Acs Jmedchem 6b00064

Article
pubs.acs.org/jmc
Discovery of Entrectinib: A New 3‑Aminoindazole As a Potent

Anaplastic Lymphoma Kinase (ALK), c‑ros Oncogene 1 Kinase (ROS1),
and Pan-Tropomyosin Receptor Kinases (Pan-TRKs) inhibitor
Maria Menichincheri,*,† Elena Ardini,† Paola Magnaghi,† Nilla Avanzi,† Patrizia Banfi,† Roberto Bossi,†,§
Laura Buffa,† Giulia Canevari,† Lucio Ceriani,† Maristella Colombo,† Luca Corti,† Daniele Donati,†
Marina Fasolini,† Eduard Felder,† Claudio Fiorelli,†,∥ Francesco Fiorentini,‡ Arturo Galvani,†
Antonella Isacchi,† Andrea Lombardi Borgia,† Chiara Marchionni,† Marcella Nesi,† Christian Orrenius,†
Achille Panzeri,† Enrico Pesenti,‡ Luisa Rusconi,† Maria Beatrice Saccardo,† Ermes Vanotti,†
Ettore Perrone,† and Paolo Orsini†
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Oncology, Nerviano Medical Sciences Srl, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
‡
Accelera Srl, Viale Pasteur 10, 20014 Nerviano, Milan, Italy
*
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S Supporting Information
ABSTRACT: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different
tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the
emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery
program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its
optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines,
efficiently penetrant the blood−brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models.
Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found
constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of
patients affected by ALK-, ROS1-, and TRK-positive tumors.
■ INTRODUCTION
Oncogenic forms of protein kinases have been described in
sequently, additional ectopically expressed, activated fusion
forms of ALK have been identified and recognized as potent
different human tumor types and acting as drivers in specific oncogenic drivers in other tumor types such as inflammatory
genetic contexts define subsets of patients that are highly myofibroblastic tumors (IMT)6 and, most importantly, in a
responsive to kinase inhibitors such as imatinib in BCR-ABL subset of NSCLC patients where they account for nearly 3−7%
positive chronic myelogenous leukemia (CML) and erlotinib in of cases.7,8 The majority of ALK-positive NSCLC harbors the
epidermal growth factor receptor (EGFR)-mutated nonsmall same intrachromosomal rearrangement that results in the
cell lung cancer (NSCLC).1,2 The anaplastic lymphoma kinase expression of EML4−ALK chimeric protein, generated from
(ALK), a receptor tyrosine kinase (RTK) belonging to the the fusion of the N-terminal portion of echinoderm micro-
insulin receptor superfamily, is a well validated target based on tubule-associated protein-like-4 (EML4) and the ALK kinase
preclinical and clinical studies.3,4 domain. ALK fusion partners invariably provide a protein/
A constitutively activated, oncogenic form of ALK has been protein interaction domain that induces ALK oligomerization
first found in a subset of anaplastic large cell lymphoma and leads to constitutive activation of the tyrosine kinase.7
(ALCL), namely the chimeric protein NPM-ALK, resulting Other tumorigenic forms of the ALK kinase are due to the
from a chromosomal translocation, which causes the fusion of
the N-terminal portion of the normally expressed protein Received: January 14, 2016
nucleophosmin (NPM) to the ALK kinase domain.5 Sub- Published: March 22, 2016
© 2016 American Chemical Society 3392 DOI: 10.1021/acs.jmedchem.6b00064

J. Med. Chem. 2016, 59, 3392−3408
Journal of Medicinal Chemistry Article
Figure 1. Structures of the most advanced ALK inhibitors.
acquisition of single point mutations that lead to the expression investigation (Figure 2). This compound showed a good
of activated forms of the full-length receptor, as reported for biochemical potency against the ALK kinase (IC50 = 0.073 μM)
example in both familial and sporadic neuroblastoma.9−12
With ALK being a well validated target in oncology for many
years, several ALK inhibitors have been identified and entered
clinical trials such as crizotinib from Pfizer,13 ceritinib from
Novartis,14 alectinib from Chugai/Roche,15 and AP26113 from
Ariad16 (Figure 1). All these compounds demonstrated striking
clinical efficacy in ALK-positive selected patient population
already during the initial phases of clinical development, and
the most advanced ones have reached the registration for ALK-
dependent cancers. First, crizotinib has been approved in 2011
for the treatment of ALK-positive NSCLC patients, followed by
ceritinib and alectinib. Ceritinib has been approved in 2014 for
the treatment of ALK-positive, metastatic NSCLC patients
relapsed from or intolerant to crizotinib and alectinib has been
granted accelerated approval in 2015 by the Food and Drug Figure 2. Structure of the starting hit compound 1.
Administration (FDA) for the treatment of ALK-positive
NSCLC patients who progressed after crizotinib treatment. and an appreciable antiproliferative activity on the ALK-
However, despite the initial clinical benefit demonstrated in dependent ALCL Karpas-299 cell line (IC50 = 0.253 μM).
specific clinical settings with ALK inhibitors, relapse was The chemical structure of 1 was considered amenable to
invariably observed in all treated patients. On the basis of structural modifications, which we believed to be potentially
clinical experience accumulated to date with crizotinib, it clearly suitable for improvement of its biological activity. Among the
emerged that low brain penetration represents a major available options, we focused our attention on the substitution
weakness of this drug and appearance of brain metastases is a pattern of the fluorinated phenyl ring B, the introduction of
demonstrated cause of disease recurrence in crizotinib-treated substituents on ring A, and the modification of the N-
patients.17 In addition, the emergence of mutations in the ALK methylpiperazinyl solubilizing group at the para position of ring
kinase domain that confer resistance to the drug was reported A.
for the first as well as the second generation ALK Here we describe the optimization process of compound 1
inhibitors.18−20 toward our final candidate compound 2 (entrectinib, Figure 3),
Because of the relevant role of the ALK oncogene in different a potent ALK inhibitor characterized by good oral absorption in
tumor settings, we performed a high-throughput screening rodent and nonrodent animal species and good efficacy in in
(HTS) of our Corporate Compound Collection with the aim to vivo animal tumor models. Most importantly, compound 2
identify suitable hits for further optimization as ALK kinase turned out to be a highly potent inhibitor of the closely related
inhibitors. Among different hits, the 3-amino-5-substituted RTKs ROS1 and TRKs that were recently found rearranged in
indazole 1 emerged as a promising starting point for further different tumor types. The expression of oncogenic fusion
3393 DOI: 10.1021/acs.jmedchem.6b00064
J. Med. Chem. 2016, 59, 3392−3408
Synthesis of 3-acylamino-5-benzyl indazoles is outlined in

Scheme 2 and Scheme 3. Activation of properly protected
carboxylic acids III with oxalyl chloride, followed by reaction
with intermediates I or II at low temperature, gave
regioselective acylation at the 3-amino group of the scaffold.32
Removal of the trifluoroacetamido protecting group provided
the desired target molecules 2, 3, 5−10, 12−16, 18, 20−25,
and 28−31. Amino group in compound 4 was obtained by
reduction of the corresponding nitro derivative 3. Compound
11 required an additional deprotection step to remove the
silyloxy protecting group by using TBAF in THF. Compounds
17 and 19 were obtained by hydrolysis of the benzoic group
Figure 3. Modification of the starting hit 1 toward the final candidate under standard basic conditions (LiOH, water, methanol).
2. Enantiomers 26 and 27 were separated by preparative chiral
HPLC from racemic derivative 25, but the absolute
configuration of the stereocenter has not been evaluated.
proteins containing constitutively activated ROS1 kinase
domain have been identified in a subset of NSCLC patients
(1−2% of cases).21 Analogously chromosomal rearrangements
■ RESULTS AND DISCUSSION
Structure Based Design. To design new ALK inhibitors
involving the neurotrophic tyrosine receptor kinase 1 gene starting from compound 1, we took advantage of the in-house
(NTRK1), which encodes for the high affinity nerve growth available structure of the ALK kinase in complex with the weak
factor (NGF) receptor tropomyosin receptor kinase A prototype ALK inhibitor PHA-E42933 (see Figure 4, right
(TRKA), have been found in around 12% of papillary thyroid panel).
carcinoma22 and very recently reported in a subset of NSCLC23 Analysis of this structure suggests that appropriately mono
and colorectal carcinoma (CRC) patients24 as well as in substituted A ring at position 2′ (Figure 4, left panel) would
additional tumor types (Spitz tumors,25 intrahepatic cholangio- yield derivatives occupying an area in and around the adenosine
carcinoma,26 glioblastoma,27 and pediatric high grade glio- triphosphate (ATP) sugar pocket while displacing the observed
ma28). Although the frequency of such events in these water molecule W1 (Figure 4, right panel). In particular, NH-R
neoplastic diseases remains to be defined due to the low substituents at that position of ring A were predicted to achieve
number of patients screened so far, these findings support the this result while stabilizing the bioactive conformation through
rationale for targeted therapy with TRKA inhibitors in selected an intramolecular hydrogen bond involving the hydrogen of the
patient populations. Thus, the activity of compound 2 on these ortho amino group and the carbonyl of the adjacent
kinases represents a major opportunity for clinical development carboxamido group (Figure 4, left panel).
of the compound in additional ROS1- and TRK-dependent As far as the nature of the group R is concerned, a combined
clinical settings. On the basis of these findings, together with medicinal chemistry and docking strategy using sugar-
the preclinical data in different models, compound 2 is resembling substituents led to the prioritization of saturated
currently undergoing phase I/II clinical trials for the treatment aliphatic rings containing nitrogen, oxygen, or hydroxyl groups.
of patients affected by ALK-, ROS1-, and TRK-positive tumors Such cyclic R substituents were expected to rotate with respect
where it has already shown remarkable activity.29,30 to the plane of the hinge binder optimally filling the available
■ CHEMISTRY
Target compounds were obtained by coupling either scaffold I
space, thus making contact interactions with both the catalytic
loop “floor” and the Gly-rich loop “ceiling” of the ATP-binding
site. Branched aliphatic chains were also considered but were
or II (Scheme 1) with benzoic acid derivatives of general expected to be less promising due to their flexibility. This
formula III (Scheme 2 and Scheme 3). Key step in the approach produced a subset of new compounds driving both
synthesis of intermediates I and II is the Suzuki−Miyaura cross- biochemical and cellular potencies in the desired direction (see
coupling reaction between different substituted benzyl Table 1 and further discussion). In particular, compound 2
bromides and commercially available 3-cyano-4-fluorophenyl- emerged as one of the most interesting compounds in terms of
boronic acid in the presence of Pd(Ph3P)4 complex as a catalyst biochemical potency, with the tetrahydropyranyl ring at
and K3PO4 as a base.31 position 2′ of phenyl ring A displaying an optimal fitting
Methyl group in scaffold II has been introduced by reaction within the ATP sugar pocket.
of the corresponding diarylmethane with methyl iodide and The 2.2 Å structure of ALK in complex with compound 2
LiHMDS. (Figure 5) was indeed in agreement with the above
Scheme 1. Synthesis of 5-Benzyl-indazoles I and IIa
a
Conditions: (a) Pd(PPh3)4, K3PO4, toluene, 100 °C; (b) NH2NH2.H2O, n-BuOH, 120 °C; (c) CH3I, LiHMDS, THF then NH2NH2.H2O, n-
BuOH, 120 °C.

J. Med. Chem. 2016, 59, 3392−3408
Scheme 2. Synthesis of Benzoyl-Modified 3-Acylamino-5-benzyl Indazoles 2−24a
a
Conditions: (a) (COCl)2, DCM, DMF; (b) DIPEA, THF, −20 °C; (c) MeOH, TEA; (d) cyclohexene. Pd/C, dioxane; (e) TBAF, THF;( f) LiOH·
H2O, MeOH, water.
Scheme 3. Synthesis of Benzyl-Modified 3-Acylamino-5-benzyl Indazoles 25−31a
a
Conditions: (a) (COCl)2, DCM, DMF; (b) DIPEA, THF, −20 °C; (c) MeOH, TEA; (d) chiral HPLC.
expectations based on the modeling prediction. The compound core of the compound also makes favorable contacts with
is anchored to the hinge region via the canonical donor− Leu1256, Leu1122, Val1130, and the gatekeeper Leu1196. The
acceptor−donor hydrogen bonding motif between the nitro- glycine-rich loop adopts a peculiar “collapsed” conformation,
gens on the aminoindazole moiety and the backbone of which was not previously reported in publicly available ALK
residues Glu1197 and Met1199. The difluorobenzyl-indazole structures, so that the 3,5-difluorobenzyl moiety is favorably
J. Med. Chem. 2016, 59, 3392−3408
Figure 4. (left) Predicted H-bond interaction suitable to stabilize the bioactive conformation of this chemical template. (right) ATP-binding site
occupied by PHA-E429 (PDB ID 2XBA) with the observed water molecule W1 (red sphere).
stacked between Leu1256 and Phe1127 from the glycine-rich is “decorated” with a hydroxyl group as in 17, good biochemical
loop. Moreover, the fluorine atom pointing toward the interior and cellular activity are restored. In particular, the trans
of the pocket is involved in multipolar interactions with the diastereoisomer 17 is better than the cis one 19 in both
backbone carbonyls of Gly1269 (DFG-1 residue) and Asn1254. enzymatic and cellular assays. The crystal structure of 17 (see
The phenyl ring A makes a hydrophobic contact with the Supporting Information, Figure S1) revealed a hydrogen bond
glycine-rich loop residue Leu1122, while the methylpiperazine interaction mediated by a water molecule of the hydroxyl group
moiety protrudes into the solvent. As expected, the partially with a carbonyl of His1124 in the glycine-rich loop, thus
solvent-exposed tetrahydropyranyl moiety adopts a roughly potentially explaining the observed activity of this compound.
orthogonal orientation with respect to the scaffold, in order to When R3 is a 4-amino-N-methylpiperidinyl or a 4-amino-
optimally fill the space underneath the hydrophobic glycine-rich tetrahydropyranyl ring, compounds 20 and 2 display a very
loop. good biochemical potency on ALK with IC50 values of 0.015
SAR Study. We started the optimization study of compound and 0.012 μM, respectively. However, the former showed weak
1 by varying the substitution pattern on ring A. This cellular activity, which might be due to low cell permeability, as
investigation was mainly focused on R3 substituent (see observed in a Caco-2 permeability experiment (Papp A−B = 1.5
Table 1) at position 2′, according to the evaluation deriving × 10−6 cm/s).
from the structural and computational studies previously Structure−activity relationship (SAR) Tables 2, 3, and 4
discussed. In Table 1, the biochemical IC50 values (μM) for show results where the 4-aminotetrahydropyranyl ring was
ALK and for the most critical kinases among the hit ones, i.e., maintained at position 2′ of ring A, and the other regions of the
IR (insulin receptor) and its closest family member IGF1R structure were modified.
(insulin-like growth factor 1 receptor), are reported along with As reported in Table 2, the introduction of a methyl onto
the antiproliferative activity on the ALK-dependent ALCL linker L afforded a racemic mixture, which upon chiral column
Karpas-299 cell line (IC50 evaluated after 72 h treatment).
chromatography separation yielded enantiomers 26 and 27
Generally, a nearly 10-fold or greater selectivity versus IR and
whose absolute stereochemistry has not been assigned. The two
IGF1R is observed for all the compounds. In the toxicological
compounds have a moderately different activity on ALK with
studies performed later on our candidate compound 2, this
27 showing the best potency. Compared to compound 2, it
selectivity ratio was considered safe based on the experimental
determination of glucose and insulin levels. displays a stronger activity in cells. However, the labor-intensive
The introduction of an unsubstituted amino group at R3 led chiral separation and the labile nature of the benzhydryl
to compound 4, which displays an unchanged potency respect stereogenic center discouraged any further development of this
to 1. This was in line with modeling predictions as compound 4 compound.
lacks the crucial substituent suitable to fill the sugar pocket. The exploration of ring B confirmed the contribution of the
R3 aminoaliphatic chains substituted with a methoxy group fluorine interaction with the protein observed in the crystal
generally led to a moderate loss in ALK activity (see structure. As depicted in Table 3, removal of both fluorines
compounds 5, 7, and 8). At best, activity was conserved (see caused a nearly 10-fold decrease of biochemical activity on ALK
compound 6) without any improvement with the exception for and, correspondingly, of antiproliferative activity in Karpas-299
the good cellular activity of 6 in the double-digit nanomolar cells (see compound 28). The presence of a single fluorine as in
range. compound 29 was sufficient to restore the activity on ALK
Compounds 9 and 11, bearing shorter two carbon aliphatic analogously to the introduction of both halogens with the
chains decorated with a fluorine and a hydroxyl group, different 2,5-regiochemistry (see compound 30). Both
respectively, display a surprisingly good biochemical potency compounds display an activity profile similar to compound 2.
on ALK. Compound 9 also shows good antiproliferative cellular A decrease in the potency was observed when replacing one
activity (IC50 = 0.049 μM). The data related to 12, 13, and 14 fluorine with a methyl group in the 2,5-substituted ring B (see
suggest that they cannot properly accommodate the ortho derivative 31). This substitution is likely to negatively impact
substituent of ring A within the sugar pocket. Compound 15, the geometry of the stacking interaction of phenyl B with the
with an aminocyclohexyl ring in R3, is poorly active, but when it phenylalanine 1127 of the glycine-rich loop.
J. Med. Chem. 2016, 59, 3392−3408
Table 1. SAR: Substitution on Ring A (IC50, μM)a S2 and Figure S2). It ranges as the second best compound after
derivative 22, which, however, is characterized by an
unsatisfactory pharmacokinetic profile as discussed below.
On the basis of the in vitro biological data reported in the
previous SAR tables, we selected a subset of the most
interesting compounds and profiled them for their in vitro
absorption, distribution, metabolism, and excretion (ADME)
properties, with the aim to anticipate potential liabilities in in
vivo studies. The results of this profiling are shown in Table 5,
where medium-throughput solubility data at physiological pH,
compound permeability evaluation in the Caco-2 cell
permeability assay, and intrinsic clearance both in human
liver microsomes (HLM) and in rat hepatocytes are reported.
Compounds 21, 22, 23, and 24 display a moderate to good
solubility with an improvement relative to 2, as expected. The
Caco-2 permeability appears generally low, with compound 21
showing higher B−A Papp compared to A−B, while the
remaining derivatives show quite similar values. As for the
intrinsic clearance in human liver microsomes and in rat
hepatocytes, the data range from low to moderate except for
23, which shows a higher value of intrinsic clearance in rat
hepatocytes. As the in vitro ADME parameters of the overall
compounds were quite similar, we decided to evaluate the
pharmacokinetic profile in mouse for all of them and the
corresponding data are reported in Table 6.
The mice were treated with the compounds by iv and os
administration at the dose of 10 mg/kg. Among them, 9 and 2
clearly emerged as the most interesting derivatives with very
high oral bioavailability with low clearance and good volume of
distribution and half-life values. The remaining compounds
generally display much lower oral bioavailability with low
exposure in most cases. However, compound 9 turned out to
be less potent in following in vivo efficacy experiments, thus we
focused our attention on 2.
Compound 2 Characterization. An in-depth profiling of
compound 2 was undertaken, including its potency on both
kinase and cellular panels.
The compound has been profiled on a highly diverse panel of
56 biochemical kinase assays (kinase selectivity screening,
KSS), and key data are reported in Table 7. We found that
compound 2 has an average IC50 of 12 nM on the ALK kinase.
To better characterize the inhibitor potency on the target, we
then calculated a Ki of 6.2 nM, which is in line with the
calculated IC50. Compound 2 was even more potent on the
kinases ROS1 (IC50 = 0.007 μM) and on TRKA (IC50 = 0.001
μM). Only on two additional kinases, JAK2 and ACK1, the
a
Biochemical IC50 values are reported as the mean (n ≥ 2). bIC50 compound shows a less than 10-fold selectivity. For 12
values as determined in an antiproliferative activity assay (72 h additional kinases, a selectivity ratio equal or greater than 10
treatment) are reported as the mean of 2−3 experiments with a
is observed, while for the other 40 kinases at least a 100-fold
coefficient of variation below 35%.
selectivity is observed.
On the basis of the observed activity on TRKA, the
Finally, we addressed the solubilizing group with variations in biochemical potencies on the related kinases TRKB and
the R4 position as reported in Table 4. All of these compounds TRKC were also evaluated and found to be in the single-digit
compare favorably with derivative 2, in terms of potency on nanomolar range (TRKB IC50 = 0.002 μM and TRKC IC50 =
ALK, of selectivity versus IGF1R and IR and of antiproliferative 0.005 μM).
activity in Karpas-299. Among them, analogue 22 was from 3- Then compound 2 was profiled on a panel of nearly 200
to 5-fold more potent on ALK than the others and more active human tumor cell lines (see Table 8). The antiproliferative
on Karpas-299 cells. All of these compounds showed an activity observed is consistent with the compound kinase
improved aqueous solubility with respect to 2 (see Table 5) selectivity profile, as compound 2 displays an IC50 below 0.1
and appeared worth of further study. μM on different ALK-dependent cancer cells, such as ALCL
In terms of lipophilic ligand efficiency, a significant cell lines Karpas 299, SU-DHL-1, SUP-M2, and SR-786 and
improvement has been observed for compound 2 compared NSCLC cell line NCI-H2228. In addition, it also potently
to the starting compound 1 (see Supporting Information, Table inhibits (IC50 = 0.017 μM) the proliferation of KM12, a human
J. Med. Chem. 2016, 59, 3392−3408
Figure 5. (left) Binding mode of compound 2 (yellow carbon atoms) into the ALK active site (light-blue carbon atoms; PDB 5FTO). The gray
dashed line indicates the intramolecular hydrogen bond, whereas black dashed lines represent hydrogen bonds and multipolar interactions. (right)
Surface representation of the ALK active site showing the conformation adopted by the tetrahydropyranyl ring.
Table 2. SAR: Linker Modification (IC50, μM)a Table 3. SAR: Substitution Pattern on Ring B (IC50, μM)a
compd no. R1 R2 ALKa IGF1Ra IRa Karpas-299b

compd no. L ALKa IGF1Ra IRa Karpas-299b
28 H H 0.106 0.903 1.489 0.182
26c CHCH3 0.059 ND 1.163 0.041
29 3-F H 0.030 0.206 0.427 0.018
27c CHCH3 0.019 0.136 0.189 0.016
30 2-F 5-F 0.038 ND 0.170 0.049
2 CH2 0.012 0.122 0.209 0.031
31 2-CH3 5-F 0.181 ND 0.823 0.851
a
Biochemical C50 values are reported as the mean (n ≥ 2). bIC50 2 3-F 5-F 0.012 0.122 0.209 0.031
values as determined in an antiproliferative activity assay (72 h
a
Biochemical C50 values are reported as the mean (n ≥ 2). bIC50
coefficient of variation below 35%. cAbsolute configuration unknown. values as determined in an antiproliferative activity assay (72 h
coefficient of variation below 35%.
CRC cell line whose growth was recently found to be TRKA-
dependent due to the presence of the oncogenic tropomyosin
3-NTRK1 (TPM3-NTRK1) chromosomal rearrangement,24 KM12, a TRKA-dependent cellular model. KM12 cells have
whereas the antiproliferative activity observed in MV-4-11 is been treated for 2 h with compound 2 at different
presumably due to the cross-reactivity with FLT3. The concentrations, and then the phosphorylation of TRKA and
additional not reported IC50s are comprised between 0.1 and downstream transducers has been evaluated. As reported in
1 μM for 11 cell lines and greater than 1 μM for the remaining Figure 7, a clear inhibition of target and signaling pathway
cell lines. phosphorylation is observed at low compound concentration,
In ALK-dependent models the inhibition of cell growth was supporting the rationale for considering TRKA-positive patients
correlated with the modulation of the activated target in cells. for clinical development of compound 2.
In the ALCL cell line Karpas-299 and in NSCLC cell line NCI- To evaluate and compare the inhibition by compound 2 of
H2228, the ability of the compound to modulate the its main targets in the same cellular environment, the
autophosphorylation of the ALK kinase was assessed after 2 h interleukine-3 (IL-3)-dependent Ba/F3 cell line was transfected
treatment at different concentrations. As reported in Figure 6, a and made dependent for survival on ALK, ROS1, TRKA,
complete inhibition of ALK phosphorylation can be clearly TRKB, and TRKC kinases.34 These IL-3-independent cell lines
appreciated in both cell lines at very low doses. Consistently a were treated with compound 2, which has shown the
dose-dependent inhibition of phosphorylation of the down- antiproliferative activities reported in Table 9.
stream effector STAT3 is also clearly observed. Compound 2 proved to be very potent in inhibiting the
To further explore the cellular activity on TRKA, the growth of ALK-, ROS1-, TRKA-, TRKB-, and TRKC-
modulation of target phosphorylation was also studied on dependent Ba/F3 cell lines with IC50 values in the low
J. Med. Chem. 2016, 59, 3392−3408
Table 4. SAR: Solubilizing Group Variations on Ring A Table 7. Kinase Profile of Compound 2a
(IC50, μM)a
kinase IC50 (μM) kinase IC50 (μM)
TRKA 0.001 IGF1R 0.122
TRKB 0.003 FAK 0.140
TRKC 0.005 FLT3 0.164
ROS1 0.007 BRK 0.195
ALK 0.012 IR 0.209
JAK2 0.040 AUR2 0.215
ACK1 0.070 JAK3 0.349
JAK1 0.112 RET 0.393
a
Biochemical IC50 values are reported as the mean (n ≥ 2). They have
been calculated using the KSS biochemical panel at 2Km ATP
concentration. IC50 > 1 μM were obtained for the following kinases:
FGFR1, VEGFR2, VEGFR3, LCK, KIT, AUR1, ABL, PKCβ, CDK2/
CycA, SYK; IC50 > 10 μM were obtained for: AKT1, CDC7/DBF4,
CHK1, CK2, EEF2K, EGFR1, ERK2, GSK3β, IKK2, MAPKAPK2,
MELK, MET, MPS1, MST4, NEK6, NIM1, P38α, PAK4, PDGFRβ,
PDK1, PERK, PIM1, PKAα, PLK1, SULU1, ZAP70.
Table 8. Antiproliferative Activity of Compound 2a (Cell

Lines with IC50 below 0.1 μM)
cell line cancer type IC50 (μM)a
KM12 adenocarcinoma colon TRKA + 0.017
SU-DHL-1 anaplastic large cell lymphoma ALK + 0.024
KARPAS-299 anaplastic large cell lymphoma ALK + 0.031
a
Biochemical C50 values are reported as the mean (n ≥ 2). bIC50 SUP-M2 anaplastic large cell lymphoma ALK + 0.041
values as determined in an antiproliferative activity assay (72 h NCI-H2228 nonsmall cell lung cancer ALK + 0.068
treatment) are reported as the mean of 2−3 experiments with a SR-786 anaplastic large cell lymphoma ALK + 0.081
coefficient of variation below 35%. MV-4-11 biphenotypic B myelomonocytic leukemia 0.081
a
Table 5. In Vitro ADME Parameters of Selected Derivatives IC50 values as determined in an antiproliferative activity assay (72 h
intrinsic intrinsic coefficient of variation below 35%.
solubility permeability clearance clearance
Caco-2a Papp A−B nanomolar range. As expected, compound 2 was poorly active
compd pH 7 [10−6cm/s] (Papp B− HLMb rat hepatocytes in inhibiting the proliferation of IL-3-dependent Ba/F3 cell line
no. (μM) A) (mL/min/kg) (mL/min/kg)
(IC50 = 2.1 μM).
21 51 2.7 (19.8) 57 42 As the emergence of resistance mutations is frequently
22 103 0.6 (4.5) 7.5 52 responsible for relapse in patients treated with ALK inhibitors,
23 84 1.1 (1.9) 40 229 we tested compound 2 in a series of Ba/F3 cell lines transfected
24 59 0.3 (2) 12 49 with different crizotinib or ceritinib-resistant mutated forms of
9 16 2.4 (1.5) 24 18
EML4-ALK. Entrectinib demonstrated a good antiproliferative
17 7 1.8 (5.1) 25 45
activity on EML4-ALK-wt (Ba/F3-EML4-ALK-wt IC50 = 0.028
2 20 2.4 (3.0) 33 87
a
μM) and on the gate-keeper mutant EML4-ALK-L1196M (Ba/
Caco-2 permeability assay. bHuman liver microsomes. F3-EML4-ALK-L1196M IC50 = 0.067 μM), but it proved to be
poorly active on the EML4-ALK-G1202R mutant (Ba/F3-
EML4-ALK-G1202R IC50 = 0.897 μM), as expected based on
entrectinib mode of binding.
Table 6. In Vivo ADME Parameters of Selected Derivatives (Harlan nu/nu Mice)a

PK data (iv), doseb: 10 mg/kg PK data (per os), doseb: 10 mg/kg
compd CL Fc
no. Cmax (μM) AUC∞ (μM·h) (mL/min/kg) Vss (L/kg) t1/2 (h) Cmax (μM) Tmax (h) AUC∞ (μM·h) t1/2 (h) (%)
21 10.3 ± 0.1 7.86 ± 0.95 38.8 ± 5.06 4.41 ± 1.30 2.83 ± 1.20
22 40.5 ± 8.85 42.2 ± 6.56 7.01 ± 1.08 1.40 ± 0.41 4.76 ± 0.78 0.081 ± 0.072 6 1.33 ± 0.79 9.63 ± 3.92 2.9
23 5.69 ± 0.37 7.97 ± 1.04 32.1 ± 3.75 2.63 ± 0.12 1.52 ± 0.09 0.36 ± 0.13 0.83 ± 0.29 1.51 ± 0.35 2.25 ± 0.43 18
24 6.93 ± 0.92 33.1 ± 0.72 8.16 ± 0.31 2.62 ± 0.40 4.94 ± 0.63 0.80 ± 0.05 6 11.0 ± 0.21 4.08 ± 0.37 29.3
9 7.58 ± 1.19 24.7 ± 2.17 11.7 ± 0.87 4.76 ± 0.82 5.92 ± 0.33 1.88 ± 0.40 4.33 ± 2.89 31.5 ± 3.99 9.86 ± 1.77 99
17 7.06 ± 1.67 13.1 ± 3.32 21.3 ± 4.69 3.02 ± 1.21 3.01 ± 1.32 0.36 ± 0.38 1 1.25 ± 1.01d 7.3
2 4.90 ± 1.35 19.6 ± 1.5 13.5 ± 1.05 3.34 ± 0.60 3.59 ± 0.47 1.33 ± 0.19 2.5 ± 3.04 17.4 ± 0.21 2.94 ± 0.10 77
a
n = 6 animals per study. bDosed in 10% Tween 80/saline iv = intravenous adm.; dosed in 0.5% methocel suspension per os = oral adm.
c
Bioavailability. dDetectable concentration of the compound were measured up to 6 h post dosing.

J. Med. Chem. 2016, 59, 3392−3408
was observed at both doses with complete tumor regression

achieved at the end of treatment at the dose of 60 mg/kg. At
day 90, four out of seven mice treated at the highest dose were
still tumor free and were considered cured. No body weight loss
or other signs of toxicity were observed in treated animals.
The ex vivo target modulation was evaluated by Western blot
analysis after single administration of the compound at the dose
of 60 mg/kg to tumor bearing mice. As shown in Figure 8,
NPM-ALK and STAT3 phosphorylation is completely
inhibited 12 h after compound administration and is still
Figure 6. Mechanism of action of compound 2 in Karpas-299 (A) and
NCI-H2228 (B) cell lines. Cells were treated with the compound at evident at 18 h.
the indicated concentrations for 2 h, and the levels of P-ALK and P- The in vivo efficacy of compound 2 was then evaluated in the
STAT3 were evaluated by Western blot analysis of cell lysates using ALK-dependent NSCLC NCI-H2228 xenograft model. In the
specific antibodies. experiment reported in Figure 9, the compound was
administered orally at the doses of 30 and 60 mg/kg twice a
day for 10 consecutive days to nude mice bearing NCI-H2228
xenograft tumors. At both doses, complete tumor regression
was observed in all the animals at the end of treatment. The in
vivo target modulation was also evaluated in this ALK-
dependent NSCLC model. In Figure 9, the complete inhibition
of EML4-ALK and AKT phosphorylation can be clearly
appreciated by Western blot analysis of tumors harvested 12
or 18 h after single oral administration. Because, as mentioned
above, low BBB penetration is the major weakness of crizotinib
and development of brain metastases is an important cause of
relapse in crizotinib-treated patients, we decided to investigate
the ability of compound 2 to cross the BBB. The value of brain
levels measured after oral administration of the compound to
nu/nu mice reaches nearly 50% of plasma levels, as reported in
Table 10. Such brain levels suggest the possibility to reach an
efficacious exposure of the compound also in the brain.
To further characterize compound 2, its pharmacokinetic
parameters in rat and dog have been evaluated by treating the
animals at the dose of 10 mg/kg for both iv and oral
administration as reported in Table 11. Good exposure and
moderate to good oral bioavailability have been reached in both
Figure 7. Mechanism of action of compound 2 in KM12 cell line. species, with volume of distribution indicative of tissue
Cells were treated with the compound at the indicated concentrations distribution and low clearance in both animal species. This
for 2 h, and the levels of P-TPM3-TRKA and of phosphorylated profile allowed the evaluation of the safety and tolerability of
downstream transducers were evaluated by Western blot analysis of this compound in further animal studies.
■
cell lysates using specific antibodies.
Table 9. Antiproliferative Activity of Compound 2 on Ba/F3 CONCLUSIONS

Cells Dependent on ALK, ROS1, and TRKs Kinases In conclusion, we have identified a novel 3-aminoindazole ALK
cell line IC50 (μM) a inhibitor (compound 2) through the optimization of the
starting hit compound 1. Compound 2 is a potent ALK, ROS1,
Ba/F3 2.104
and TRKs inhibitor with nanomolar activity on the
Ba/F3_ ALK 0.028
corresponding target-driven cell lines. It shows a favorable
Ba/F3_ ROS1 0.005
Ba/F3_ TRKA 0.003
pharmacokinetic profile in rodent and nonrodent animal
Ba/F3_ TRKB 0.003 species, and it induces stable tumor regression in ALK-
Ba/F3_ TRKC 0.003 dependent ALCL and NSCLC human xenograft tumor models
a
IC50 values as determined in an antiproliferative activity assay (72 h
with a clear inhibiton of ALK and downstream effector
treatment) are reported as the mean of 2−3 experiments with a phosphorylation. Compound 2, based on its permissive safety
coefficient of variation below 35%. and tolerability profile, was selected for development, and it is
currently undergoing phase I/II clinical trials for the treatment
Compound 2 was tested for evaluating its in vivo efficacy in of patients affected by ALK-, ROS1-, and TRKs-dependent
different ALK-dependent tumor models. In the experiment tumors with remarkable signs of activity.29,30 An exhaustive
reported in Figure 8, severe combined immune deficiency biological and pharmacological profile of compound 2,
(SCID) mice bearing Karpas-299 xenograft tumors were including its activity on a panel of resistant mutants identified
treated orally with the compound at the doses of 30 and 60 in patients treated with different ALK inhibitors, is under
mg/kg twice a day for 10 consecutive days. Excellent efficacy publication.35
J. Med. Chem. 2016, 59, 3392−3408
Figure 8. In vivo efficacy of compound 2 in Karpas 299 ALK-dependent xenograft model and ex vivo target modulation. (left) Nu/Nu mice bearing
established Karpas-299 tumors were treated with compound 2 at the doses of 30 and 60 mg/kg per os twice a day for 10 consecutive days. (right) To
evaluate ex vivo target modulation, mice were administered a single dose of 60 mg/kg and sacrificed 12 h or 18 h after the treatment. Levels of P-
NPM-ALK and P-STAT3 in tumor lysates were analyzed by Western blot using specific antibodies.
Figure 9. In vivo efficacy of compound 2 in NSCLC NCI-H2228 ALK-dependent xenograft model and ex vivo target modulation. (left) Nu/Nu
mice bearing established NCI-H2228 tumors were treated with compound 2 at the doses of 30 and 60 mg/kg per os twice a day for 10 consecutive
days. (right) To evaluate ex vivo target modulation mice were administered a single dose of 60 mg/kg and sacrificed 12 h or 18 h after the treatment.
Levels of P-EML4-ALK and P-STAT3 in tumor lysates were analyzed by Western blot using specific antibodies.
Table 10. Brain and Plasma Levels of Compound 2 after 2- 215−400 nm). A Waters XSelect CSH C18 column 50 × 4.6 mm, 3.5
Week Repeated Administration in Mouse μm particle size, was used. Mobile phase A was ammonium acetate 5
mM buffer (pH 4.5 with acetic acid):acetonitrile 95:5, and mobile
plasma brain brain/ phase B was ammonium acetate 5 mM buffer (pH 4.5 with acetic
species treatment dose levels levels plasma acid):acetonitrile 5:95. Gradient from 0 to 100% B in 7 min, hold
mouse 2 weeks 240 4.57 μM 1.94 μM 0.43 100% B 2 min. Flow rate 1 mL/min. Injection volume 10 μL. Full
mg/kg/day scan, mass range from 50 to 1200 amu. Heated capillary temp was 275
°C, and spray voltage value was set at 4 kV. Mass are given as m/z
■ EXPERIMENTAL SECTION
1. Chemistry. Unless otherwise noted, solvents and reagents were
ratio. Instrument control, data acquisition, and processing were
performed by using Xcalibur 1.2 software (Thermo). Column
chromatography was conducted either under medium pressure on
obtained from commercial suppliers and used without further silica gel (Merck silica gel 40−63 μm) or on prepacked silica gel
purification. All reactions involving air- or moisture-sensitive reagents cartridges (Biotage) on a Horizon system. 1H NMR spectra were
were performed under an argon atmosphere. All final compounds were acquired at 25 °C in DMSO-d6 on a Varian Inova 400 spectrometer
purified to >95% purity as determined by high-performance liquid operating at 400 MHz and equipped with a 5 mm 1H{15N-31P} Z-axis-
chromatography (HPLC). HPLC-MS/UV analyses were performed PFG indirect detection probe. Residual not-deuterated solvent signal
on a LCQ DecaXP (Thermo, San Jose, US) ion trap instrument, was used as reference with δ = 2.50 ppm for DMSO-d5. Data are
equipped with an electrospray (ESI) ion source. The mass reported as follows: chemical shift, multiplicity (s = singlet, d =
spectrometer is connected to a Surveyor HPLC system (Thermo, doublet, t = triplet, q = quartet, quint = quintet, bs = broad singlet, bd
San Jose, US) with an UV photodiode array detector (UV detection = broad doublet, dd = doublet of doublet, td = triplet of doublet, m =
Table 11. Pharmacokinetic Profile of Compound 2 in Rat (Sprague Dawley Rats)a and Dog (Beagle Dog)b
PK data (iv), dose:c 10 mg/kg PK data (per os), dose:c 10 mg/kg
species Cmax (μM) AUC∞ (μM·h) CL (mL/min/kg) Vss (L/kg) t1/2 (h) Cmax (μM) Tmax (h) AUC∞ (μM·h) t1/2 (h) F (%)d
rat 11.0 ± 1.1 15.1 ± 1.5 20.8 ± 2.1 4.0 ± 0.4 3.5 ± 0.3 0.5 ± 0.1 3.08 5.13 ± 0.18 3.8 ± 0.2 43e
dog 8.6 ± 2.5 17.6 ± 3 17.5 ± 3.6 6.7 ± 0.9 11.9 ± 6.0 0.6 ± 0.2 2.0 5.7 ± 2.6 15.2 ± 8.9 32
a
n = 6 animals per study. bn = 3 animals per study. cDosed in 10% Tween 80/saline iv = intravenous administration; dosed in 0.5% methocel
suspension per os = oral administration. dBioavailability. eF % calculated based on the actual 8 mg/kg oral dose.

J. Med. Chem. 2016, 59, 3392−3408
multiplet), coupling constants, and number of protons. As formerly (ddd, J = 11.7, 3.8, 3.8 Hz, 2 H) 4.04 (s, 2 H) 6.13 (d, J = 2.1 Hz, 1 H)
reported,36 ESI(+) high-resolution mass spectra (HRMS) were 6.23 (dd, J = 9.0, 2.2 Hz, 1 H) 6.93−7.04 (m, 3 H) 7.25 (dd, J = 8.7,
obtained on a Q-Tof Ultima (Waters, Manchester, UK) mass 1.5 Hz, 1 H) 7.40 (d, J = 8.7 Hz, 1 H) 7.48 (s, 1 H) 7.80 (d, J = 9.0
spectrometer directly connected with a Agilent 1100 micro-HPLC Hz, 1 H) 8.29 (d, J = 7.6 Hz, 1 H) 10.07 (s, 1 H) 12.62 (s, 1 H).
system (Palo Alto, CA, US). Thin-layer chromatography was LCMS (ESI) m/z 507 (M + H)+. HRMS (ESI) calcd for
performed on Merck silica gel 60 plates coated with 0.25 mm layer C31H34F2N6O2 + H+ 561.2784, found 561.2785.
with fluorescent indicator. Components were visualized by UV light (λ By employing the above-described procedure, starting from scaffold
= 254 and 366 nm) and iodine vapors. I or II and benzoic acid III2, the following compounds were prepared.
Synthesis of Scaffold I and II. General Procedure for Diaryl- N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-pipera-
methanes Preparation. 3-Cyano-4-fluorophenylboronic acid (1 zin-1-yl)-2-nitro-benzamide (3). Eluant for column chromatography:
equiv), powdered K3PO4 (2 equiv), and Pd(PPh3)4 (2% mol) were DCM/EtOH/NH3 5 N in MeOH = 100/5/1. 1H NMR (400.5 MHz,
charged in an oven-dried flask under argon atmosphere. The flask was DMSO-d6) δ ppm 2.23 (s, 3 H) 2.42−2.47 (m, 4 H) 3.33−3.38 (m, 4
evacuated and backfilled with argon three times. Toluene (3 mL/ H) 4.05 (s, 2 H) 7.01 (tt, J = 9.4, 2.4 Hz, 2H) 7.24 (dd, J = 8.7, 1.6 Hz,
mmol boronic acid) and benzyl bromide (1 equiv) were added under 1 H) 7.27 (br s, 1 H) 7.41 (d, J = 8.7 Hz, 1 H) 7.44 (br s, 1 H) 7.63 (s,
good stirring. The reaction mixture was heated to 100 °C in half an 1 H) 7.69 (br s, 1 H) 10.81 (br s, 1 H) 12.70 (s, 1 H). LCMS (ESI)
hour and maintained at that temperature for 1.5−8 h. The dark m/z 561 (M + H)+. HRMS (ESI) calcd for C26H24F2N6O3 + H+
mixture was taken up with diethyl ether, washed with saturated 507.1951, found 507.1947.
aqueous NH4Cl, and brine. The organic phase was dried over N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-methoxy-ethyla-
anhydrous Na2SO4 and evaporated to dryness. The residue was then mino)-4-(4-methyl-piperazin-1-yl)-benzamide (5). Eluant for column
purified by flash chromatography (n-hexane/ethyl acetate) to provide chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/5/1. 1H
the desired diarylmethane in yields of 60−90%. NMR (400.5 MHz, DMSO-d6) δ ppm 2.25 (s, 3 H) 2.44−2.49 (m, 4
General Procedure for 3-Aminoindazoles Preparation. A mixture H) 3.26 (s, 3 H) 3.27−3.31 (m, 6 H) 3.54 (t, J = 5.5 Hz, 2 H) 4.05 (s,
of 3-cyano-4-fluoro-diarylmethane (1 equiv) and hydrazine hydrate (5 2 H) 6.09 (d, J = 1.9 Hz, 0 H) 6.25 (dd, J = 9.0, 1.9 Hz, 0 H) 6.94−
equiv) in n-butanol (2.5 mL/mmol diarylmethane) was refluxed 7.04 (m, 3 H) 7.24 (dd, J = 8.7, 1.6 Hz, 1 H) 7.41 (d, J = 8.7 Hz, 1H)
overnight. The reaction mixture was diluted with water/ethyl acetate, 7.51 (s, 1 H) 7.79 (d, J = 9.0 Hz, 1 H) 8.23 (t, J = 4.9 Hz, 1 H) 10.06
and the organic phase was washed twice with brine, dried, and (s, 1 H) 12.63 (s, 1 H). LCMS (ESI) m/z 535 (M + H)+. HRMS
evaporated. The residue was purified by flash chromatography (ESI) calcd for C29H32F2N6O2 + H+ 535.2628, found 535.2632.
(CH2Cl2/EtOH) to provide the desired 3-aminoindazole in yields of N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-((R)-2-methoxy-1-
75−90%. methyl-ethylamino)-4-(4-methyl-piperazin-1-yl)-benzamide (6).
General Procedure for Diarylmethanes Alkylation. A mixture of 3- Eluant for column chromatography: DCM/EtOH/NH3 5 N in
cyano-4-fluoro-diarylmethane (1 equiv) and methyl iodide (1.5 equiv) MeOH = 100/10/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.14
was dissolved in THF dry (8 mL/mmol diarylmethane) under (d, J = 6.3 Hz, 3 H) 2.23 (s, 3 H) 2.41−2.47 (m, 4 H) 3.24−3.29 (m, 4
nitrogen atmosphere at −20 °C. Bis(trimethylsilyl)-lithiumamid 1.0 M H) 3.27 (s, 3 H) 3.30−3.40 (m, 2 H) 3.74−3.83 (m, 1 H) 4.05 (s, 2
in THF (2 equiv) was gradually added. After 20 min, the reaction was
H) 6.13 (d, J = 2.2 Hz, 1 H) 6.24 (dd, J = 9.0, 2.2 Hz, 1 H) 6.94−7.04
quenched by adding a solution of KHSO4 10% and extracted with
(m, 3 H) 7.25 (dd, J = 8.7, 1.6 Hz, 1 H) 7.41 (d, J = 8.7 Hz, 1 H) 7.49
ethyl acetate. The organic phase was washed with aqueous KHSO4
(s, 1 H) 7.78 (d, J = 9.0 Hz, 1 H) 8.20 (d, J = 7.7 Hz, 1 H) 10.04 (s, 1
10% and brine, dried over sodium sulfate, and evaporated to dryness.
H) 12.63 (s, 1 H). LCMS (ESI) m/z 549 (M + H)+. HRMS (ESI)
The crude was purified by flash chromatography (hexane/ethyl
acetate) to provide the desired alkylated diarylmethane in yields of calcd for C30H34F2N6O2 + H+ 549.2784, found 549.2787.
70−85%. N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(3-methoxy-propy-
lamino)-4-(4-methyl-piperazin-1-yl)-benzamide (7). Eluant for col-
Synthesis of 3-acyl-5-benzyl indazoles 2, 3, 5−10, 12−16, 18, 20−
umn chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/5/1.
25, and 28−31.
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-pipera-
1
H NMR (400.5 MHz, DMSO-d6) δ ppm 1.79 (quin, J = 6.5 Hz, 2 H)
zin-1-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide (2). To a 2.22 (s, 3 H) 2.41−2.46 (m, 4 H) 3.17 (q, J = 6.5 Hz, 2 H) 3.21 (s, 3
suspension of 4-(4-methyl-piperazin-1-yl)-2-[(tetrahydro-pyran-4-yl)- H) 3.25−3.31 (m, 4 H) 3.40 (t, J = 6.5 Hz, 2 H) 4.03 (s, 2 H) 6.05 (d,
(2,2,2-trifluoro-acetyl)-amino]-benzoic acid trifluoroacetate (10 g, 22.1 J = 2.2 Hz, 1H) 6.23 (dd, J = 9.0, 2.2 Hz, 1 H) 6.93−7.03 (m, 3 H)
mmol) in dry dichloromethane (300 mL), oxalyl chloride (3.58 mL, 7.23 (dd, J = 8.7, 1.6 Hz, 1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.50 (s, 1 H)
42.3 mmol) and N,N-dimethylformamide (1−2 drops) were added. 7.79 (d, J = 9.0 Hz, 1 H) 8.18 (t, J = 5.2 Hz, 1 H) 10.06 (s, 1 H) 12.61
The mixture was stirred at room temperature for 2 h then evaporated (s, 1 H). LCMS (ESI) m/z 549 (M + H)+. HRMS (ESI) calcd for
to dryness. The resulting crude acyl chloride was taken-up with C30H34F2N6O2 + H+ 549.2784, found 549.2780.
toluene, evaporated, and then dissolved in dry tetrahydrofuran (130 N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-methoxy-1-me-
mL) at −20 °C. A solution of 5-(3,5-difluoro-benzyl)-1H-indazol-3- thoxymethyl-ethylamino)-4-(4-methyl-piperazin-1-yl)-benzamide
ylamine (5 g. 19.28 mmol) and N,N-diisopropylethylamine (12.8 mL, (8). Eluant for column chromatography: DCM/MeOH/NH3 5N in
73.3 mmol) in dry THF (40 mL) was added to the cooled reaction MeOH = 100/5/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 2.42 (br
mixture. The mixture was stirred at −20 °C for 4 h then quenched by s, 3 H) 2.70 (br s, 4 H) 3.18−3.44 (m, 4 H) 3.26 (s, 6 H) 3.41 (d, J =
adding water/ethyl acetate. The organic phase was washed with a 5.0 Hz, 4 H) 3.80−3.88 (m, 1 H) 4.04 (s, 2 H) 6.20 (d, J = 2.1 Hz, 1
saturated solution of sodium hydrogenocarbonate, dried over sodium H) 6.26 (dd, J = 8.9, 2.1 Hz, 1 H) 6.94−7.03 (m, 3 H) 7.24 (dd, J =
sulfate, and evaporated to dryness. 8.9, 1.5 Hz, 1 H) 7.41 (d, J = 8.7 Hz, 1 H) 7.48 (s, 1 H) 7.79 (d, J =
Crude reaction mixture is dissolved in methanol (375 mL) in the 8.9 Hz, 1 H) 8.32 (d, J = 8.3 Hz, 1 H) 10.06 (s, 1 H) 12.64 (s, 1 H).
presence of triethylamine (60 mL) and stirred at 65 °C for 2 h. The LCMS (ESI) m/z 579 (M + H)+. HRMS (ESI) calcd for
solvents were removed under reduced pressure and the residue treated C31H36F2N6O3 + H+ 579.2890, found 579.2890.
with water/ethyl acetate. Organic phase was dried over sodium sulfate N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-fluoro-ethylami-
and evaporated to dryness. Purification of the crude by chromatog- no)-4-(4-methyl-piperazin-1-yl)-benzamide (9). Eluant for column
raphy over silica gel (DCM/EtOH/NH3 5 N in MeOH = 1000/50/5) chromatography: DCM/MeOH = 95/5. 1H NMR (400.5 MHz,
and crystallization of the so obtained compound from ethyl acetate/ DMSO-d6) δ ppm 2.24 (s, 3 H) 2.43−2.48 (m, 4 H) 3.26−3.31 (m, 4
hexane afforded 8.4 g of the title compound as a white solid (78% H) 3.48 (ddt, JHF = 27.7, JHH = 5.4, 4.9, 4.9 Hz, 2 H) 4.04 (s, 2 H) 4.60
yield). (dt, JHF = 47.7, JHH = 4.9, 4.9 Hz, 2 H) 6.12 (d, J = 2.2 Hz, 1 H) 6.27
1
H NMR (400.5 MHz, DMSO-d6) δ ppm 1.29−1.41 (m, 2H) (dd, J = 9.0, 2.2 Hz, 1 H) 6.94−7.04 (m, 3 H) 7.23 (dd, J = 8.5, 1.6 Hz,
1.89−1.97 (m, 2H) 2.24 (s, 3 H) 2.41−2.48 (m, 4H) 3.23−3.29 (m, 4 1 H) 7.40 (d, J = 8.5 Hz, 1 H) 7.50 (s, 1 H) 7.80 (d, J = 9.0 Hz, 1 H)
H) 3.49 (ddd, J = 11.7, 10.2, 2.3 Hz, 2 H) 3.62−3.72 (m, 1 H) 3.80 8.36 (t, J = 5.4 Hz, 1 H) 10.10 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI)

J. Med. Chem. 2016, 59, 3392−3408
m/z 523 (M + H)+. HRMS (ESI) calcd for C28H29F3N6O + H+ (m, 2 H) 2.04 (br s, 3.25 H, rotamer) 2.24 (br s, 2.75 H, rotamer) 2.39
523.2428, found 523.2427. (br s, 1.08 H, rotamer) 2.50 (overlapped by 6DMSO-d6 signal, 0.92 H,
2-[(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)amino]-N-[5-(3,5-di- rotamer) 2.91 (br s, 1.38 H, rotamer) 2.97 (br s, 1.63 H, rotamer) 3.31
fluorobenzyl)-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)- (overlapped by water signal, 1.08 H), rotamer) 3.45−3.52 (m, 2 H)
benzamide (10). ESI(+) MS: m/z 635 (MH+). 3.54 (br s, 0.92 H, rotamer) 3.62−3.72 (m, 1 H) 3.79−3.85 (m, 2 H)
N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-2-{[(1-methylazetidin- 4.05 (s, 2 H) 6.55 (d, J = 7.9 Hz, 1 H) 6.75 (s, 1 H) 6.94−7.04 (m, 3
3-yl)methyl]amino}-4-(4-methylpiperazin-1-yl)benzamide (12). H) 7.27 (dd, J = 8.6, 1.6 Hz, 1 H) 7.43 (d, J = 8.6 Hz, 1 H) 7.53 (s, 1
Eluant for column chromatography: DCM/MeOH/NH3 5 N in H) 7.92 (d, J = 7.9 Hz, 1 H) 7.95 (d, J = 7.7 Hz, 1 H) 10.55 (s, 1 H)
MeOH = 100/10/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 2.25 (s, 12.74 (s, 1 H). LCMS (ESI) m/z 591 (M + H)+. HRMS (ESI) calcd
3 H) 2.43−2.48 (m, 4 H) 2.62 (br s, 3 H) 2.85−2.95 (m, 1 H) 3.31 for C32H36F2N6O3 + H+ 591.2890, found 591.2891.
(m overlapped by water signal, 4 H) 3.37−3.42 (m, 2 H) 3.57 (br s, 2 N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-4-(piperazin-1-yl)-2-
H) 3.80−3.90 (m, 2 H) 4.03 (s, 2 H) 6.08 (d, J = 2.1 Hz, 1 H) 6.27 (tetrahydro-2H-pyran-4-ylamino)benzamide (22). Eluant for col-
(dd, J = 8.9, 2.1 Hz, 1 H) 6.93−6.99 (m, 2 H) 6.99−7.05 (m, 1 H) umn chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/10/1.
7.24 (dd, J = 8.5, 1.5 Hz, 1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.49 (s, 1 H) 1
H NMR (400.5 MHz, DMSO-d6) δ ppm 1.30−1.41 (m, 2 H) 1.88−
7.81 (d, J = 8.9 Hz, 1 H) 8.24 (t, J = 5.2 Hz, 1 H) 10.11 (s, 1 H) 12.63 1.98 (m, 2 H) 2.80−2.87 (m, 4 H) 3.17−3.22 (m, 4 H) 3.45−3.54 (m,
(s, 1 H). LCMS (ESI) m/z 560 (M + H)+. HRMS (ESI) calcd for 2 H) 3.62−3.72 (m, 1 H) 3.78−3.84 (m, 2 H) 4.04 (s, 2 H) 6.11 (d, J
C31H35F2N7O + H+ 560.2944, found 560.2954. = 2.2 Hz, 1 H) 6.22 (dd, J = 9.0, 2.2 Hz, 1 H) 6.94−7.04 (m, 3 H) 7.25
2-Benzylamino-N-[5-(3,5-difluoro-benzyl)-1H-indazol-3-yl]-4-(4- (dd, J = 8.6, 1.6 Hz, 1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.48 (s, 1 H) 7.79
methyl-piperazin-1-yl)-benzamide (13). Eluant for column chroma-
(d, J = 9.0 Hz, 1 H) 8.28 (d, J = 7.7 Hz, 1 H) 10.06 (s, 1 H) 12.62 (s, 1
tography: DCM/EtOH/NH3 5 N in MeOH = 100/5/1. 1H NMR
H). LCMS (ESI) m/z 547 (M + H)+. HRMS (ESI) calcd for
(400.5 MHz, DMSO-d6) δ ppm 2.21 (s, 3 H) 2.36−2.44 (m, 4 H)
C30H32F2N6O2 + H+ 547.2628, found 547.2628.
3.19−3.24 (m, 2 H) 4.03 (s, 2 H) 4.38 (d, J = 5.6 Hz, 2 H) 6.08 (d, J =
1-[4-{[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]carbamoyl}-3-(tet-
2.2 Hz, 1 H) 6.25 (dd, J = 9.0, 2.2 Hz, 1 H) 6.92−7.03 (m, 3 H) 7.21− rahydro-2H-pyran-4-ylamino)benzyl]piperidine (23). Eluant for
7.27 (m, 2 H) 7.30−7.36 (m, 2 H) 7.35−7.38 (m, 2 H) 7.39 (d, J = 8.5 column chromatography: DCM/MeOH/NH3 5 N in MeOH = 100/
Hz, 1 H) 7.50 (s, 1 H) 7.80 (d, J = 9.0 Hz, 1 H) 8.59 (t, J = 5.6 Hz, 1 5/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.32−1.44 (m, 3 H)
H) 10.10 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 567 (M + H)+. 1.67−1.87 (m, 5 H) 1.95−2.02 (m, 2 H) 2.82−2.93 (m, 2 H) 3.32 (br
HRMS (ESI) calcd for C33H32F2N6O + H+ 567.2679, found 567.2680. s, 2 H) 3.44−3.54 (m, 2 H) 3.63−3.74 (m, 1 H) 3.82−3.91 (m, 2 H)
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-pipera- 4.06 (s, 2 H) 4.23 (d, J = 5.37 Hz, 2 H) 6.75−6.81 (m, 1 H) 6.94−7.06
zin-1-yl)-2-phenylamino-benzamide (14). Eluant for column chro-
matography: DCM/EtOH/NH3 5 N in MeOH = 100/5/1. 1H NMR (m, 2 H) 7.13 (s, 1 H) 7.29 (dd, J = 8.66, 1.46 Hz, 1 H) 7.45 (d, J =
(400.5 MHz, DMSO-d6) δ ppm 2.23 (s, 3 H) 2.46 (br s, 4 H) 3.18− 8.54 Hz, 1 H) 7.50 (s, 1 H) 7.96 (d, J = 8.05 Hz, 1 H) 8.00 (br s, 1 H)
3.24 (m, 4 H) 4.04 (s, 2 H) 6.52 (dd, J = 9.0, 2.2 Hz, 1 H) 6.73 (d, J = 10.14 (br s, 1 H) 10.54 (s, 1 H) 12.77 (br s, 1 H)). LCMS (ESI) m/z
2.2 Hz, 1 H) 6.93−7.02 (m, 4 H) 7.16−7.20 (m, 2 H) 7.24 (dd, J = 560 (M + H)+. HRMS (ESI) calcd for C32H35F2N5O2 + H+ 560.2832,
8.6, 1.5 Hz, 1 H) 7.28−7.34 (m, 2 H) 7.41 (d, J = 8.6 Hz, 1 H) 7.54 (s, found 560.2831.
1 H) 7.90 (d, J = 9.0 Hz, 1 H) 10.02 (s, 1 H) 10.38 (s, 1 H) 12.68 (s, 1 N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-4-[(1-methylpiperidin-
4-yl)oxy]-2-(tetrahydro-2H-pyran-4-ylamino)benzamide (24).
Eluant for column chromatography: DCM/EtOH/NH3 5 N in
C32H30F2N6O + H+ 553.2522, found 553.2499.
MeOH = 100/10/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm
2-Cyclohexylamino-N-[5-(3,5-difluoro-benzyl)-1H-indazol-3-yl]-
4-(4-methyl-piperazin-1-yl)-benzamide (15). Eluant for column 1.30−1.41 (m, 2 H) 1.62−1.75 (m, 2 H) 1.87−2.00 (m, 4 H) 2.20−
chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/5/1. 1H 2.87 (br s, 5 H) 2.69 (br s, 2 H) 3.44−3.53 (m, 2 H) 3.63 (m, 1 H)
NMR (400.5 MHz, DMSO-d6) δ ppm 1.16−1.30 (m, 3 H) 1.33−1.45 3.78−3.85 (m, 2 H) 4.04 (s, 2 H) 4.50 (br s, 1 H) 6.23−6.28 (m, 2 H)
(m, 2 H) 1.48−1.58 (m, 1 H) 1.57−1.69 (m, 2 H) 1.83−1.92 (m, 2 6.95−7.04 (m, 3 H) 7.26 (dd, J = 8.7, 1.6 Hz, 1 H) 7.42 (d, J = 8.6 Hz,
H) 2.24 (s, 3 H) 2.41−2.48 (m, 4 H) 3.22−3.27 (m, 4 H) 3.40−3.52 1 H) 7.49 (s, 1 H) 7.85−7.89 (m, 1 H) 8.21 (d, J = 7.7 Hz, 1 H) 10.24
(m, 1 H) 4.03 (s, 2 H) 6.08 (d, J = 2.1 Hz, 1 H) 6.21 (dd, J = 9.0, 2.1 (s, 1 H) 12.67 (s, 1 H). LCMS (ESI) m/z 576 (M + H)+. HRMS
Hz, 1 H) 6.93−7.03 (m, 3 H) 7.25 (dd, J = 8.7, 1.6 Hz, 1 H) 7.40 (d, J (ESI) calcd for C32H35F2N5O3 + H+ 576.2781, found 576.2773.
= 8.6 Hz, 1 H) 7.48 (s, 1 H) 7.77 (d, J = 9.0 Hz, 1 H) 8.27 (d, J = 7.8 N-{5-[1-(3,5-Difluoro-phenyl)-ethyl]-1H-indazol-3-yl}-4-(4-meth-
Hz, 1 H) 10.04 (s, 1 H) 12.61 (s, 1 H). LCMS (ESI) m/z 559 (M + yl-piperazin-1-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide (25).
H)+. HRMS (ESI) calcd for C32H36F2N6O + H+ 559.2992, found Eluant for column chromatography: DCM/EtOH/NH3 5 N in MeOH
559.2992. = 100/5/2. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.30−1.40 (m, 2
trans-4-{[2-{[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]carbamoyl}- H) 1.60 (d, J = 7.2 Hz, 3 H) 1.89−1.98 (m, 2 H) 2.28 (br s, 3 H) 2.50
5-(4-methylpiperazin-1-yl)phenyl]amino}cyclohexyl Benzoate (16). (overlapped by DMSO-d6 signal, 4 H) 3.30 (overlapped by water
ESI(+) MS: m/z 679 (MH+). signal, 4 H) 3.46−3.54 (m, 2 H) 3.64−3.74 (m, 1 H) 3.78−3.85 (m, 2
cis-4-{[2-{[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]carbamoyl}-5- H) 4.31 (q, J = 7.2 Hz, 1 H) 6.15 (d, J = 2.0 Hz, 1 H) 6.25 (dd, J = 9.0,
(4-methylpiperazin-1-yl)phenyl]amino}cyclohexyl Benzoate (18). 2.0 Hz, 1 H) 6.95−7.03 (m, 3 H) 7.27 (dd, J = 8.7, 1.6 Hz, 1 H) 7.40
ESI(+) MS: m/z 679 (MH+). (d, J = 8.7 Hz, 1 H) 7.51 (s, 1 H) 7.80 (d, J = 9.0 Hz, 1 H) 8.31 (d, J =
N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-4-(4-methylpiperazin- 7.7 Hz, 1 H) 10.08 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 575 (M
1-yl)-2-[(1-methylpiperidin-4-yl)amino]benzamide (20). Eluant for + H)+. HRMS (ESI) calcd for C32H36F2N6O2 + H+ 575.2941, found
column chromatography: DCM/MeOH/NH3 5 N in MeOH = 100/ 575.2935.
10/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.35−1.48 (m, 2 H) N-[5-Benzyl-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-yl)-2-(tet-
1.87−1.97 (m, 2 H) 2.18 (br s, 3 H) 2.21 (br s, 2 H) 2.23 (s, 3 H) rahydro-pyran-4-ylamino)-benzamide (28). Eluant for column
2.41−2.46 (m, 4 H) 2.61 (br s, 2 H) 3.22−3.27 (m, 4 H) 3.45 (br s, 1 chromatography: DCM/EtOH/NH3 5N in MeOH = 100/10/1. 1H
H) 4.04 (s, 2 H) 6.08 (d, J = 2.0 Hz, 1 H) 6.22 (dd, J = 9.0, 2.0 Hz, 1 NMR (400.5 MHz, DMSO-d6) δ ppm 1.30−1.41 (m, 2 H) 1.89−1.98
H) 6.94−7.03 (m, 3H) 7.24 (dd, J = 8.6, 1.5 Hz, 1 H) 7.40 (d, J = 8.5 (m, 2 H) 2.23 (s, 3 H) 2.40−2.46 (m, 4 H) 3.23−3.30 (m, 4 H) 3.46−
Hz, 1 H) 7.49 (s, 1 H) 7.78 (d, J = 9.0 Hz, 1 H) 8.26 (d, J = 7.4 Hz, 1 3.54 (m, 2 H) 3.63−3.73 (m, 1 H) 3.78−3.85 (m, 2 H) 4.01 (s, 2 H)
H) 10.06 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 574 (M + H)+. 6.13 (d, J = 2.2 Hz, 1 H) 6.23 (dd, J = 9.0, 2.2 Hz, 1 H) 7.13−7.29 (m,
HRMS (ESI) calcd for C32H37F2N7O + H+ 574.3101, found 574.3099. 6 H) 7.37 (d, J = 8.9 Hz, 1 H) 7.43 (s, 1 H) 7.78 (d, J = 9.0 Hz, 1 H)
N1-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-N4-[2- 8.27 (d, J = 7.7 Hz, 1 H) 10.04 (s, 1 H) 12.58 (s, 1 H). LCMS (ESI)
(dimethylamino)ethyl]-N 4 -methyl-2-(tetrahydro-2H-pyran-4- m/z 525 (M + H)+. HRMS (ESI) calcd for C31H36N6O2 + H+
ylamino)benzene-1,4-dicarboxamide (21). Eluant for column 525.2973, found 525.2983.
chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/10/1. 1H N-[5-(2-Fluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-
NMR (400.5 MHz, DMSO-d6) δ ppm 1.31−1.42 (m, 2 H) 1.87−1.95 yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide (29). Eluant for

J. Med. Chem. 2016, 59, 3392−3408
column chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/ N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-2-[(trans-4-

10/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.29−1.40 (m, 2 H) hydroxycyclohexyl)amino]-4-(4-methylpiperazin-1-yl)benzamide
1.89−1.98 (m, 2 H) 2.23 (s, 3 H) 2.40−2.48 (m, 4 H) 3.22−3.30 (m, (17). Crude trans-4-{[2-{[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]-
4 H) 3.45−3.53 (m, 2 H) 3.63−3.73 (m, 1 H) 3.77−3.85 (m, 2 H) carbamoyl}-5-(4-methylpiperazin-1-yl)phenyl]amino}cyclohexyl ben-
4.03 (s, 2 H) 6.14 (d, J = 2.2 Hz, 1 H) 6.24 (dd, J = 9.0, 2.2 Hz, 1 H) zoate (16, 1.0 mmol) was dissolved in MeOH (100 mL) and water
6.95−7.02 (m, 1 H) 7.04−7.09 (m, 1 H) 7.10−7.12 (m, 1 H) 7.24 (10 mL) and treated at 60 °C with LiOH hydrate (125 mg, 3.0 mmol)
(dd, J = 8.6, 1.5 Hz, 1 H) 7.27−7.34 (m, 1 H) 7.40 (d, J = 8.9 Hz, 1 for 4 h. MeOH was evaporated, and the resulting acqueous phase was
H) 7.46 (s, 1 H) 7.79 (d, J = 9.0 Hz, 1 H) 8.28 (d, J = 7.8 Hz, 1 H) extracted with ethyl acetate. Collected organic phases were dried over
10.07 (s, 1 H) 12.61 (s, 1 H). LCMS (ESI) m/z 543 (M + H)+. Na2SO4, filtered, and evaporated to dryness. Residue was purified by
mHRMS (ESI) calcd for C31H35FN6O2 + H+ 543.2879, found column chromatography over silica gel (DCM/EtOH/NH3 5N in
543.2864. MeOH = 100/10/2), affording 233 mg of title compound (40% yield).
N-[5-(2,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-pipera-
1
H NMR (400.5 MHz, DMSO-d6) δ ppm 1.10−1.22 (m, 2 H) 1.28−
zin-1-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide (30). Eluant 1.42 (m, 2 H) 1.75−1.86 (m, 2 H) 1.94−2.03 (m, 2 H) 2.23 (s, 3 H)
for column chromatography: DCM/EtOH/NH3 5 N in MeOH = 2.41−2.46 (m, 4 H) 3.22−3.28 (m, 4 H) 3.34−3.42 (m, 1 H) 3.43−
100/5/2. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.28−1.41 (m, 2 3.52 (m, 1 H) 4.03 (s, 2 H) 4.52 (d, J = 4.1 Hz, 1 H) 6.08 (d, J = 2.1
H) 1.88−1.98 (m, 2 H) 2.26 (br s, 3 H) 2.42−2.48 (m, 4 H) 3.23− Hz, 1 H) 6.21 (dd, J = 9.0, 2.1 Hz, 1 H) 6.94−7.03 (m, 3 H) 7.24 (dd,
3.31 (m, 4 H) 3.44−3.55 (m, 2 H) 3.63−3.73 (m, 1 H) 3.78−3.85 (m, J = 8.5, 1.6 Hz, 1 H) 7.40 (d, J = 8.5 Hz, 1 H) 7.47 (s, 1 H) 7.76 (d, J =
2 H) 4.03 (s, 2 H) 6.13 (d, J = 2.2 Hz, 1 H) 6.24 (dd, J = 9.0, 2.2 Hz, 1 9.0 Hz, 1 H) 8.16 (d, J = 7.8 Hz, 1 H) 10.03 (s, 1 H) 12.61 (s, 1 H).
H) 7.03−7.11 (m, 1 H) 7.14−7.22 (m, 2 H) 7.24 (dd, J = 8.6, 1.0 Hz, LCMS (ESI) m/z 575 (M + H)+. HRMS (ESI) calcd for
1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.45 (s, 1 H) 7.79 (d, J = 9.0 Hz, 1 H) C32H36F2N6O2 + H+ 575.2941, found 575.2949.
8.29 (d, J = 7.7 Hz, 1 H) 10.07 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) By employing the above-described procedure, the following
m/z 561 (M + H)+. HRMS (ESI) calcd for C31H34F2N6O2 + H+ compound was prepared.
561.2784, found 561.2790. N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-2-[(cis-4-
N-[5-(2-Methyl-5-fluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-pi- hydroxycyclohexyl)amino]-4-(4-methylpiperazin-1-yl)benzamide
perazin-1-yl)-2-(tetrahydro-pyran-4-ylamino)-benzamide (31). (19). Eluant for column chromatography: DCM/EtOH/NH3 5N in
Eluant for column chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/10/2. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.41−
MeOH = 100/5/1. 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.28− 1.70 (m, 8 H) 2.23 (s, 3 H) 2.40−2.47 (m, 4 H) 3.20−3.28 (m, 4 H)
1.40 (m, 2 H) 1.89−1.97 (m, 2 H) 2.21 (s, 3 H) 2.24 (br s, 3 H) 3.50−3.64 (m, 2 H) 4.04 (s, 2 H) 4.42 (d, J = 3.8 Hz, 1 H) 6.08 (d, J =
2.41−2.48 (m, 4 H) 3.23−3.29 (m, 4 H) 3.45−3.53 (m, 2 H) 3.63− 1.9 Hz, 1 H) 6.21 (dd, J = 8.9, 2.2 Hz, 1 H) 6.94−7.04 (m, 3 H) 7.24
3.73 (m, 1 H) 3.78−3.85 (m, 2 H) 4.02 (s, 2 H) 6.12 (d, J = 2.1 Hz, 1 (dd, J = 8.6, 1.6 Hz, 1 H) 7.40 (d, J = 8.5 Hz, 1 H) 7.50 (s, 1 H) 7.78
(d, J = 9.0 Hz, 1 H) 8.38 (d, J = 7.7 Hz, 1 H) 10.03 (s, 1 H) 12.62 (s, 1
H) 6.22 (dd, J = 9.0, 2.1 Hz, 1 H) 6.88−6.97 (m, 2 H) 7.14−7.20 (m,
2 H) 7.37 (s, 1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.78 (d, J = 9.0 Hz, 1 H)
C32H36F2N6O2 + H+ 575.2941, found 575.2943.
8.30 (d, J = 7.8 Hz, 1 H) 10.06 (s, 1 H) 12.60 (s, 1 H). LCMS (ESI)
(S)- or (R)-N-[5-(1-(3,5-Difluorophenyl)ethyl)-1H-indazol-3-yl]-4-
m/z 557 (M + H)+. HRMS (ESI) calcd for C32H37FN6O2 + H+ (4-methylpiperazin-1-yl)-2-(tetrahydro-2H-pyran-4-ylamino)-
557.3035, found 557.3024. benzamide (26) and (27). Single enantiomers (26 and 27) have been
2-Amino-N-[5-(3,5-difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl- obtained from racemate 25 by preparative chiral-HPLC by using
piperazin-1-yl)-benzamide (4). A mixture of N-[5-(3,5-difluoro- Daicel Chiralpak AD 250 mm × 20 mm 10 μm as column system and
benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-yl)-2-nitro-benza- n-hexane/2-propanol 40:60 as eluant (flow rate 6 mL/min): first peak
mide (4, 3.21 g, 6.33 mmol), cyclohexene (20 mL), dioxane (200 mL), eluted = 26, second peak eluted = 27.
and 10% Pd/C (0.8 g) was stirred at 100 °C for 2 h. The reaction 26: 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.30−1.40 (m, 2 H)
mixture was filtered over a Celite pad washing thouroughly with THF 1.60 (d, J = 7.2 Hz, 3 H) 1.89−1.98 (m, 2 H) 2.27 (br s, 3 H)) 2.50
and MeOH. After evaporation of the organic phase, purification of the (overlapped by DMSO-d6 signal, 4 H) 3.30 (overlapped by water
crude by chromatography over silica gel (DCM/MeOH 95/5) gave signal, 4 H) 3.46−3.54 (m, 2 H) 3.64−3.74 (m, 1 H) 3.78−3.85 (m, 2
2.51 g of title compound (83% yield). 1H NMR (400.5 MHz, DMSO- H) 4.31 (q, J = 7.2 Hz, 1 H) 6.15 (d, J = 2.0 Hz, 1 H) 6.25 (dd, J = 9.0,
d6) δ ppm 2.23 (s, 3 H) 2.40−2.47 (m, 4 H) 3.17−3.22 (m, 4 H) 4.04 2.0 Hz, 1 H) 6.95−7.03 (m, 3 H) 7.27 (dd, J = 8.7, 1.6 Hz, 1 H) 7.40
(s, 2 H) 6.18 (d, J = 2.4 Hz, 1 H) 6.24 (dd, J = 9.0, 2.4 Hz, 1 H) 6.53 (d, J = 8.7 Hz, 1 H) 7.51 (s, 1 H) 7.80 (d, J = 9.0 Hz, 1 H) 8.31 (d, J =
(br s, 2 H) 6.93−7.03 (m, 3H) 7.22 (dd, J = 8.7, 1.6 Hz, 1 H) 7.39 (d, 7.7 Hz, 1 H) 10.08 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 575 (M
J = 8.7 Hz, 1 H) 7.52 (br s, 1 H) 7.72 (d, J = 9.0 Hz, 1 H) 10.01 (s, 1 + H)+. HRMS (ESI) calcd for C32H36F2N6O2 + H+ 575.2941, found
H) 12.60 (s, 1 H). LCMS (ESI) m/z 477 (M + H)+. HRMS (ESI) 575.2944.
calcd for C26H26F2N6O + H+ 477.2209, found 477.2207. 27: 1H NMR (400.5 MHz, DMSO-d6) δ ppm 1.30−1.40 (m, 2 H)
N-[5-(3,5-Difluorobenzyl)-1H-indazol-3-yl]-2-[(2-hydroxyethyl)- 1.60 (d, J = 7.2 Hz, 3 H) 1.89−1.98 (m, 2 H) 2.25 (s, 3 H) 2.43−2.50
amino]-4-(4-methylpiperazin-1-yl)benzamide (11). Crude 2-[(2- (m, 4 H) 3.24−3.31 (m, 4 H) 3.46−3.54 (m, 2 H) 3.64−3.74 (m, 1
{[tert-butyl(dimethyl)silyl]oxy}ethyl)amino]-N-[5-(3,5-difluoroben- H) 3.78−3.85 (m, 2 H) 4.31 (q, J = 7.2 Hz, 1 H) 6.15 (d, J = 2.0 Hz, 1
zyl)-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)benzamide (10, 0.2 H) 6.25 (dd, J = 9.0, 2.0 Hz, 1 H) 6.95−7.03 (m, 3 H) 7.27 (dd, J =
mmol) was dissolved in dry THF (3 mL), and 1 M TBAF in THF 8.7, 1.6 Hz, 1 H) 7.40 (d, J = 8.7 Hz, 1 H) 7.51 (s, 1 H) 7.80 (d, J =
(0.24 mL) was added at 0 °C. The resulting solution was stirred 9.0 Hz, 1 H) 8.31 (d, J = 7.7 Hz, 1 H) 10.08 (s, 1 H) 12.62 (s, 1 H).
overnight at room temperature. Reaction was quenched with water LCMS (ESI) m/z 575 (M + H)+. HRMS (ESI) calcd for
and extracted with ethyl acetate. Collected organic phases were dried C32H36F2N6O2 + H+ 575.2941, found 575.2943.
over Na2SO4, filtered, and evaporated to dryness. Residue was purified 2. High-Throughput Screening. More than 50000 compounds
by column chromatography over silica gel (DCM/EtOH/NH3 5N in of our Corporate Compound Collection were tested at 10 μM using
MeOH = 85/15/1), affording 83 mg of title compound (80% yield). KinaseGlo assay format (Promega). Recombinant ALK protein was
1
H NMR (400.5 MHz, DMSO-d6) δ ppm 2.32 (br s, 3 H) 2.54 (br s, 4 tested at low ATP concentration (1/8 fold appKm ATP) and at 2 μM
H) 3.19 (td, J = 5.6, 5.2 Hz, 2 H) 3.30 (m overlapped by water signal, MBP (Myelin Basic Protein). After an incubation time at room
4 H) 3.58 (td, J = 5.5, 5.1 Hz, 2 H) 4.04 (s, 2 H) 4.74 (t, J = 5.1 Hz, 1 temperature for 90 min, KinaseGlo reagent was added and the
H) 6.08 (d, J = 2.1 Hz, 1 H) 6.24 (dd, J = 8.9, 2.1 Hz, 1 H) 6.94−7.04 luminescent signal was read using ViewLux Reader (PerkinElmer).
(m, 3 H) 7.23 (dd, J = 8.6, 1.6 Hz, 1 H) 7.40 (d, J = 8.6 Hz, 1 H) 7.50 3. Kinase Assays. The potency of entrectinib toward ALK and its
(s, 1 H) 7.77 (d, J = 8.9 Hz, 1 H) 8.21 (t, J = 5.2 Hz, 1 H) 10.05 (s, 1 selectivity toward additional kinases was tested using a highly diverse
H) 12.61 (s, 1 H). LCMS (ESI) m/z 521 (M + H)+. HRMS (ESI) panel of tyrosine and serine-threonine kinases called kinase selectivity
calcd for C28H30F2N6O2 + H+ 521.2471, found 521.2465. screening (KSS), based on measurement of phosphorylated substrate

J. Med. Chem. 2016, 59, 3392−3408
after capture of non reacted γ33-ATP with a strong anion exchanger rapid in-frame cloning of the kinase domains downstream of a Tel
(Dowex 1-X8 resin, formate form), as previously described.37 For each cassette using a Gateway system. The constructs obtained were the
enzyme, the absolute Km values for ATP and the specific substrate result of the 336 NH2-residues of TEL fused to the amino acids
were initially determined, and each assay is run at optimized ATP (2 residues of ROS1 (1891−2347), TRKA (440−796), TRKB (455−
Km) and substrate (5 Km) concentrations. Because under these 822), and TRKC (454−825). The pcDNA expression vectors were
conditions IC50 = 3βKi according to the Cheng−Prusoff relation for introduced into the murine IL-3 dependent pro-B Ba/F3 cells
competitive inhibitors, these conditions enable direct comparison of (DSMZ) by electroporation (Amaxa Nucleofector II device) using
IC50 values among the different enzymes of the KSS panel for the the “Amaxa Cell Line Nucleofector Kit V” (VCA-1003), according to
evaluation of inhibitor biochemical selectivity. All assays were manufacturer protocol. Stable strains of IL-3 independent Ba/F3 cells
performed in house, with the exception of TRKB and TRKC, for were established by post-transfection selection of cells for 2 weeks with
which IC50s were extrapolated from percentage inhibition values 800 μg/mL G418 (Enzo Life Sciences) and subsequent growth in
obtained at 10 and 100 nM of inhibitor in duplicate employing the medium lacking IL-3.
SelectScreen Kinase Profiling Services from Life Technologies using 12. Analysis of Cell Proliferation. To evaluate the antiprolifer-
the equation: IC50extrap = ([I] × 100/% inhibition) − [I], with the ative activity of test compounds cells were seeded into 384-well plates
assumption that Hill slope = 1. in appropriate complete medium. Twenty-four hours after seeding,
Ki determination: Enzymatic activity at different ATP and Substrate cells were treated with serial dilutions of test compounds in medium at
concentrations was evaluated in the presence of different concen- a final concentration of 0.1% DMSO and incubated for additional 72 h.
trations of entrectinib. The experiment has been done at initial At the end of treatment, cell viability was assessed using the CellTiter-
velocity, substrates consumption <10%, in 96-well plate. The data Glo luciferase-based ATP detection assay (Promega) following
obtained were analyzed using different models and a statistical manufacturer’s instructions. CellTiter-Glo is a homogeneous assay
approach was used in order to choose the most probable mechanism method based on the quantification of ATP present, an indicator of
of inhibition to calculate the Ki. the number of metabolically active cells. Growth inhibitory activity was
4. Crystallization and Structure Determination. Crystals of the evaluated comparing data for treated versus control samples, using
two ALK−inhibitor complexes here presented were obtained following Accelrys Assay Explorer software. IC50 values were calculated using
the procedures previously described.33 X-ray diffraction data from the sigmoidal interpolation curve fitting.
ALK−2 crystal were collected in house using a Rigaku Micromax-007 13. Western Blot Analysis. Cellular mechanism of action of
HF X-ray generator and Mar345 image plate detector (Marresearch), compound 2 was investigated by treating cells with different
while data from the ALK−17 crystal were collected at ESRF in concentrations of the compound for 2 h at 37 °C. Treated cells
Grenoble, on beamline ID23-1. Indexing, integration, and scaling were were washed twice with ice-cold phosphate buffered saline (PBS) and
performed using MOSFLM and SCALA.38 Both ALK-KD structures lysed in a buffer containing containing 100 mM Tris-HCl pH 7.4, 2%
were determined by molecular replacement using Phaser.39 The search SDS, 1 mM DTT, 1 mM sodium orthovanadate, and 1 mM EDTA.
model was the structure of the ALK-KD-PHA-E429 complex (PDB ID Cell extracts were immediately boiled for 10 min, briefly sonicated,
2XBA). Model building was done using Coot,40 and refinement was clarified by centrifugation, and analyzed for protein content (BCA
done with RefMac.41 Crystallographic data are listed in Table S1 (see Protein Assay, Pierce). Protein extracts were separated by SDS-PAGE
Supporting Information). Structural images have been generated with and analyzed by immunoblotting following standard procedures.
PyMol v1.3.42 Staining was performed with the following antibodies: Phospho-
5. High-Throughput Solubility. Solubility at pH 7 was TRKA-Tyr490 and TRKA from Calbiochem, Phospho-ALK-Tyr1604,
performed as previously described.43 ALK, Phospho-PLCγ1-Tyr783, PLCγ1, Phospho-STAT3-Tyr705,
6. Cell Permeability. Caco-2 permeability assay was performed as Phospho-AKT-Ser473, AKT, Phospho-MAPK-Thr202/Tyr204, and
previously described.44,45 MAPK from Cell Signaling, STAT3 from BD Biosciences.
7. Intrinsic Clearance Determination in HLM (Human Liver 14. Efficacy Studies and ex Vivo Target Modulation
Microsomes). Intrinsic clearance in human liver microsomes was Analysis. All procedures adopted for housing and handling of animals
determined as previously described.45 were in strict compliance with Italian and European guidelines for
8. Intrinsic Clearance Determination in Rat Hepatocytes. Laboratory Animal Welfare. A total of 107 NCI-H2228 nonsmall cell
Intrinsic clearance in human liver microsomes was determined as lung cancer cells were transplanted subcutaneously in athymic nu/nu
previously described.45 mice (Harlan). For the ALCL model, a total of 107 Karpas-299 cells
9. In Vivo Pharmacokinetics. Pharmacokinetic properties were were transplanted subcutaneously in SCID mice (Harlan) previously
investigated in mouse (nu/nu), rat (Han Wistar), and beagle dogs pretreated with total body irradiation at 2 Gy. Mice bearing a minimal
after single intravenous and oral administration. Plasma levels of the tumor mass (150−250 mm3) were randomized into vehicle and
compounds were determined by protein precipitation in a 96-well treated groups. Oral treatments started the day after randomization,
plate format followed by LC-MS/MS analysis. The limits of with different doses as described. Tumor dimensions were measured
quantification were 5−20000 ng/mL. Pharmacokinetic data analysis regularly using Vernier calipers, and tumor volume was calculated
was carried out using a noncompartmental approach (linear according to the following formula: length (mm) × width2 (mm)/2.
trapezoidal rule) with the aid of WinNonlin software (v3.1; Pharsight, The percentage of tumor inhibition (%TI) was calculated as follows: %
Inc.). TI = 100 − (mean tumor volume of treated group/mean tumor
For the analysis of brain levels, entrectinib was orally administered volume of control group) × 100. Toxicity was evaluated on the basis of
for 14 consecutive days and 6 h after last administration mice were body weight reduction. At the end of the experiment, mice were
eutanized, plasma and brain were collected and analyzed as previously sacrificed and gross autopsy findings were reported. Tumor-free
reported.46 animals at 90 days after tumor implant were considered cured. For ex
10. Cell Culture. Human cancer cell lines were obtained from vivo target modulation analysis, animals bearing established xenograft
ATCC, from ECACC, from Interlab Cell Line Collection (ICLC) and tumors were treated with a single orally administered dose and
from NCI. Cells were maintained in the media recommended by the sacrificed at 12 and 18 h following treatment. Xenograft tumor samples
suppliers, in a humidified 37 °C incubator with 5% CO2. The identity were snap frozen in liquid nitrogen immediately after excision and
of all cell lines used in this study was verified using DNA fingerprinting stored at −80 °C until analyzed. The frozen samples were
technology (AmpFlSTR Identifiler Plus PCR amplification kit, Applied homogenized using an Ultra Turrax T25 potter (Janke and Kunkel)
Biosystems). at a 5:1 ratio (v/w) in a lysis buffer containing 100 mM Tris-HCl pH
11. Generation of Oncogene-Driven Ba/F3 Cells. Ba/ 7.4, 2% SDS, 1 mM EDTA, 1 mM sodium orthovanadate, and 1 mM
F3_EML4/ALK were generated as previously reported.34 To generate DTT and immediately boiled. Tumor lysates were briefly sonicated,
tyrosine kinases fused to the human ETV6/TEL partner, a plasmid clarified by centrifugation, assayed for protein content, and used for
based on a pcDNA3.1 DEST backbone was constructed that allowed SDS-PAGE and Western blotting analysis.

J. Med. Chem. 2016, 59, 3392−3408
Journal of Medicinal Chemistry
■
Article
ASSOCIATED CONTENT fluoride; TEA, triethylamine; THF, tetrahydrofuran; TPM3,

* tropomyosin 3; TRK, tropomyosin receptor kinase
■
S Supporting Information
The Supporting Information is available free of charge on the
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J. Med. Chem. 2016, 59, 3392−3408

Acs Jmedchem 6b00064

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Article

Discovery of Entrectinib: A New 3‑Aminoindazole As a Potent

© 2016 American Chemical Society 3392 DOI: 10.1021/acs.jmedchem.6b00064

Figure 1. Structures of the most advanced ALK inhibitors.

Synthesis of 3-acylamino-5-benzyl indazoles is outlined in

Scheme 1. Synthesis of 5-Benzyl-indazoles I and IIa

3394 DOI: 10.1021/acs.jmedchem.6b00064

Scheme 2. Synthesis of Benzoyl-Modiﬁed 3-Acylamino-5-benzyl Indazoles 2−24a

Scheme 3. Synthesis of Benzyl-Modiﬁed 3-Acylamino-5-benzyl Indazoles 25−31a

compd no. R1 R2 ALKa IGF1Ra IRa Karpas-299b

Table 8. Antiproliferative Activity of Compound 2a (Cell

Table 6. In Vivo ADME Parameters of Selected Derivatives (Harlan nu/nu Mice)a

3399 DOI: 10.1021/acs.jmedchem.6b00064

was observed at both doses with complete tumor regression

Table 9. Antiproliferative Activity of Compound 2 on Ba/F3 CONCLUSIONS

3401 DOI: 10.1021/acs.jmedchem.6b00064

3402 DOI: 10.1021/acs.jmedchem.6b00064

3403 DOI: 10.1021/acs.jmedchem.6b00064

column chromatography: DCM/EtOH/NH3 5 N in MeOH = 100/ N-[5-(3,5-Diﬂuorobenzyl)-1H-indazol-3-yl]-2-[(trans-4-

3404 DOI: 10.1021/acs.jmedchem.6b00064

3405 DOI: 10.1021/acs.jmedchem.6b00064

ASSOCIATED CONTENT ﬂuoride; TEA, triethylamine; THF, tetrahydrofuran; TPM3,

3406 DOI: 10.1021/acs.jmedchem.6b00064

3407 DOI: 10.1021/acs.jmedchem.6b00064

3408 DOI: 10.1021/acs.jmedchem.6b00064

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