TPX-0005 | Mirna-2

Discovery of the Next-Generation Pan-TRK Kinase Inhibitors for the Treatment of Cancer

Cite This: J. Med. Chem. 2021, 64, 10286−10296
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Zongliang Liu, Pengfei Yu, Lin Dong, Wenyan Wang, Sijin Duan, Bingsi Wang, Xiaoyan Gong, Liang Ye, Hongbo Wang,* and Jingwei Tian*

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ABSTRACT: The neurotrophic receptor tyrosine kinase (NTRK) genes including NTRK1, NTRK2, and NTRK3 encode the tropomyosin receptor kinase (Trk) proteins TrkA, TrkB, and TrkC, respectively. So far, two TRK inhibitors, larotrectinib sulfate (LOXO-101 sulfate) and entrectinib (NMS-E628, RXDX-101), have been approved for clinical use in 2018 and 2019, respectively. To overcome acquired resistance, next-generation Trk inhibitors such as selitrectinib (LOXO-195) and repotrectinib (TPX-0005) have been developed and exhibit eﬀectiveness to induce remission in patients with larotrectinib treatment failure. Herein, we report the identiﬁcation and optimization of a series of macrocyclic compounds as potent pan-Trk (WT and MT) inhibitors that exhibited excellent physiochemical properties and good oral pharmacokinetics. Compound 10 was identiﬁed via optimization from the aspects of chemistry and pharmacokinetic properties, which showed good activity against wild and mutant TrkA/TrkC in in vitro and in vivo studies.

⦁ INTRODUCTION
In order to improve the eﬃcacy of anti-tumor drugs and
reduce their side eﬀects, cancer treatment is shifting toward precision oncology. As such, patients receive individualized therapy based on the prescreening of predictive biomarkers, such as oncogenic mutations, rather than using empiric chemotherapy. It was reported in 2007 that gene fusions account for 20% of human cancer morbidity.1 The neuro- trophic receptor tyrosine kinase (NTRK) genes including NTRK1, NTRK2, and NTRK3 encode the tropomyosin receptor kinase (Trk) proteins TrkA, TrkB, and TrkC, respectively.2 NTRK rearrangements and fusion gene products have been observed in numerous tumor types.3 For example, NTRK fusions are found in about 50% of pediatric diﬀuse intrinsic pontine glioma and non-brainstem glioblastoma,4 16.7% of thyroid carcinoma,5 7.1% of pediatric gliomas,6 3.3% of lung cancers,7 2.5% of glioblastomas, and 2.2% of colorectal cancer.6,8
The growing number of identiﬁed gene fusions involving the NTRK gene drew the interest of the scientiﬁc community to develop gene-speciﬁc anti-cancer drugs. So far, two TRK inhibitors, larotrectinib sulfate (LOXO-101 sulfate) and entrectinib (NMS-E628, RXDX-101), have been approved for clinical use in 2018 and 2019, respectively, and many are currently under clinical trials (Figure 1). Entrectinib is a potent, orally available, and central nervous system-active pan- Trk inhibitors approved and in clinical trials.

Received: April 19, 2021
Published: July 13, 2021

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© 2021 American Chemical Society

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https://doi.org/10.1021/acs.jmedchem.1c00712

J. Med. Chem. 2021, 64, 10286−10296

Trk, ROS1, and ALK inhibitor. Entrectinib inhibits TrkA, TrkB, TrkC, ROS1, and ALK with IC50 values of 1, 3, 5, 12, and 7 nM, respectively.9 Larotrectinib is an adenosine triphosphate (ATP)-competitive orally active pan-Trk inhib- itor, with IC50 values of 2−20 nmol/L against cancer cells and between 5 and 11 nM against all three isoforms, and with 1000-fold or greater selectivity relative to other kinases.10 The ﬁrst 55 patients with solid tumors with the conﬁrmed NTRK gene fusion were enrolled in one of the three multicenter, open-label, single-arm clinical trials (NCT02122913, NCT02637687, and NCT02576431). Of all the enrolled
patients with conﬁrmatory response data available, the objective response rate was 76, with 12% complete response and 64% partial response (information from the drug label).

One major limitation to maintain durable therapeutic eﬃcacy for all kinase inhibitors is the acquired resistance. To overcome acquired resistance, next-generation Trk inhibitors such as selitrectinib (LOXO-195) and repotrectinib (TPX- 0005) have been developed, which exhibited eﬀectiveness to induce remission in patients with larotrectinib treatment failure (Figure 1).11,12 LOXO-195 shows strong binding to the wild- type TrkA, TrkB, and TrkC kinase domains and has potent (IC50 < 1 nM) inhibitory activity in kinase enzyme assays.11 Importantly, LOXO-195 achieves low nanomolar inhibitory activity against TrkAG595R, TrkAG667C, TrkCG623R, and TrkCG696A, with IC50 values being 2.0, 9.8, 2.3, and 2.5 nM, respectively, but showed little eﬀect on 228 other diﬀerent kinases. In an in vitro eﬃcacy study, LOXO-195 demonstrated potent inhibition of cell proliferation in Trk fusion-containingKM12, CUTO-3, and MO-91 cell lines (IC50 ≤ 5 nM).11 TPX- 0005 is a multi-kinase inhibitor which shows potent eﬀects
Imageagainst ROS1 (IC50 = 0.07 nM), Trk (IC50 = 0.83/0.05/0.1 nM for TrkA/B/C), WT ALK (IC50 = 1.01 nM), and JAK2 (IC50 = 1.04 nM).12 Both LOXO-195 and TPX-0005demonstrated signiﬁcant eﬃcacy in clinical trials in patients who were resistant to the ﬁrst-generation Trk inhibitors.11−14

In this study, we report the identiﬁcation and optimization of a series of macrocyclic compounds as potent pan-Trk (WT and MT) inhibitors that exhibited excellent physiochemicalproperties and good oral pharmacokinetics.

RESULTS AND DISCUSSION
Docking of LOXO-195 and TPX-0005 with TrkA. Currently, the cocrystal structures of LOXO-101 or LOXO- 195 with Trk protein remain unknown. We docked LOXO-195 and TPX-0005 to explore their binding conformation with the TrkA protein by using Discovery Studio 2018 software (CDOCKER) and the existing eutectic complex (PDB ID: 4YNE).15 From the docking test, we found that the binding modes of LOXO-195 and TPX-0005 with TrkC are nearly

Docking of LOXO-195 and TPX-0005 with TrkA.identical (Figure 2). The nitrogen atom on the pyrazole ring
can form a hydrogen bond with Met592 in the hinge area. Theﬂuorophenyl/ﬂuoropyridine moiety that was well integrated into the hydrophobic pocket maintained a vertical conforma- tion with the entire skeleton and had hydrophobic interactions with the surrounding Phe521, Leu657, Gly667, and Asp668. Simultaneously, the ﬂuorine atom formed a key interaction force with Asn655. The oxygen atom on the amide formed a hydrogen bond with Met592 in the hinge region through the water molecule. There was a hydrophobic eﬀect between the tetrahydropyrrole of LOXO-195 with the gatekeepers Phe589 and Phe521. The methyl group at the same position of TPX- 0005 also had a hydrophobic eﬀect with Phe521. The samehydrophobic interaction existed between the remaining methyl groups of LOXO-195 and TPX-0005 with P-loop Val524 and Gly517. The structures of the hinge region and the ﬂuoropyridine/ﬂuorobenzene region (marked in red) were relatively conservative, which plays a decisive role in the activity of Trk kinase. The tetrahydropyrrole region and the amide alkyl chain (marked in blue) were more ﬂexible and there was chemical space for structural modiﬁcation. There- fore, we mainly modiﬁed the blue area of the structure in order to obtain preclinical drug candidates with better activity and selectivity.
Structure−activity relationship of TPX-0005.

Table 1. PD Data of Compounds 8, 9, and 10 In Vitro

compound (IC50 nM)
LOXO-195 TPX-0005 8 9 10
6.7 ± 0.5 2.5 ± 0.4 3.2 ± 0.6 4.9 ± 0.4 2.4 ± 0.3
6.2 ± 0.5 1.4 ± 0.2 2.1 ± 0.4 9.4 ± 1.0 3.5 ± 0.5
110.0 ± 10.1 8.7 ± 0.8 0.7 ± 0.1 1.7 ± 0.3 2.3 ± 0.3

enzymatic TrkA TrkAG595R TrkAG667C
TrkC 0.5 ± 0.1 0.01 ± 0.00 0.2 ± 0.0 0.2 ± 0.1 0.2 ± 0.1
ALK 274.0 ± 16.4 2.3 ± 0.4 18.5 ± 0.7 88.9 ± 12.5 182.0 ± 14.2
Ros1 1.15 ± 0.07 0.17 ± 0.05 0.2 ± 0.1 1.2 ± 0.3 1.0 ± 0.3
cellular Ba/F3 TrkA 4.9 ± 0.3 1.8 ± 0.3 0.2 ± 0.0 0.6 ± 0.1 0.6 ± 0.1
Ba/F3 TrkAG595R 6.8 ± 0.7 8.0 ± 0.6 0.4 ± 0.0 7.7 ± 0.5
Ba/F3 TrkAF589L 25.4 ± 4.5 0.7 ± 0.2 4.9 ± 0.2
Ba/F3 TrkAG667A 24.4 ± 2.5 0.8 ± 0.1
Ba/F3 TrkCG623R 2.7 ± 0.5 4.8 ± 0.4 5.8 ± 0.3 2.7 ± 0.2
Ba/F3 SLC34A2-Ros1 36.7 ± 3.6 2.2 ± 0.4 12.6 ± 0.7 2.3 ± 0.3

Structure−Activity Relationship of TPX-0005. We ﬁrst studied the structure−activity relationship of TPX-0005. The amide group in TPX-0005 was replaced with a sulfonamidegroup (compound 1, Figure 3), which led to the loss of its activity. Maintaining the chain length between the amide and ﬂuorophenyl groups, the structure modiﬁcation was carried out to obtain compounds 2, 3, 4, 5, and 6 (Figure 3). Compounds 2 and 3 with imide and sulfonimide were unstable likely due to the increased ring tension. Changing the ether bond to an amide group (compound 4) reduced the TrkA inhibitory activity by 300−400 times. When piperazine or piperidine was used for bonding, the TrkA inhibitory activity disappeared.
This could be due to the fact that after the chain was replaced with a ring, the spatial conformation of the molecule was altered.
Design of Compounds with One-Chiral Atom. There are two chiral centers in LOXO-195 and TPX-0005, which posed great challenges for the synthesis and separation of suchcompounds. Compounds 7 and 8 were designed and synthesized, which contained only one chiral atom (Figure 3). Excitingly, both compounds had good inhibitory activity, particularly compound 8, which showed a similar activity against wild TrkA/C with IC50 ranging from 0.2 to 3.2 nM to LOXO-195 and TPX-0005.

In Vitro PD and PK Data of Compound 8. To determine the impact of TRK kinase resistance mutations on the activity of inhibitors, compound 8 was tested against puriﬁed kinase domains in vitro. Compound 8 achieved low nanomolar inhibitory activity against TrkA, TrkAG595R, TrkAG667C, TrkC, Alk, and Ros1, with IC50 values being 3.2, 2.1, 0.7, 0.2, 18.5, and 0.2 nM, respectively (Table 1). In an enzyme inhibition study, compound 8 also had good activity against mutant TrkA, especially its inhibitory activity against TrkAG667C, which was 10−100 times higher than those of LOXO-195 and TPX-
0005. The anti-tumor activity of compound 8 was evaluated
using Ba/F3 cells, and the IC50 values were 0.2 and 5 nM in

LOXO-195 TPX-0005 8 9 10
cLog P 1.86 2.96 1.97 0.57 0.58
LLEa 6.3 5.6 6.5 7.7 8.0
PappA→B (10−6 cm/s)/ER 8.4/4.1 0.9/45.3 16.3/1.4 3.9/9.5 12.0/2.8
KSb (μM) 65.8 192.9
MMS (mL/min/kg) 76/92/1351/38 29/49/982/21/ 242/980/1010/268 43/29/342/74 17.7/28.8/1504/<10.7
PPB (Fu %) H/R/M/D 34/3/2/20 5/6/5/9 2/2/2/3 36/30/28/34 16/12/11/21
CYP (IC50, μM): >50/>50/>50/>50/>50 >50/6.4/12.3/28.5/>50 >100/18.1/89.0/>100/>50
1A2/2C9/2C19/2D6/3A4
hERG (IC50, μM) 6.7 13

Table 2. PK Data of Compounds 8, 9, and 10 In Vitro
LLE, ligand lipophilicity eﬃciency. bKS, kinetic solubility. cH/R/M/D, human/rat/mouse/dog.

Design of compounds with one-chiral atom.wild and mutant TrkA/C cells, respectively. In Ba/F3 cells with SLC34A2-Ros1 fusion, the activity of compound 8 was slightly lower (IC50 value: 12.6 nM).

Due to its good kinase inhibitory activity and cellular pharmacodynamics, the pharmacokinetic evaluation of com- pound 8 was carried out in vitro (Table 2). Most of the pharmacokinetic parameters of compound 8 were similar to those of LOXO-195 or TPX-0005 except for MMS (mL/min/ kg). From the perspective of MMS, compound 8 was metabolized faster in humans, rats, and dogs. The oxidation on the piperidine ring may be the reason for the faster metabolism of compound 8. Based on this ﬁnding, the structural optimization was continued.
In Vitro PD and PK Data of Compounds 9 and 10.

Substituting piperidine in compound 8 with piperazine gave compound 9 and replacing it with morpholine gave compound
10. From the results of PD data in vitro, we can see that both compounds 9 and 10 maintained good inhibitory activity against WT and MT TrkA/TrkC, and IC50 values were essentially the same as those of compound 8 (Table 1). It is worth noting that the selectivity of compounds 9 and 10 to

ALK was greatly improved, with IC50 values of 88.9 and 182.0 nM, respectively, while the IC50 value of compound 8 was 18.5 nM. This selectivity may be beneﬁcial in reducing oﬀ-target side eﬀects.The pharmacokinetic evaluation of compounds 9 and 10 was carried out in vitro (Table 2). These results demonstrated that the drugability of compounds 9 and 10 was further improved compared to that of compound 8. The ligand lipophilicity eﬃciencies (LLEs) of compounds 9 and 10 were7.7 and 8.0, respectively, while the LLE of compound 8 was 6. The metabolic stability was greatly improved, and the MMS in humans, rats, and dogs were all less than 100 mL/min/Kg, which was not only superior to compound 8 but also better than LOXO-195 and TPX-0005. It was worth noting that the MMS values of these compounds in mice were signiﬁcantly higher than those of other species, which may be caused by special metabolic enzymes. The inhibitory activity of compound 10 on the human ether-a-go-go-related gene (hERG) was much lower than that of compound 9, with IC50 values of 13 and 6.7 μM, respectively. Therefore, compound 10 as a preclinical drug candidate continued to
undergo eﬃcacy and pharmacokinetic evaluation in vivoof repeated administrationtoxicity
study in Cynomolgus

Kinase Assay Data of Compound 10. Kinase assays (Table 3) showed that compound 10 could inhibit the

Table 3. Kinase Assay Data of Compound 10

kinases activity (% control)
kinases activity (% control)
ALK 26 Met(M1268T) 86
A-Raf 103 Met(Y1248C) 85
Blk 46 Met(Y1248D) 96
BTK(R28H) 74 Met(Y1248H) 89
B-Raf 111 PDGFRα 106
B-Raf(V599E) 87 PDGFRα(D842V) 102
cKit 100 PDGFRα(V561D) 103
cKit(D816V) 101 Ret(h) 64
cKit(D816H) 96 Ret(V804L) 59
cKit(V560G) 109 Ret(V804M) 54
cKit(V654A) 105 RIPK1 102
c-RAF 103 RIPK2 93
EGFR 108 Ron 112
EGFR(L858R) 91 Ros1 16
EGFR(L861Q) 95 Src(1−530) 33
EGFR(T790M) 105 Src(T341M) 55
FAK 72 TrkA 2
FGFR1 92 TrkB −1
FGFR2 93 TrkC −3
FGFR3 96 JAK1 91
FGFR4 114 JAK2 44
Met 98 JAK3 94
Met(D1246H) 91 KDR 108

activities of TrkA, TrkB, and TRC with high selectivity at 0.5 μM (kinase activity remaining, <10%) and slightly inhibit the activities of ALK and ROS1 (kinase activity remaining, 10− 30%), and the inhibitory activities of other kinases were not signiﬁcantly aﬀected by compound 10.
In Vivo PD and PK Data of Compound 10. After a single intravenous administration of compound 10 at a dose of 2 mg/ kg, the mean terminal half-life (t1/2) was 0.87 h for male rats and 2.21 h for female rats. The exposure area under the curve (AUC0−t) in female rats was signiﬁcantly higher (2.5-fold) than that in male rats. After a single intragastric administration of 10 mg/kg, the time to peak (Tmax) concentrations was 0.667 h. The AUC0−t were 11,720 nM*h in male and 31,676 nM*h infemale rats, respectively. The bioavailability was 56.0−61.9%.
Four engineered BaF3-NTRK xenograft tumor models were used to assess the therapeutic eﬀect of compound 10 on tumor growth. Mice bearing 100−200 mm3 tumors were treated orally at doses of 5, 10, or 20 mg/kg, once daily for 21 days. As shown in Figure 5, compound 10 produced signiﬁcant inhibition of tumor growth both in wild-type NTRK fusion mutation cells (Ba/F3 LMNA-NTRK1cells and Ba/F3 ETV6- NTRK3 cells) and drug-resistant mutation cells (Ba/F3 ETV6- NTRK3-G623R cells and Ba/F3 LMNA-NTRK1-G595R) atall three doses, and the anti-tumor eﬀect of compound 10 at 10 mg/kg was similar to that of LOXO-195 at 100 mg/kg. In addition, some side eﬀects were observed in the LOXO-195 group, including unkempt appearance, weight loss, or even death. In contrast, animals that received the highest dose of compound 10 only showed slight weight loss and all animals survived during the treatment period. In our previous 4 weeksmonkeys, treatment-related changes included gait disturbance, impaired balance, and poor coordination, which could be attributed to excessive on-target Trk receptor inhibition. In addition, no signiﬁcant changes in food consumption, electrocardiogram, blood pressure, hematological and bio- chemical parameters, organ weight, or bone marrow parameters were observed.16 All these data suggest that compound 10 was more eﬃcacious and safer against tumor growth than LOXO-195 (Table 4).

Chemistry. A typical synthetic route toward macrocyclic compound 10 is described in Scheme 1. Compound 10-1 was protected by Boc2O (Boc, tert-butyloxycarbonyl) with N,N- dimethylpyridin-4-amine in THF to aﬀord 10-2. Enol phosphate 10-3 was synthesized from compound 10-2 by reaction with diphenyl phosphorochloridate under basic conditions in THF. Subsequent Suzuki−Miyaura coupling was performed with borate 10-4 to give 10-5. Compound 10-5 was reduced with H2 and Pd(OH)2/C in EtOH to aﬀord 10-6. Compound 10-7 was obtained from 10-6 via deprotection under acid conditions. Compounds 10-3, 10-5, 10-6, and 10-7did not need to be puriﬁed, and the crude product was directly subjected to the next reaction. Compound 10-7 was alkylated by ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate 10-8 with N,N-diisopropylethylamine in NMP to aﬀord 10-9. Compound 10-9 was demethylated with chlorotrimethylsilane to obtain compound 10-10, and the hydrolysis was continued to aﬀord compound 11-11. Compounds 10-11 and 10-12 were condensed with HATU to give compound 10-13. After the intramolecular Mitsunobu reaction, compound 10-14 was obtained. The racemic material was puriﬁed by chiral chromatography to give the target compound 10. As a result, compound 10 was found to be an R-isomer by single-crystal X- ray analysis, as shown in Figure 5.

CONCLUSIONS
■Due to its high clinical eﬃcacy, there is substantial interest in the development of small-molecule inhibitors of TrkA for the treatment of tumors. We have successfully discovered a novel, highly potent, and selective second-generation Trk inhibitor compound 10 (LPM4870108). Although initial eﬀorts using a structural modiﬁcation strategy was largely futile, compound 10 was found via optimization from the aspects of chemistry and pharmacokinetic properties, which had good activity against wild-type and mutant TrkA/TrkC. Moreover, the high potency, high oﬀ-target selectivity, good in vivo toxicity proﬁle, as well as good absorption and human CL predictions made compound 10 an exciting and promising structurally diﬀer- entiated asset suitable for advancement into a variety of clinical anti-tumor studies (Figure 6).

EXPERIMENTAL SECTION
General. The recorded 1H nuclear magnetic resonance (NMR) spectra were found to be consistent with the proposed structures. Characteristic chemical shifts (δ) are given in parts-per-million (ppm) (δ relative to the residual solvent peak) using conventional abbreviations for the designation of major peaks: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad. The mass spectra (m/ z) were recorded using the electrospray ionization technique. The following abbreviations were used for common solvents: CDCl3, deuterochloroform; DMSO-d6, deuterodimethyl sulfoxide; and CD3OD, deuteromethanol. All solvents were of reagent grade and, when necessary, were puriﬁed and dried by standard methods. The concentration of solutions after reactions and extractions involved the In vivo eﬃcacy of compound 10 against four engineered BaF3-NTRK tumor models.

Table 4. PK Data of Compound 10 in Sprague−Dawley Rats

LOXO-195 TPX-0005 10
dose (mpk) 3 3 2 (M) 2 (F)
C0 (nM)
T1/2 (h)
Vd (L/kg)
Cl (mL/kg/min) 23,779 ± 1526
1.2 ± 0.1
0.5 ± 0.1
11.8 ± 0.8 12,870 ± 831
2.8 ± 0.1
1.1 ± 0.2
9.4 ± 0.5 4118 ± 833
0.872 ± 0.0156
1.39 ± 0.229
19.3 ± 0.624 3509 ± 719
2.21 ± 0.215
1.46 ± 0.224
8.19 ± 1.96
AUC0−t (nM*h) 11,146 ± 656 15,002 ± 859 4191 ± 139 10,282 ± 2298
dose (mpk) 10 10 10 (M) 10 (F)
Cmax (nM)
Tmax (h) AUC0−t (nM*h) F % 12,700 ± 1121
0.4 ± 0.0
27,344 ± 1590
74 ± 3 16,900 ± 1173
0.8 ± 0.0
59,724 ± 1434
119 ± 4 6384 ± 1525
0.667 ± 0.289
11,720 ± 1749
56.0 ± 8.4 6628 ± 626
0.667 ± 0.289
31,676 ± 6628
61.9 ± 13.0
use of a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Anhydrous solvents were obtained from commercial sources and used as received. The chemical yields reported below are unoptimized. Purity criteria: ﬁnal compounds isolated as singletons are >95% based on LCMS and/or HPLC.

tert-Butyl 3-Oxomorpholine-4-carboxylate (10-2). 4-Dimethyla-
minopyridine (12.08 g, 98.91 mmol, 0.1 equiv) was added to the solution of compound 10-1 (morpholin-3-one) (100 g, 989.08 mmol, 1 equiv) in tetrahydrofuran (1000 mL) and then Boc2O (280.62 g,
1.29 mol, 295.39 mL, 1.3 equiv) was dropped. The mixture was stirred at 30 °C for 2 min and poured into an aqueous sodium chloride solution (1 L). The reaction was extracted with EtOAc. The combined organic phases were added with 1% HCl to adjust the pH to 5−6 in an ice bath, washed with water, dried over Na2SO4, ﬁltered,and concentrated to obtain compound 10-2 (185 g, crude product) as a yellow oily compound.
tert-Butyl 5-((Diphenoxyphosphoryl)oxy)-2,3-dihydro-4H-1,4-ox- azine-4-carboxylate (10-3). Under nitrogen, LiHMDS (1 M, 546.67 mL, 1 equiv) was slowly dropped into the solution of compound 10-2 (110 g, 546.67 mmol, 1 equiv) in tetrahydrofuran (1000 mL) and the temperature of the reaction was controlled between −60 and −30 °C. The reaction mixture was stirred for 1 h and then diphenyl chlorophosphate (146.85 g, 546.67 mmol, 112.96 mL, 1 equiv) in tetrahydrofuran (120 mL) was added dropwise. After stirring for 3 h, the reaction mixture was poured into water (1000 mL) and extracted with EtOAc. The organics were washed with water, dried over Na2SO4, ﬁltered, and concentrated. The crude material was puriﬁed
by SiO2 column chromatography to aﬀord 10-3 (127 g, 293.3 mmol, yield 52.87%).

aReagents and conditions: (a) Boc2O, DMAP, THF, RT, 16 h, 93%; (b) ClPO(OPh)2, LiHMDS, THF; (c) Pd(PPh3)4, NaHCO3, THF/H2O, reﬂux, 16 h; (d) H2, Pd(OH)2/C (5% wt), EtOH, 96 h; (e) HCl/EA, the total yield is 44% from step b to e; (f) DIPEA, NMP, 75 °C, 16 h, 85.3%;
Image(g) TMSCl, NaI, ACN, 75 °C, 2 h; (h) NaOH, H2O/MeOH, 60 °C, 2 h, total yield of steps g and h is 98.6%; (i) HATU, DIPEA, DMF, RT, 72.4%; (j) PPh3, DEAD, THF.

ORTEP of 10.tert-Butyl 5-(5-Fluoro-2-methoxypyridin-3-yl)-2,3-dihydro-4H- 1,4-oxazine-4-carboxylate (10-5). Acetonitrile (1000 mL), water
(250 mL), compound 10-3 (70 g, 161.52 mmol, 1 equiv), potassiumphosphate (68.57 g, 323.04 mmol, 2 equiv), and compound 10-4(81.75 g, 323.04 mmol, 2 equiv) were sequentially added to the ﬂask. After nitrogen replacement, tri-tert-butylphosphine palladium (6 g,
11.74 mmol) was added under a nitrogen ﬂow, and the reaction mixture was heated at 75 °C for 10 h. The reaction mixture was poured into water (1000 mL) and extracted with EtOAc. The organics were washed with water, dried over Na2SO4, ﬁltered, and concentrated. The crude material was puriﬁed by SiO2 column chromatography to aﬀord 10-5 (39.6 g, 127.8 mmol, yield 78.9%). LCMS [M + 1] 311.1; 1H NMR (400 MHz, chloroform-d): δ ppm 7.87 (d, J = 2.8 Hz, 1H), 7.24−7.19 (m, 1H), 6.30−6.22 (m, 1H),4.26−4.19 (m, 2H), 3.90 (s, 3H), 3.83−3.75 (m, 2H), 1.15 (s, 9H).
tert-Butyl 3-(5-Fluoro-2-methoxypyridin-3-yl)morpholine-4-car- boxylate (10-6). Compound 10-5 (30 g, 96.67 mmol, 1 equiv) was dissolved in ethanol (1000 mL) and then dry palladium hydroxide on carbon (9.62 g, 13.69 mmol, 20% purity) was added under an argon atmosphere. Under a hydrogen atmosphere (50 psi), the reaction was heated at 80 °C for 24 h. The reaction solution was ﬁltered, and the ﬁlter cake was washed with ethanol (50 mL). The ﬁltration was concentrated in vacuo to obtain compound 10-6 as a white solid (27.8 g, 89.1 mmol, yield 91.95%). LCMS [M + 1] 313.2; 1H NMR (400
MHz, chloroform-d): δ ppm 7.87 7.83 (d, J = 2.8 Hz, 1H), 7.24 (dd, J
= 2.4, 8.7 Hz, 1H), 5.06 (br d, J = 3.8 Hz, 1H), 4.19 (br d, J = 12.0
Hz, 1H), 3.91−3.79 (m, 5H), 3.72 (dd, J = 4.3, 12.0 Hz, 1H), 3.51
(dt, J = 3.4, 11.6 Hz, 1H), 3.36−3.23 (m, 1H), 1.33 (s, 9H).

3-(5-Fluoro-2-methoxypyridin-3-yl)morpholine (10-7). HCl/ ethyl acetate (4 M, 750.69 mL, 10.91 equiv) was added to the solution of compound 10-6 (86 g, 275.34 mmol, 1 equiv) in ethyl acetate (300 mL), and the reaction mixture was stirred at 25 °C for 16
h. A large amount of white solid precipitated. Under a nitrogen atmosphere, the reaction solution was ﬁltered, washed with ethyl acetate (300 mL), and the ﬁlter cake was vacuum-dried without further puriﬁcation to obtain compound 10-7 as a white solid (57.46 g, 271 mmol, yield 98.33%). LCMS [M + 1] 213.1; 1H NMR (400 MHz, chloroform-d): δ ppm 7.88 (d, J = 3.0 Hz, 1H), 7.62−7.57 (m, 1H), 4.17 (dd, J = 3.0, 9.2 Hz, 1H), 3.96 (dd, J = 3.2, 10.8 Hz, 1H), 3.92 (s, 3H), 3.89−3.84 (m, 1H), 3.61 (dt, J = 2.6, 11.0 Hz, 1H), 3.24
(dd, J = 9.2, 10.4 Hz, 1H), 3.15−3.06 (m, 1H), 3.03−2.96 (m, 1H).

Ethyl 5-(3-(5-Fluoro-2-methoxypyridin-3-yl)morpholino)- pyrazolo[1,5-a]pyrimidine-3-carboxylate (10-9). N,N-Diisopropyle-
thylamine (88.09 g, 681.58 mmol, 118.72 mL, 2.53 equiv) and
compound 10-8 (50.37 g, 223.24 mmol, 0.83 equiv) were added to
the mixture of compound 10-7 (67 g, 269.42 mmol, 1 equiv, HCl) in 1-methyl-2-pyrrolidone (350 mL) and then stirred at 70 °C for 16 h. The reaction solution was poured into ice water (1000 mL) and stirred for 30 min. A large number of solids precipitated out, which were then ﬁltered and washed with water (400 mL). The ﬁlter cake was dissolved in dichloromethane (1.5 L), dried with anhydrous sodium sulfate, and concentrated in vacuo to obtain compound 10-9 (92.2 g, crude product, yield 85.33%).

Ethyl 5-(3-(5-Fluoro-2-hydroxypyridin-3-yl)morpholino)- pyrazolo[1,5-a]pyrimidine-3-carboxylate (10-10). The crude com- pound 10-9 (90 g, 224.77 mmol, 1 equiv) was dissolved in the acetonitrile solvent (900 mL), then sodium iodide (101.08 g, 674.32 mmol, 3 equiv) and trimethylchlorosilane (73.26 g, 674.32) mmol,85.58 mL, (3 equiv) were added, and the reaction solution was stirred at 25 °C for 24 h. The reaction solution was poured into an aqueous sodium bicarbonate solution (prepared with 65 g of sodium bicarbonate and 1.2 L of water), then the pH was adjusted to 7−8, and extracted with dichloromethane (500 mL). The organic phase was washed with saturated sodium chloride (150 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a crude product. The crude product was slurried (ethyl acetate−methyl tert-butyl ether = 1:1, 400 mL) for 1 h, ﬁltered, and the ﬁlter cake was dried in vacuo to obtain compound 10-10 as a pale yellow solid (86 g, crude product).
5-(3-(5-Fluoro-2-hydroxypyridin-3-yl)morpholino)pyrazolo[1,5- a]pyrimidine-3-carboxylic Acid (10-11). Compound 10-10 (85 g,
219.43 mmol, 1 equiv) was dissolved in methanol and then sodium hydroxide (3 M, 292.58 mL, 4 equiv), water (292 mL), and methanol (850 mL) were added. The reaction mixture was stirred at 60 °C for 2
h.

After the reaction mixture was cooled in an ice water bath, 3 mol/L dilute hydrochloric acid was slowly added to adjust the pH to 2. After stirring for 0.5 h, a solid precipitated out. It was ﬁltered and the ﬁlter cake was dried in concentrated vacuum to obtain compound 10-11 as a light brown solid (60.5 g, 168.38 mmol, yield 76.73%). LCMS [M + 1] 360.9; 1H NMR (400 MHz, DMSO-d6): δ ppm 11.75 (br s, 1H), 8.74 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.56 (br s, 1H), 7.44 (br d, J = 8.0 Hz, 1H), 6.72 (br d, J = 8.0 Hz, 1H), 5.31 (br s, 1H), 4.51 (br s, 1H), 4.37 (br d, J = 12.0 Hz, 1H), 4.07 (br d, J = 8.4 Hz, 1H), 3.84 (br dd, J = 4.0, 11.8 Hz, 1H), 3.66−3.45 (m, 3H).
5-(3-(5-Fluoro-2-hydroxypyridin-3-yl)morpholino)-N-(1-
(hydroxymethyl)cyclopropyl)pyrazolo[1,5-a]pyrimidine-3-carboxa- mide (10-13). Compound 10-11 (53.5 g, 148.90 mmol, 1 equiv) and compound 10-12 (20.24 g, 163.79 mmol, 1.1 equiv) were dissolved in N,N-dimethylformamide (530 mL) and then 2-(7-benzotriazole oxide)-N,N,N′,N′-tetramethylurea hexaﬂuorophosphate (62.28 g,163.79 mmol, 1.1 equiv) was added. After N,N-diisopropyl ethyl-
amine (67.35 g, 521.14 mmol, 90.77 mL, 3.5 equiv) was added dropwise and the reaction solution was heated at 15 °C for 16 h. The reaction was quenched by adding 16 mL of saturated aqueous ammonium chloride solution to the reaction solution and concentrated in vacuo at 60 °C. After concentration, acetonitrile (400 mL) was added and it was stirred for 1 h. A large amount of solids precipitated. This solid was ﬁltered to obtain compound 10-13

(72 g). LCMS [M + 1] 429.1; 1H NMR (400 MHz, DMSO-d6): δ
ppm 8.80 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.87 (s, 1H), 7.56 (s, 1H),
7.43 (d, J = 6.4 Hz, 1H), 6.81 (s, 1H), 5.38 (s, 1H), 4.69 (s, 1H), 4.31
(d, J = 19.2 Hz, 1H), 4.22 (s, 1H), 4.08−4.02 (m, 2H), 3.89−3.85
(m, 2H), 3.66 (d, J = 6.8 Hz, 3H), 0.75−0.71 (m, 3H), 0.60 (br s, 1H).
(3′E,4′E)-5′-Fluorospiro[cyclopropane-1,6′-4-oxa-7-aza-2(4,3)- morpholina-1(5,3)-pyrazolo[1,5-a]pyrimidina-3(3,2)-pyridinacy- clooctaphan]-8′-one (10-14). Compound 10-13 (25 g, 58.35 mmol,
1 equiv) was dissolved in tetrahydrofuran (250 mL), then
tributylphosphorus (17.71 g, 87.53 mmol, 21.60 mL, 1.5 equiv) was added dropwise, followed by azodimethyl bis(piperidine) (22.09 g,
87.53 mmol, 1.5 equiv), and it was stirred at 15 °C for 16 h. The reaction solution was directly ﬁltered, and the obtained white ﬁlter cake was compound 10-14 (35 g, crude product). The crude product was dissolved in dichloromethane (1000 mL) and methanol (100 mL), washed twice with 1 M HCl (250 mL), the organic phase was dried and concentrated in vacuo to obtain the crude product. The crude product was slurried with ethanol (500 mL) and stirred for 1 h, ﬁltered, and the ﬁlter cake was vacuum-dried to obtain compound 10- 14 (22 g). LCMS [M + 1] 411.1; 1H NMR (400 MHz, CDCl3): δ ppm 8.98 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.26 (s, 1H), 7.89 (d, J = 2.4 Hz, 1H), 7.77−7.75 (m, 1H), 6.36 (d, J = 7.6 Hz, 1H), 6.15 (s,
1H), 5.07 (d, J = 10.8 Hz, 1H), 4.39 (d, J = 9.6 Hz, 1H), 4.12−3.88
(m, 4H), 3.76 (d, J = 10.0 Hz, 1H), 3.60 (d, J = 10.8 Hz, 1H), 2.27−
2.22 (m, 1H), 1.07−0.94 (m, 2H), 0.84−0.79 (m, 1H).
(S,3′E,4′E)-5′-Fluorospiro[cyclopropane-1,6′-4-oxa-7-aza-2(4,3)- morpholina-1(5,3)-pyrazolo[1,5-a]pyrimidina-3(3,2)-pyridinacy- clooctaphan]-8′-one (10). Column: CHIRALPAK IG-3 (IG30CD- WE016), 0.46 cm ID × 15 cm L; mobile phase: MeOH/DCM = 60/
40 (V/V); ﬂow rate: 1.0 mL/min; wavelength: UV 220 nm; Temperature: 35 °C; HPLC equipment: Shimadzu LC-20AT CP- HPLC-06. LCMS [M + 1] 411.1; 1H NMR (400 MHz, CDCl3): δ ppm 8.98 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.26 (s, 1H), 7.89 (d, J = 2.4 Hz, 1H), 7.77−7.75 (m, 1H), 6.36 (d, J = 7.6 Hz, 1H), 6.15 (s,
1H), 5.07 (d, J = 10.8 Hz, 1H), 4.39 (d, J = 9.6 Hz, 1H), 4.12−3.88
(m, 4H), 3.76 (d, J = 10.0 Hz, 1H), 3.60 (d, J = 10.8 Hz, 1H), 2.27−
2.22 (m, 1H), 1.07−0.94 (m, 2H), 0.84−0.79 (m, 1H). 13C NMR (100 MHz, CDCl3): δ ppm 163.47, 157.17, 156.25, 155.36, 146.03,
145.60, 138.58, 132.71, 125.85, 125.06, 103.55, 96.29, 75.25, 70.91,
66.09, 46.33, 42.40, 33.55, 11.91, 8.29. HRMS for C20H20FN6O3 m/z:
[M +H]+ calcd, 411.15754; found, 411.15746. HPLC 99.64%, chiral
HPLC 98.84%.

Computational Modeling Methods. All molecules were prepared for docking simulation using Discovery Studio 2018 to consider proper protonation states and tautomers. The docking simulation was performed with CDOCK (Discovery Studio 2018) using protein models based on X-ray structures (PDB ID: 4YNE) downloaded from RCSB PDB (http://www.rcsb.org/).

Permeability Experiment. A permeability experiment was conducted with the Caco-2 cell model. Caco-2 cells were seeded on 96-well transport inserts and cultured for 28 days before being used for the experiment. The apical-to-basolateral (A to B) transport of the test compound was measured by adding a transport buﬀer solution (HBSS, 25 mM HEPES, pH 7.4) containing the test compound (or rosuvastatin or propranolol as the control) to the apical and transport buﬀer solution (HBSS, 25 mM HEPES, pH 7.4) to basolateral. The basolateral-to-apical (B to A) transport of the test compound was measured by adding transport buﬀer solution (HBSS, 25 mM HEPES, pH 7.4) containing the test compound to the basolateral and transport buﬀer solution (HBSS, 25 mM HEPES, pH 7.4) to apical. After incubating at 37 °C for 2 h, the cell plates were taken out and samples on both sides were obtained. The concentrations of the test compound were determined by LC−MS/MS.
According to theconcentrations of the substrate at the donor compartment and the
receiver compartment, the apparent permeability of substrate transport from the apical to the basolateral and from the basolateral to the apical in the monolayer were calculated, and the eﬄux ratio was calculated.

Metabolism Stability in Microsomes. The test compound was incubated in liver microsomes at a ﬁnal concentration of 1 μM, and the protein concentration was 0.5 mg mL−1. The reactions were terminated at serial time points after initiation of the reaction. The test compound was analyzed using an optimized LC−MS/MS method. The percentages of the remaining test compound were measured and the in vitro intrinsic clearance was calculated accordingly. Hepatic intrinsic clearance (CLint, in vivo) was evaluated based on the in vitro intrinsic clearance.
Plasma Protein Binding Assay. Plasma protein binding studies were performed using a 96-well HTDialysis equilibrium dialysis chamber apparatus. Spiked plasma and phosphate buﬀer were added to the donor side and the receiver side, respectively. The plate was covered with an adhesive sealing ﬁlm and placed in a water bath at 37°C with shaking. After a certain time, the samples in the donor side and the receiver side were taken out and analyzed using an optimized LC−MS/MS method. The values of unbound fraction (fu) were calculated.
CYP Isoenzyme Inhibition Experiment. Serial concentrations of the test compound were incubated with each probe substrate (phenacetin for CYP1A2, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, dextromethorphan for CYP2D6, and midazolam and testosterone for CYP3A4) in pooled human liver microsomes.

The reaction was initiated by the addition of the coenzyme NADPH. The reaction was stopped by adding the ice-cold acetonitrile-containing internal standard to the incubation system. The generation amounts of probe drug metabolites (acetaminophen, OH-diclofenac, OH-S- mephenytoin, dextrorphan, OH-midazolam, and 6-OH-testosterone) at diﬀerent test compound concentrations were measured by the LC−
MS/MS method to calculate the remaining activity percentage of liver
microsomal enzymes, and ﬁnally the IC50 values of test compounds for the cytochrome P450 (CYP) isoenzyme were obtained using Graphpad Prism 6.02 software. Selective inhibitors (α-naphthoﬂavone for CYP1A2, sulfaphenazole for CYP2C9, nootkatone for CYP2C19, quinidine for CYP2D6, and ketoconazole for CYP3A) were used as the positive controls to validate the incubation system.
Kinase Assay. Compound 10 was tested against each of the selected kinases using Euroﬁns standard KinaseProﬁler assays. The compounds supplied were prepared to a working stock of 50× ﬁnal assay concentration in 100% DMSO. The required volume of the 50× stock of the test compound was added to the assay well before a reaction mix containing the enzyme and the substrate was added. The reaction was initiated by the addition of ATP at the selected concentration. Data were handled using custom-built in-house analysis software. The results were expressed as kinase activity remaining, as a percentage of the DMSO control, which were calculated using the following formula (mean of sample counts − mean of blank counts)/mean of control counts.

In Vitro PD Assay. The indicated kinases (TrkA, TrkAG595R, TrkAG667C, TrkC, ALK, or Ros1) were delivered into the substrate solution and gently mixed. Then, the compounds in DMSO were delivered to the kinase reaction mixture utilizing the acoustic technology (Echo 550). 33P-ATP (speciﬁc activity, 0.01 μCi/μL ﬁnal) was added to the reaction mixture to initiate the reaction. The kinase reaction was incubated for 120 min at room temperature. Reactions were spotted onto P81 ion-exchange paper (Whatman # 3698-915). The ﬁlters were washed extensively in 0.75% phosphoric acid. The radioactive phosphorylated substrate remaining on the ﬁlter paper was measured. Kinase activity data were expressed as the percent remaining kinase activity in the test samples compared to vehicle (dimethyl sulfoxide) reactions. IC50 values and curve ﬁts were obtained using GraphPad Prism 5.

The cell viability was evaluated using the cytotoxicity assay, as
described previously.17 Brieﬂy, cells were plated at 5000 per well into 96-well plates and then incubated with compounds 8−10 at the desired concentrations. The MTT solution (5 mg/mL) was added to each well and incubated continuously for 4 h. DMSO was added to dissolve the MTT formazan product, and the absorbance was measured at 570 nm using a Molecular Devices SpectraMax M5
(Molecular Devices, USA). IC50 values were calculated using GraphPad Prism 5.

In Vivo PD Study. Animals were provided free access to food and water in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23). All animal protocols were approved by the Ethics Committee of Yantai University (no. 011 in 2020 for Animal Ethics Approval). All surgeries were performed under anesthesia, and all eﬀorts were made to minimize suﬀering. Twelve Sprague−Dawley rats (six males and six females) were divided into two groups (three males and three females) randomly. Animals in group A were administered with the test compound by single intravenous administration at 2 mg/kg. Animals in group B were administered with the test compound by single oral administration at 10 mg/kg. Blood samples were collected
from the jugular vein cannula at a pre-dose, and 0.083, 0.25, 0.5, 1, 2,
4, 6, 8, 12, and 24 h after ﬁnishing the dose for group A and collected
at pre-dose and 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after ﬁnishing the dose for group B. The concentration of the test compound was analyzed using an optimized LC−MS/MS method. The pharmaco- kinetic (PK) parameters were calculated using a non-compartmental model of WinNonlin version 8.0 (Pharsight, St. Louis, USA).

■Xenograft models (eight animals/group) were established following a subcutaneous injection of 3 × 106 Ba/F3 LMNA-NTRK1cells, Ba/ F3 ETV6-NTRK3 cells, Ba/F3 ETV6-NTRK3-G623R cells, or Ba/F3 LMNA-NTRK1-G595R cells into the right ﬂank of each animal. When tumors in all animals ranged from 100 to 200 mm3 in size, compound 10 was administered orally once daily for 11 days at doses of 5, 10, or 20 mg/kg, and LOXO-195 was administered at 100 mg/ kg, respectively. Tumor dimensions and body weights were recorded twice weekly starting from the ﬁrst day of treatment, and tumor volumes were calculated using the formula (l × (w)2)/2, where l was the longest dimension of the tumor and w was the width, respectively.

ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00712.

Crystallographic data of compounds (CSV)
1H NMR, 13C NMR, COSY, HSQC, HMBC, 19F NMR,
HRMS, and single-crystal X-ray diﬀraction experiment report of compound 10 (PDF)
Predicted binding mode of LOXO-195 and TPX-0005 binding to the active site of TrkC (PDF)

⦁ AUTHOR INFORMATION
Corresponding Authors
ImageHongbo Wang − School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China; orcid.org/0000-0001-5055-4261;
Phone: 86-535-6706060; Email: hongbowangyt@ gmail.com
Jingwei Tian − School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai
University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China; Email: [email protected]

Authors
Zongliang Liu − School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugsin Universities of Shandong, Yantai University, Yantai 264005, China
Pengfei Yu − Department of Clinical Medicine, Binzhou Medical College, Yantai 256603, China
Lin Dong − Luye Pharma Group, Yantai 264005, China Wenyan Wang − School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai
University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
Sijin Duan − Luye Pharma Group, Yantai 264005, China Bingsi Wang − Luye Pharma Group, Yantai 264005, China Xiaoyan Gong − Luye Pharma Group, Yantai 264005, China Liang Ye − Department of Clinical Medicine, Binzhou Medical
College, Yantai 256603, China
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.1c00712

Author Contributions
Z.L. and P.Y. contributed equally. H.W. and J.T. designed the research. Z.L., P.Y., L.D., W.W., S.D., B.W., X.G., and L.Y. performed the experiments. H.W., J.T., and L.Y. performed data analyses. H.W. and J.T. wrote the paper.

Funding
This work was partially supported by grants from the National Natural Science Foundation of China (82073888), the Science and Technology Support Program for Youth Innovation in Universities of Shandong (2019KJM009 and 2020KJM003), The Medical and Health Science and Technology Develop- ment plan project of Shandong (2017WS691), the Top Talents Program for One Case Discussion of Shandong Province, and the Taishan Scholar Project.

Notes
The authors declare no competing ﬁnancial interest.
⦁ ABBREVIATIONS
ATP, adenosine triphosphate; Boc, tert-butyloxycarbonyl;
CYP, cytochrome P450; ER, eﬄux ratio; F, oral bioavailability; hERG, human ether-a-go-go-related gene; IC50, half-maximum inhibitory concentration; MW, molecular weight; PDB, Protein Data Bank; PK, pharmacokinetics; T1/2, half-life; Trk, tropomyosin-related kinase; Vd, volume of distribution at steady state

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