Targeting tropomyosin receptor kinase for cancer therapy

European Journal of Medicinal Chemistry 175 (2019) 129e148

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Targeting tropomyosin receptor kinase for cancer therapy Qi Miao a, Kun Ma b, Dong Chen a, Xiaoxing Wu a, **, Sheng Jiang a, * a

State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China b Center for Drug Evaluation, China Food and Drug Administration, Beijing, 100038, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 February 2019 Received in revised form 17 April 2019 Accepted 18 April 2019 Available online 30 April 2019

NTRKs and their expression product tropomyosin receptor kinases (Trks) are widely distributed in mammals. While neural growth factor (NGF)-induced normal Trk activation plays a key role in nerve growth, NTRK alternations occurring in tumor cells were highly correlated to tumor progression and invasion. Recent clinical data from several pan-Trk inhibitors have demonstrated potential and broad applications in various cancers. This intrigues us to summarize the development of inhibitors targeting Trks with different mechanisms of action and their applications in cancer therapy. We believe that this perspective would be of great help in investigating novel anticancer drugs with better therapeutic index. © 2019 Published by Elsevier Masson SAS.

Keywords: Tropomyosin receptor kinase Neoplasm Receptor kinase inhibitor Gene alterations

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Current reported compounds with Trk inhibitory potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 2.1. ATP-competitive inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 2.2. Non-ATP competitive inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 2.2.1. Allosteric inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 2.2.2. Trk downstream pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 2.3. Trk inhibitors with unknown mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Abbreviations used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

1. Introduction Cancer is the second leading cause of death worldwide according to the latest Cancer Report by WHO [1]. Considerable costs

* Corresponding author. Department of Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, China. ** Corresponding author. Department of Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing, 210009, China. E-mail addresses: [email protected] (X. Wu), [email protected] (S. Jiang). https://doi.org/10.1016/j.ejmech.2019.04.053 0223-5234/© 2019 Published by Elsevier Masson SAS.

for the treatment of cancer have been witnessed and have brought heavy burden on society in the past few decades. Although substantial advances have been made in the development of anticancer drugs with novel mechanisms, drug effectiveness and resistance remain as a challenging task for pharmaceutical researchers. Owing to the development of human genome project and related gene sequencing tools, current cancer therapy has entered a new era e precision medicine [2,3]. It is important as well as reasonable for clinicians to prescribe medications for patients based on individual gene expression. In 1982, a high-molecular weight DNA was discovered from two

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colon carcinoma patients, leading to the identification of tropomyosin receptor kinase A (TrkA) [4]. Although Trk is initially identified in oncological transformation, this family is ubiquitously distributed in nervous systems and highly related to nerve development. Trk family includes TrkA, TrkB, and TrkC, which were encoded by NTRK1, NTRK2, and NTRK3, respectively. The kinase domains of TrkA-C share a sequence identity of more than 70%. Apo-TrkA-C displayed a similar pattern: phenylalanine in DFG motif occupies the ATP binding site and formed a T-stacking interaction with gatekeeper phenylalanine [5]. In normal cells, NGFs can firstly induce homo-dimerization of Trks which further bind and phosphorylate the tyrosine residues in NGFs, followed by autophosphorylation of each Trks monomer. Subsequently, the NPXY motif in the juxtamembrane domain and the YLDIG motif in the C-terminus are phosphorylated, creating attachment sites for downstream proteins [6]. The preferentially endogenous ligands for TrkA-C are slightly different. After binding with the preferred ligand NGF or NT-3, TrkA activates the pathway of PI3k/Akt and RAS/RAF/MAPK to induce nerve cell proliferation. TrkB tends to bind with BDNF or NT-3/4 and phosphorylate at Tyr515 residue. When Src-homology 2-domain containing adaptor protein (Shc) is fused into TrkB, the neural cell could survive via activation of PI3K/ Akt pathway which further regulates bioactivities of several proteins such as Bcl-2 antagonist of cell death (BAD) and glycogen synthase kinase 3b (GSK-3b) [7]. TrkC commonly binds with NT-3 and can activate PI3K/Akt pathway to promote neuronal differentiation and inhibit cell apoptosis. The aforementioned Trk activation and signaling transduction can also proceed in oncological cells when NTRK gene alterations occur in these cells. Generally speaking, there are three main types of gene alterations associated with NTRK. The most common one in cancer is NTRK gene fusion. Different from normal expression of NTRK, when 30 portion of NTRK fuses the 5’ portion of another gene, the corresponding expression products would lead to a ligandindependent kinase activation and subsequent oncogenic singling [8]. Generally, gene fusion products could activate same pathways compared with normal expression products with one exception so far. In ETV6eNTRK3 fusion, due to the lack of Tyr485 in the fusion breakpoint, a critical binding site for SHC-transforming protein 1, the corresponding expression product would utilize an alternate homologous protein, Insulin receptor substrate 1 (IRS-1) to activate downstream pathways (Fig. 1) [9]. As shown in Table 1, Trk fusion widely distributed in different cancer types. The most noticeable cases are mammary analogue secretory carcinoma [10] and congenital fibrosarcoma, with 100% NTRK fusion detected in the two cancer types [11]. Several investigations have showed that NTRK gene fusions are the driving factors in certain acute myeloid leukemia (AML) cases [12]. Besides, NTRK fusion have also been reported in other cancers, such as lung adenocarcinoma and colorectal cancer, etc. [13]. Apart from gene fusion, upregulation of NTRK copies in nerve cells would also lead to abnormal overexpression of Trk kinases. For example, Bourhis et al reported that the level of TrkA and phosphoTrkA in MDA-MB-231 cells were distinctly higher than that in normal breast tissues. They further discovered that the overexpression of TrkA was highly related to cell proliferation and tumor metastasis [54]. Although single-point mutation occurring in Trks is rarely related to cancer development, but several reports proposed that mutation of single amino acid in Trk was highly correlated with drug resistance against Trk kinase inhibitors [55]. Since most of the cancer-related NTRK gene alterations are associated with abnormal and uncontrolled kinase activation [56,57], Trk kinase inhibitors could function as anticancer agents for personalized drug therapy. There have been some successful examples in this field. The currently existed reviews mainly focused

on Trk inhibitors in literature with anticancer [58] or analgesic [59] effects. While the most recent patent reviews summarized Trk inhibitors claimed before 2016 [60,61]. Herein, in this review, we mainly summarize recent literature and patents of Trk inhibitors for the treatment of cancer. 2. Current reported compounds with Trk inhibitory potency 2.1. ATP-competitive inhibitors Soon-Sun Hong and Sungwoo Hong reported a series of 7azaindole derivatives as novel Trk inhibitors by extensive kinase cross-screening (Fig. 2). Lead compound 1 was found to have a moderate inhibitory effect against TrkA but a potent inhibition against PI3Ka. By analyzing the structures of TrkA and PI3Ka, they found the substitution at 5 position of 1 may influence the selectivity between these two kinases. After several SAR optimizations, the final compound 2 can inhibit TrkA with an IC50 value of 1.67 nM. The KD of 2 against TrkA was 16 nM and 2900 nM against PI3Ka, indicating a good selectivity. Antiproliferation investigations showed that 2 could inhibit cell growth with an IC50 value of 4.2 mM via inducing nuclei condenses and fragment in MCF-7 cell line. It was found later that 2 could effectively suppress the phosphorylation of TrkA and Akt and induce the apoptosis of MCF-7 by regulating the Trk and PI3k/Akt pathway. Additionally, 2 could induce the expression of cytochrome C and Bax, induce cleavage of caspase-3 and reduce expression of Bcl-2. It was also observed in MCF-7 that HIF-1a expression was increased and VEGF protein level was reduced. Hence, 2 was further tested in HUVEC cell and showed inhibitory effect against migration of by blocking VEGF-induced angiogenesis [62]. J€ anne and Doebele et al. reported the discovery of larotrectinib (3), a pan-Trk inhibitor against with IC50 value varying from 2 to 20 nM with good selectivity against other kinases (IC50 > 1 mM) (Fig. 3). Larotrectinib could not prevent Ba/F3 cells from expressing other oncogene targets, such as EGFR, ALK and ROS1, but selectively inhibit autophosphorylation of NTRK1 fusion cells. Meanwhile, MAPK and Akt pathways were also inhibited with the presence of 3. In KM12 cell line, 3 could inhibit cell growth with IC50 value of 1.9 nM. 3 could induce cell-cycle arrest in G1 and subsequent apoptosis of KM12 cell [22,23]. Pharmacokinetic data of larotrectinib were shown in Table 2. In intent-to-treat analyses (n ¼ 55) at primary data cut-off, the centrally-assessed overall response rate was 75%. Notably, Larotrectinib (3) was approved by FDA (Brand name: Vitrakvi) for the treatment of adult and pediatric patients with solid tumors that have a NTRK gene fusion, other than a specified cancer type [63,64]. Despite the potent inhibitory effect of 3, further investigations showed that duration of response eventually diminished by acquired resistance, due to the mutation of Trk. To overcome this limitation, Rothenberg and Hyman et al. reported the discovery of LOXO-195 (4). First, TrkA-C inhibitory ability on LOXO-195 was tested, indicating similar inhibition and selectivity as 3. Most importantly, the inhibitory effect of LOXO-101 on mutated TrkA was greatly reduced, while LOXO-195 still strongly inhibited this mutant with IC50 values varying from 2 to 9 nM. Besides, LOXO-195 showed potent inhibition on cell proliferation in Trk fusioncontaining KM12, CUTO-3, and MO-91 cell lines (IC50  5 nM). LOXO-195 suppressed tumor growth in Trk kinase-mutated tumor models, while LOXO-101 did not show good inhibitory ability [55]. Dr. Reddy's Laboratories Ltd. claimed a series of imidazo [1,2-a] pyridine derivatives (5e7, Fig. 4), the inhibition rates of this series of compounds could reach the level of <50 nM [65]. Chia Tai Tianqing pharmaceutical group co ltd also claimed a series of pyrazolo [1,5-a]pyrimidine derivatives (8 and 9, Fig. 5). In

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Fig. 1. Trk pathway.

this series of compounds, urea group in LOXO-101 was replaced by amide group. Besides, a primary amine was substituted at 2position. This series of compounds have a pan-Trk inhibitory ability with IC50 values against TrkA-C could reach <10 nM, while JAK2 was not influenced (Table 3) [66]. TP therapeutics also claimed a series of macrocyclic compounds (10 and 11, Fig. 6). The biggest difference compared with LOXO-195 is that non-ring amine between aryl and pyrazolo [1,5-a]pyrimidine group and an oxygen atom was inserted. This series of compounds displayed potent inhibitory ability against TrkA (Table 4) [67]. Molteni reported (R)-2-phenylpyrrolidine substituted imidazopyridazines as potent and selective pan-Trk inhibitors (Fig. 7). Cellular Ba/F3 assay indicated lead compound 12 could inhibit TrkA-C with IC50 values varying from 0.1 to 0.8 mM. X-ray co-crystal structure of 12 with TrkC indicated that 12 bound to DFG-out kinase domain. A hydrogen-bond interaction was detected between N1 of 12 and Met620. Benzontrile motif could deeply bind to the pocket formed by Phe617, Phe698 and benzyl ring of 12 (Fig. 8a). Based on this co-crystal structure, they initially performed structural modifications on the benzylamino group. It was found that benzylamino group could be replaced by ring system, enhancing the interactions between ligand and protein. Hence, 13 was synthesized and cocrystalized with TrkB. This indicated that R-enantiomer preferentially induced protein conformation change (from DFG-out to the DFG-in state, Fig. 8b). Further, they attempted to change the eCN

group and produced 14. Interestingly, co-crystal structure of 14 and TrkA demonstrated that the conformation of 14 flipped around the imidazopyridazine core while maintaining key interaction with the hinge region. This structural flip was only observed in R-enantiomer in which fluorophenyl group inserted into hydrophobic pocket originally occupied by Phe669. Meanwhile, the 3-position of pyridine motif in 14 exposed to solvent, indicating that polar group could be introduced to the structure. In this series, GNF-8625 (15) was found to have excellent Trk inhibitory activity with IC50 value varying from 2 to 4 nM (Fig. 8c). Ba/F3 cellular kinase panel displayed good selectivity of GNF-8625 over other kinases. GNF-8625 inhibited proliferation of Ba/F3 cell and KM12 cell lines with IC50 value of 3 nM and 10 nM, respectively. Further, pharmacokinetics investigations of GNF-8625 were performed. High plasma clearance, moderate volume distribution and short half-life were observed for GNF-8625. In KM12 cell-derived xenograft model, GNF-8625 dose-dependently inhibited tumor growth when administered at ascending doses twice daily for 14 days in rats [68]. Bristol-Myers Squibb disclosed a series of pyrrolo [2,1-f][1,2,4] triazine derivatives as TrkA/B inhibitor for the cancer treatment (16e20, Fig. 9). The IC50 values of these compounds against TrkA/B could reach below 1 nM [69]. Array Biopharma Inc. claimed a series of N, N0 -biarylurea derivatives with good inhibitory ability against TrkA as well as good selectivity among other kinases (Fig. 10, Table 5). Merck sharp & dohme corp. claimed a series of N,N0 -biarylurea

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Table 1 Reported NTRK gene fusion cases concerning with neoplasms. NTRK

Fusion Protein Partner

Cancer Type

Frequency

NTRK1

ARHGEF2 BCAN

Glioblastoma Glioblastoma

CD74 CHTOP LMNA

Lung adenocarcinoma Glioblastoma AYA sarcoma Colorectal Congenital infantile fibrosarcoma Spitzoid neoplasms Lung adenocarcinoma Glioblastoma

1/115 [14] 1/162 [15] 1/185 [16] 2/36 [17] 2/115 [14] 1 case report [18] 1 case report [19] 1 case report [20] a [21] 3/91 [17] 1/162 [15] 4/162 [16] 1 case report [22] 1/182 [23] 1 case report [24] 1/1378 [25] 28/228 [26] a [21] 1/1378 [25] 1/408 [27] 1 case report [28] 2/185 [29] 2/52 [30] 5/81 [31] 2 case reports [32] 1 case report [33] 1/408 [27] 1 case report [34] 2/461 [35] 1 case report [29] 1 case report [36] 1 case report [29] 1/335 [35] 1 case report [36] 1 case report [29] 1/513 [35] 1 case report [29] 1 case report [29] 1 case report [37] 1 case report [38] 1 case report [39] 1 case report [40] 10/11 [11] 1 case report [39] 6/11 [41] 3/6 [42] 1/751 [43] 1/202 [44] 1 case report [45] 3/10 [46] 1 case report [47] 1 case report [48] 1/5 [49] 2/5 [29] 12/12 [50] 25/121 [51] 1/11 [52] 9/62 [53]

MPRIP NFASC PPL RABGAP1L RFWD2 SQSTM1 TFG TP53 TPM3

Thyroid carcinoma Intrahepatic cholangiocellular carcinoma Large cell neuroendocrine tumor Lung adenocarcinoma Papillary thyroid carcinoma Spitzoid neoplasms NSCLC Colorectal cancer Glioblastoma Papillary thyroid carcinoma

NTRK2

NTRK3

TPR

Papillary thyroid carcinoma

SCYL3 AFAP1 AGBL4 NACC2 PAN3

Colorectal cancer Colorectal cancer Low-grade glioma Glioblastoma Pilocytic astrocytoma Head and neck squamous cell carcinoma

QKI TRIM24

Pilocytic astrocytoma Lung adenocarcinomas

VCL BTBD1 ETV6

Glioblastoma Glioblastoma Acute myelogenous leukemia

Congenital fibrosarcoma Congenital mesoblastic nephroma

Colorectal cancer Ductal carcinoma

Fibrosarcoma Gastrointestinal stromal carcinoma Glioblastoma Mammary analogue secretory carcinoma

Papillary thyroid carcinoma a

The total frequency of NTRK1-LMNA and NTRK1-TP53 detected in spitzoid neoplasm patients is 23/140.

derivatives that possess strong inhibitory ability against TrkA. IC50 values of examples claimed in WO2015042085 could reach the level of 50 nM (24 and 25, Fig. 11) [70]. In another patent, they fixed one aryl group with 9H-fluorenyl group. Inhibitory ability of the resulting compounds could be raised a bit with IC50 values at about 30 nM (26e28, Fig. 12) [71]. Afterwards, in another patent claimed by Merck sharp & dohme corp., they deleted a nitrogen atom in urea hence formed an amide linker. Compounds in this series could inhibit TrkA at the level of 1 nM (29e31, Fig. 13) [72]. In the meantime, they explored another similar core skeleton, pyrazolo [4,3-b]pyridine. This series of compounds also displayed potent

TrkA inhibitory ability (32e34, Fig. 14) [73]. Apart from fusion of two rings, 4 fused rings could be seen in some patents. For example, Merck Sharp & Dohme Corp. claimed a series of 4-ring-fused amide derivatives which could inhibit TrkA with IC50 value below 1 nM (35e37, Fig. 15) [74]. Instead of fused rings, compounds claimed by Pfizer inc. chose the N-phenylpyrazolo ring as the core skeleton. Amide substitutions at 3- position are aliphatic chains. This series of compounds could reach the IC50 value of 2 nM (38e40, Fig. 16) [75]. In the further study, they designed and synthesized a series of arylsubstituted 3-amidepyrazolo derivatives. This change of

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Fig. 2. Chemical structure of lead compound 1 and modified compound 2.

Fig. 3. Chemical structures of LOXO-101 (3) and LOXO-195 (4).

Table 2 Pharmacokinetic data of larotrectinib. Recommended adult dosage Cmaxa tmaxa AUC0e24hra Fb Vssb CL/Fb t1/2b Main metabolism product

100 mg twice daily 788 ng/mL 1h 4351 ng*h/mL 32%e37% 48 L 98 L/h 2.9 h O-linked glucuronide

a

Data were acquired from adult patients with a dosage of 100 mg twice daily. Data were acquired from healthy volunteers with a dosage of 100 mg twice daily. b

substitution could retain the inhibitory ability (41 and 42, Fig. 17) [76]. GVK Biosciences claimed a series of urea derivatives, different from ureas derivatives mentioned above, one of the substitutions were replaced by tetrahydropyrrole group. This series of compounds also possess good inhibitory ability against TrkA (43e45, Fig. 18) [77]. Shionogi & co., ltd. also claimed a series of tetrahydropyrrolesubstituted urea derivatives, this series could also reach IC50 values < 100 nM (46 and 47, Fig. 19) [78]. In 2018, they also claimed

a series of 3-ring-fused urea derivatives. This series of compounds also possess good inhibitory ability (48e50, Fig. 20) [79]. Blueprint medicines disclosed a series of pyrazolo [1,5-a]pyrimidine derivatives for the treatment of disorders related to NTRK. Examples shown in Fig. 21 (51e53) were reported to have inhibitory potency against NTRK1 and KM12 cell line with IC50 values of lower than 10 nM. More importantly, compounds from these series demonstrated good inhibitory against G595R-KM12, a cell line that has drug resistance against Crizotinib [80]. In 2017, they claimed a series of pyrazolo [3,4-d]pyrimidine derivatives (54e56, Fig. 22). They also explored substitutions on the 2- and 4- position. These compounds could also reach the IC50 value of below 10 nM [81]. CMG Pharmaceutical & Handok also disclosed pyrazolo [1,5-a] pyrimidine derivatives as the Trk inhibitors (57e59, Fig. 23.). Different from Blueprint medicines, the chemical structures were incorporated with heterocycles in place of cyano group. This class of compounds possessed strong pan-Trk inhibitory effect, which can be potentially used for the treatment of cancer and pain, etc. [82]. Thress et al. reported a novel, potent, and selective Trk inhibitor AZ-23 (60, Figs. 24e25) [83]. AZ-23 showed strong inhibitory activity against human Trk on both enzymatic and cellular levels with IC50 values of 2 nM and 8 nM, respectively. The inhibitory ability of AZ-23 was not attractive against Trk-independent cell line. Furtherly, anti-proliferation effect of AZ-23 was tested on 3T3-TrkA-D allograft, an engineered subcutaneous model designed to be

Fig. 4. Chemical structures claimed in patent US20150368238.

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Fig. 5. Chemical structures claimed in WO2018077246. Table 3 IC50 values of 8 and 9 against TrkA-C and JAK2.

8 9

TrkA IC50 (nM)

TrkB IC50 (nM)

TrkC IC50 (nM)

JAK2 IC50 (nM)

<1 <1

<10 <10

<10 <10

>2000 >500

Fig. 6. Chemical structures claimed in WO2018022911. Table 4 IC50 values of 10 and 11 against TrkA, TrkB and BTK.

10 11

TrkA IC50 (nM)

TrkB IC50 (nM)

BTK IC50 (nM)

0.2 0.2

5000 1500

9.67 0.536

dependent on Trk signaling, and SK-N-SH cell line, a natural neuroblastoma model with endogenously expressing functional Trk receptors. Growth of both cells was strongly inhibited. Pharmacokinetic study indicated that the free concentration of AZ-23 in tumors is greatly higher than that needed for Trk inhibition, even two days after the administration [83]. In 2012, the same group reported two series of inhibitors structurally derived from AZ-23 (61 and 62). Both ring fusion patterns improved pharmacokinetic properties, while keeping the same inhibition against TrkA. These compounds showed high inhibitory ability against Trk-driven cell line (MCF10A-TrkA-D) but less potency against MCF10A parental cells with more than 3800-fold selectivity [84]. AstraZeneca has claimed a variety of patented structures shown in Fig. 26 since 2005. The N-pyrimidine-1H-pyrazol-3-amines were first identified as Trk inhibitor for the treatment of cancer. (63e65, Fig. 27) [85]. However, the replacement of pyrimidine core to substituted phenyl ring decreased the inhibitory effect against Trks (66e68, Fig. 28) [86]. In 2006, AstraZeneca attempted to replace the pyrimidine ring to pyridine ring in subsequent patent. The position of N in pyridine ring (In this markush structure, one of the X stood for N, the others were carbon with substitutions) was extensively studied, while only pyridines with 1,5-diamine retained the inhibitory potency (69e71, Fig. 29) [87]. Compared with previously reported, the

Fig. 7. Chemical structure of 12e15 and its optimal compound GNF-8625 (15).

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Fig. 8. a) X-ray crystal structure of 12 bound to TrkC (PDB ID code 4YMJ); b) X-ray crystal structure of 13 bound to TrkB (PDB ID code 4YPS); c) X-ray crystal structure of 14 bound to TrkA (PDB ID code 4YNE). Backbones of protein are shown in grey. Carbons of ligands are shown in green. Nitrogens are shown in purple. Oxygens are shown in red. Sulfurs are shown in yellow. Fluorides are shown in bright blue. Hydrogen bonds are shown in green dashed line. pcharge interactions are shown in yellow dash lines. p-p interactions are shown in pink dashed lines. Hydrophobic contacts are shown in bright blue dashed line. Color coding are the same in the following figures.

structures in patent WO2006123113 were modified by deleting of substitution on amine (72e74, Fig. 30). Further introduction of 5fuloropyridine-2-yl into these structures strongly enhance the inhibitory effect against Trks (Fig. 31) [88]. Similarly, in patent WO2007049041, the inhibitory activity of Example 7 (75) was 2fold stronger than the original one. Further, this compound inhibited against JAK2 with an IC50 value of 3.9 nM (Fig. 31) [89]. However, the change of pyrimidine ring into pyrazine led to increased JAK inhibition (76 and 77, Fig. 32) [90].

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Sasaki et al. reported a pan-Trk inhibitor, AZD-7451 (78, Fig. 33), showing potent anticancer activity against non-small-cell lung cancer. The cell growth of KM12 bearing NTRK-fusion was greatly inhibited at a concentration of 2 nM. 78 also showed antiproliferation on other NTRK-fusion cell lines such as KM12 and H460 cells. Interestingly, in H810 cell lines, AZD-7451 did not showed inhibitory effect against expression of wild-type NTRK1-3 [91]. Lee designed and synthesized an array of salicyl-hydrazone analogues as TrkA inhibitors (Fig. 34). The most potent compound 79 could inhibit TrkA up to 98% at the concentration of 1 mM. In the antiproliferation test, 79 also demonstrated good inhibitory activity against proliferation of HCT15, A549, SK-OV-3, SK-MEL-2 and MIAPaCA-2 with IC50 value of 0.89 mM, 2.46 mM, 1.43 mM and 0.48 mM, respectively [92]. K-252a (80) was proven to be a potent inhibitor of Trk tyrosine receptor in vitro and showed angiostatic effect in murine brain capillary endothelial cells [93]. However, no inhibitory effect of K252a was found against P388 leukemia cell line [94]. Dionne et al. reported that a K-252a derivative, CEP-751 (82), suppressed prostatic cancer growth in various animal models with good selectivity between cancerous and normal prostate cells. Biological evaluation proved that CEP-751 could inhibit Trk tyrosine receptor via inducing inhibition of tyrosine phosphorylation of Trk tyrosine receptor. Subsequent cellular assays indicated that CEP-751 inhibited growth of different prostatic cancer cell lines in a cycledependent manner, suggesting the potential combination of hormone-dependent and hormone-independent therapy against prostatic cancer [95e97]. Suppress of tumor growth on OVCAR-3 and SK-Mel-5 was efficiently detected when administration of 82 s.c. to adult rodents with 21 mg/kg BID for a period of 30 days. The hydroxyl form of CEP-751, commonly known as CEP-701 (81), can be orally administrated, exhibiting similar inhibitory activity on both enzyme and cell assays as CEP-751 [98]. To improve solubility, Vogelzang et al. developed a lysinyl-b-alanyl ester of CEP-751, namely CEP-2563 (83). Preclinical studies and phase I clinical trial indicated that CEP-2563 showed antitumor activity in a variety of tumors and was reliably converted to CEP-751 in vivo (Fig. 35) [99,100]. Arfini et al. has reported entrectinib targeting Trks, protooncogene tyrosine-protein kinase (ROS), and anaplastic lymphoma kinase (Alk) with inhibitory activities against multiple cancer indications (84, Figs. 36e37). Steady-state kinetic experiments proved entrectinib is a competitor for ATP binding, with <10 nM IC50 against TrkA-C. In anti-proliferation test, entrectinib was found to exhibit inhibitory activity on three different kinds of cancer cell lines: TrkA-driven cell line (KM12, IC50 ¼ 17 nM), Alkdependent cell lines (SU-DHL-1, IC50 ¼ 20 nM; Karpas-299, IC50 ¼ 31 nM; SUP-M2, IC50 ¼ 41 nM; SR786, IC50 ¼ 81 nM and NCI-H2228, IC50 ¼ 68 nM) and Flt3-dependent AML cell line (MV-411, IC50 ¼ 81 nM). In vitro assessment of entrectinib against KM12 showed accumulation of cells in the G1 phase and apoptosis. Further pharmacokinetic and efficacy studies in mouse demonstrated that entrectinib could be orally administrated and resulted in no weight loss at efficacious concentration. When dosed at 15 mg/kg, distinct decrease of tumor volume was observed [101]. Entrectinib could also inhibit growth of SY5Y-TrkB and NLF-TrkB both in vitro and in vivo. [102] Phase I trial indicated that entrectinib was safe and well-tolerated. Additionally, entrectinib could be beneficial to a broad range of solid tumors, such as non-small cell lung cancer (NSCLC), mammary analogue secretory carcinoma (MASC), melanoma, glioneuronal tumor, colorectal cancer and renal cell carcinoma. Especially entrectinib demonstrated excellent antitumor activity against neoplasm in central nervous system [103].

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Fig. 9. Examples in WO2007061882 and their IC50 values against TrkA/B.

Fig. 10. Examples in WO2015175788.

Table 5 IC50 values of 21e23 against TrkA.

21 22 23

TrkA Enzyme IC50 (nM)

TrkA Cell IC50 (nM)

1.1 1.5 1.7

1.9 1.3 6.1

Fig. 11. Examples in WO2015042085 and their IC50 values against TrkA.

Milciclib (85, Fig. 38) was first reported as a cyclin-dependent kinase (CDK) inhibitor with IC50 value of 45 nM against CDK2/ cyclin A complex kinase by Brasca et al. [104] Albanese proved that milciclib could also function as a Trk inhibitor. Kinase profiling screen showed milciclib inhibited TrkA and TrkC with IC50 values of 85 nM and 134 nM, respectively, while the inhibition against TrkB is slightly weaker (IC50 ¼ 745 nM). Further, cellular assay indicated that proliferation of cancer cells were suppressed with less than 1 mM IC50 values against 96 kinds of cell lines. Moreover, the antiproliferation of milciclib was not significantly influenced by P-gp over expression, DNA repair deficiency, and p53 activity [105]. To date, milicicib is in phase II trial for the treatment of patients with recurrent or metastatic hepatocellular carcinoma. Notably, milciclib have received orphan drug designation in the U.S. for the treatment of thymic epithelial tumors. Dalton reported small-molecule Trk and ROS1 tyrosine kinase inhibitors, crizotinib (86, Fig. 39), and its (S)-isomer, GTx-186 (87, Fig. 39). GTx-186 selectively inhibited TrkA, TrkB, TrkC, ROS1, ALK, and RET-mediated phosphorylation with IC50 values in the picomolar to low nanomolar range. GTx-186 showed strong antiproliferative activity against IMR-32 with IC50 value of about 100 nM and completely inhibited NGF-dependent migration of IMR-32 cells. Under the same circumstance, GTx-186 showed no inhibition against TrkA-null SH-SY5Y cells. Further, GTx-186 was found to function as a strong anti-inflammatory agent via PMA- and

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Fig. 12. Examples in WO2015042088 and their IC50 values against TrkA.

Fig. 13. Examples in WO2015143652 and their IC50 values against TrkA.

Fig. 14. Examples in WO2015143854 and their IC50 values against TrkA.

NGF-induced MMP-3 expression and effectively inhibit key inflammatory mediators such as cFos, IL-8, and IL-1b with ED50 at nanomolar range [106]. Gyochang Keum reported a series of urea derivatives as a multikinase inhibitor (Fig. 40). In this series, KST016366 (88) was found to have the most potent inhibitory activity against TrkA and TrkB with IC50 values of 3.81 nM and 4.42 nM. It could also inhibit the proliferation of leukemia K-562 and colon carcinoma KM12 cell lines with GI50 values of 51.4 nM and 19 nM. Docking study suggests that KST016366 could bind into the site that adjacent to ATP binding site. Additionally, the interaction of KST016366 and TrkA hinge region was observed, which revealed that KST016366 might function as a type II inhibitor [107]. Molteni reported the discovery of GNF-5837 (89, Figs. 41e42), which is a potent, selective, and orally bioavailable pan-Trk inhibitor with in vitro activities around 10 nM. Ba/F3 Cellular Kinase Panel indicated good selectivity of Trk kinases over other kinases. GNF-5837 showed strong antiproliferative activites in Ba/F3 and RIE (rat intestinal epithelial) cells with IC50 values of 42 nM and

17 nM, respectively. X-ray crystallography showed that GNF-5837 could bind to the inactivated kinase, indicating that GNF-5837 functions as a type II kinase inhibitor. In vivo efficacy evaluation in mice led to decreased tumor volume when administered at 50 mg/kg or more. However, pharmacokinetic study of GNF-5837 afforded moderate bioavailability (18% in BalB/C mice and 19% in Sqrague-Dawley rats) due to poor permeability and low solubility [108]. IRM and LLC claimed a series of indolinone derivatives as Trk inhibitors. Example 1 (90, Fig. 43) was reported to have inhibitory activity against TrkB with IC50 value of 14 nM. At the concentration of 10 mM, 53 could inhibit TrkA-C with by more than 50%. Additionally, the IC50 values of 53 to inhibit Aurora-A, C-RAF and cSRC were 15 nM, 116 nM and 53 nM, respectively [109]. 2.2. Non-ATP competitive inhibitors 2.2.1. Allosteric inhibitor Soon-Sun Hong and Gyochang Keum reported a novel TrkA

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Fig. 15. Examples in WO2016054807 and their IC50 values against TrkA.

Fig. 16. Examples in WO2015159175 and their IC50 values against TrkA.

Fig. 17. Examples in WO2015170218 and their IC50 values against TrkA.

inhibitor, KK5101 (91), for the treatment of pancreatic cancer (Fig. 44). KK5101 could strongly inhibit the cell growth of two kinds of pancreatic cancer cell line, MIAPaCa-2 and PANC-1, at the dose of 5 mM. In mechanic investigation, it was found that KK5101 could inhibit the expressions of downstream signaling pathways via inhibition of TrkA phosphorylation. Besides, KK5101 could also exhibit strong anticancer activity in MIAPaCa-2 xenograft mouse models. With the administration of 5 mg/kg of KK5101 for 28 days, obvious decrease of tumor volume could be seen with no adverse effects and obvious loss of body weight. Docking analysis suggests that KK5101 bound to an allosteric site of TrkA but lack of interaction with the hinge region. Hence, KK5101 may function as a type III inhibitor of TrkA [110].

Fig. 18. Examples in WO2016116900 and their IC50 values against TrkA.

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Fig. 19. Examples in WO2017135399 and their IC50 values against TrkA.

Fig. 20. Examples in WO2018079759 and their IC50 values against TrkA.

Fig. 21. Examples in WO2017087778.

Fig. 22. Examples in US20160168156 and their IC50 values against TrkA-C.

2.2.2. Trk downstream pathway Rende first reported GW441756 (92) that exhibited strong antiproliferative and proapoptotic effects via Trk inhibition (Fig. 45). When 20 mM were administrated, GW441756 completely

inhibited proliferation and induced apoptosis against all the tested cancer cell lines including HTB-114, HTB-115, HTB-82, TE-671 and PC-3. In mechanism investigation, GW441756 was found to upregulate P75NTR and caspase-3, as well as shift the TrkA/P75NTR

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Fig. 23. Examples in WO2017035354.

Fig. 24. Chemical structures of AZ-23 (60) and its derivatives (61, 62).

Fig. 25. X-ray crystal structure of AZ-23 bound to TrkA (PDB ID code 4AOJ).

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Fig. 26. Skeletons comprising of 1H-pyrazol-3-amine claimed by AstraZeneca in patents.

Fig. 27. Examples of Trk inhibitors in WO2005049033 and their IC50s against Trk.

Fig. 28. Examples of Trk inhibitors in WO2005103010 and their IC50s against Trk.

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Fig. 29. Examples of Trk inhibitors in WO2006082392 and their IC50s against Trk.

Fig. 30. Examples of Trk inhibitors in WO2006123113 and their IC50s against Trk.

Fig. 33. Chemical structure of AZD7451 (78).

Fig. 31. Example of Trk inhibitors in WO2007049041 and IC50 against Trk.

Fig. 34. Chemical structure of 79.

Fig. 32. Examples in WO2008117050 and their IC50 values against JAK or Trk.

receptor balance to a more proapoptotic phenotype. Interestingly, GW441756 did not directly affect TrkA or AKT. The apoptosis was actually mediated by a functional downregulation of TrkA/AKT pathway [111].

Hong et al. reported HS-345 (93, Fig. 46), a novel TrkA inhibitor. KINOMEscan indicated that HS-345 displayed tight binding with 3 kinases: TrkA, TrkB and CDK11. HS345 inhibited cell growth in a dose-dependent manner. When administrated with 10 mM, viability of MIA PaCa-2 and BxPC-3 decreased to <20% and viability of PANC-1 reduced to around 30%. Subsequent human phosphokinase array analysis indicated that HS-345 functioned as anticancer agent via the inhibition of TrkA/AKT signaling pathway. In addition, HS-345 induced apoptosis in PANC-1 and MIA PaCa-2 cells. Increased level of cleaved PARP, cleaved caspase-3, and Bax, and decreased expression of Bcl-2 were observed in both two cells. HS345 both in vitro and in vivo inhibited angiogenesis of HUVECs and pancreatic cancer cells [112]. Giving BDNF is the natural ligand of TrkB, Gheibi designed and synthesized a library of TrkB small peptides based on the region III of the BDNF containing the sequence of N0 -TKCNPMGYTKE-C’. Five

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Fig. 35. Chemical structures of K-252a (80) and its derivatives 81e83.

Fig. 38. Chemical structure of milciclib (85). Fig. 36. Chemical structure of Entrectinib (84).

mostly stable peptides were docked with TrkB to find lowest energy value for 94 and 95. These two peptides can reduce the cell variability of ov-car-3 and sk-ov-3 with the IC50 level of 200 nM. Further, 94 and 95 can effectively decrease phosphorylation on TrkB, MAPK3 and eIF4E to further induce apoptosis in the treated cells (Fig. 47) [113]. 2.3. Trk inhibitors with unknown mechanism Croucher, Iyer and Brodeur et al. reported GNF-4256 as a novel pan-Trk inhibitor, that could inhibit the growth of neuroblastoma xenografts. GNF-4256 was shown to inhibit Ba/F3-Tel-TrkA, TrkB and TrkC with an IC50 less than 20 nM, while there was no activity in the parental Ba/F3 cells grown in the presence of IL-3. Kinase

Fig. 39. Chemical structure of (dl)-crizotinib (86) and its (S)-isomer, GTx-186 (87).

Fig. 37. X-ray crystal structure of entrectinib (84) bound to TrkA (PDB ID code 5KVT).

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Fig. 43. Example 1 in WO2006052936.

Fig. 40. Chemical structure of KST016366 (88).

Fig. 44. Chemical structure of KK5101 (91).

Fig. 41. Chemical structure of GNF-5837 (89).

profiling test indicated high selectivity of Trk over ROS (IC50 ¼ 3.8 mM). In the GNF-4256-treated samples, there was a dose-dependent inhibition of ligand-induced phosphorylation with IC50 between 3 and 10 nM. GNF-4256 was tested on SY5Y Trk-null and SY5Y-TrkB cells to confirm its TrkB specificity. While GNF4256 did not obviously inhibit the cell growth of SY5Y-Trk-null cells, proliferation of SY5Y-TrkB cell could be inhibited with IC50 value of 100 nM. When BDNF was added, GNF-4256 could inhibit the cell growth of SY5Y-TrkB cell with the IC50 value of 50 nM.

Compared between above inhibitory concentrations, GNF-4256 could be recognized as a potent and selective inhibitor of TrkBexpression NB cells. While single use of GNF-4256 decreased the tumor volume, the combination use of GNF-4256 with Irino and TMZ greatly enhanced antineoplastic effect and thus were better than single use of GNF-4256, Irino or TMZ [114]. Onishi reported a novel selective pan-Trk inhibitor ONO-7579 with potent antitumor activity (structure not yet disclosed). ONO7579 inhibited BDNF-induced TrkB phosphorylation and downstream activation of AKT, ERK and HIF-1a in TYGBK-1 and NOZ cells. Besides, ONO-7579 also inhibited MEK phosphorylation in TYGBK1 cells with wild-type KRAS. In NOZ cell, MEK phosphorylation was not observed after treatment with ONO-7579 because of KRAS mutation. In migration study, ONO-7579 was found to show

Fig. 42. X-ray crystal structure of GNF-5837 (52) bound to TrkC (PDB ID code 3V5Q).

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Fig. 45. Chemical structure of GW441756 (92).

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find wide application in other cancers that are induced by abnormal Trk activation [91]. Although it is of high value to carry forward the drug development of Trk inhibitor, there are still some unneglectable aspects associated with Trk inhibition as for cancer treatment. Firstly, normal NTRK is widely expressed in brain tissue, targeting Trks without affecting CNS Trks would be a tricky problem. Thus, Trk inhibitors for the treatment of peripheral tumor should have poor permeability towards blood-brain barrier. Secondly, drug resistance, the common failure for all RTKs, still remains a trouble. Although aforementioned LOXO-195 could inhibit acquired resistance induced by LOXO-101, reports with inhibitory activity against mutated Trks were rarely witnessed. Since acquired mutations are frequently observed in ATP-competitive kinase inhibitors, much effort should be paid on the development of non-ATP competitive inhibitors based on known co-crystal structures of Trks [5]. Abbreviations used

Fig. 46. Chemical structure of HS345 (93).

Fig. 47. Chemical structures of 94 and 95.

inhibitory ability against both GBC cells, especially TYGBK-1 cell line with wild-type KRAS. [115].

Trk: tropomyosin receptor kinase; WHO: world health organization; GAB1: GRB2-associated-binding protein 1: GRB2: Growth factor receptor-bound protein 2: SOS: Son of Sevenless expression product, a set of genes encoding guanine nucleotide exchange factors that act on the Ras subfamily of small GTPases; NGF: neural growth factor; NT: neurotrophins; PI3k/Akt pathway: phosphoinositide 3-kinase/protein kinase B pathway; MAPK/ERK pathway: mitogen-activated protein kinases pathway; BDNF: brain derived neurotrophic factor; Shc: Src-homology 2-domain containing adaptor protein; Bcl-2: B-cell lymphoma-2; BAD: Bcl-2 antagonist of cell death; GSK-3b: glycogen Synthase Kinase 3b; IRS-1: Insulin receptor substrate 1; Pi3K: phosphatidylinositol 3-kinase; KD: affinity constant; AML: acute myeloid leukemia; Bax: Bcl-2associated X protein; HIF-1a: hypoxia-inducible factor 1a; VEGF: vascular endothelial growth factor; ALK: anaplastic lymphoma kinase; ROS: proto-oncogene tyrosine-protein kinase; Cmax: peak plasma concentration; tmax: time that reach Cmax; AUC0e24h: area under curve (0e24 h); F: Bioavailability; Vss: volume of distribution; CL/F: clearance; t1/2: half-life; JAK: janus kinase; CDK: cyclindeoendent kinase; P-gp: P-glycoprotein; RET: rearranged during transfection; c-Fos: expression product of human homolog of the retroviral oncogene v-fos; IL: Interleukin; eIF4E: eukaryotic initiation factor 4E

3. Concluding remarks Author contributions With the rapid development of precision medicine, more and more clinical drug use for cancer treatment depends on the personal gene test. Among these oncological gene expression-induced tumor progressions, NTRK alteration has gained much attention due to its high correlation with tumor metastasis that was considered as the major death factor in cancer treatment. Actually, NTRK genes and their expression proteins Trks function nearly the same between CNS cells and oncological cells. Trks broadly distributes in brain tissue and function as growth modulator in the central nervous system. However, in aberrant conditions, such as NTRK fusion-induced auto-activation or NTRK gene overexpression, Trks would activate oncological signaling pathway to accelerate the growth and transformation of preneoplastic cells towards neoplastic cells. Delightfully, the selectivity across TrkA-C seems not so be essential because all Trk members are involved in tumor development and evasion. To date, NTRK alteration has been detected in various cancer cells. For example, gene sequence has shown that approximately 15% of NSCLC correlates with NTRK fusion [17]. Giving the major treatment for NSCLCs is still chemotherapy; the development of Trk inhibitors will provide a novel cancer therapy with higher efficacy and lower toxicity. This will also

Qi Miao and Kun Ma contributed equally. Acknowledgements This work was supported by the National Natural Science Foundation of China (81773559, 21807114, 21472191), the Double First-Class University Project (CPU2018GY03), the National Major Scientific and Technological Program for Drug Discovery Grant (2018ZX09301045002), the International Cooperation Grant of Guangzhou (201704030099) of Guangzhou, the Project of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZRC201810), and Chinese Pharmaceutical AssociationYiling Biopharmaceutical Innovation Foundation. References [1] B.W. Stewart, C.P. Wild, World Cancer Report 2014, International Agency for Research on Cancer, Lyon, 2014. [2] A.A. Friedman, A. Letai, D.E. Fisher, K.T. Flaherty, Precision medicine for cancer with next-generation functional diagnostics, Nat. Rev. Canc. 15 (2015) 747e756.

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