Discovery of Allosteric, Potent, Subtype Selective, and Peripherally

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Discovery of Allosteric, Potent, Subtype Selective and Peripherally Restricted TrkA Kinase Inhibitors Sharan K Bagal, Kiyoyuki Omoto, David C Blakemore, Peter J. Bungay, James G Bilsland, Philip J Clarke, Matthew S. Corbett, Ciarán N Cronin, Jingrong Jean Cui, Rebecca Dias, Neil J Flanagan, Samantha E Greasley, Rachel Grimley, Eric Johnson, David Fengas, Linda Kitching, Michelle L Kraus, Indrawan McAlpine, Asako Nagata, Gareth J. Waldron, and Joseph S Warmus J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00280 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Journal of Medicinal Chemistry

Discovery of Allosteric, Potent, Subtype Selective and Peripherally Restricted TrkA Kinase Inhibitors Sharan K. Bagal,a* Kiyoyuki Omoto,a David C. Blakemore,a Peter J. Bungay,b James G. Bilsland,c Philip J. Clarke,f Matthew S. Corbett,d Ciaran N. Cronin,e J. Jean Cui,e Rebecca Dias,c Neil J. Flanagan,c Samantha E. Greasley,e Rachel Grimley,c Eric Johnson,e David Fengas,f Linda Kitching,c Michelle L. Kraus, e Indrawan McAlpine, e Asako Nagata, e Gareth J. Waldron,c Joseph S. Warmus.d a

Worldwide Medicinal Chemistry, Pfizer Global R&D UK, The Portway Building, Granta Park,

Cambridge, CB21 6GS, UK b

Pharmacokinetics, Dynamics & Metabolism, Pfizer Global R&D UK, The Portway Building,

Granta Park, Cambridge CB21 6GS, UK c

Pfizer Global R&D UK, The Portway Building, Granta Park, Cambridge CB21 6GS, UK

d

Pfizer Global R&D, Groton Laboratories, Eastern Point Road, Groton, Connecticut 06340, USA

e

Pfizer Global R&D, La Jolla Laboratories, 10770 Science Center Drive, San Diego, California,

92121, USA f

Peakdale Molecular, Discovery Park House, Ramsgate Road, Sandwich, Kent CT13 9ND, UK

ACS Paragon Plus Environment

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KEYWORDS Nerve growth factor receptor, NTRK1, NGF, TrkA, Tropomyosin Receptor Kinase, Pain, Allosteric, Type III inhibitor, Kinase inhibitor

ABSTRACT Tropomyosin receptor kinases (TrkA, TrkB, TrkC) are activated by hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4). Moreover, the NGF antibody tanezumab has provided clinical proof of concept for inhibition of the TrkA kinase pathway in pain leading to significant interest in the development of small molecule inhibitors of TrkA. However, achieving TrkA subtype selectivity over TrkB and TrkC via a Type I and Type II inhibitor binding mode has proven challenging and Type III or Type IV allosteric inhibitors may present a more promising selectivity design approach. Furthermore, TrkA inhibitors with minimal brain availability are required to deliver an appropriate safety profile. Herein we describe the discovery of a highly potent, subtype selective, peripherally restricted, efficacious and well-tolerated series of allosteric TrkA inhibitors that culminated in the delivery of candidate quality compound 23.


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INTRODUCTION Tropomyosin receptor kinases are a family of three tyrosine kinases (TrkA, TrkB, TrkC) activated by peptidic hormones of the neurotrophin family: nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4) (Figure 1).1 Preclinical and clinical studies have identified a crucial role for NGF in the pathogenesis of inflammatory pain,2 with neutralizing antibodies such as tanezumab demonstrating efficacy in preclinical pain models and in clinical trials for osteoarthritis, chronic low back pain and interstitial cystitis.

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Since NGF binds to the TrkA kinase receptor this has provided clinical proof of

concept for inhibition of the TrkA pathway in pain and has also led to significant interest in the development of small molecule modulators of TrkA.

Figure 1. Neurotrophins and their receptors. The neurotrophins exhibit specific interactions with the three Trk receptors: NGF binds TrkA, BDNF and NT-4 binds TrkB, and NT-3 binds TrkC.

A key concern in the development of TrkA kinase inhibitors for the treatment of chronic pain is achieving sufficient TrkA selectivity over TrkB and TrkC. TrkB is expressed throughout the body


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with the BDNF/TrkB axis involved in excitatory signaling, long-term potentiation and feeding behavior.7,8,9-10 Moreover, human genetic data has linked decreased function of TrkB with obesity and developmental delay.11 BDNF has been shown to play a role in regulating the arterial baroreceptor reflex and in modulating heart contraction force.12-14 TrkC is widely expressed in neural and non-neural tissues and has wide physiological functions that include the development and survival of the sympathetic nervous system.15-16 Most small molecule kinase inhibitors are ATP competitive Type I or Type II binders, by reference to the highly conserved aspartate-phenylalanine-glycine (DFG) motif in the beginning of the activation loop in the C-lobe of the kinase domain. Type I inhibitors such as the JAK inhibitor tofacitinib (recently approved for rheumatoid arthritis) binds in the ATP pocket in a DFGin conformation,17 whilst Type II inhibitors such as imatinib bind in the ATP pocket and extend into a less conserved allosteric region that is formed following a conformational change to the DFG-out structure.18 Whilst Type I and II inhibitors can display high levels of selectivity, TrkA, TrkB and TrkC kinases have no residue difference in the ATP binding site, suggesting that achieving TrkA selectivity over TrkB/C in this site may be extremely challenging. Moreover, our previous work on the pan-Trk inhibitor program where isoform non-selective TrkA/B/C inhibitors were progressed into clinical development, suggested that Type I and Type II binders tend to exhibit pan-Trk activity rather than subtype selectivity.19 One known method for achieving isoform selectivity within closely related kinases is the identification of Type III and Type IV allosteric ligands.20 Type III ligands bind in an adjacent site to the ATP binding site without making any interactions with the hinge region of the ATP binding site. Trametinib is a Type III inhibitor of MEK1/2 kinases and is the first allosteric kinase inhibitor approved by the FDA, being accepted as a single-agent for the treatment of patients with either B-Raf V600E or V600K mutated


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metastatic melanoma (Figure 2).21 Other allosteric kinase inhibitors that bind in a pocket adjacent to the ATP binding site include allosteric Akt inhibitors such as MK-2206. MK-2206 targets a unique allosteric pocket at the catalytic kinase domain and the regulatory pleckstrin homology (PH) domain of the inactive conformation of Akt and is currently under clinical investigation in numerous Phase I and II oncology trials.22 Type IV inhibitors bind to a site that is distant from the ATP binding site, exemplified by GNF-2 that binds the myristoyl pocket of the C-lobe of the kinase domain of Abl (Figure 2).23-24 An advantage of Type III and Type IV inhibitors is that the comparatively low sequence homology of allosteric sites provides unique opportunities for more specific inhibition and minimal off-target pharmacology, for example Type I and Type II Akt inhibitors tend to exhibit relatively poor kinome selectivity whereas allosteric Akt inhibitor MK2206 is reported to be more than 100-fold selective over 256 kinases screened.22, 25

Figure 2. Allosteric inhibitors Trametinib, MK-2206 and GNF-2 Inspection of an apo TrkA protein crystal structure generated at Pfizer indicated TrkA protein prefers to adopt a DFG-out autoinhibitory conformation. The DFG-out conformation is stabilized


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by edge to face interactions between three phenylalanines: DFG motif Phe669, gatekeeper Phe589 and back pocket Phe575 (Figure 3). Moreover, the presence of these residues serve to illustrate the uniqueness of TrkA since according to our understanding only five kinases exhibit the FFF motif, three of which are the Trks. This motif combined with Leu564 from the α-C helix creates a hydrophobic binding pocket behind the gatekeeper Phe589 that could, in theory, accommodate a Type III allosteric ligand. Moreover this is a similar allosteric pocket to that formed by the kinases IGFR-1R and c-Src.26-27 In these cases the kinase adopts a DFG-out conformation revealing a hydrophobic pocket adjacent to the ATP binding site and Type III ligands have been shown to bind to these allosteric sites by X-ray crystallography.26-27 Hence, the apo TrkA protein crystal structure suggested that an allosteric approach utilizing a Type III binder to achieve TrkA selectivity over TrkB/C was a reasonable proposition.

Figure 3. Apo crystal structure of TrkA protein highlighting a hydrophobic pocket potentially suitable for an allosteric Type III binder.


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The high level of expression of TrkA in cholinergic neurons of the central nervous system (CNS), and the effect of ablation of the TrkA gene that leads to dysfunction in cholinergic circuitry in preclinical species raised the risk of CNS adverse events in the development of TrkA inhibitors for the treatment of chronic pain.28-30 Furthermore, clinical studies on CE-245677, a pan-Trk/Tie2 kinase inhibitor identified by Pfizer as a potential treatment of certain cancers, identified CNS side-effects leading to the suspension of Phase I multiple dose trials. Adverse events included cognitive deficits, personality changes and sleep disturbances that fully resolved upon cessation of dosing.31 Preclinical studies with CE-245677 demonstrated both efficacy in pain models and CNS side-effects (changes in electroencephalography (EEG), cognitive function, body weight and hypothalamic mRNA).32 The efficacy of TrkA kinase inhibitors is expected to be driven by target engagement in peripheral neurons. Therefore, as safety and toleration risks are associated with CNS Trk receptor occupancy, safety risks can be addressed by restricting TrkA inhibitors to the peripheral compartment. Experience within Pfizer and the wider pharmaceutical community suggests that small molecules can be developed that possess acceptable oral bioavailability but are also peripherally restricted.33-37 This profile can be delivered by designing compounds within physicochemical space appropriate for absorption across the gastrointestinal epithelium (e.g. molecular weight (MW) 80-fold TrkA selectivity over TrkB/C in cell based assays (Scheme 1).41, 43 There was no strong correlation between docking score and potency but it was clear that the hit rate of the virtual library was enhanced. Note that ligand 3 wherein an aminopyridine group was appended at the meta position of the dichlorophenyl unit of 2 was ranked 15th in the 100 molecule set for docking score. Docking of 3 suggested that this fragment not only formed a hydrogen bond with NH in Asp668 using the pyridyl N atom, but also formed a hydrogen bond with the main chain C=O Asp668 via the amino hydrogen bond donor (Figure 8A-B). Furthermore, the aminopyridine fragment displaced some of the unstable waters identified in Figure 6B (Figure 8AB). The methyl pyrazole was substituted with a primary carboxamide in order to decrease LogD and interact with the side chain of Arg673 via a hydrogen bond with the carboxamide C=O (Figure 8B). The amide moiety also replaced some of the moderately stable waters identified in Figure 6B and interacted with the water network in this region. Lead molecule 3 exhibited a significant improvement in potency (TrkA cell IC50 50nM) and LipE (5), and maintained >80-fold TrkA selectivity over TrkB/C in cell based assays (Scheme 1).41, 43 The improvement in potency and LipE achieved in 3, whilst retaining TrkA subtype selectivity suggested that design of candidate quality molecules binding to the allosteric site of TrkA was an achievable prospect. Ligand 3 was screened for protein kinase selectivity at ca. 400 biochemical kinase assays (including TrkA) from Invitrogen™ at a concentration of 1µM (see supporting information). Pleasingly, 3 exhibited excellent TrkA selectivity with >80% inhibition of TrkA and 50% at 10µM, all of which corresponded with IC50 or EC50 >6µM). Lead molecule 3 was evaluated by surface plasmon resonance (SPR) binding methods with immobilised TrkA protein (residues 441-796) using Biacore instrumentation.38 With non-activated unphosphorylated TrkA protein, ligand 3 demonstrated fast association and dissociation kinetics (Ka 1.2 x107 M-1s-1; Kd 1s1

) and KD (80nM) consistent with TrkA cell potency. When activated phosphorylated TrkA protein

was used in the SPR assay there was a substantial reduction in signal from ligand 3 binding, indicating that allosteric ligands prefer to bind to the non-activated unphosphorylated state of TrkA. This finding was further corroborated by screening in non-activated TrkA and activated phosphorylated TrkA biochemical enzyme assays where 3 was approximately 6-fold more potent against the non-activated TrkA construct (TrkA non-activated enzyme IC50 25nM, TrkA activated enzyme IC50 150nM) (see supporting information).41, 43 The preference of allosteric ligands for the inactive state of TrkA protein can be rationalised by consideration of protein conformation. In the autoinhibitory DFG-out conformation the allosteric binding site can be formed, whereas in the phosphorylated state the JM domain will move to allow the activation loop to access an active DFG-in conformation. In the latter case the allosteric binding site is blocked by the activation loop making the allosteric binding site less readily accessible.46 Further in vitro studies were carried out to understand metabolic liabilities of ligands 2 and 3. Hit molecule 2 exhibited moderate metabolic turnover in human liver microsomes (HLM) and human hepatocytes (hHep) (intrinsic clearance (CLint) in HLM 35L/min/mg protein, hHep 48µL/min/million cells, see experimental section). Lead molecule 3 is a unit lower in LogD when


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compared with 2 resulting in greater metabolic stability in human in vitro systems (HLM 2.5. Whilst hit 2 was not a P-gp substrate, ligand 3 was a substrate of both P-gp and BCRP which is consistent with the higher MW and PSA of 3 relative to 2 (Scheme 1).33-34, 39 The CNS penetration of 3 was assessed in vivo in rat and was confirmed to be peripherally restricted with Cb,u/Cp,u of 0.04. Further oral and i.v. rat PK studies revealed that whilst 3 exhibited relatively low clearance (CL) in vivo it also had low oral bioavailability (6%) (Table 1). The measured passive permeability of 3 assessed in the RRCK transcellular flux assay was consistent with good absorption (RRCK Papp 17 x10-6 cms-1),54-55 suggesting that low oral bioavailability could have resulted from pre-systemic metabolism. Incubation of 3 with rat intestinal microsomes in the


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presence of UDPGA and NADPH revealed high turnover (CLint 175L/min/mg), presumably via glucuronidation on the amino group (as identified via metabolite identification). Turnover of 3 was also detectable in human intestinal microsomes (HIM) (CLint 4.5L/min/mg) and in HLM supplemented with UDPGA (HLMUGT, CLint 29L/min/mg). The most effective method of reducing intestinal metabolism (and hence improving oral bioavailability) whilst maintaining TrkA potency, was the removal of the amino unit as exemplified by ligands 4-10 (Tables 1 and 2). N-acyl derivative 4 (TrkA cell IC50 0.034µM, LogD 2.7, LipE 4.8) is likely to have a reduced glucuronidation liability. However, the acetyl group was chemically unstable and ligand 4 underwent N-deacetylation in buffer systems at pH 7.5. N-acetyl amide stability can be improved by reversing the amide,56 and in this case reversing the amide to yield 5 improved chemical and metabolic stability (HLMUGT CLint 1000-fold weaker at TrkA, possibly due to a repulsive interaction between the C=O of 5 and main chain C=O of Asp668. Introduction of a polarized CH in 6 using a CHF2 moiety resulted in a 20-fold decrease in potency at TrkA (TrkA cell IC50 1.1µM) when compared with 3. Introduction of a lipophilic group on the 3-position of the aminopyridine as in 9 (R2=Cl, Table 2) improved TrkA potency by 3-fold (TrkA cell IC50 16nM) but also increased the glucuronidation rate significantly (HLMUGT CLint 209µL/min/mg). Removal of the amino group in 9 to give 8 reduced glucuronidation to a minimum (HLMUGT CLint

Discovery of Allosteric, Potent, Subtype Selective, and Peripherally

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