Tropomyosin receptor kinase inhibitors

EXPERT OPINION ON THERAPEUTIC PATENTS, 2017 http://dx.doi.org/10.1080/13543776.2017.1297797

REVIEW

Tropomyosin receptor kinase inhibitors: an updated patent review for 2010-2016 – Part II Justin J. Bailey, Ralf Schirrmacher, Kristen Farrell and Vadim Bernard-Gauthier Faculty of Medicine & Dentistry, Department of Oncology, University of Alberta, Edmonton, Canada ABSTRACT

ARTICLE HISTORY

Introduction: TrkA/B/C receptor activation supports growth, survival, and differentiation of discrete neuronal populations during development, adult life, and ageing but also plays numerous roles in human disease onset and progression. Trk-specific inhibitors have therapeutic applications in cancer and pain and thus constitute a growing area of interest in oncology and neurology. There has been substantial growth in the number of structural classes of Trk inhibitors and the number of industrial entrants to the Trk inhibitor field over the past six years. Areas covered: In Part II of this two-part review, the discussion of recent patent literature covering Trk family inhibitors is continued from Part I and clinical research with Trk inhibitors is considered. Expert opinion: Trk has been molecularly targeted for over a decade resulting in the progressive evolution of structurally diversified Trk inhibitors arising from scaffold hopping and HTS efforts. Correspondingly, there have been a growing number of clinical investigations utilizing Trk inhibitors in recent years, with a particular focus on the treatment of NTRK-fusion positive cancers and chronic pain. The observed potential of Trk inhibitors to cause adverse CNS side effects however suggests the need for a more rigorous consideration of BBB permeation capabilities during drug development.

Received 21 October 2016 Accepted 17 February 2017

1. Introduction The tropomyosin receptor kinase (Trk) family encompasses the three structurally homologous receptors, TrkA, TrkB, and TrkC, which control the spatiotemporal regulation of growth, differentiation, and survival of central and peripheral neurons [1]. Extracellular interaction of Trk receptors with their preferential ligands, nerve growth factor (NGF) for TrkA [2,3], brain-derived neurotrophic factor for TrkB [4], and neurotrophin-3 for TrkC [5], induces receptor dimerization and autophosphorylation of tyrosines in the intracellular kinase domain, successively activating downstream signaling cascades. Changes in normal expression levels, ectopic Trk expression, oncogenic Trk activation, and the ensuing signaling alterations linked to those changes can trigger neurological disorders or contribute to the genesis of both neural and non-neural neoplasms. Significant pharmaceutical effort has been focused on the development of inhibitors that target the Trk kinase domain, yet isoform-specific inhibitors of Trk remain scarce owing to the high degree of sequence homology in the kinase domain of the Trk family. Herein is a continuation of the review Tropomyosin receptor kinase inhibitors: an updated patent review for 2010–2016 – Part I [6], opening with the continued deliberation of patents published within the last 6 years.

2. Patent evaluations (a continuation from Part I) Patent applications published between 2010 and mid-2016 claiming compounds for the inhibition of Trk are summarized

CONTACT Justin J. Bailey

[email protected]; Vadim Bernard-Gauthier

© 2017 Informa UK Limited, trading as Taylor & Francis Group

KEYWORDS

Tropomyosin receptor kinase; TrkA; TrkB; TrkC; NTRK1/2/3; tropomyosin receptor kinase inhibitor; oncology; cancer treatment; chronic pain; pruritis; entrectinib; LOXO-101; targeted therapy

in this review. Patents are also included in this review if Trk is not the main kinase target of interest but where either noteworthy Trk inhibition data are included or insight into the structural origins of the Trk inhibitor series is provided. Patents prior to this date which include inhibitors of Trk or other kinases are mentioned in support of these current patents. References to primary literature and public meetings are also described if relevant. Representative compounds from each patent are chosen to illustrate key structural features and/or highlight Trk inhibition values. In some instances, the primary sites of structural exploration in each patent series are denoted in red on representative compounds for clarity. Figures containing structures originating from more than one patent reference their respective patents in the figure legend while figures focusing on one patent are referenced in the body of the text. Compound numbers are retained from the originating patent or are numbered sequentially throughout the review if no label was found. Patents are organized in alphabetical order of companies claiming Trk inhibitors during the reviewed time period, starting with Merck (Kenilworth, NJ, USA). Companies alphabetized before Merck can be found in Part I of this review [6].

2.1. Merck Merck has published multiple patent applications in recent years describing the development of pan-Trk inhibitors covering an extensive breadth of structural and type I/II/III (juxtamembrane domain (JM)-interacting) binding mode diversity [7,8]. In line with promising preclinical and clinical data [email protected]

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Article highlights ●

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Many novel Trk inhibitor scaffolds have been identified through HTS and repositioning of inhibitors developed for kinase targets other than Trk. Characterization of NTRK1/2/3 fusion oncogenic drivers as low frequency occurrences across multiple human cancer subtypes and the unambiguous clinical validation of the pan-Trk inhibitors entrectinib and LOXO-101 from phase I study reports in NTRK fusion-positive patients have enticed an increase in interest in Trk inhibitors. The potential for neurological side effects induced by CNS Trk inhibition is beginning to influence Trk inhibitor design with the inclusion of screening assays to assess BBB penetration and tailoring inhibitor design to reduce passive diffusion or increase efflux. Trk inhibitors are also gaining interest as modulators of the nervous system and are currently under clinical investigation for the treatment of osteoarthritis pain.

This box summarizes key points contained in the article.

ensuing from anti-TrkA and anti-NGF monoclonal antibody therapeutic approaches [9–11], Merck has largely emphasized the treatment of inflammatory and neuropathic pain for Trk inhibitors in preclinical work [7], while all patented inhibitors over the last 4 years acknowledge and claim potential utility for a variety of Trk-related conditions. Similar to the contributions by GNF/Novartis (San Diego, CA, USA) and Array (Boulder, CO, USA), as discussed in Part I of this review, Merck released a series of pyrido[3,2-d]pyrimidine and quinazoline inhibitors appended with racemic or (R)-enantiomer halogenated 2-phenylpyrrolidines, including the prominent 2,5-difuorophenyl pyrrolidine moiety [12]. Potency data provided for selected compounds reveal low nanomolar to high picomolar activities (Figure 1(b)). Moving from a 6,6-fused heterocyclic hinge binder to the largely explored 6,5-bicyclic nitrogen-containing core, a concomitant application describes analogous triazolo[4,3-b]pyridazine and triazolo[4,3-a]pyridine inhibitors [13]. Compounds with exemplified biological data from this series displayed moderate TrkA potencies (Figure 1 (c)). Inhibitors covered within these applications originate from an early high-throughput screening (HTS) effort identifying a pyrido[3,2-d]pyrimidine low potency hit bearing a 3picolylamine substituent, which was refined in structure–

a

Hinge Region

Met592

b

c

N

N

N N

NH

activity relationship (SAR) development. Crystal structure determination of 1 with TrkA (Figure 1(a)) clarified the overlap of the 3-picolylamine within the ribose-binding pocket, confirming the type I binding mode of the inhibitors from the resultant series [7]. Interestingly, these data contrast the crystallography results obtained by GNF/Novartis with a related benzylamine-bearing pan-Trk inhibitor (Part I, Figure 27) [14], which was shown to bind the inactive kinase conformation of TrkC in reverse orientation with respect to the hinge. Although no TrkB/C or kinome data were described within these two applications, Merck independently reported data indicating high kinome selectivity and pan-Trk activity for these two early series [7]. Merck’s HTS campaign also identified structurally original hits from non-kinase programs. Notable examples initially described in patent applications include bicyclic carboxylic acids [15] and tri-substituted ureas [16] exemplified in Figures 2 and 3, respectively. Examples from the carboxylic acid series fall within either a 4-substituted 5-azaindole-2carboxylic acid or 5-substituted indole-2-carboxylic acid general chemotype. All examples bear a lipophilic linker fragment at the N1 position. The carboxylic acid substituent is a conserved feature in all but one example in this application. The initial hit (Figure 2, compound 2) from this series was shown to exhibit an unprecedented binding mode to TrkA, where the carboxylate engages the Lys544 side chain and the amide protons from the Gly671 and Met670 backbone, and the naphthalene substituent is concealed within the hydrophobic region facing the hinge. The inhibitor interacts with TrkA in a DFG-out conformation, orientating the N1 substituent toward the allosteric pocket without direct hinge interaction (Figure 2) [7]. SAR exploration and substituent replacement on the indole core led to a shift from lipophilic moieties, such as the naphthyl groups or the 3-ethoxyphenyl moiety exemplified in most compounds, for more compact and hydrophilic motifs such as the pyrazole in Example 76, which enables backbone interaction with the hinge (Figure 2). In addition to their unique binding mode, derivatives from this application were reported in a later study displaying a unique phosphorylation state-dependent binding which clarifies the modest potencies of selected examples from the patent application [15]. Marked discrepancies

N H

N

N S N

Ribose pocket

N O

F

N

N

N

N N

N

N

N

N

N

F N

N

NH

NH

F

F F

HO

F

N O

1 TrkA IC50 = 997 nM (Caliper) Cell IC50 = >10000 nM

Example 12 TrkA IC50 = 1.5 nM (Caliper) Cell IC50 = 13 nM

Example 27 TrkA IC50 = 0.7 nM (Caliper)

Example 24 TrkA IC50 = 27 nM (Caliper)

Figure 1. Binding mode of compound 1 with TrkA (a) (PDB: 4PMT) and Merck pyrido[3,2-d]pyrimidines ((b): WO2012125668) and triazole[4,3-b]pyridazines ((c): WO2012125667).

EXPERT OPINION ON THERAPEUTIC PATENTS

Asp590

Met592 H2O

HN N

N

Selectivity pocket

N

N

N N

N O

N O

O

CO2H

N

3

N

O

N O CO2H OCF3

Lys544

Met670 Gly671 2 HTS TrkA IC50 = 163 nM (HTRF) TrkA IC50 = 17130 nM (Caliper) TrkA (non-phos) IC50 = 2300 nM (Caliper) Cell IC50 = 1162 nM KD = 7.9 nM (SPR)

Example 16 TrkA IC50= 5358 nM (Caliper)

Example 76 TrkA IC50 = 28 nM (Caliper)

Figure 2. Binding mode of compound 2 with TrkA (PDB: 4PMS) and Merck 6,5-bicyclic heterocyclic carboxylic acids.

Met592

Asp668

S

OCF3

O

N N

Selectivity pocket

N H

OCF3

O N

N

N H

HN

SCF 3

O N

N

N H

HN

Hydrophobic region II (Val524) 3 TrkA IC50 = 4233 nM (Caliper) TrkA IC50 = 549 nM (DiscoverX PathHunter)



Figure 3. Binding mode of compound 3 with TrkA (PDB: 4PMP) and Merck tri-substituted ureas.

in TrkA activity between the HTS assay (HTFR), which utilizes a non-phosphorylated target, and the caliper assay (also used in the patent application), which uses phosphorylated TrkA, illustrate a phosphorylation-dependent binding mode of these inhibitors to Trk which displays a bias toward the inactive non-phosphorylated form of the kinase. This characteristic was also confirmed by surface plasmon resonance (SPR) analyses [7]. The cases of inconspicuous TrkA potencies in the caliper assay therefore may not reflect the activity of those inhibitors under physiological conditions, and the cellbased assay data likely provide more suitable insight for this inhibitor class. The trisubstituted ureas mentioned prior are derived from the HTS lead compound 3 (Figure 3) [16]. This work covers an extensive series of 163 compounds including selected examples assessed toward TrkA with potencies varying widely from low micromolar to low nanomolar. Here again, this application has been substantiated by a separate report discussing binding mode, crystallographic analysis, and SAR study. Compounds from this series were shown to bind TrkA receptors in a DFG-out conformation with minimal hydrogen bonding interactions between the ureayl moiety, DFG triad, and adjoining residues. The most potent examples from this application combine a 7-azaindole hinge binder motif along with a crucial hydrophobic isopropyl urea substituent which fills a cleft surrounded by Val524, Ala542, Lys544, and the gatekeeper Phe589 from the β-strand of the N-lobe. Removal of this moiety completely abrogates the activity of this series.

Pan-Trk activity, along with clean profiling in a 92-membered kinase panel, is reported for this series [7]. Two simultaneously filed patents [17,18] further elaborate on the urea TrkA inhibitors exploring substituted heterocyclic fused ring systems opposite a conserved 1-(substituted)-1Hpyrazol-5-yl or 2-(substituted)-pyridin-3-yl motifs (Figures 4 and 5). Merck has separately provided crystallographic data showing that representative compounds from these series bind as a unique type III inhibitor wherein the folded over JM domain plays a key role in allowing binding and in some instance, inducing TrkA selectivity (see Part I). In one application, these motifs are used in conjunction with 9H-fluoren-9-yl or 9H-xanthen-9-yl groups as the explored urea substituent (Figure 4) [17]. TrkA potency is provided for all compounds described and varies widely from 27 to 4800 nM. TrkB/C data are not provided but it should be expected that all of those examples display some level of TrkA selectivity. The majority of compounds from the second of these urea patents display low TrkA activity except for the two examples presented in Figure 5. Even assuming a type III JM-interacting binding mode, it is remarkable that the structurally simple symmetric urea, Example 1 (Figure 5), is the second most potent inhibitor enumerated with an IC50 of 82 nM for TrkA. Analogous, semisymmetrical modestly potent ureas are also found in Array’s recent patent filings [19]. Notably, these applications also include the appearance of the 2-phenylimidazo[1,2-a]pyridine-3-yl surrogate of the 1-phenyl-1H-pyrazol-5-yl moiety which is re-explored and refined in subsequent applications

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JM domain

Selectivity pocket

Ile490 N

O

O N H

N H

O

O

N

N H

Asp668 Example 4 TrkA IC50 = 99 nM TrkB IC50 > 81000 nM TrkC IC50 = 25000 nM

O N H

N N

N

N H

N H

Example 11 TrkA IC50 = 27 nM


(DiscoverX PathHunter)


(DiscoverX PathHunter) Figure 4. Binding mode of Example 4 with TrkA (PDB: 5KMI) and Merck non-symmetrical 1-(9H-fluoren-9-yl)ureas.

N

N

N

O N H

O

S

N

N

N

N N H

N H

N H





Figure 5. Merck symmetric and non-symmetric ureas.

by Merck (vide infra). Depending on substituent patterns, various examples originating from this application were reported as either type I pan-Trk inhibitors (similar to IRAK inhibitor, see Part I) or type III allosteric pan-Trk inhibitors (with different JM domain interactions, see Part I). Merck published five additional patent applications in 2015 for TrkA inhibitors featuring substituted six-membered heterocyclic benzamides (Figures 6 and 7) [20,21], substituted fivemembered heterocyclic benzamides (Figure 8) [22], and bicyclic heteroaryl benzamides (Figures 9 and 10) [23,24]. These compounds represent close structural analogs to the most recent TrkA inhibitor series from Pfizer (New York, NY, USA) (Figure 17, vide infra). In light of the information gained from

the Trk X-ray data by Merck, the IRAK4 X-ray data (Part I), and the SAR from the tri- and bis-substituted urea series, these novel Trk amide inhibitors may appear as analogous type I or III inhibitors [25] (this assumption is also valid for Pfizer benzamide inhibitors, see Figure 17). These five applications describe a total of 622 inhibitors all assessed toward TrkA using DiscoverX PathHunter assay. Neither selectivity data nor TrkB/C potencies are presented but highly potent TrkA inhibitors emerge from this broad compound triage and SAR effort. With few exceptions, these inhibitors display TrkA potency in the low nanomolar range and even break into the picomolar range. The 2-fluoro-5-(1methyl-1H-pyrazol-3-yl)-4-(trifluoromethyl)benzamide (and minor variations thereof) surfaces as a constant and key complementary motif. Highly potent examples derived from the substituted six-membered heterocyclic benzamides bear substituents of various sizes and compositions at the meta/ortho positions and are not affected by nitrogen positional changes of the N-heteroaryl ring (Figure 6) [20]. Compounds introducing fused ring systems or large linear or cyclic polar substituents at the para position of the aforementioned ring achieve remarkable picomolar potencies (Figure 7) [21]. Similar trends in the substituent orientation are also observed within the five-membered (fused) heterocyclic benzamides (Figures 8– 10) [22–24]. It is interesting to note that while large polar substituents are favorable, certain compounds with smaller

OH O N N

O N H

F 3C

NH2

OH

N

F

N

N N

N N

O N H

O

N

F 3C

N N H

N N N

F3C

N H F3C

Example 29 TrkA IC50 = 1.0 nM




Figure 6. Merck substituted six membered heterocyclic benzamides.

O

Example 60 TrkA IC50 = 1.3 nM (DiscoverX PathHunter)

F


N


O HN N N

O N N

O

F

F3C


N N

O

N

N H

H N

O

O

N

N

O

N

N H F3C

HN

O O S

N

N H F3C

F

5

F





Figure 7. Merck substituted six membered heterocyclic benzamides.

O NH2

N

N O NH N N

N N

O

N

O

N N H F3 C

N

N F3C

Cl

F

O N

N

N

N H HN

N

N H F3C

F

N F Example 36 TrkA IC50 = 0.37 nM






Figure 8. Merck substituted five membered heterocyclic benzamides.

N N

O

N N H

O

HO

OH

N

F3C

N N

N

O N H

HN

N N N

F3C


CN

O

N N

O

N N H

N

O

N F3C

F

N H

N

F3C

Example 41 (S) TrkA IC50 = 0.96 nM Example 42 (R) TrkA IC50 = 0.79 nM


Example 111 TrkA IC50 = 1000 nM (DiscoverX PathHunter)


Figure 9. Merck bicyclic heteroacryl benzamides.

substituents, such as Example 111 (Figure 9) bearing a cyano group, are associated with a complete loss of TrkA activity. The most recent Merck patent for Trk inhibitors [26] describes at first glance what appears to be a more conventional type II inhibitor series which borrows structural features from the previous series including the bicyclic heteroaryl benzamide compounds (Figure 11) [7]. The inhibitors described also share the overall topology of compound 4, e.g. an amide centering on a long para-substituted ring system expanding toward the hinge on one side and an

allosteric pocket-occupying group, inspired by the classic N-phenylpyrazole motif, on the other side. Interestingly, Merck has separately shown that the replacement of the N-phenylpyrazole group in compound 4 with naphthalene or simple aryl groups, while keeping the rest of the molecule entirely intact, affords selective GPR142 agonists as potential therapeutics for the treatment of type 2 diabetes [27]. Compounds from the Trk patent described here differ from compound 4 in that they bear a bicyclic ring chain as opposed to a tricyclic arrangement with the additional

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O HO

HO

N

HN N

N

N N

O

N

N F3C

N N

N H

F

F3C

F

O

N

N H

N

N H

O

N

N N

O

N

F

F3C

N

N

N N

O

N N H

NH

HO

F

F3C

Example 21 (R or S) TrkA IC50 = 0.69 nM

Example 33 (R or S) TrkA IC50 = 0.25 nM







Figure 10. Merck bicyclic heteroaryl benzamides.

Lys544

Met592 N

N N N

N

Asp668 H 2O

O N H

N

O

O

N

N

N

N

N H

N N

MeO O S HN

Glu560

F

F

Allosteric pocket

4 TrkA IC50 = 662 nM (Caliper)

N N

O HN


N

N H

S Example 82 TrkA IC50 = 0.7 nM (DiscoverX PathHunter)

F

JM domain Ile490

Selectivity pocket

O N

S O

O N

N

N H O

F

O

O HN

N H

O N

N N

S

O S O OH

N H

N O

N

Asp668 Example 1 TrkA IC50 = 607 nM TrkB IC50 > 81000 nM TrkC IC50 = 21000 nM


Example 112 TrkA IC50 = 1590 nM (DiscoverX PathHunter)


Figure 11. Binding mode of compound 4 with TrkA (PDB: 4PMM), binding mode of Example 1 with TrkA (PDB: 5KMK) and Merck 2-substituted and fused tetracyclic 2 (1H-indol-1-yl)acetamides.

triazole. However, Merck also showed that the initial truncated hits at the origin of this series (Example 1, Figure 11) act as a type III inhibitor with TrkA selectivity ensuing from the interaction with the JM domain around Ile490 (Part I, Figure 1). It is therefore unclear if the importance of the extended ring motifs serves the purpose of engaging the hinge or is involved in the type III binding interaction (Figure 11). Compounds bearing negatively charged sulfonic acids in place of the preferable methylsulfonimidoyl group lead to a significant abrogation of potency (Figure 11, Examples 71 and 112). Of interest, the most potent inhibitors exemplified bear a fused polycyclic ring system which may serve the purpose of displacing an unfavorable water molecule within the allosteric pocket (Figure 11, Examples 80 and

82). In all examples, the phenyl ring found in compound 4 is replaced by more compact five-membered rings with hydrogen bond-accepting heteroatoms. An optimized derivative of compound 4 was also shown to display prolonged residence time in SPR analysis compared to other leads from Merck, especially when compared to type I inhibitors, and proven efficacious in preclinical inflammatory and neuropathic pain mice models following oral administration [7].

2.2. Nerviano The pyrazolo[4,3-h]quinazoline-3-carboxamide inhibitor milciclib (formerly PHA-848125) was initially developed as a CDK2/ cyclin A inhibitor by Nerviano (Nerviano, Italy) which


Hinge region

Leu83

Lys33

N HN

O

Glu1197 Hinge region

N

Met1199

N N N

Hydrophobic pocket II (Leu1256/Phe1127)

F

H N

7

F

N H

HN

O H N

N N

N

Adenosine pocket

O

N Milciclib (PHA-848125) TrkA IC50 = 53 nM CDK2/A IC50 = 45 nM

Entrectinib (RXDX-101) TrkA/B/C IC50 = 1/3/5 nM ROS1 IC50 = 7 nM ALK IC50 = 12 nM

Figure 12. Nerviano’s clinical leads milciclib and entrectinib and respective binding modes with CDK2/cyclin A (PDB: 2WIH) and ALK (PDB: 5FTO). IC50 values determined by scintillation proximity assay.

fortuitously displayed nanomolar TrkA inhibition during kinase profiling (Figure 12) [28]. An application patent was filed in 2010 claiming milciclib for the treatment of thymic carcinoma based on the positive responses observed in patients with the disease during a concurrent phase I clinical study (study identifier NCT01300468) [29]. Milciclib is currently in phase II clinical trials (study identifiers NCT01011439 and NCT01301391) for the treatment of malignant thymoma. Milciclib readily penetrates the blood-brain barrier (BBB), sustaining brain/ plasma ratios >1 for over 24 h in rat models [30,31]. A formulation patent for entrectinib (formerly RXDX-101) was released in 2013, just prior to the start of the STARTRK-1/ 2 phase I/II clinical trials (study identifiers NCT02097810 and NCT02568267) (vide infra) [32]. Discovered via HTS within Nerviano’s ALK drug discovery program, the antecedent 3amino-5-benzyl-substituted indazole acquired only the addition of the aminotetrahydropyranyl group during structural optimization to yield the clinical inhibitor entrectinib [33]. Meant to resemble ribose, the aminotetrahydropyran occupies the ATP pocket of ALK, displacing a resident water molecule and stabilizing the bioactive conformation through an intramolecular hydrogen bond between the NH of the amino linkage and the adjacent carbonyl (Figure 12). Subsequent kinase profiling revealed that entrectinib is an even more potent inhibitor of ROS1 and TrkA/B/C than the originally targeted kinase, ALK.

2.3 Ono Pharmaceutical Ono (Osaka, Japan) disclosed two patents in 2013 and 2014, describing an extensive series of N-(2-phenoxypyrimidin-5-yl)-N ′-aryl ureas for the treatment of pain and conditions arising from NGF-mediated vascular hyperpermeability [34,35]. Structurally similar N-(4-aryloxyphenyl)-N′-aryl ureas have been previously described by Bayer (Leverkusen, Germany) [36] and Kalypsys (San Diego, CA, USA) [37] for targeting RAF kinase, though Trk data were not described. The binding mode of these compounds is likely analogous to pan-Trk inhibitor GNF-5837 unveiled in 2012 [38] which features a structurally comparable N-phenyl-N

′-[4-(phenylamino)phenyl] ureayl core (Figure 13(a)). Substituted trifluoromethylpyridinyls and tert-butylpyrazoles are explored as allosteric pocket motifs, although a 6-substituted 3-trifluorophenyl group is prominent. Triazoles, pyrazolo[1,5-a]pyrimidines, and imidazo[1,2-b]pyridazines hinge-binding motifs are utilized along with amino-substituted pyridines and pyrimidines (Figure 13(b,c)). Biochemical assays are provided only for a representative set of compounds which exhibit low nanomolar TrkA inhibitory activity. A phase I clinical trial (study identifier NCT02454387) for the treatment of osteoarthritis using the Trk inhibitor ONO-4474 was completed in 2016, and while the structure of ONO-4474 has not yet been disclosed, it ostensibly originates from the described urea series.

2.4. Pfizer In 2012, Pfizer released a patent [39] containing 591 compounds resulting from their efforts to refine the pharmacokinetics of the Tie-2/TrkA inhibitor series developed in 2005 [40]. The candidate compound of the 2005 series, CE-245677 (Figure 14), displayed adverse neurological effects during a phase I clinical trial and was subsequently discontinued in 2007 owing to these side effects. The ensuing lead optimization effort focused on peripheral restriction by purposely targeting P-gp recognition through structural design to minimize CNS exposure. The urea from the original series was replaced with an acetamide, and modification of the pyridine-3-yl(7Hpyrrolo[2,3-d]pyrimidin-5-yl)methanone template yielded the lead PF-06278121 bearing optimal efflux transport (Figure 14). A multitude of functional groups are appended to the N7 nitrogen of the hinge binder while the distal allosteric pocket-binding motif bears largely (hetero)aryl functionalities. Enzymatic and cell-based assays reveal uniformly dispersed inhibition values spanning the low micromolar to high picomolar range. MDCK permeability data are also included in this patent to assess brain uptake potential. Brain penetration assays are subsequently included in all Pfizer’s proceeding Trk inhibitor patents described herein.

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Hinge Region

a

Met620

Glu618

NH O

Hydrophobic region II (Phe617/698) Asp697

NH

Allosteric pocket

CF3

O N H

N H

N H

F

Glu588 DFG Motif GNF-5837 TrkA IC50 = 11 nM (BAF3 Tel-TrkA) TrkA IC50 = 8 nM (Caliper) b

CF3 N

O H N

O

N

S

N H

N H

Cl

N Example 85-117 TrkA IC50 = 2 nM (CellSensor) TrkA IC50 = 20 nM (LanthaScreen) c

CF3 N

O

NH2

O

N

N

N H

N H

N N

Cl Example 8-1 TrkA IC50 = 1 nM (CellSensor) Figure 13. Binding mode of the GNF-5837 series with TrkC (a) (PDB: 3V5Q) and Ono ureas ((b): WO2013161919, (c): WO2014129431).

Cl N

O

O

N H

H2N N N

O N H

Cl

N

CE-245677 TrkA IC50 = 48 nM (enzymatic assay)

N O

N

O

CF3

N H N7

N

The acetamide series bearing the pyrrolopyrimidine hinge binder is continued in a 2014 publication [41]. Sets of small alkyl and hydroxyl-bearing alkyl groups adorning the pyrrolopyrimidine N7 nitrogen are explored as the allosteric pocket-binding moieties are cycled through (Figure 15(a)). The addition of substituents such as a methyl group to the acetamide methylene carbon is not tolerated. Preclinical candidate PF-06273340 [42] is also described in this patent, although development was discontinued from the Pfizer pipeline shortly after the completion of a phase I study. The acetamide series was concurrently extended in two additional patents [43,44] utilizing bioisosteric 1H-pyrrolo [3,2-c]pyridine and 1H-pyrazolo[4,3-c]pyridine hinge-binding motifs (Figure 15(b,c)). Structural exploration is comparable to that seen in the previous patents, although only three compounds are included in the 1H-pyrazolo[4,3-c]pyridine series. A later iteration of the acetamide series departs from the well-used heterobicyclic hinge binders [45], adopting an alternative arylamide motif (Figure 16(a,b)). Impressively, 140 of the 183 compounds in this series exhibit TrkA IC50 values ≤10 nM, illustrating the degree of structural tolerance at the terminal ends of the core structure. Example 114 is the only compound displaying an IC50 > 20 nM owing to the detrimental (S)-methylation of the acetamide. Interestingly, the (R)enantiomer is tolerated (Figure 16(a)). Fluorination of the 3position of the piperidine ring is prominent, and only the 3R,4R stereoconfiguration with adjacent ether decreases potency (Figure 16(b)). This series is continued in another small patent containing only 25 compounds [46], carrying over the piperidine fluorination and trifluoromethoxy substitution of the peripheral phenyl group (Figure 16(c)). This investigation focuses on 4-substitution of the hinge binder with Oand N-linked alkyl groups bearing primarily hydrogen bonddonating groups such as alcohols and amines. An equally short companion publication [47] describes pyrrolidine analogs which utilize a 6-aminonicotinamide hinge binder adorned with a variety of amide substitutions (Figure 16(d)). Compounds within this series are presented as sets of enantiomers and trend for a preference of the (R)-enantiomer. A 2015 patent application [48] discloses an array of 841 3(pyridin-2-yl)benzamides bearing an N-phenylpyrazole allosteric pocket motif (Figure 17(a)), resembling compounds released by Merck later in the year (Figure 8). A structurally diverse collection of N-linked substituents is described for the 3-amido group of the phenylpyrazole with the N-(2-aminopyridin-4-yl)methylene group featured in the most potent compounds against TrkA. The hinge-binding pyridine is decorated with small functionalities such as halogens, alcohols, ethers, amines, and alkyl groups with various patterns of substitution although the structural correlation with potency is ambiguous. Similar structural explorations are observed in a concurrent disclosure of analogous N-(phenylpyrazolyl)benzamides featuring 2-aminopyridine substituents (Figure 17(b)) [49], providing similar inhibitory results.

OH PF-06278121 (Example 9) TrkA/B IC50 = 4/94 nM (enzymatic assay) TrkA/B/C IC50 = 13/7/6 nM (PathHunter cell assay) MDCK-MDR1 Papp (A-B) = < 1×10-6 cm/s Figure 14. Pfizer pyrrolo[2,3-d]pyrimidines.

2.5. Pierre Fabre Medicament A 2014 patent from Pierre Fabre (Paris, France) [50] expands on the pyrazolo[4,3-h]quinazoline-3-carboxamides inhibitors seen previously in a patent from Nerviano [51] where the clinical lead entrectinib was first described (Figure 12). This


a

N O

N H2N

Cl

O

b

N

N

N

N O

O

N H

c

CF3

O

N

N

O N H

N H

9

N N

CF3

N N

N F OH Example 5 TrkA IC50 = 4.41 nM (enzymatic assay)

OH PF-06273340 (Example 48) TrkA IC50 = 7.29 nM (enzymatic assay)

Example 3 TrkA IC50 = 16.6 nM (enzymatic assay)

Figure 15. Pfizer pyrrolo[2,3-d]pyrimidines ((a): WO2014053967), pyrrolo[3,2-c]pyridines ((b): WO2014053968), and pyrazolo[4,3-c]pyridines ((c): WO2014053965).

a

O

O N O

CF3

b

O

O

O

N N

N N Enantiomer IC50 (nM) R 6.0 285.7 S

c O

O

Example Diastereomer IC50 (nM) 2 1.7 3S,4S 3 3R,4S 1.3 9 3S,4R 1.5 164 3R,4R 12.3 d

CF3

N O F

H2N

F

N

H2N

H2N

O

CF3

N

R or S

O

Example 113 114

O

F R or S O H2N

Example 15 TrkA IC50 = 4.9 nM (enzymatic assay)

O

O

CF3

N

H N

N O

O HO

F

Example 11 12

Enantiomer IC50 (nM) R 11.6 1250 S

Figure 16. Pfizer N-acylpiperidine ethers (a,b: WO2015092610; c: WO2016009296), and N-acylpyrrolidine ethers (d: WO2016020789).

inhibitor series replaces the indazole hinge binder utilized by entrectinib with (di)azaindazoles (Figure 18). Structural diversification is primarily centered on the thiophenyl ring, exploring various amino/thio linkages and chloro/fluoro/ trifluoromethyl aryl-substitution patterns. A few examples replacing the piperazine and tetrahydropyran rings and modifying the indazole core with methyl, methoxy, and hydroxyl substitutions at the 6-postion are provided. Details for three biological assays are included but results are reported for only a few selected compounds. A phase I clinical study in France (identifier EudraCT 2013-003009-24) has commenced using the Trk inhibitor F17752 of undisclosed structure for the treatment of solid tumors with ALK, ROS, or NTRK gene fusions. F17752 may originate from the described patent.

2.6. Plexxikon Plexxikon (Berkeley, CA, USA), a subsidiary of Daiichi Sankyo (Tokyo, Japan), filed an investigational new drug application for PLX-7486 in March 2013 for an orally available pan-Trk inhibitor which also targets CSF-1R. It is currently in a phase I study (study identifier NCT01804530) for the treatment of solid tumors including pancreatic cancer with future plans to explore efficacy in patients with oncogenic Trk fusion and point mutations. While the structure of PLX-7486 has not yet been disclosed, TrkA inhibition data are included in patents detailing CSF-1R and c-kit inhibitors from 2008 [52] and RAF kinase inhibitors from 2010 [53]. A series of 1H-pyrrolo(2,3-b)pyridines bearing 3-mono and 3,5-disubstitutions, as well as 7H-pyrrolo(2,3-d)pyrimidines substituted in

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J. J. BAILEY ET AL.

a

2.7. Dr. Reddy’s Laboratories

NH2

Dr. Reddy’s Laboratories (Hyderabad, India) disclosed a series 3,6disubstituted pyrazolo[1,5-a]pyridines in 2013 [54], similar to those described by GNF/Novartis a year earlier (Part I, Figure 26) [55]. Slight alterations to the pyrrolidinylphenyl ring with small alkyl or alkoxy group are explored analogously to GNF/Novartis’ series (Figure 20(a)). The amide functionality is heavily diversified, and the majority of the 306 compounds in the series are described as possessing sub-micromolar IC50 for TrkA via a time-resolved fluorescence resonance energy transfer assay. A separate patent focusing on an acylsulfonamide sub-series was released in tandem (Figure 20(b)) [56]. Various amine, alkyl, and (hetero)aryl additions to the sulfonamide are investigated. A third of the sulfonamide series are described as possessing 80 min. The (sulfon)amide series was continued in a 2015 patent using a imidazo[1,2-a]pyridine hinge binder isostere [57]. Amino sulfonamides represent half of the inhibitor series, adorned with tertiary distal amines in all cases (Figure 20(c)). The aromatic ring is consistently ornamented with cycloalkoxy rings in this series revealing remarkable tolerance for these substitutions.

N

O NH N

O N N H

F Cl

b

N

Cl

Example 557 TrkA IC50 = 1.02 nM (enzymatic assay) H N

N

O

NH2

NH

N

O N N

N H Cl

Cl

Example 157 TrkA IC50 = 1.55 nM (enzymatic assay) Figure 17. Pfizer 3-(6-aminopyridin-2-yl)benzamides (a: WO2015170218; b: WO2015159175).

H N

F N

2.8. VM Discovery/Purdue Pharma

N S HN

F

O H N O

N N

Example 30 TrkA IC50 = 36 nM (KM12 proliferation) TrkA/B IC50 = 45/31 nM (enzymatic assay) TrkA/B/C IC50 = 94/84/64 nM (KINOMEscan)

Figure 18. Pierre Fabre medicament amido azaindazoles.

the 5-position, are exemplified as the top TrkA inhibitors in terms of potency (Figure 19).

a N

H N

N H N

S

b

H N

N H N

N N

N

VM Discovery (Fremont, CA, USA) published a patent in 2010 [58], detailing a series of branched bi/tri/tetra(hetero)cycles as part of their TrkA receptor antagonist research program (Figure 21). The structures are exceedingly varied and functionalized with a multitude of substituted alkyl and aromatic groups. An enzymatic scintillation assay and SHC1-Trk proliferation assay is described, but data are only provided for three compounds which display low nanomolar IC50 for TrkA inhibition, and only compound 201 is revealed by structure. Interestingly, kinase profiling reveals over 100-fold selectivity for TrkA in the selected panel of kinases, including TrkB/C. These compounds also bind TrkA non-competitively with respect to ATP concentration. A formulation patent from 2014 [59] covers the composition and crystalline forms of compound 701, which may be the peripherally acting candidate and allosteric TrkA inhibitor VM-902A used in a completed 72-patient study for pain. Purdue Pharma (Stamford, CT, USA) acquired the intellectual property rights in 2015 to VM-902A and associated compounds and has since initiated a phase II clinical trial (study

N

N N

Cl P-0171 TrkA IC50 < 1 µM

P-0180 TrkA IC50 < 1 µM

Figure 19. Plexxikon pyrrolo[2,3-b]pyridines (a: WO2008063888, b: WO2010129570).

H N F

F

O

O

S NH

O O F

P-2061 TrkA IC50 < 10 µM

H N N N


a

b

N N

c

N N

N F

N NH

O

O

O O

F

O

OH

F

N

N

N F

NH

O

O S O

O

F

NH O S N O N N

O O

Example 240 WO2013088257

11

Example 59 WO2013088256

Example 96 WO2015200341

Figure 20. Dr. Reddy’s disubstituted pyrazolo[1,5-a]pyridines and imidazo[1,2-a]pyridines.

O N H

N

O S N

F

N F

O

Compound 103

Compound 109

O

Compound 481 F

F F O

N

O N

O

N

N

S

Compound 495

N

Compound 803

Compound 811

O O

H N N

N

N

O

N N

N H

N N

Cl O

HN OH

Compound 201 TrkA IC50 = 85 nM (scintillation assay)

O Compound 701

Figure 21. VM Discovery/Purdue Pharma bicyclic (a) and tri/tetracyclic (b) Trk inhibitors.

identifier NCT02847702) for the treatment of pain related to osteoarthritis of the knee (OAK).

derived inhibitors over the past 6 years, with urea and amide inhibitors becoming more prevalent. HTS and rational design has introduced many new and interesting Trk inhibitor scaffolds.

3. Conclusion Interest in Trk and Trk-related diseases has increased substantially in the last decade, as evidenced by the number of companies exploring Trk inhibitors in this review, especially in the most recent years. As noted in Part I of the review, there has been a shift away from the archetypal indazole-

4. Clinical applications of Trk inhibitors Various Trk kinase inhibitors, most of which are delineated in the patent literature covered in Part I and Part II of this review, are currently under clinical evaluation for the treatment of

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solid tumors (Table 1) and chronic pain (Table 2). It is notable that while trk has been one of the first transforming genes characterized more than three decades ago (the TPM3-NTRK1 – OncD – oncogene) [60,61], and both Trk fusion proteins and proto-oncoproteins have been the center of sustained preclinical research since then, demonstrations of the clinical potential of Trk inhibition in human cancer have remained latent until 2015 [62–65]. For over 20 years, accumulating preclinical evidence and in vitro human data point toward the involvement of neurotrophin-mediated autocrine/paracrine signaling involving Trk proto-oncoproteins in the aggressiveness and metastatic potential of different human neoplasms. Examples of cancers where full-length Trk receptors were shown to play a pro-tumorigenic role include breast [66] and prostate cancers [67] (TrkA/B) as well as neuroblastoma (TrkA/C and TrkB associated with good and poor prognoses, respectively) [68,69]. Potentially clinically relevant oncogenic mutations

were also characterized such as the NGF-unresponsive TrkA splice variant (TrkAIII) in neuroblastoma [70] and activating TrkA deletion (ΔTrkA) in acute myeloid leukemia [71]. Based on those observations and robust preclinical data [72–75], an early clinical trial using the multitargeted pan-Trk inhibitor lestaurtinib (formerly CEP-701, a K252a derivative) was conducted within the context of refractory neuroblastoma (study identifier NCT00084422). Of the 46 patients evaluated (median age 10.7 years), 2 showed partial responses and 9 displayed stable disease at higher-dose regimens [76]. At that point, the lack of Trk selectivity of lestaurtinib combined with the unspecified Trk expression status from responsive versus unresponsive patient groups emerged as conspicuous limitations which prevented unambiguous validation of the anti-Trk approach in the clinical setting. The development of lestaurtinib in the patent literature largely predates the scope of the current review and has been described previously [77].

Table 1. Clinical trials focusing on cancer treatment. Compound ID Altiratinib (DCC-2701, DCC-270, DP-5164) Belizatinib (TSR-011)

O

O

N H

O

F

Phase I/II in patients with advanced solid tumors Selective and lymphomas (including Trk-positive) ALK, TrkA/B/C

O F N

N

N

H N O

O

O

F

MeO OMe

H N

O H N

NH2 N

F

N

N

(structure undisclosed)

Entrectinib (RXDX-101)

N

F

H N

N N

F HN

HN

O

F17752

O


LOXO-101 (ARRY-470)

F OH

N N

F N

N HN

N O

Milciclib (PHA848125AC)

N N

H N

O

N

PLX7486

Sitravatinib (MGCD516)


O N

N

HN

S O F

O N H

H N

N N

N

F

O N H

NCT02048488 (ongoing)/Tesaro, Inc.

NH

H N

N

Dovitinib (TKI-258, CHIR-258) DS-6051b

O

F

H N

HO

H N

H N

F

N

O

Cabozantinib (XL-184, BMS907351)

Representative clinical trial ID/ Current stage (overview) Kinase targets sponsor Phase I in patients with locally advanced tumors Multi-targeted, including: NCT02228811 and metastatic solid tumors including TRK TrkA/B/C, c-Met, Tie-2 (recruiting)/Deciphera genomic alterations and VEGFR Pharmaceuticals LLC

Structure

Multiple ongoing phase II trials including in patients with RET fusion-positive advanced onsmall cell lung cancer and those with other genotypes: ROS1 or NTRK fusions or increased c-Met or AXL activity Multiple ongoing trials including phase II in patients with tumor pathway activations including mutations or translocations of Trk Phase I first-in-human in patients with advanced solid tumors; phase I study in Japanese subjects with advanced solid malignant tumors with NTRK and ROS1 fusions Phase II in patients with locally advanced or metastatic solid tumors with rearrangement including for NTRK1/2/3 (STARTRK-2); phase I/Ib in pediatric patients with relapsed refractory solid tumors/primary CNS tumors/ neuroblastoma/non-neuroblastoma Phase I/II study in patients with advanced solid tumors including NTRK fusion-positive tumors Phase I in patients with advanced adult solid tumors with NTRK1/2/3 genetic alterations; pediatric solid or primary central nervous system tumors (SCOUT); phase II in patients with NTRK fusion-positive tumors (NAVIGATE)

Multi-targeted, including: N/A, approved in 2016 for kidney c-Met, RET, VEGFR, cancer treatment; KIT, CSF-1R, FLT3, TieNCT01639508 (recruiting)/ 2 TrkA/B/C, AXL Memorial Sloan Kettering Cancer Center Multi-targeted, including: NCT01831726 (completed in KIT, FLT3, FGFR, 2016)/Novartis VEGFR, TrkA/B/C Pharmaceuticals Selective ROS1, TrkA/B/C

NCT02279433 (recruiting)/Daiichi Sankyo Co., Ltd.; NCT02675491 (recruiting)/Daiichi Sankyo Co., Ltd. Multi-targeted, including: NCT02568267 (recruiting)/Ignyta, ROS1, ALK, TrkA/B/C Inc.; NTC02650401 (recruiting)/ Ignyta, Inc.; NCT02097810 (recruiting)/Ignyta, Inc.

Multi-targeted, including: EudraCT 2013-003009-24 ALK, ROS1, TrkA/B/C (recruiting)/Pierre Fabre Selective TrkA/B/C NCT02576431 (recruiting)/Loxo Oncology, Inc.; NCT02122913 (recruiting)/Loxo Oncology, Inc.; NCT02637687 (recruiting)/Loxo Oncology, Inc. Phase II in patients with malignant thymoma; Multi-targeted, including: NCT01301391 (recruiting)/Tiziana phase II in patients with unresectable thymic CDK, TrkA Life Sciences, PLC; carcinoma NCT01011439 (ongoing)/ Tiziana Life Sciences, PLC Phase I in patients with advanced solid tumors as Multi-targeted, including: NCT01804530 (recruiting)/ a single agent and with gemcitabine plus nabCSF-1R, TrkA/B/C Plexxikon paclitaxel (including with NTRK1/2/3 point or fusion mutations) Phase I in patients with advanced cancer Multi-targeted, including: NCT02219711 (recruiting)/Mirati including with genetic alteration of NTRK1/2/3 c-Met, AXL, MER, Therapeutics Inc. VEGFR, PDGFR, DDR2, Eph, TrkA/B/C


13

Table 2. Clinical trials focusing on pain. Compound ID ASP7962

Structure (structure undisclosed)

GZ389988

Kinase targets

Representative clinical trial ID/ sponsor

Phase II for analgesic efficacy in patients with pain due to osteoarthritis of the knee Phase II for analgesic efficacy in patients with pain due to osteoarthritis of the knee (intra-articular dose)

TrkA/B/C

NCT02611466 (recruiting) /Astellas Pharma Europe B.V. NCT02611466 (recruiting) /Astellas Pharma Europe B.V.

Current stage overview

TrkA/B/C

ONO-4474


Phase I for safety, tolerability, pharmacokinetics, and pharmacology for the treatment of osteoarthritis

TrkA/B/C

VM902A


Phase I for analgesic efficacy in patients with pain due to osteoarthritis of the knee

TrkA

The accumulation of evidences accelerating in recent years, in part fueled by the rise of next-generation sequencing (NGS) techniques, has revealed that oncogenic NTRK1/2/3 gene fusions are recurrent occurrences in human cancer, well beyond the initially described TPM3-NTRK1 mutation in colorectal cancer [61,78] and ETV6-NTRK3 identified in congenital mesoblastic nephroma and secretory breast cancer [64,79,80]. The diversity and incidence of NTRK1/2/3 oncogenic fusions, which are now recognized in approximately 20 cancer types, have been reviewed recently [81,82]. Taken together, the characterization of Trk chimeric protein expression as widespread low-frequency events in several human cancers, combined with the discovery of novel pan-Trk inhibitors with favorable kinome selectivity profiles, constitutes the mainstay of current clinical trials. We identified 11 distinct Trk kinase inhibitors currently investigated in several recruiting or active phase I and II trials for cancer treatment (Table 1). In contrast to the early lestaurtinib trial, all inhibitors currently under investigation except one (milciclib) are included in genomically driven trials focused around NTRK fusion-positive patient subpopulations. As described, milciclib (Tiziana Life Sciences) is a dual Trk/CDK inhibitor which has been previously investigated in patients with advanced malignancies in phase I (study identifier NCT01300468) [31]. Milciclib has progressed to phase II evaluations based on promising phase I results (including a thymic carcinoma-specific trial – study identifier NCT01011439). Yet, as for lestaurtinib, no data on Trk expression have been provided in the phase I report. The authors have, however, described reversible dose-limiting neurological toxicities (e.g. Grade 2–4 ataxia and Grade 2–3 tremors) which may be attributed to CNS Trk engagement at higher doses. The other 10 Trk inhibitors currently under evaluation can be divided into either a multitargeted or a selective inhibitor category. The ostensible proof-of-concept of the clinical relevance of Trk inhibition for human cancer has been provided consecutively by the early disclosure of phase I study results from the investigation of the multitargeted inhibitor entrectinib (Ignyta) [83,84] and the Trk-selective inhibitor LOXO-101 (formerly ARRY-470, Loxo Oncology) [85] for NTRK fusion-positive solid cancers. Interestingly, 2 years prior to those reports, a study which compared the inhibitory activities of LOXO-101 (then identified as ARRY-470 – first structural disclosure) with lestaurtinib and the ALK inhibitor crizotinib (also displaying

NCT02454387 (completed in 2016)/Ono Pharmaceutical Co. Ltd. NCT02847702 (recruiting) /Purdue Pharma LP

moderate pan-Trk activity) described the off-label use of crizotinib in a lung cancer patient presenting with a MPRIPNTRK1 fusion-expressing tumor [86]. In spite of the modest clinical activity observed then, likely due in part to the suboptimal Trk activity of crizotinib, it is reasonable to assume that this result catalyzed the increasing interest in clinical Trk inhibitors for cancers harboring NTRK oncogenic drivers. Favorable results from two early phase trials with entrectinib were first reported at the 2015 ASCO Annual Meeting (ALKA372-001 and STARTRK-1, study identifier NCT02097810) [83,84]. Entrectinib was initially described and optimized as part of an ALK inhibitor program from the Nerviano pipeline (Figure 9) [33]. The compound was later shown to display stronger pan-Trk inhibition compared to ALK as well as ROS1 activity – all relevant molecular targets in the context of fusion oncogenic drivers – and showed promising preclinical activities for all relevant molecularly defined targets including intracranial EML4-ALK-rearrenged NCI-H228 tumor-bearing mice [87,88]. Further kinase profiling revealed ≥10-fold selectivity for all targets tested except JAK2 and ACK1. The ALKA372-001 and STARTRK-1 trials are evaluating the safety and dose escalation responses of entrectinib in adult patients with advanced solid tumors presenting oncogenic alterations (fusion, amplification, and SNP for NTRK1/2/3, ROS1, and ALK) and have enrolled 119 patients so far. Most subjects in those trials are non-small cell lung cancer (NSCLC) patients presenting ALK alterations. Antitumor response in the phase II-eligible patient population has been reported. Within those 25 patients treated at or above the recommended phase II dose (RP2D), 100% objective response rate was achieved in three patients with NTRK-rearranged tumors (3/3). These three cases were for a cancer stem cell (TPM3-NTRK1), a mammary analog secretory carcinoma (MASC) (ETV6-NTRK3), and an NSCLC (SQSTM1-NTRK1) for which separate detailed accounts have been published [89–91]. For the NSCLC case study, the patient showed complete and durable response of brain metastasis upon treatment, which is in line with appropriate CNS penetration initially described in preclinical investigations and the fact that entrectinib was purposely designed in order to permeate the BBB [33,87]. Further indication of the potential of entrectinib for the treatment of intracranial tumors has been provided by the favorable response of a patient of young age with a recurrent metastatic infantile fibrosarcoma

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(ETV6-NTRK3, primary tumor). So far in early studies, entrectinib has been demonstrated to be safe and well-tolerated under RP2D. Three occurrences of dose-limiting toxicities were described at higher dose. Pediatric patients presenting with recurrent or refractory solid tumors (including neuroblastoma) and primary CNS tumors are currently being recruited for a new trial (study identifier NCT02650401) based on the aforementioned CNS-related results [88]. An important corollary of the deep molecular profiling dimension of those studies has been the rapid characterization of the first resistance mechanisms from phase I case study patients. Russo et al. [92] described the emergence and detailed biochemical characterization of p.G595R (solvent front) and p.G667C (activation loop) TrkA mutations following 4 months of treatment in the colorectal cancer case study. While the p.G595R mutation completely abrogated the activity of entrectinib, p.G667C only partially affected its inhibitory effect (as for LOXO-101). Those residues are conserved outside the Trk family and have been previously linked with secondary resistance for other tyrosine kinase inhibitors (TKIs). A similar mutation to the paralog solvent front Gly623 TrkC residue has been described in the MASC case report. The investigation of entrectinib is currently pursued in the ongoing STARTRK-2 trial (phase II, study identifier NCT02568267). In parallel, summaries from the evaluation of LOXO-101 from a phase I trial have also been provided (study identifier NCT02122913) [85,93]. LOXO-101 is a type I low nanomolar pan-Trk inhibitor with >1000-fold selectivity over all off-targets tested so far and was initially described in an Array Biopharma patent application (Part I, Figure 9) (selectivity in a 226 kinases panel and an 82-non-kinase targets screen). With respect to Trk selectivity, LOXO-101 stands alone amongst all clinical Trk inhibitors currently under investigation. The clinical efficacy and safety of this inhibitor has been tested in seven NTRKpositive patients so far, identified from a larger 43-patient group enrolled in the phase I trial. Tumor regression was observed in all six patients evaluable. Those cases were a sarcoma (LMNA-NTRK1), a gastrointestinal stromal tumor (ETV6-NTRK3), an MASC (ETV6-NTRK3), a papillary thyroid cancer (ETV6-NTRK3), an NSCLC (TPR-NTRK1), and an infantile fibrosarcoma (ETV6-NTRK3) [94,95]. Treatment with LOXO-101 was overall well-tolerated. LOXO-101, contrary to entrectinib, was selected based on its modest CNS exposure in order to contain potential toxicities associated with the inhibition of Trk signaling in the brain and balance possible CNS efficacy. Early data indicate potential utility for CNS tumors as the NSCLC case report also mentioned brain metastasis regression upon treatment in addition to modest primary tumor reduction. Overall, patients lacking NTRK fusions did not experience a response to LOXO-101. Accordingly, the NAVIGATE phase II basket trial is currently enrolling patients with NTRK-fusionpositive cancer exclusively (study identifier NCT02576431). No LOXO-101 responders have been reported to have experienced secondary resistance upon prolonged treatment as of yet. However, the potency of this inhibitor has already been shown to be dramatically affected by point mutations in vitro as previously described in the context of entrectinib. With this perspective, Loxo Oncology is already developing a second-

generation Trk inhibitor, LOXO-195 (undisclosed structure, Array’s pipeline) which is claimed to retain activity against all resistance mutations characterized so far for Trk. This inhibitor is expected to enter phase I in 2017. The additional pan-Trk inhibitors with claimed kinome selectivity currently under investigation are the dual Trk/ ROS1 inhibitor DS-6051a (Daiichi Sankyo, study identifier NCT02279433/NCT02675491) and the dual Trk/Alk inhibitor belizatinib (formerly TSR-011, Amgen/Tesaro (Thousand Oaks, CA, USA/Waltham, MA, USA), study identifier NCT02048488). Both compounds have demonstrated promising preclinical and in vitro activities [96,97]. Belizatinib is developed as a second-generation ALK inhibitor, affording better retention of potency upon point mutations during secondary resistance compared to crizotinib. Early phase I/IIa trial results for patients with ALK-driven tumors have been described [98]. Although 11 patients with NTRK-positive molecular drivers have reportedly been enrolled, no data on this patient subpopulation are currently available. Similarly, no data have arisen so far from clinical studies of DS-6051a. The multitarget ALK/ROS1/Trk inhibitor F17752 is in a solid tumor study in France with a particular focus on targeting gene rearrangements and patients who are resistant to a prior ALK inhibitor, although no data from the trial are yet available. The remaining five clinical leads are multitargeted inhibitors which show some level of anti-Trk activities. Some of the corresponding trials aim at diversifying or repurposing otherwise clinically approved (e.g. cabozantinib) or heavily investigated (e.g. dovitinib) inhibitors initially developed for other kinase targets which are or have been included in several trials unrelated to Trk. Altiratinib, PLX7486, and sitravatinib are inhibitors undergoing a first-time clinical setting investigation, and the corresponding trials capitalize on a basket design to gain insights on potentially useful indications for those novel multitargeted agents. No data from those clinical trials are available at the moment. Apart from antineoplastic applications, Trk inhibitors are currently screened as putative therapeutics for the treatment of chronic pain (Table 2). The TrkA/NGF pathway plays a central role in nociceptive and neuropathic pain and as such represents a promising target for the development of a novel potential NSAID/non-narcotic analgesic drug class [99,100]. The demonstration of the validity of NGF/TrkA signaling blockade has been provided clinically by the NGFneutralizing antibody approach for the management of osteoarthritis pain (e.g. tanezumab) [10]. While being intrinsically less selective, small molecule Trk inhibitors are actively pursued as an alternative approach in this context [101]. The analgesic efficacy of three distinct Trk inhibitors is currently being investigated in phase II trials focused on the management of pain in OAK (Table 2). A fourth compound, ONO4474 (Ono Pharmaceutical), was assessed in a recently completed phase I trial (study identifier NCT02454387). Detailed results from early trial rounds have not been rendered public for any of the investigated compounds. Of those compounds, the inhibitor GZ389988 (Genzyme/Sanofi (Cambridge, MA, USA/Paris, France)) is the only one being developed for intra-articular delivery. This inhibitor is also the one of the


four for which a structure has been released. Particular attention within the patent literature was spent, detailing the kinetics of polymer-based microspheres loaded with GZ389988 as a slow release formulation. A single intra-articular injection of drug-laden microspheres achieved a steady therapeutic release of GZ289988 over and beyond 3 months from the time of injection in rat models. A microcrystalline suspension formulation of GZ389988 monohydrate was concurrently used in a phase I clinical trial (study identifier NCT02424942) for the treatment of painful OAK. This formulation relied on the poor solubility of GZ389988 monohydrate, and dose was chosen to control the duration of release, achieving sustained drug delivery for approximately 1 month in vivo [102]. GZ389988 is currently in a phase II trial in OAK (study identifier NCT02845271). ASP7962 (Astellas Pharma) is an orally available unspecified Trk inhibitor in a phase II trial for OAK and back pain following the phase I trial’s completion in healthy volunteers (study identifier NCT02611466). The current phase II trial has enrolled about 410 patients. VM902A (Purdue Pharma), prospectively compound 701 from VM Discovery’s TrkA receptor antagonist program (Figure 20), is also undergoing a phase II trial as an orally available analgesic treatment for OAK patients. Importantly, while all other inhibitors discussed in the context of pain treatment are likely pan-Trk inhibitors, indications from VM Discovery’s patent suggest that VM902A may be TrkA selective while the uncommon structures released point toward non-TKI TrkA antagonism.

5. Expert opinion More than 30 years after the initial characterization of the OncD oncogene, pan-Trk inhibitors now show promise in phase I clinical trials with NTRK fusion-positive patient subpopulations. These long-awaited clinical proof-of-concept studies were enabled by the success of TKIs as an antineoplastic drug class stimulating research in the field, as well as by the emergence of NGS techniques facilitating the identification of relevant biomarkers in responsive patients and narrowing prospective patient’s subgroups in clinical trials, and by the complex medicinal chemistry developments which are highlighted in the patent literature covered herein for Trk. Current results and efforts point toward Trk inhibitors reaching the market for cancer treatment in the forthcoming future. Advances in this context will largely benefit from the so-called basket trial design [103] which have been critically lacking in early anti-Trk therapy trials and should rapidly enable the precise delimitation of conditions, especially in the context of oncology, where these novel inhibitors may be beneficial. NTRK gene fusions are rare occurrences in common neoplasms, such as NSCLC, and are displayed in high frequency in a number of rare cancers such as MASC [81]. The recent curvature in the discovery rate of novel NTRK fusions in humans suggests further discoveries in the coming years on this front. Globally, based on current knowledge, Trk inhibitors already bear potential for a significant patient base assuming that such patients can be readily identified. As patient groups increase within phase II/III trials and trial focus tightens exclusively around NTRK-fusion position cases,

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patient identification and recruitment may become challenging for biomarkers such as diversified NTRK fusions which may not be routinely included in genomic screening. The awareness of the relevance of those alterations will likely co-evolve positively with the clinical progression of current Trk clinical inhibitor leads. Outside antineoplastic applications, it is interesting to see the emergence of the first trials for the evaluation of Trk inhibitors for the treatment of pain – mostly in the context of osteoarthritis pain thus far. It is of notice that none of the current trials originate from the pipelines of Merck or Array which have a comprehensive patent profile on Trk inhibitors and have encouraging preclinical results in this matter [7,104]. It should be expected that more trials sponsored by these very companies will begin shortly, especially with the preclinical demonstration by Array of the benefits gained with regard to reduced neurotoxicity for the now disclosed TrkA-selective inhibitors which raise anticipations for the clinical application of more efficacious and safer Trk inhibitors. It will also be interesting to see how the progression of this new drug class unfolds with respect to NGF-neutralizing antibodies now in phase III trials [10,105,106]. At the moment, inhibitors for cancer or pain treatment have remained mutually exclusively used although we may expect convergence in use if one of those compounds progresses rapidly toward approval for a given indication. Cases of rapidly progressive osteoarthritis have been described during the development of anti-NGF biologics which has led to the halt of such programs in the past [107,108]. Examination of such potential side effects may be pertinent in the development of small molecule inhibitors with anti-Trk activity owing to the shared target NGF/TrkA axis between the two drug classes. The majority of current clinical leads have emerged from recent patent literature either from Trk inhibitor-specific programs or through repurposing within programs aimed at closely related targets, notably ALK, ROS1, and Met. A central observation from applications filed in recent years is the rapidly increasing interest in Trk inhibitors, selective and multitargeted, bringing forth significant structural diversification as observed in the patent literature – including the identification of the first TrkA isoform-specific inhibitors. It is also remarkable that Trk inhibitors now routinely achieve picomolar potencies in various assays. As for kinase inhibitors in general, the question of selectivity remains central, and to a certain extent, an unresolved conundrum as far as efficacy and safety goes. The early Trk clinical inhibitor lestaurtinib likely did not achieve sustained Trk inhibition in vivo at clinically relevant doses [72]. The discovery phases for early Trk inhibitors were followed by the disclosure and pursuit of more selective compounds (panTrk), albeit in many instances claims of selectivity are only accompanied with screening of less than 5% of the kinome. Even LOXO-101, by all measurable means the most selective pan-Trk inhibitor in clinical trials at the moment, has only been evaluated for selectivity in