also leads to a fully bent helix-I (Figure 5D).30

Figure 5. The NMR-based conformational assay allows the conformation of the C-terminal helix I to be monitored, particularly distinguishing the linear (light blue) and bent (orange) states.32 The principle of the assay is depicted in panel (A), which shows a superposition of two ABL1 structures, one with linear helix-I (light blue) and one with bent helix-I (orange). Panels (B-F) show results of the HSQC NMR conformational assay with the myristate pocket in various states: with unliganded myristate pocket (B), with the myristate pocket complexed with a myristoylated peptide (C),22 or in complex with GNF-2 (D).4 Panel (E) shows that compound 4 does not bend helix-I, although it was designed to do so. However, optimized compound 5


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induces bending of helix-I (F) and consequently displays BCR-ABL1wt inhibition in cellular assays.

To our surprise, follow-up compounds 3 and 4, although designed to bind to the bent conformation of helix-I, were shown by the conformation assay not to bind to this conformation (Figure 5E). This explains why those compounds, although binding to the myristate pocket of ABL1, cannot induce the assembled inactive state and are therefore not BCR-ABL1 inhibitors. In GNF-2, the CF3O- group attached at the para-position of the aniline was found to be essential for inhibitory activity. Our fragment hits lacked a CF3O- group in this position. We reasoned that the introduction of a CF3O- group could induce helix-I bending and impart inhibitory activity. Indeed, the resulting compound 5, having a Kd of 10 µM, showed a bent helix I in the conformation assay (Figure 5F). Furthermore, this profile translated into a GI50 value of 8 µM in BCR-ABL1wt Ba/F3 cells (ATPLite™).6 Removal of the hydroxy group and the chlorine atom from the benzamide core and replacement of the N-methyl-piperazine by a morpholine ring led to compound 6 having Kd = 2 µM and GI50 = 2 µM in the same BCR-ABL1wt Ba/F3 derived cell line. Thus, with the help of the NMR conformational assay, we understood the molecular requirements for allosteric inhibition of BCR-ABL1, and were able to rationalize the design and synthesis of allosteric BCR-ABL1 inhibitors. Compound 6 provided the basis for further optimization. While the NMR assay was used to generate early SAR, the development of cell-free biochemical assays capable of quantifying inhibitory effects of myristate ligands was critical to optimize activity. Thus, we developed a radiometric filter binding assay6 and later a microfluidic capillary electrophoresis (Caliper) assay with the minimum autoinhibitory construct (comprising


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the SH3-SH2-kinase domain) which could form the assembled inactive state. It proved important to not add any surfactants to these assays since, due to their structural similarity with myristate, they could block the myristate binding site and prevent the detection of myristate ligands. Using the radiometric filter binding assay without detergent, the inhibitory potencies of myristate ligands could be measured, as for example with compound 6, having an IC50 value of 0.55 µM in the Caliper assay (Table 1).


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A

B

C

D

E

F

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Figure 6. (A) Crystal structure of ABL1-imatinib complex bound to compound 6 (view of the myristate binding pocket; PDB code 6HD6, 2.30 Å). H-bonds are indicated by black dotted lines. The orthogonal polar interaction between one of the fluorine atoms of the CF3O- group and the


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carbon atom of the backbone carbonyl of leucine L359 is indicated by a red dotted line. The same applies for the other figures. (B) The two possible orientations of the morpholine ring of 6 in the crystal structure. (C) Binding model of designed compound 8 in the myristate pocket. (D) Binding model of designed compound 7 in the myristate pocket. (E) Crystal structure of ABL1imatinib complex bound to compound 7 (PDB code 6HD4, 2.03 Å). (F) Crystal structure of ABL1-imatinib complex bound to compound 1 (PDB code 5MO4, 2.17 Å). The crystal structure of compound 6 (Figure 6A) shows the ligand bound in the myristate pocket of the ABL1-imatinib complex. Notably, the O- group of 6 binds deeply into the narrow lipophilic channel, with space only available for substitution in the para-position of the aromatic ring, as seen in crystal structure of GNF-2 bound to the ABL1-imatinib complex.5 There is a favorable polar interaction between one of the fluorine atoms and the carbon atom of the backbone carbonyl group of leucine L359. While the carbonyl of the amide bond in compound 6 does not make any interaction with the protein, the NH is involved in water-mediated H-bonding interactions to the backbone carbonyls of alanine A452 and glutamic acid E481. The crystal structure of 6 shows very good electron density of the aromatic core, but the 3morpholinomethyl is disordered and shows only weak interactions within the pocket, most of it protruding into the solvent. The electron density is best rationalized by two different binding modes of the morpholine ring where in one binding mode the morpholine is pointing into the solvent, in another binding mode the morpholine is flipped by almost 180°, binding along a lipophilic cleft at the exit of the myristate channel (Figure 6B). The existence of two different binding modes revealed by the co-crystal structure pointed to some opportunities to increase the affinity of 6 by better filling the lipophilic cleft revealed by the alternative conformation of the morpholine. We also noticed that an accessible hydrophobic


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surface, formed by the side chain of leucine L529 on the other side of the myristate channel opening, was not leveraged by the inhibitor. To exploit the latter observation analogues were designed having an aryl or heteroaryl group directly attached at the phenyl position-3 in place of the basic morpholine. In modeling experiments, such groups could form favorable hydrophobic contacts with the side chain of leucine L529 as illustrated in Figure 6C. A small set of biaryls designed according to this concept, in which the 3-morpholinomethyl substituent of 6 was replaced by lipophilic, neutral or weakly basic, unsubstituted diazines, substantially improved potency. For example compound 8, having a pyrimidine attached to the meta-position of the benzamide had an IC50 value of 18 nM in the Caliper assay. Compound 8 was a milestone for the project since it not only had remarkable biochemical potency, but also inhibited the proliferation of Luc-Ba/F3 BCR-ABL1wt cells (GI50 = 150 nM; in addition to being transfected to express wild-type BCR-ABL1, these cells were engineered to express a puromycin-luciferase fusion cassette to enable the direct detection of cell numbers using the BriteLite™ proliferation assay) and Luc-Ba/F3 BCR-ABL1T315I cells (GI50= 1.8 µM; Table 1). In vitro profiling of 8 revealed low solubility (HT-Sol FaSSIF = 0.01 mM, Table 1), and PK studies in mice indicated high clearance (75 mL/min/kg) and negligible exposure after oral administration (Table 2). To impart in vivo activity, our strategy focused on increasing the potency by picking up further interactions and optimizing the physicochemical properties of compound 8. To improve solubility we employed three approaches (Table 1): i) reducing clogP by incorporating nitrogen at various positions in ring-B, ii) introducing an R2 substituent ring-B ortho to R1, with the aim to push aromatic residues in R1 out-of-plane to avoid intermolecular π-π stacking, iii) adding a solubilizing group to R2. By incorporating a nitrogen in ring-B, the logP of 8 decreased from 4.3 to 2.7 in 9 and solubility (high-throughput kinetic solubility)33 increased


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from 0.01 mM to 0.026 mM. Overall, compound 9 showed the best balance of solubility, biochemical potency (IC50 0.024 µM) and antiproliferative activity against Luc-Ba/F3 BCRABL1WT cells (GI50 0.078 µM). Compound 9 also showed low µM activity against Luc-Ba/F3 BCR-ABL1T315I (GI50 1.65 µM) and did not show hERG binding up to 30 µM in a highthroughput dofetilide binding screening assay (this research assay was not compliant with Good. Laboratory Practice (GLP) regulations).34


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Table 1. From hit to lead: Comparison of effects of BCR-ABL1 in biochemical assay (IC50) and in cellular proliferation assays (GI50) with BCRABL1wt and BCR-ABL1T315I dependent Luc-Ba/F3 cells (data is mean of n ≥ 2, ± SD). Comparison of selected physicochemical and in vitro pharmacological properties of compounds 6-14.

Cpd

R2

X

-

6

N

7

ABL1164-515 IC50 (µ µM)

Luc-Ba/F3 BCR-ABL1wt GI50 (µ µM)

Luc-Ba/F3 BCR-ABL1T315I GI50 (µ µM)

clogP / logP

pKa

FASSIF (mM)

HTperm calc FA (%)

Rat liver microsome CL (mLmin-1 kg-1)

hERG dof. Binding IC50 (µ µM)

0.55 ± 0.69

0.253; 0.341

2.93; > 10

3.6 / 4.2

6.2

0.499

100

147

3.7

0.0023 ±

0.0017 ±

0.073 ± 0.006

2.0 / 3.0

3.3

0.59

56

40

9.6

0.0009

0.0001

8

H

CH

0.018 ± 0.004

0.117 ± 0.065

1.80 ± 0.44

3.3 / 4.3

3.3

0.01

100

28

> 10

9

H

N

0.024 ± 0.004

0.078 ± 0.018

1.65 ± 0.44

2.5 / 2.7

n/a

0.026

98

20

>30

10

OMe

CH

0.011 ± 0.001

0.004 ± 0.001

0.80 ± 0.09

2.9 / 4.6

n/a

0.16

100

16

1.1

CH

0.019 ± 0.004

0.020 ± 0.003

1.82; 1.59

3.7 / >3.4

8.9

0.5

99

60

0.009

N

0.018 ± 0.008

0.004; 0.007

0.511; 0.543

2.7 / 3.2

3.7

0.013

36

60

1.5

13

N

0.007 ± 0.001

0.005 ± 0.0001

0.396 ± 0.043

3.1 / n/a

7.4

0.151

98

34

0.31

14

N

0.004 ± 0.001

0.004 ± 0.001

0.294 ± 0.066

2.0 / 3.5

3.7

>1

40

50

17

11

12

NH(CH2)3OH


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For further optimization, the pyrimidine core of 9 was kept constant and variations of the substituent R2 were explored by employing structure-guided design (e.g. targeting H-bonding at the mouth of the pocket to further enhance binding), and adding groups to further improve solubility. It was hypothesized that a substituent R2 ortho to R1 might improve potency through having an improved topological fit to the binding site, while also promoting a torsion angle between ring-B and the pyrimidine and thereby increase solubility by impacting crystal packing. Compound 10 incorporated a CH3O-group, substantially improved solubility from 0.01 mM to 0.16 mM and this was further enhanced by linking a tertiary amine substituent as in compound 11 (0.5 mM), which also exhibited good cellular potency (Table 1). Pyridine derivatives with appropriate R2 substituents had improved solubility without possessing a strongly basic group, and compound 12 potently inhibited the growth of Luc-Ba/F3 cells dependent upon BCRABL1wt and BCR-ABL1T315I with GI50 values of 5 and 527 nM, respectively. Incorporating basic groups, such as in the methylpiperazine analog 13, substantially enhanced solubility, but imparted submicromolar affinity to hERG. In an attempt to eliminate the hERG signal, molecules were designed to maintain the polarity of the solubilizing group without incorporating a basic center. Molecular modeling studies suggested that a 3-hydroxy-pyrrolidine group would make hydrophobic contacts with the side chain of Leu529 and form an H-bond with the side-chain of Glu481, located at the entrance of the cavity. However, we realized that if we wanted to keep the pyrimidine ring, this had to bind on the other side of the cavity, when compared to compound 8. In fact, the presence of the hydroxy-pyrrolidine group increased the pyridine-pyrimidine torsion angle resulting in a steric clash with Leu529 in the binding orientation hypothesized for this ring in compound 8 (Figure 6C), To our satisfaction, molecular modeling indicated that the pyrimidine ring could be


21


Page 22 of 56

accommodated in this new orientation, where it made favorable hydrophobic contacts with threonine T453, methionine M456, proline P480 (the residues forming the hydrophobic cleft revealed by the alternative conformation of the morpholine group of 6 in the X-ray structure) and possibly a H-bond with the side-chain of arginine R351 as depicted in Figure 6D.

Indeed, adding a 3-hydroxy-pyrrolidine at R2 provided another boost in antiproliferative activity (3, GI50 2 nM). Compound 7 also displayed reduced hERG binding (9.6 µM) compared to the methylpiperazine 13 (0.31 µM), although this still translated into activity in non-GLP hERG Qpatch (IC50 4.2 µM) and patch clamp (IC50 4.5 µM) assays. On comparing both enantiomers the S-enantiomer, 7, exhibited marginally better biochemical and cellular potency compared to 14, and with this the project reached a milestone by clearly demonstrating the potential of the series to serve as a starting point towards a clinical candidate. The next section documents the in vitro and in vivo profiles of lead compound 7, followed by its optimization culminating in the discovery of the clinical candidate 1.

Lead Characterization The crystal structure of compound 7 bound to the ABL1-imatinib complex showed a welldefined electron density for the entire complex, confirming that the pyrimidine ring occupied the hydrophobic channel formed by threonine T453, methionine M456 and proline P480 of the myristate binding pocket (Figure 6E). In addition, the pyrimidine participated in water-mediated H-bonds with the backbone nitrogen of glutamate E481, of the carbonyl oxygen with the sidechain hydroxyl group of the tyrosine Y454 and from the nitrogen atom of the central pyridine. As anticipated, the hydroxy-pyrrolidine group made favorable van der Waals interactions with


22

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the surface formed by the side-chains of E481 and L529, but did not participate in polar interactions with the protein. The hydroxyl group remained solvent exposed rather than forming an H-bond with E481 as hypothesized in the design.

Replacing the typical phenylaminopyrimidine TKI pharmacophore as present in GNF-2, with a N-phenylnicotinamide scaffold largely abrogated the risk of off-target TKI activity, as demonstrated by profile of 7 in the KINOMEscan® (DiscoveRx, San Diego, U.S.A.) active ATP-site-directed competition binding assay, which contained more than 440 kinases.35 However, it should be noted that a limitation of this platform is that it is based upon recombinant proteins containing just the catalytic portion of the SH-1 kinase domain, e.g. ABL1(229-512) compared to ABL1(64-515) used in the present publication, and therefore potential myristoyl binding sites are not present.


23


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Table 2. Comparison of pharmacokinetic data for compounds 1, 7, 8 and 18-20. Compounds were administered to mice as solutions for intravenous and oral use, and/or respectively as *supensions (amorphous material) for oral use. AUC and Cmax values are dose normalized. Cpd 1 7

8 18 19 20

Route (dose; mg/kg) i.v. (1)

AUC (µmol.h/L) 3.2

Cmax (µmol/L)

Tmax (hr)

F (%)

p.o. (30)

1.7

0.4

2.0

53

i.v. (1)

4.2

p.o. (10)

4.2

1.2

1

99

*p.o. (10)

3.2

0.8

0.8

76

i.v. (1)

0.61

*p.o. (10)

0.005

i.v. (1)

1.9

p.o. (30)

2.5

i.v. (1)

3.3

p.o. (30)

2.4

i.v. (1)

1.4

p.o. (30)

2.8

0.01 0.6 0.6 0.7

0.33 1.3 1.7 2.0

CL (mL/min/kg) 12

t½ (h) 1.1

8.3

2

75

0.2

22

1.1

11

1.0

26

0.8

0.8 >100 73 >100

Although replacing the benzyl-morpholine moiety of 6 with a biaryl ring system markedly reduced the intrinsic clearance in rat liver microsomes (Tables 1 and 2), it was necessary to demonstrate that compounds had adequate pharmacokinetic properties in mice before proceeding with efficacy studies in murine models of leukemia. The pharmacokinetic properties of 7 compared to 8 supported the potential of the former compound to serve as a high quality lead (Table 2). Thus, mice were treated with either 7 or 8 at 1 mg/kg i.v. and 10 mg/kg p.o.: Following i.v. administration the clearance of compound 7 was substantially slower than that of 8, resulting in a longer half-life. Most strikingly, when 7 was administered orally it afforded excellent bioavailability and exposure, with the mean plasma level 7 hours after administration


24

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of 10 mg/kg (C7hr 3.1 ± 1.0 µM) still remaining >600-fold the GI50 value (4.8 ± 0.5 nM) in our Luc-KCL22 BCR-ABL1wt cell-line engineered from cells derived from a patient with blast crisis disease36. Considering the mouse plasma protein binding of 96% and a maximal fraction unbound of 100% in the cell proliferation assay (incubation medium contained 10% fetal calf serum) the concentration remaining after 7 h would be at least >20-fold over the GI50 value. The overall profile of 7 translated into efficacy in a murine, subcutaneous xenograft model of CML, using the above-mentioned cell-line, where once daily oral administration of 7 to tumorbearing mice resulted in dose-dependent growth inhibition after 7 days. At a dose of 30 mg/kg afforded 15% tumor growth compared to that of vehicle control (T/C 15%), with no loss in body weight (Supplementary Figure 1). 36

From lead compound 7 to clinical candidate 1 Although modifications of the CF3O-group had been unsuccessful for GNF-2 related compounds,26 in order to further optimize the potency and in vivo efficacy of lead compound 7, the effect of modifications to this group in our chemotype were examined. Analysis of the crystal structures of compound 6 or 7 bound to the ABL1-imatinib complex showed the CF3O-group to sit in the deepest part of the myristate pocket, adopting its characteristic orthogonal orientation37 with respect to the plane of the phenyl ring, and with one fluorine atom making a highly directional polar interaction with the carbonyl carbon of leucine L359 deep in the pocket. Molecular modeling indicated that a slightly larger group could be accommodated in this part of the cavity leading to the idea of either replacing the oxygen atom by a sulfur, or replacing one of


25


Page 26 of 56

the fluorine atoms with chlorine. Ab initio calculations predicted that such modifications should not alter the preference for the desired orthogonal conformation of the group, and even suggested an increased stabilization in the case of the oxygen-sulfur exchange (Supplementary Figure 2). Remarkably, compounds 15 and 16, which incorporated these modifications, provided an additional order of magnitude gain in potency over compound 7 in biochemical and cellular assays. To further optimize these extremely potent allosteric ABL1 kinase inhibitors the introduction of an additional hydroxyl group in the pyrrolidine ring of 16, a variation designed to further improve solubility (compound 17), weakened the cellular activity.


26

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Table 3. Comparison of ABL1 / BCR-ABL1 inhibition of compounds progressing from the lead pyrimidine series A to the pyrazoles B (lead compound 7 to clinical candidate 1). ABL1 inhibition was assessed in the biochemical transphosphorylation caliper assay (IC50) and BCR-ABL inhibition was assessed in cell proliferation assays (GI50) with either BCR-ABL1wt of BCR-ABL1T315I transformed Luc-Ba/F3 cells (n ≥ 3). hERG activity and selected physicochemical parameters of compounds 1 and 15-20 for dofetilide RLB (# n = 1, * n ≥ 2) Qpatch and patch clamp assays.

64-615

Cpd

ABL1 IC50 (nM)

Structure

Luc-Ba/F3 BCR-ABL1 GI50 (nM) wt

T315I

hERG IC50 (µM) or % inh. @ (µM) clogP / logP

pKa

Dofetilide RLB*

Qpatch

Patch clamp

1

B R2 = R3 = H

X = Cl

0.5 ± 0.1

1.0 ± 0.1

25 ± 3

3.2 / 3.9

4.0

>30

26

15 @ 30 -1 @ 10

15

A

X = F, Y=S

0.4 ± 0.05

< 0.3

12.4 ± 3.0

2.6 / 4.6

3.5

10.5

1.5

n/a

16

A

X = Cl, Y=O

< 0.3

0.4 ± 0.05

12.8 ± 1.6

2.4 / 3.7

3.8

3.6

1.8

5.0

17

A R1 = OH

X = Cl, Y=O

0.5 ± 0.04

9.0 ± 1.4

213 ± 45

1.9 / 3.3

3.3

>30#

>30

n/a

18

B R2 = R3 = H

X=F

1.1 ± 0.1

2.6 ± 0.5

90 ± 16

2.8 / 3.9

4.0

>30, 23

>30

66 @ 30 4 @ 10

19

B R2 = Me, R3 = H

X=F

1.6 ± 0.3

2.7 ± 0.5

94 ± 17

3.3 / 4.4

3.8

>30

>30

1 @ 10

20

B R2 = H, R3 = F

X = Cl

0.7 ± 0.2

0.6 ± 0.1

11 ± 3

3.5 / 5.1

3.6

18.5, >30

>30

16 @ 10


27


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Lead compound 7, together with 15 and 16, showed good potency and acceptable physicochemical properties (Table 1 and 3), although their hERG activity in our highthroughput, non-GLP screening assays was of concern. Compound 7 inhibited dofetilide binding to hERG with an IC50 of 9.6 µM in a radioligand binding assay (RLB) assay,34 inhibited K+ flux through hERG with an IC50 of 4.2 µM in an automated patch clamp (QPatch) assay38 and with an IC50 of 4.5 µM in a manual patch clamp assay39 (Table 3). Reduction of the potential cardiac safety risk associated with hERG inhibition was considered a key priority.40 Although several ion currents and channels are involved in the cardiac action potential,41 we will here only discuss the de-risking strategy for QT prolongation based on minimization of hERG inhibition, since it is well established that preclinical studies measuring hERG current are predictive for QT prolongation in patients.42,

43

With the aim of reducing the hERG activity of compound 7,

without reducing potency, we synthesized analogs in which the pyrimidine ring was replaced by other moieties. From the crystal structure of 7 (Figure 6E) we discerned that the pyrimidine was in close proximity to the backbone carbonyl group of glutamic acid E481. This suggested that this carbonyl group could be a target for H-bonding which might be achieved by replacing the pyrimidine with a five-membered heterocycle bearing an H-bond donor functionality. Docking studies indicated that a pyrazole ring was suitable for this purpose and compounds 1 and 18-20 were synthesized to explore this concept (Tables 2 and 3). Although the pyrazole derivatives (1 and 18-20) were slightly less potent as ABL1 kinase inhibitors compared to their pyrimidine counterparts (3, 15 and 16), their hERG activity was markedly reduced. This finding cannot simply be attributed to changes in lipophilicity and ionization state44, since replacing the pyrimidine with pyrazole only slightly increased logP (compare the matched pair, 7 and 18) and all of the compounds are weakly basic and largely


28

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unionized at physiological pH.44 Although hERG activity could also be reduced in the pyrimidine series, as in the dihydroxy-pyrrolidine derivative 17, this was associated with a marked decrease in cellular potency and this approach was not pursued. Our strategy of replacing pyrimidine with pyrazole would appear not to be a general strategy to reduce hERG activity, since such replacement has been reported not to influence hERG activity.45 The pyrazole derivatives combined good antiproliferative activity with strongly reduced hERG inhibition and lower QT prolongation risk, and consequently these were further profiled. Compounds 1 and 18-20 all had similar cellular potency (GI50 0.6-2.7 nM in the Luc-Ba/F3 BCR-ABL1WT cell line; Table 3) similar in vitro clearance and permeability in liver microsome46 and Caco-2 assays47 (Supplementary Table 1) and similar PK profiles in mice (Table 2). However, the compounds were differentiated in terms of efficacy in our KCL-22 mouse xenograft model: When administered by oral gavage at 3 mg/kg BID for 1 week, compounds 1 and 20 afforded 68-70% tumor growth inhibition, whereas compounds 18 and 19 showed no efficacy at this dose and schedule (Figure 7A). The increased efficacy of 1 and 20 over 18 and 19 might possibly result from the higher tumor concentrations achieved at 7 h for 1 and 20 (C7hr 0.16 ± 0.04 and0.20 ± 0.03 µM) compared to 18 and 19 (C7hr 0.08 ± 0.01 and 0.10 ± 0.02 µM); though all compounds 1, 19-20 showed overall similar tumor exposures at 1 h (~C1hr 0.2 µM) (Supplementary Table 3). Consequently, compounds 1 and 20 were regarded as being viable development candidates.

Formulation of compound 1 Compound 1 is weakly basic (pKa 4.0) and exhibits a typical pH-dependent solubility profile, with high solubility at acidic pH and low thermodynamic solubility at neutral pH


29


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(Supplementary Figure 4A). As an anhydrous crystalline solid, 1 has a melting point of 197199°C, with a high crystal-lattice energy of 97.3 J/g indicating a well-ordered lattice, and a glass transition temperature of 130°C. Compound 1 exhibits two additional properties which were important for formulation development. First was the influence of bile, where the addition of 50 mM sodium taurocholate at pH 6.8 increased solubility in aqueous buffer (pH 6.8) from 0.001 mg/mL to 0.82 mg/mL (>800-fold). Secondly, in a solvent-shift assay48 the compound gave a stable solution of 0.36 mg/mL in fasted state simulated intestinal fluid (FaSSIF) media at 15 minutes, representing a ~40-fold improvement over the equilibrium solubility. Considering the likely effect of intestinal bile on drug absorption, a solid dispersion (SD) formulation strategy was pursued to minimize the effect of bile by capitalizing on the propensity for supersaturation to increase the fraction absorbed in the fasted state. Formulation screening provided a formulation comprising of 25% compound 1 in an amorphous polymer matrix consisting of 37.5% Pharmacoat® 603 and 37.5% PVP VA 64. The SD had a glass transition temperature of 118°C and solubility of 260 µg/mL in FaSSIF. In order to help evaluate the potential of the SD, two-step dissolution experiments were performed (Supplementary Figure 4B). The SD and the crystalline form dissolved completely in simulated gastric fluid (SGF) within 60 minutes, whereupon concentrated FaSSIF media and bile salts were added to adjust the pH to that of the fasted intestinal environment in human. Whereas crystalline 1 rapidly precipitated to undetectable solution levels at 60 minutes post pH-switch, the SD had a sustained solubility of ~0.3 mg/mL 120 minutes post pH switch. On the basis of this data, both formulations were administered by oral gavage at 60 mg/kg in fasted beagle dogs, whereupon the SD gave a ~6-fold improvement in Cmax and AUC compared to the crystalline free-form suspension (Supplementary Table 2). Absorption was complete for


30

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the SD, but incomplete for the crystalline free-form suspension (~30%). Based on this data, the SD was adopted for in vivo studies.

Characterization of clinical candidate 1 as an ABL1 tyrosine kinase inhibitor. Compound 1 is a highly potent ABL1 inhibitor at the biophysical, biochemical and cellular levels. Isothermal titration calorimetry gave a binding affinity, KD of

0.5 nM, with

thermodynamic parameters (∆H = -72.8 kJ/mol; ∆S = -65.3 J/mol/K; ∆G = -53.2 kJ/mol) being consistent with strong enthalpy-driven, entropically unfavorable binding. The co-crystal structure of compound 1 liganded to the ABL1-imatinib complex is illustrated in Figure 6F. As an allosteric inhibitor it adopts the anticipated binding mode, with the pyrazole ring participating in a H-bond interaction with the backbone carbonyl group of glutamic acid E481 and making hydrophobic contact with threonine T453. The chlorine atom of the CF2ClO- group makes van der Waals contacts with residues I508, V487 and L448 located deep within the pocket. The biochemical activity translated into cellular activity, with the compound inhibiting the proliferation of Luc-Ba/F3 cells transformed to express BCR-ABL1WT and BCR-ABL1T315I with mean GI50 values of 1.0 ± 0.1 and 25 ± 3 nM, respectively. In the parental human CML KCL-22 cells, which were employed for in vivo studies (unfortunately the luminescence response from the luciferase-expressing cell lines was insufficiently robust to use light-emission as a measure of tumor size), the mean GI50 value was 1.6 ± 0.3 nM. The ability to maintain activity against mutant forms of BCR-ABL which impart resistance to ATP-competitive drugs used to treat CML was evaluated in a panel of Luc-Ba/F3 cells engineered to express mutant forms of BCRABL. In direct comparison with the ATP-competitive drugs, compound 1 potently inhibited with GI50 values 20

Theoretical hepatic extraction ratio ER [%] Microsomes Rat/Hu/Ms/Dog

48 / 44 / 60 / 74

Hepatocytes

65 / 64 / 63 / 64


36

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Table 7. Pharmacokinetics of compound 1 in preclinical species, comparison of PK parameters of compound 1 in male mice (C57BL/6), rats (sprague-dawley) and dogs (beagle). Species Route Formulation Feeding status Dose (mg/kg) Tmax (h) Tlast (h) Cmax (ng/mL)

Mouse i.v. A Nonfasted 1 7

Rat

p.o. A Nonfasted 3 2 7 295

i.v. A Nonfasted 2 7

Dog p.o. A Fasted

i.v. B Fasted

p.o. C Fasted

3 4 7 60

1

3 0.5 - 1 24 1258

24

98 20 419 Cmax/dose (ng/mL)/(mg/kg ) 1404 849 1888 268 2812 5552 AUClast (ng.h/mL) 1404 283 944 89 2812 1851 AUClast/dose . (ng h/mL)/(mg/kg) CL (mL/min/kg) 11.7 16.3 6 Vss (L/kg) 0.5 2.2 1 t½(h) 1.1 2.7 3.7 F (%) 21 9 66 Formulation: A = HCl, PEG, solutol, PBS, B = HCl, PEG300, solutol HS 15, citrate buffer, C = SD in 100% PBS.


37


Page 38 of 56

Synthesis of lead compounds and clinical candidate 1 The syntheses of trisubstituted B-ring derivatives and the CF3O- analogs exemplified in Tables 1 and 3 are illustrated in Schemes 1 and 2. 4-(Trifluoromethoxy)aniline 22a was coupled with iodo-benzoic acid 21 and the resulting amide 23 was subjected to a Suzuki reaction50 with pyrimidin-5-ylboronic acid 24a to afford the pyrimidine-benzamide 8 (Scheme 1). The nicotinic acid 25 was elaborated to give the pyrimidine-nicotinamide 9 using an analogous reaction sequence. The alkoxy-substituted benzamides, 10 and 11, were prepared in a similar fashion, with the latter compound being prepared from the corresponding phenol 30, by a nucleophilic displacement reaction with 1-(2-chloroethyl)pyrrolidine 31. The 5,6-disubstituted nicotinamides were prepared by the sequence shown in Scheme 2. Treatment of the acid chloride, generated from 32, with aniline 22a provided the pivotal intermediate 33a. Subsequent SNAr reaction with the appropriate amine followed by Suzuki coupling then provided the target compounds. The pyrazole analog of 7, compound 18, was prepared from the bromo intermediate via Suzuki reaction with (1-(tetrahydro-2H-pyran-2-yl)1H-pyrazol-5-yl)boronic acid pinacol ester 24b and subsequent deprotection of the tetrahydro2H-pyran-2-yl moiety with TFA to afford the pyrazole analog 18. The pyrazole derivative 19 was prepared analogously to 18, however rather than employing the enantiomerically pure methylpyrrolidin-3-ols as used for compounds 7 and 14, racemic 3-methylpyrrolidin-3-ol 34e was employed to give a racemic mixture with the enantiomers being separated prior to the Suzuki reaction.


38

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Compound 127 and the trifluoromethylthio- and chlorodifluoromethoxy-analogs 15-17 were prepared by similar routes to those discussed above as depicted in Scheme 3. Thus, 5-bromo-6chloronicotinic acid (32) was treated with thionyl chloride in the presence of DMF and the acid chloride was condensed in situ with the appropriate aniline 22b or 22c, in the presence of DIPEA. The nicotinamides 33b and 33c were then reacted with the enantiomerically pure pyrrolidin-3-ol derivatives 34d or 34f to afford the substituted bromo-nicotinamides in high yield. These intermediates were then coupled with 5-pyrimidinyl boronic acid (24a) or the pinacol ester of the protected (1H-pyrazol-5-yl)boronic acid 24b, using a standard SuzukiMiyaura coupling reaction to afford the pyrimidines 15-17 and the tetrahydropyranyl protected pyrazole analog of compound 1. The protecting group was then cleaved under acidic condition in DCM in a further step to afford compound 1 in 47% yield over 4 steps. For compound 20 the displacement with (R)-pyrrolidin-3-ol 34d and the coupling reaction step were reversed: the iodine analog of nicotinic acid derivative 32, 6-chloro-5-iodonicotinic acid, and 4-fluoro-3(tributylstannyl)-1H-pyrazole were first coupled using Stille reaction51 conditions (Pd(PPh3)4 in DMSO and then subsequently condensed with (R)-pyrrolidin-3-ol using the same conditions as described for compound 1. Further details for the synthesis of compounds 1 and 7-20, characterization of compounds and intermediates are given in the supporting information.


39


Page 40 of 56

Scheme 1. Synthesis of compounds 8-11. F O

F

I

HO

F

O F

O

F I

N H

(a)

N

N H 8

23 F

O

F

Br N

F

O F

O

F

Br

N H N

(c)

25

O F

F F

Br

F

N 9

O N H

R2

N

N H

(d) F

O

N

O

26

O HO

F

N

O

(b)

21

HO

O

F

Br

O F

N

O

N

N H

R2

R2 '

27 R2 = OMe

(e)

28

(f)

29 R2 = OH

(g)

30

(h), (i)

10 R2' = OMe 11 R2' = O

N

Reagents and conditions: (a) 4-(trifluoromethoxy)aniline (22a), HCTU, DIPEA, THF. MeCN, RT; (b) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME, H2O, EtOH, 80°C; (c) (1) SOCl2, DMF, toluene, RT then 80°C 1.5 h; (2) 4-(trifluoromethoxy)aniline (22a), DIPEA, THF, -15°C then

RT (d) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3,

DME/H2O/EtOH (7.5:1.5:1) 125°C MW, 15 min; (e) (1) SOCl2, DMF, toluene, 80°C, 1 h; (2) 4(trifluoromethoxy)aniline (22a), DIPEA/THF, 0°C, 1 h; (f) pyrimidin-5-ylboronic acid (24a), PdCl2(dtbpf), TEA, H2O, 2.5% TPGS-750-M, RT, 40°C, 42 h; (g) (1) SOCl2, DMF, toluene, 80°C, 2.5 h; (2) 4-(trifluoromethoxy)aniline (22a), DIPEA, THF, 0°C to RT, 1 h, reflux, 2 h; (iii) NaOH, H2O, reflux, 3 h; (h) 1-(2-chloroethyl)pyrrolidine 31, K2CO3, KI, acetone, 80°C 16 h; (i) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH (7:2:1) 80°C, 16 h.


40

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Scheme 2. Synthesis of 5-6-di-substituted N-(4-(trifluoromethoxy)phenyl)nicotinamide analogs 7, 12-14, 18 and 19. F O Br

HO N

O

F

O

F

(a)

Cl

Br

N H

Cl

N

32

33a F F

O

O

F

R1

N H N

(b), (c)

7

(d), (e)

12

(f), (g)

13

(h), (g)

14

(b), (i), (j)

18

R2 =

N

19

OH

N

N R1 =

OH

N H

N " "

N N

OH

N

OH

N (k), (i), (j)

R2

OH

" HN N

"

Reagents and conditions (a) (1) SOCl2, DMF, toluene, RT then 85°C 2 h; (2) 4(trifluoromethoxy)aniline (22a), DIPEA, THF, -25°C then 10°C, 30 min; (b) (R)-pyrrolidin-3-ol 34d, DIPEA, i-PrOH, MW, 140 °C, 1 h; (c) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH (7:2:1) 80 °C, 2 h; (d) 3-aminopropan-1-ol 34a, DIPEA, i-PrOH, MW, 140°C, 2.5 h; (e) pyrimidin-5-ylboronic acid (24a),

Pd(PPh3)2Cl2, Na2CO3,

DME/H2O/EtOH (7:2:1) MW, 125°C, 20 min; (f) 1-methylpiperazine 34b, DIPEA, i-PrOH, MW, 140°C, 1 h; (g) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH (7:2:1) 80°C, 2 h; (h) (S)-pyrrolidin-3-ol 34c, DIPEA, i-PrOH, MW, 140°C, 0.5 h; (i) (1-


41


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(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)boronic acid pinacol ester (24b), Pd(PPh3)4, K3PO4, toluene, 110°C, 2.5 h; (j) TFA, DCM, 10°C to RT; (k) (1) 3-methylpyrrolidin-3-ol 34e, DIPEA, i-PrOH, 140°C, 1 h; (2) Chiral separation.


42

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Scheme 3. Synthesis of compounds 1 and 15-17

Reagents

and

conditions

(a)

(1)

SOCl2,

DMF,

toluene,

80°C,

1

h;

(2)

4-

((trifluoromethyl)thio)aniline (22b), DIPEA, THF, 0°C 2 h; (b) (R)-pyrrolidin-3-ol 34d, DIPEA, i-PrOH, 140°C, 1 h; (c) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH,

125°C,

30

min;

(d)

(1)

SOCl2,

DMF,

toluene,

80°C;

(2)

4-

(chlorodifluoromethoxy)aniline (22c), DIPEA, THF, -16°C then RT 1 h; (e) (R)-pyrrolidin-3-ol 34d, DIPEA, i-PrOH, 140°C, 1 h; (f) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH, 125°C, 30 min; (g) (3S,4S)-pyrrolidine-3,4-diol 34f, DIPEA, i-PrOH, 140°C, 1 h; (h) pyrimidin-5-ylboronic acid (24a), Pd(PPh3)2Cl2, Na2CO3, DME/H2O/EtOH, 125°C, 10 min; (i) (1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)boronic acid pinacol ester (24b), Pd(PPh3)4, K3PO4, toluene, 110°C, 4 h; (j) TFA, DCM, 10°C to RT.


43


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Conclusions This paper describes the discovery and preclinical profile of asciminib (ABL001, 1). Because this compound potently inhibits the tyrosine kinase activity of the wild-type BCR-ABL1, as well as drug-resistant mutant forms of the enzyme that can emerge under treatment with ATPcompetitive drugs, it has the potential to combat TKI-resistant CML. The compound acts via an allosteric mechanism and can be used in combination with nilotinib and therefore, probably also with other ATP-site-directed BCR-ABL1 inhibitors, such as imatinib or dasatinib. The discovery of this compound started with a fragment-based approach for hit-finding. Then, following the development of an NMR-based conformational assay which elucidated the conformational requirements for allosteric BCR-ABL1 inhibition, extensive structure-based design was employed to assist optimization of lead compounds for activity in biochemical and cellular assays for ABL1 kinase inhibition. These studies culminated with compound 1, which exhibits strong antiproliferative activity in cells where proliferation is driven by the oncogenic fusion protein BCR-ABL1 and crucially this activity is maintained in cells driven by mutations which can render currently available CML drugs ineffective in CML. The in vitro activity of the compound translated into anti-tumor activity in murine models of CML and commensurate with its attractive preclinical profile it was progressed into full development as a potential drug for the treatment of CML. Although resistance has been reported in preclinical studies with allosteric ABL inhibitors 27, the resistance mutation patterns of ATP-site inhibitors, such as nilotinib, and compound 1 are orthogonal, the emergence of resistance is dramatically reduced upon simultaneous treatment with both inhibitors. Compound 1 is currently being evaluated in clinical trials as a single agent and in combination with ATP-competitive TKIs in CML and ALL patients.


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ASSOCIATED CONTENT Supporting Information. Synthesis procedures for compounds 1 and 6- 24 (PDF); molecular formula string (CSV). The supporting information is available free of charge on the ACS Publication website at DOI: 10.10201/acs.jmedchem.xxxxxxx. “A brief statement in non-sentence format listing the contents of material supplied as Supporting Information should be included, ending with “This material is available free of charge via the Internet at http://pubs.acs.org.” For instructions on what should be included in the Supporting Information, as well as how to prepare this material for publication, refer to the journal’s Instructions for Authors.”

AUTHOR INFORMATION Corresponding Author *(A.M.) Tel: +41795581186. E-mail: [email protected] ORCID Andreas L. Marzinzik: 0000-0003-0829-0412 Notes The authors declare no competing financial interests.


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ACKNOWLEDGMENT We acknowledge and thank all colleagues involved in the discovery of ABL001. We specially thank the following scientists: Hongbo Cai, Giorgio Caravatti, Gabriele Fendrich, Geeti Gangal, Markus Gaugler, Tami Hood, Marc Lang, Danuta Lubicka, Roberto Martinez, Jürgen Mestan, Andrew W. Pape, Gisela Scheffel, Egge Seeber, Sreenath Sharma, Erich Spieser, André Strauss, Sanjeev Thohan, Susanne Vollmer, Nigel Waters, Andrea Wiget, Wenjing Zhu and Robin Fairhurst for their help reviewing parts of the manuscript.

ABBREVIATIONS ABL, Abelson murine leukemia viral oncogene homolog; AUC, area under the curve; BCR, breakpoint cluster region protein; BID, bis in die, twice a day; CL, clearance; Cmax, maximum concentration; CML, chronic myelogenous leukemia; DCM, dichloromethane; DIPEA, N,Ndiisopropylethylamine; DMF, dimethyl formamide; DMSO, dimethyl sulfoxide; ER, extraction ratio; F, fraction absorbed; FaSSIF, fasted state simulated intestinal fluid, GLP, good laboratory practice; HCl , hydrochloric acid; hERG, human ether-à-go-go-related gene (protein KV11.1); IC50, half maximal inhibitory concentration; i-PrOH, isopropyl alcohol; ITC, isothermal titration calorimetry; K3PO4, tripotassium phosphate; NMR nuclear magnetic resonance; PK, pharmacokinetic; RLB, radio ligand binding assay; TKI, tyrosine kinase inhibitor; Papp; apparent

permeability;

PBS,

phosphate-buffered

saline;

Pd(PPh3)4,

tetrakis(triphenylphosphine)palladium(0); PEG, polyethylene glycol; Qpatch, Automated patch clamp; QT (interval), time between the start of the Q wave and the end of the T wave in the heart's electrical cycle; RT, room temperature; SD, solid dispersion, standard deviation; SGF,


46

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simulated gastric fluid; SOCl2, thionyl chloride; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Tlast, the last time point at which a quantifiable plasma drug concentration can be measured; Tmax, time at which Cmax is observed; Vss, volume of distribution; WT, wild type.

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Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine

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