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

Article abstract of DOI:10.1021/acs.jmedchem.1c00712

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 ent

Full text of DOI:10.1021/acs.jmedchem.1c00712

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Article
Discovery of the Next-Generation Pan-TRK Kinase Inhibitors for the
Treatment of Cancer
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
Figure 1. Trk inhibitors approved and in clinical trials.
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
Received: April 19, 2021
Published: July 13, 2021
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-
https://doi.org/10.1021/acs.jmedchem.1c00712
© 2021 American Chemical Society
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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-containing
KM12, CUTO-3, and MO-91 cell lines (IC50 ≤ 5 nM).11 TPX-
0005 is a multi-kinase inhibitor which shows potent effects
against 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-0005
demonstrated 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 physiochemical
properties 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
identical (Figure 2). The nitrogen atom on the pyrazole ring
can form a hydrogen bond with Met592 in the hinge area. The
fluorophenyl/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 same
Figure 2. Docking of LOXO-195 and TPX-0005 with TrkA.
hydrophobic 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.
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Figure 3. 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
enzymatic
cellular
TrkA
6.7 0.5
6.2 0.5
110.0 10.1
0.5 0.1
274.0 16.4
1.15 0.07
4.9 0.3
2.5 0.4
1.4 0.2
8.7 0.8
0.01 0.00
2.3 0.4
0.17 0.05
1.8 0.3
8.0 0.6
0.7 0.2
3.2 0.6
2.1 0.4
0.7 0.1
0.2 0.0
18.5 0.7
0.2 0.1
0.2 0.0
0.4 0.0
4.9 0.4
9.4 1.0
1.7 0.3
0.2 0.1
88.9 12.5
1.2 0.3
0.6 0.1
2.4 0.3
3.5 0.5
2.3 0.3
0.2 0.1
182.0 14.2
1.0 0.3
0.6 0.1
7.7 0.5
4.9 0.2
0.8 0.1
2.7 0.2
2.3 0.3
TrkAG595R
TrkAG667C
TrkC
ALK
Ros1
Ba/F3 TrkA
Ba/F3 TrkAG595R
Ba/F3 TrkAF589L
Ba/F3 TrkAG667A
Ba/F3 TrkCG623R
Ba/F3 SLC34A2-Ros1
6.8 0.7
25.4 4.5
24.4 2.5
2.7 0.5
4.8 0.4
2.2 0.4
5.8 0.3
12.6 0.7
36.7 3.6
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 sulfonamide
group (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.
compounds. 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
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 such
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Table 2. PK Data of Compounds 8, 9, and 10 In Vitro
LOXO-195
TPX-0005
8
9
10
cLog P
LLE
1.86
6.3
8.4/4.1
2.96
5.6
0.9/45.3
65.8
1.97
6.5
16.3/1.4
0.57
7.7
3.9/9.5
0.58
8.0
12.0/2.8
192.9
a
PappA→B (10−6 cm/s)/ER
b
KS (μM)
MMS (mL/min/kg)
76/92/1351/38
29/49/982/21/
242/980/1010/268
2/2/2/3
43/29/342/74
36/30/28/34
17.7/28.8/1504/<10.7
c
H/R/M/D
PPB (Fu %) H/R/M/D
34/3/2/20
5/6/5/9
16/12/11/21
CYP (IC50, μM):
1A2/2C9/2C19/2D6/3A4
>50/>50/>50/>50/>50
>50/6.4/12.3/28.5/>50
>100/18.1/89.0/>100/>50
hERG (IC50, μM)
6.7
13
a
b
c
LLE, ligand lipophilicity efficiency. KS, kinetic solubility. H/R/M/D, human/rat/mouse/dog.
Figure 4. 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).
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.
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
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 were
7.7 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
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undergo efficacy and pharmacokinetic evaluation in vivo
(Figure 4).
Kinase Assay Data of Compound 10. Kinase assays
of repeated administration toxicity study in Cynomolgus
monkeys, 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 10-7
did 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.
(Table 3) showed that compound 10 could inhibit the
Table 3. Kinase Assay Data of Compound 10
activity
activity
kinases
ALK
A-Raf
Blk
BTK(R28H)
B-Raf
(% control)
kinases
(% control)
26
103
46
74
111
87
100
101
96
109
105
103
108
91
Met(M1268T)
Met(Y1248C)
Met(Y1248D)
Met(Y1248H)
PDGFRα
PDGFRα(D842V)
PDGFRα(V561D)
Ret(h)
86
85
96
89
106
102
103
64
59
54
102
93
112
16
33
55
B-Raf(V599E)
cKit
cKit(D816V)
cKit(D816H)
cKit(V560G)
cKit(V654A)
c-RAF
Ret(V804L)
Ret(V804M)
RIPK1
RIPK2
EGFR
Ron
Ros1
EGFR(L858R)
EGFR(L861Q)
EGFR(T790M)
FAK
95
105
72
Src(1−530)
Src(T341M)
TrkA
2
FGFR1
FGFR2
FGFR3
92
93
96
TrkB
TrkC
JAK1
−1
−3
91
FGFR4
Met
114
98
JAK2
JAK3
44
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.
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).
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 in
female 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) at
all 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 weeks
EXPERIMENTAL SECTION
General. The recorded H nuclear magnetic resonance (NMR)
■
1
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
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Figure 5. 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)
C0 (nM)
T1/2 (h)
3
3
2 (M)
2 (F)
23,779 1526
1.2 0.1
0.5 0.1
11.8 0.8
11,146 656
10
12,700 1121
0.4 0.0
12,870 831
2.8 0.1
1.1 0.2
9.4 0.5
15,002 859
10
16,900 1173
0.8 0.0
59,724 1434
4118 833
0.872 0.0156
1.39 0.229
19.3 0.624
4191 139
10 (M)
6384 1525
0.667 0.289
11,720 1749
56.0 8.4
3509 719
2.21 0.215
1.46 0.224
8.19 1.96
10,282 2298
10 (F)
6628 626
0.667 0.289
31,676 6628
61.9 13.0
Vd (L/kg)
Cl (mL/kg/min)
AUC0−t (nM*h)
dose (mpk)
Cmax (nM)
Tmax (h)
AUC0−t (nM*h)
F %
27,344 1590
74
3
119
4
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%).
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a
Scheme 1. Synthetic Route for Compound 10
a
Reagents 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%;
(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.
(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%).
1
LCMS [M + 1] 311.1; H 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
Figure 6. ORTEP of 10.
concentrated in vacuo to obtain compound 10-6 as a white solid (27.8
1
g, 89.1 mmol, yield 91.95%). LCMS [M + 1] 313.2; H 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).
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), potassium
phosphate (68.57 g, 323.04 mmol, 2 equiv), and compound 10-4
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1
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
(72 g). LCMS [M + 1] 429.1; H 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-
1
g, 271 mmol, yield 98.33%). LCMS [M + 1] 213.1; H 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).
1
14 (22 g). LCMS [M + 1] 411.1; H 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-
1
HPLC-06. LCMS [M + 1] 411.1; H 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 the
concentrations 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.
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
1] 360.9; H 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
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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.
(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
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00712.
■
sı
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.
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
■
In Vitro PD Assay. The indicated kinases (TrkA, TrkAG595R
,
Hongbo 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
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.
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: tianjingwei@luye.cn
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
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 Drugs
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Article
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Ercan, D.; Mahale, S.; Davies, K. D.; Aisner, D. L.; Pilling, A. B.;
Berge, E. M.; Kim, J.; Sasaki, H.; Park, S.-i.; Kryukov, G.; Garraway, L.
A.; Hammerman, P. S.; Haas, J.; Andrews, S. W.; Lipson, D.;
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(8) Ardini, E.; Bosotti, R.; Borgia, A. L.; De Ponti, C.; Somaschini,
A.; Cammarota, R.; Amboldi, N.; Raddrizzani, L.; Milani, A.;
Magnaghi, P.; Ballinari, D.; Casero, D.; Gasparri, F.; Banfi, P.;
Avanzi, N.; Saccardo, M. B.; Alzani, R.; Bandiera, T.; Felder, E.;
Donati, D.; Pesenti, E.; Sartore-Bianchi, A.; Gambacorta, M.; Pierotti,
M. A.; Siena, S.; Veronese, S.; Galvani, A.; Isacchi, A. The TPM3-
NTRK1 rearrangement is a recurring event in colorectal carcinoma
and is associated with tumor sensitivity to TRKA kinase inhibition.
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in 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.
(9) Ardini, E.; Menichincheri, M.; Banfi, P.; Bosotti, R.; De Ponti,
C.; Pulci, R.; Ballinari, D.; Ciomei, M.; Texido, G.; Degrassi, A.;
Avanzi, N.; Amboldi, N.; Saccardo, M. B.; Casero, D.; Orsini, P.;
Bandiera, T.; Mologni, L.; Anderson, D.; Wei, G.; Harris, J.; Vernier,
J.-M.; Li, G.; Felder, E.; Donati, D.; Isacchi, A.; Pesenti, E.; Magnaghi,
P.; Galvani, A. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor
with Activity in Multiple Molecularly Defined Cancer Indications.
Mol. Cancer Ther. 2016, 15, 628−639.
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.
(10) Berger, S.; Martens, U. M.; Bochum, S. Bochum, S.
Larotrectinib (LOXO-101). Recent Results Cancer Res. 2018, 211,
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B.; Ebata, K.; Smith, S.; Lauriault, V.; Kolakowski, G. R.; Brandhuber,
B. J.; Larsen, P. D.; Bouhana, K. S.; Winski, S. L.; Hamor, R.; Wu, W.-
I.; Parker, A.; Morales, T. H.; Sullivan, F. X.; DeWolf, W. E.;
Wollenberg, L. A.; Gordon, P. R.; Douglas-Lindsay, D. N.; Scaltriti,
M.; Benayed, R.; Raj, S.; Hanusch, B.; Schram, A. M.; Jonsson, P.;
Berger, M. F.; Hechtman, J. F.; Taylor, B. S.; Andrews, S.;
Rothenberg, S. M.; Hyman, D. M. A Next-Generation TRK Kinase
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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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