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

Article abstract of DOI:10.1021/acs.jmedchem.6b00064

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.

Full text of DOI:10.1021/acs.jmedchem.6b00064

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Article
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 T. Bossi,
Laura Buffa, Giulia Canevari, Lucio Ceriani, Maristella Colombo, Luca Corti, Daniele Donati, Marina
Fasolini, Eduard R. Felder, Claudio Fiorelli, Francesco Fiorentini, Arturo Galvani, Antonella Isacchi,
Andrea Lombardi Borgia, Chiara Marchionni, Marcella Nesi, Christian Orrenius, Achille Panzeri, Enrico
Pesenti, Luisa Rusconi, Beatrice Maria Saccardo, Ermes Vanotti, Ettore Perrone, and Paolo Orsini
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00064 • Publication Date (Web): 22 Mar 2016
Downloaded from http://pubs.acs.org on March 23, 2016
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Discovery of Entrectinib: a new 3-aminoindazole as a
potent Anaplastic Lymphoma Kinase (ALK), c-ros
Oncogene 1 Kinase (ROS1), and Pan-Tropomyosin
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Receptor Kinases (Pan-TRKs) inhibitor
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,†
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Maria Menichincheri, Elena Ardini, Paola Magnaghi, Nilla Avanzi, Patrizia Banfi,
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Roberto Bossi, Laura Buffa, Giulia Canevari, Lucio Ceriani, Maristella Colombo,
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Luca Corti, Daniele Donati, Marina Fasolini, Eduard Felder, Claudio Fiorelli,
‡
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Francesco Fiorentini, Arturo Galvani, Antonella Isacchi, Andrea Lombardi Borgia,
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Chiara Marchionni, Marcella Nesi, Christian Orrenius, Achille Panzeri, Enrico
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†
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Pesenti, Luisa Rusconi, Maria Beatrice Saccardo, Ermes Vanotti, Ettore Perrone,
†
and Paolo Orsini .
†
Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano (MI), Italy.
‡
§
Accelera Srl, Viale Pasteur 10, 20014 Nerviano (MI), Italy. Current address: Tecan
||
Italia S.r.l., Via Brescia 39, 20063 Cernusco sul Naviglio (MI), Italy. Current address:
Chiesi Farmaceutici S.p.A., Nuovo Centro Ricerche, Largo Belloli 11/a, 43122 Parma,
Italy.
Abstract
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The 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
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(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 different human tumor types,
and acting as drivers in specific genetic contexts define subsets of patients that are highly
responsive to kinase inhibitors such as imatinib in BCR-ABL positive chronic
myelogenous leukemia (CML) and erlotinib in epidermal growth factor receptor (EGFR)-
1
,2
mutated non-small cell lung cancer (NSCLC). The anaplastic lymphoma kinase (ALK),
a receptor tyrosine kinase (RTK) belonging to the insulin receptor superfamily, is a well
3,4
validated target based on preclinical and clinical studies.
A constitutively activated, oncogenic form of ALK has been firstly found in a subset of
anaplastic large cell lymphoma (ALCL), namely the chimeric protein NPM-ALK,
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resulting from a chromosomal translocation, which causes the fusion of the N-terminal
portion of the normally expressed protein nucleophosmin (NPM) to the ALK kinase
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domain. Subsequently additional ectopically expressed, activated fusion forms of ALK
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have been identified and recognized as potent oncogenic drivers in other tumor types
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such as inflammatory myofibroblastic tumors (IMT) and, most importantly in a subset of
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NSCLC patients where they account for nearly 3-7% of cases. The majority of ALK-
positive NSCLC harbors the same intrachromosomal rearrangement that results in the
expression of EML4-ALK chimeric protein, generated from the fusion of the N-terminal
portion of echinoderm microtubule-associated protein-like-4 (EML4) and the ALK
kinase domain. ALK fusion partners invariably provide a protein/protein interaction
domain that induces ALK oligomerization and leads to constitutive activation of the
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tyrosine kinase. Other tumorigenic forms of the ALK kinase are due to the acquisition of
single point mutations that lead to the expression of activated forms of the full-length
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receptor, as reported for example in both familial and sporadic neuroblastoma.
Being ALK a well validated target in oncology since many years, several ALK inhibitors
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have been identified and entered clinical trials, such as crizotinib from Pfizer, ceritinib
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from Novartis, alectinib from Chugai/Roche, and AP26113 from Ariad (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
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has been granted accelerated approval in 2015 by the Food and Drug Administration
(FDA) for the treatment of ALK-positive NSCLC patients who progressed after
crizotinib treatment.
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However, despite the initial clinical benefit demonstrated in specific clinical settings with
ALK inhibitors, relapse was invariably observed in all treated patients. Based on clinical
experience accumulated to date with crizotinib it clearly emerged that low brain
penetration represents a major weakness of this drug and appearance of brain metastases
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is a demonstrated cause of disease recurrence in crizotinib-treated patients. In addition,
the emergence of mutations in the ALK kinase domain that confer resistance to the drug
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was reported for the first as well as the second generation ALK inhibitors.
Due to the relevant role of the ALK oncogene in different tumor settings, we performed a
high-throughput screening (HTS) of our Corporate Compound Collection with the aim to
identify suitable hits for further optimization as ALK kinase inhibitors. Among different
hits, the 3-amino-5-substituted indazole 1 emerged as a promising starting point for
further investigation (Figure 2). This compound showed a good biochemical potency
against the ALK kinase (IC = 0.073 ꢀM) and an appreciable antiproliferative activity on
5
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the ALK-dependent ALCL Karpas-299 cell line (IC = 0.253 ꢀM).
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The chemical structure of 1 was considered amenable to structural modifications, which
we believed to be potentially suitable for improvement of its biological activity. Among
the available options, we focused our attention on the substitution pattern of the
fluorinated phenyl ring B, the introduction of substituents on ring A and the modification
of the N-methylpiperazinyl solubilizing group at the para position of ring A.
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Here we describe the optimization process of compound 1 towards our final candidate
compound 2 (entrectinib, Figure 3), a potent ALK inhibitor characterized by good oral
absorption in rodent and non rodent animal species, and good efficacy in in vivo animal
tumor models. Most importantly, compound 2 turned out to be a highly potent inhibitor
of the closely related RTKs ROS1 and TRKs, that were recently found rearranged in
different tumor types. The expression of oncogenic fusion proteins containing
constitutively activated ROS1 kinase domain have been identified in a subset of NSCLC
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patients (1-2% of cases). Analogously chromosomal rearrangements involving the
neurotrophic tyrosine receptor kinase 1 gene (NTRK1), that encodes for the high affinity
nerve growth factor (NGF) receptor tropomyosin receptor kinase A (TRKA), have been
2
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found in around 12% of papillary thyroid carcinoma and very recently reported in a
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subset of NSCLC and colorectal carcinoma (CRC) patients as well as in additional
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tumor types (Spitz tumors, intrahepatic cholangiocarcinoma, glioblastoma and
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pediatric high grade glioma ). Although the frequency of such events in these neoplastic
diseases remains to be defined due to the low number of patients screened so far, these
findings support the rationale for targeted therapy with TRKA inhibitors in selected
patient populations. Thus the activity of compound 2 on these kinases represents a major
opportunity for clinical development of the compound in additional, ROS1- and TRK-
dependent clinical settings. Based on these findings together with the preclinical data in
different models, compound 2 is currently undergoing phase I/II clinical trials for the
treatment of patients affected by ALK-, ROS1- and TRK-positive tumors where it has
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already shown remarkable activity.
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Chemistry
Target compounds were obtained by coupling either scaffold I or II (Scheme 1) with
benzoic acid derivatives of general formula III (Scheme 2 and Scheme 3). Key step in the
synthesis of intermediates I and II is the Suzuki-Miyaura cross-coupling reaction
between different substituted benzyl bromides and commercially available 3-cyano-4-
fluorophenylboronic acid in the presence of Pd(Ph P) complex as a catalyst and K PO
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as a base.
Methyl group in scaffold II has been introduced by reaction of the corresponding
diarylmethane with methyl iodide and LiHMDS.
Synthesis of 3-acyl-5-benzyl 3-aminoindazoles 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
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-amino group of the scaffold.
Removal of the trifluoroacetamido protecting group provided the desired target molecules
, 3, 5-10, 12-16, 18, 20-25 and 28-31. Amino group in compound 4 was obtained by
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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 under standard
basic conditions (LiOH, water, methanol). 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.
Results and Discussion
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Structure Based Design. In order to design new ALK inhibitors starting from compound
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, we took advantage of the in-house available structure of the ALK kinase in complex
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with the weak prototype ALK inhibitor PHA-E429 (see Figure 4, right panel).
Analysis of this structure suggests that appropriately mono substituted A ring at position
2’ (Figure 4, left panel) would yield derivatives occupying an area in and around the
adenosine triphosphate (ATP) sugar pocket while displacing the observed water molecule
W1 (Figure 4, right panel). In particular, NH-R substituents at that position of ring A
were predicted to achieve this result while stabilizing the bioactive conformation through
an intramolecular hydrogen bond involving the hydrogen of the ortho amino group and
the carbonyl of the adjacent carboxamido group (Figure 4, left panel).
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As far as the nature of the group R is concerned, a combined medicinal chemistry and
docking strategy using sugar-resembling substituents led to the prioritization of saturated
aliphatic rings containing nitrogen, oxygen or hydroxyl groups. Such cyclic R
substituents were expected to rotate with respect to the plane of the hinge binder
optimally filling the available 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 expected to be less promising due to their
flexibility. This approach produced a subset of new compounds driving both biochemical
and cellular potencies in the desired direction (see Table 1 and further discussion). In
particular compound 2 emerged as one of the most interesting compounds in terms of
biochemical potency, with the tetrahydropyranyl ring at position 2’ of phenyl ring A
displaying an optimal fitting within the ATP sugar pocket.
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The 2.2 Å structure of ALK in complex with compound 2 (Figure 5) was indeed in
agreement with the above expectations based on the modeling prediction. The compound
is anchored to the hinge region via the canonical donor-acceptor-donor hydrogen bonding
motif between the nitrogens on the aminoindazole moiety and the backbone of residues
Glu1197 and Met1199. The difluorobenzyl-indazole core of the compound also makes
favorable contacts with Leu1256, Leu1122, Val1130 and the gatekeeper Leu1196. The
Glycine-rich loop adopts a peculiar “collapsed” conformation, which was not previously
reported in publicly available ALK structures, so that the 3,5-difluorobenzyl moiety is
favourably stacked between Leu1256 and Phe1127 from the Glycine-rich loop.
Moreover, the fluorine atom pointing towards the interior of the pocket is involved in
multipolar interactions with the backbone carbonyls of Gly1269 (DFG-1 residue) and
Asn1254. The phenyl ring A makes a hydrophobic contact with the Glycine–rich loop
residue Leu1122, while the methylpiperazine moiety protrudes into the solvent. As
expected, the partially solvent-exposed tetrahydropyranyl moiety adopts a roughly
orthogonal orientation with respect to the scaffold, in order to optimally fill the space
underneath the hydrophobic Glycine-rich loop.
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SAR study. We started the optimization study of compound 1 by varying the substitution
pattern on ring A. This investigation was mainly focused on R substituent (see Table 1)
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at position 2’, according to the evaluation deriving from the structural and computational
studies previously discussed. In Table 1, the biochemical IC values (ꢀM) for ALK and
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for the most critical kinases among the hit ones, i.e. IR (insulin receptor) and its closest
family member IGF1R (insulin-like growth factor 1 receptor), are reported along with the
antiproliferative activity on the ALK-dependent ALCL Karpas-299 cell line (IC50
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evaluated after 72 h treatment). Generally, a nearly 10-fold or greater selectivity versus
IR and IGF1R is observed for all the compounds. In the toxicological studies performed
later on our candidate compound 2, this selectivity ratio was considered safe based on the
experimental determination of glucose and insulin levels.
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The introduction of an unsubstituted amino group at R led to compound 4, which
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displays an unchanged potency respect to 1. This was in line with modeling predictions
as compound 4 lacks the crucial substituent suitable to fill the sugar pocket.
R aminoaliphatic chains substituted with a methoxy group, generally led to a moderate
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loss in ALK activity (see compounds 5, 7, and 8). At best, activity was conserved (see
compound 6) without any improvement with the exception for the good cellular activity
of 6 in the double-digit nanomolar range.
Compounds 9 and 11, bearing shorter two carbon aliphatic chains decorated with a
fluorine and a hydroxyl group respectively, display a surprisingly good biochemical
potency on ALK. Compound 9 also shows good antiproliferative cellular activity (IC =
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.049 ꢀM). The data related to 12, 13 and 14 suggest that they cannot properly
accomodate the ortho substituent of ring A within the sugar pocket. Compound 15, with
an aminocyclohexyl ring in R is poorly active, but when it is “decorated” with a
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hydroxyl group as in 17, good biochemical and cellular activity are restored. In particular,
the trans diastereoisomer 17 is better than the cis one 19 in both enzymatic and cellular
assays. The crystal structure of 17 (see Supporting Information, Figure S1) revealed a
hydrogen bond interaction mediated by a water molecule of the hydroxyl group with a
carbonyl of His1124 in the glycine-rich loop, thus potentially explaining the observed
activity of this compound. When R is a 4-amino-N-methylpiperidinyl or a 4-amino-
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tetrahydropyranyl ring, compounds 20 and 2 display a very good biochemical potency on
ALK with IC values of 0.015 ꢀM and 0.012 ꢀM, respectively. However, the former
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a Caco-2 permeability experiment (Papp A-B = 1.5 10 cm/s).
Structure-activity relationship (SAR) Tables 2, 3 and 4 show results where the 4-
aminotetrahydropyranyl ring was maintained at position 2’ of ring A, and the other
regions of the structure were modified.
As reported in Table 2, the introduction of a methyl onto linker L afforded a racemic
mixture which upon chiral column chromatography separation, yielded enantiomers 26
and 27 whose absolute stereochemistry has not been assigned. The two compounds have
a moderately different activity on ALK with 27 showing the best potency. Compared to
compound 2 it displays a stronger activity in cells. However, the labour-intensive chiral
separation and the labile nature of the benzhydryl stereogenic center discouraged any
further development of this compound.
The exploration of ring B confirmed the contribution of the fluorine interaction with the
protein observed in the crystal structure. As depicted in Table 3, removal of both
fluorines caused a nearly 10-fold decrease of biochemical activity on ALK and,
correspondingly, of antiproliferative activity in Karpas-299 cells (see compound 28). The
presence of a single fluorine as in compound 29 was sufficient to restore the activity on
ALK analogously to the introduction of both halogens with the different 2,5-
regiochemistry (see compound 30). Both compounds display an activity profile similar to
compound 2. A decrease in the potency was observed when replacing one fluorine with a
methyl group in the 2,5-substituted ring B (see derivative 31). This substitution is likely
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to negatively impact the geometry of the stacking interaction of phenyl B with the
phenylalanine 1127 of the glycine-rich loop.
Finally, we addressed the solubilizing group with variations in the R position as reported
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in Table 4. All of these compounds compare favorably with derivative 2, in terms of
potency on ALK, of selectivity versus IGF1R and IR and of antiproliferative activity in
Karpas-299. Among them analog 22 was from 3- to 5-fold more potent on ALK than the
others and more active on Karpas-299 cells. All of these compounds showed an improved
aqueous solubility with respect to 2 (see Table 5) and appeared worth of further study.
In terms of lipophilic ligand efficiency, a significant improvement has been observed for
compound 2 compared to the starting compound 1 (see Supporting Information, Table 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.
Based on 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 parallel artificial membrane permeability assay (PAMPA), 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
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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.
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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 IC of 12 nM on the ALK kinase. To better
5
0
characterize the inhibitor potency on the target, we then calculated a Ki of 6.2 nM, which
is in line with the calculated IC . Compound 2 was even more potent on the kinases
5
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ROS1 (IC50 = 0.007 ꢀM) and on TRKA (IC50 = 0.001 ꢀM). Only on two additional
kinases, JAK2 and ACK1, the compound shows a less than 10-fold selectivity. For
additional twelve kinases a selectivity ratio equal or greater than 10 is observed, while for
the other 40 kinases at least a 100-fold selectivity is observed.
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Based on the observed activity on TRKA, the biochemical potencies on the related
kinases TRKB and TRKC were also evaluated and found to be in the single-digit
nanomolar range (TRKB IC = 0.002 ꢀM and TRKC IC = 0.005 ꢀM).
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Then compound 2 was profiled on a panel of nearly 200 human tumor cell lines (see
Table 8). The anti-proliferative activity observed is consistent with the compound kinase
selectivity profile, as compound 2 displays an IC50 below 0.1 ꢀM on different ALK-
dependent cancer cells, such as ALCL cell lines Karpas 299, SU-DHL-1, SUP-M2 and
SR-786 and NSCLC cell line NCI-H2228. In addition it also potently inhibits (IC =
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0
.017 ꢀM) the proliferation of KM12, a human CRC cell line whose growth was recently
found to be TRKA-dependent due to the presence of the oncogenic tropomyosin 3-
24
NTRK1 (TPM3-NTRK1) chromosomal rearrangement whereas the antiproliferative
activity observed in MV-4-11 is presumably due to the cross-reactivity with FLT3. The
additional not reported IC s are comprised between 0.1 and 1 ꢀM for 11 cell lines and
50
greater than 1 ꢀM for the remaining cell lines.
In ALK-dependent models the inhibition of cell growth was correlated with the
modulation of the activated target in cells. In the ALCL cell line Karpas-299 and in
NSCLC cell line NCI-H2228 the ability of the compound to modulate the
autophosphorylation of the ALK kinase was assessed after 2 hrs treatment at different
concentrations. As reported in Figure 6, a complete inhibition of ALK phosphorylation
can be clearly appreciated in both cell lines at very low doses. Consistently a dose-
dependent inhibition of phosphorylation of the downstream effector STAT3 is also
clearly observed.
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To further explore the cellular activity on TRKA, the modulation of target
phosphorylation was also studied on KM12, a TRKA-dependent cellular model. KM12
cells have been treated for 2 hrs with compound 2 at different concentrations and then the
phosphorylation of TRKA and downstream transducers has been evaluated. As reported
in Figure 7, a clear inhibition of target and signaling pathway phosphorylation is
observed at low compound concentration, supporting the rationale for considering
TRKA-positive patients for clinical development of compound 2.
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To evaluate and compare the inhibition by compound 2 of its main targets in the same
cellular environment, the interleukine-3 (IL-3)-dependent Ba/F3 cell line was transfected
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and made dependent for survival on ALK, ROS1, TRKA, TRKB, and TRKC kinases.
These IL-3-independent cell lines were treated with compound 2, which has shown the
antiproliferative activities reported in Table 9.
Compound 2 proved to be very potent in inhibiting the growth of ALK-, ROS1-, TRKA-,
TRKB-, and TRKC-dependent Ba/F3 cell lines with IC values in the low nanomolar
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range. As expected, compound 2 was poorly active in inhibiting the proliferation of IL-3-
dependent Ba/F3 cell line (IC = 2.1 ꢀM).
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As the emergence of resistance mutations is frequently responsible for relapse in patients
treated with ALK inhibitors, we tested compound 2 in a series of Ba/F3 cell lines
transfected with different crizotinib or ceritinib-resistant mutated forms of EML4-ALK.
Entrectinib demonstrated a good antiproliferative activity on EML4-ALK-wt (Ba/F3-
EML4-ALK-wt IC = 0.028 ꢀM), and on the gate-keeper mutant EML4-ALK-L1196M
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(
Ba/F3-EML4-ALK-L1196M IC = 0.067 ꢀM), but it proved to be poorly active on the
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EML4-ALK-G1202R mutant (Ba/F3-EML4-ALK-G1202R IC50 = 0.897 ꢀM), as
expected based on entrectinib mode of binding.
Compound 2 was tested for evaluating its in vivo efficacy in different ALK-dependent
tumor models. In the experiment reported in Figure 8 severe combined immune
deficiency (SCID) mice bearing Karpas-299 xenograft tumors were treated orally with
the compound at the doses of 30 and 60 mg/kg twice a day for ten consecutive days.
Excellent efficacy 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.
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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
hours after compound administration and is still evident at 18 hrs.
The in vivo efficacy of compound 2 was then evaluated in the ALK-dependent NSCLC
NCI-H2228 xenograft model. In the experiment reported in Figure 9 the compound was
administered orally at the doses of 30 and 60 mg/kg twice a day for ten 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 hrs after single oral administration.
Since, as mentioned above, low BBB penetration is the major weakness of crizotinib and
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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.
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In order 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 i.v. and
oral administration as reported in Table 11. Good exposure and moderate to good oral
bioavailability have been reached in both species, with volume of distribution indicative
of tissue distribution and low clearance in both animal species. This profile allowed the
evaluation of the safety and tolerability of this compound in further animal studies.
Conclusions
In conclusion we have identified a novel 3-aminoindazole ALK inhibitor (compound 2)
through the optimization of the starting hit compound 1. Compound 2 is a potent ALK,
ROS1 and TRK inhibitor with nanomolar activity on the corresponding target-driven cell
lines. It shows a favourable pharmacokinetic profile in rodent and non-rodent animal
species, it induces stable tumor regression in ALK-dependent ALCL and NSCLC human
xenograft tumor models with a clear inhibiton of ALK and downstream effector
phosphorylation. Compound 2, based on its permissive safety and tolerability profile, was
selected for development and it is currently undergoing phase I/II clinical trials for the
treatment of patients affected by ALK-, ROS1- and TRKs-dependent tumors with
29,30
remarkable signs of activity.
An exhaustive biological and pharmacological profile of
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compound 2, including its activity on a panel of resistant mutants identified in patients
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treated with different ALK inhibitors, is under publication.
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Experimental section
1. Chemistry
Unless otherwise noted, solvents and reagents were obtained from commercial suppliers
and used without further purification. All reactions involving air- or moisture-sensitive
reagents were performed under an argon atmosphere. All final compounds were purified
to >95% purity as determined by high-performance liquid chromatography (HPLC).
HPLC-MS/UV analyses were performed on a LCQ DecaXP (Thermo, San Jose, US) ion
trap instrument, equipped with an electrospray (ESI) ion source. The mass spectrometer
is connected to a Surveyor HPLC system (Thermo, San Jose, US) with an UV photodiode
array detector (UV detection 215-400 nm). A Waters XSelect CSH C18 column 50x4.6
mm, 3.5 ꢀm particle size was used. Mobile phase A was ammonium acetate 5 mM buffer
(pH 4.5 with acetic acid):acetonitrile 95:5, and mobile phase B was ammonium acetate 5
mM buffer (pH 4.5 with acetic acid): acetonitrile 5:95. Gradient from 0 to 100 % B in 7
minutes, hold 100% B 2 minutes. Flow rate 1 mL/min. Injection volume 10 ꢀL. Full scan,
mass range from 50 to 1200 amu. Heated capillary temp was 275 °C and Spray voltage
value was set at 4kV. Mass are given as m/z 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 silica gel (Merck silica
gel 40-63 ꢀm) or on prepacked silica gel cartridges (Biotage) on a Horizon system. 1H
NMR spectra were acquired at 25° C in DMSO-d6 on a Varian Inova 400 spectrometer
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operating at 400 MHz and equipped with a 5 mm 1H{15N-31P} Z-axis-PFG Indirect
Detection Probe. Residual not-deuterated solvent signal was used as reference with δ =
2
.50 ppm for DMSO-d5. Data are reported as follows: chemical shift, multiplicity (s =
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singlet, d = doublet, t = triplet, q = quartet, quint = quintet, bs = broad singlet, bd = broad
doublet, dd = doublet of doublet, td = triplet of doublet, m = multiplet), coupling
3
6
constants, and number of protons. As formerly reported, ESI(+) high-resolution mass
spectra (HRMS) were obtained on a Q-Tof Ultima (Waters, Manchester, UK) mass
spectrometer directly connected with a Agilent 1100 micro-HPLC system (Palo Alto,
CA, US). Thin-layer chromatography was performed on Merck silica gel 60 plates coated
with 0.25 mm layer with fluorescent indicator. Components were visualized by UV light
(λ = 254 and 366 nm) and iodine vapors.
Synthesis of Scaffold I and II:
General procedure for diarylmethanes preparation: 3-Cyano-4-fluorophenylboronic acid
(
1 equiv), powdered K PO (2 equiv) and Pd(PPh ) (2% mol) were charged in an oven-
3 4 3 4
dried flask under argon atmosphere. The flask was evacuated and back-filled with argon
three times. Toluene (3 mL/mmol boronic acid) and benzyl bromide (1 equiv) were added
under good stirring. The reaction mixture was heated to 100 °C in half an hour and
maintained at that temperature for 1.5 - 8 hours. The dark mixture was taken up with
diethyl ether, washed with saturated aqueous NH Cl and brine. The organic phase was
4
dried over anhydrous Na SO and evaporated to dryness. The residue was then purified
2
4
by flash chromatography (n-hexane/ethyl acetate) to provide the desired diarylmethane in
yields of 60-90%.
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General procedure for 3-aminoindazoles preparation: A mixture of 3-cyano-4-fluoro-
diarylmethane (1 equiv) and hydrazine hydrate (5 equiv) in n-butanol (2.5 mL/mmol
diarylmethane) was refluxed overnight. The reaction mixture was diluted with
water/ethyl acetate and the organic phase was washed twice with brine, dried and
evaporated. The residue was purified by flash chromatography (CH Cl /EtOH) to provide
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the desired 3-aminoindazole in yields of 75-90%.
General procedure for diarylmethanes alkylation: A mixture of 3-cyano-4-fluoro-
diarylmethane (1 equiv) and methyl iodide (1.5 equiv) was dissolved in THF dry (8
mL/mmol diarylmethane) under nitrogen atmosphere at -20 °C. Bis-(trimethylsilyl)-
lithiumamid 1.0 M in THF (2 equiv) was gradually added. After 20 minutes the reaction
was quenched by adding a solution of KHSO 10% and extracted with ethyl acetate. The
4
organic phase was washed with aqueous KHSO 10% and brine, dried over sodium
4
sulfate and evaporated to dryness. The crude was purified by flash chromatography
(hexane/ethyl acetate) to provide the desired alkylated diarylmethane in yields of 70-
8
5%.
Synthesis of 3-acyl-5-benzyl indazoles 2, 3, 5-10, 12-16, 18, 20-25 and 28-31
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-yl)-2-
(tetrahydro-pyran-4-ylamino)-benzamide (2)
To a suspension of 4-(4-methyl-piperazin-1-yl)-2-[(tetrahydro-pyran-4-yl)-(2,2,2-
trifluoro-acetyl)-amino]-benzoic acid trifluoroacetate (10 g, 22.1 mmol) in dry
dichloromethane (300 mL) oxalyl chloride (3.58 mL, 42.3 mmol) and N,N-
dimethylformamide (1-2 drops) were added. The mixture was stirred at room temperature
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for 2 hours then evaporated to dryness. The resulting crude acyl chloride was taken-up
with toluene, evaporated and then dissolved in dry tetrahydrofuran (130 mL) at –20 °C. A
solution of 5-(3,5-difluoro-benzyl)-1H-indazol-3-ylamine (5 g. 19.28 mmol) and N,N-
diisopropylethylamine (12.8 mL, 73.3 mmol) in dry THF (40 mL) was added to the
cooled reaction mixture. The mixture was stirred at –20 °C for 4 hours then quenched by
adding water/ethyl acetate. The organic phase was washed with a saturated solution of
sodium hydrogenocarbonate, dried over sodium sulfate and evaporated to dryness.
Crude reaction mixture is dissolved in methanol (375 mL) in the presence of
triethylamine (60 mL) and stirred at 65 °C for 2 hours. The solvents were removed under
reduced pressure and the residue treated with water/ethyl acetate. Organic phase was
dried over sodium sulfate and evaporated to dryness. Purification of the crude by
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chromatography over silica gel (DCM/EtOH/NH 5N in MeOH = 1000/50/5) and
3
crystallisation of the so obtained compound from ethyl acetate/hexane afforded 8.4 g of
the title compound as a white solid (78% yield).
1
H NMR (400.5 MHz, DMSO-d ) δ ppm 1.29 - 1.41 (m, 2H) 1.89 – 1.97 (m, 2H) 2.24 (s,
6
3 H) 2.41 - 2.48 (m, 4H) 3.23 - 3.29 (m, 4 H) 3.49 (ddd, J=11.7, 10.2, 2.3 Hz, 2 H) 3.62 -
3
.72 (m, 1 H) 3.80 (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)
.23 (dd, J=9.0, 2.2 Hz, 1 H) 6.93 - 7.04 (m, 3 H) 7.25 (dd, J=8.7, 1.5 Hz, 1 H) 7.40 (d,
6
J=8.7 Hz, 1 H) 7.48 (s, 1 H) 7.80 (d, J=9.0 Hz, 1 H) 8.29 (d, J=7.6 Hz, 1 H) 10.07 (s, 1
+
H) 12.62 (s, 1 H). LCMS (ESI) m/z 507 (M + H) ; HRMS (ESI) calcd for C H F N O
31
34
2
6
2
+
+ H 561.2784, found 561.2785.
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
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4
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5
5
5
5
5
5
5
5
5
5
6
By employing the above-described procedure, starting from scaffold I or II and benzoic
2
acid III , the following compounds were prepared:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-yl)-2-nitro-
benzamide (3)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400.5 MHz, DMSO-d ) δ ppm 2.23 (s, 3 H) 2.42 - 2.47 (m, 4 H) 3.33 - 3.38
6
(m, 4 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, 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, 1 H) 7.69 (br. s., 1 H) 10.81 (br.
+
s., 1 H) 12.70 (s, 1 H). LCMS (ESI) m/z 561 (M + H) ; HRMS (ESI) calcd for
+
C H F N O + H 507.1951, found 507.1947.
26
24
2
6
3
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-methoxy-ethylamino)-4-(4-methyl-
piperazin-1-yl)-benzamide (5)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400.5 MHz, DMSO-d ) δ ppm 2.25 (s, 3 H) 2.44 - 2.49 (m, 4 H) 3.26 (s, 3 H)
6
3
.27 - 3.31 (m, 6 H) 3.54 (t, J=5.5 Hz, 2 H) 4.05 (s, 2 H) 6.09 (d, J=1.9 Hz, 0 H) 6.25
dd, J=9.0, 1.9 Hz, 0 H) 6.94 - 7.04 (m, 3 H) 7.24 (dd, J=8.7, 1.6 Hz, 1 H) 7.41 (d, J=8.7
Hz, 1H) 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 (s, 1 H) 12.63
(
+
+
(
s, 1 H). LCMS (ESI) m/z 535 (M + H) ; HRMS (ESI) calcd for C H F N O + H
29 32 2 6 2
535.2628, found 535.2632.
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8
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N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-((R)-2-methoxy-1-methyl-
ethylamino)-4-(4-methyl-piperazin-1-yl)-benzamide (6)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/10/1.
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
H NMR (400.5 MHz, DMSO-d ) δ ppm 1.14 (d, J=6.3 Hz, 3 H) 2.23 (s, 3 H) 2.41 - 2.47
6
(m, 4 H) 3.24 - 3.29 (m, 4 H) 3.27 (s, 3 H) 3.30 - 3.40 (m, 2 H) 3.74 - 3.83 (m, 1 H) 4.05
(
(
(
(
s, 2 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 (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 (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 H) 12.63 (s, 1 H). LCMS (ESI) m/z 549 (M + H) ; HRMS
+
ESI) calcd for C H F N O + H 549.2784, found 549.2787.
3
0
34
2
6
2
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(3-methoxy-propylamino)-4-(4-
methyl-piperazin-1-yl)-benzamide (7)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.79 (quin, J=6.5 Hz, 2 H) 2.22 (s, 3 H) 2.41 -
6
2.46 (m, 4 H) 3.17 (q, J=6.5 Hz, 2 H) 3.21 (s, 3 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, J=2.2 Hz, 1H) 6.23 (dd, J=9.0, 2.2 Hz, 1 H) 6.93 – 7.03 (m, 3
H) 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) 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 (s, 1 H). LCMS (ESI) m/z 549 (M + H) ;
+
HRMS (ESI) calcd for C H F N O + H 549.2784, found 549.2780.
3
0
34
2
6
2
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-methoxy-1-methoxymethyl-
ethylamino)-4-(4-methyl-piperazin-1-yl)-benzamide (8)
Eluant for column chromatography: DCM/MeOH/NH 5N in MeOH = 100/5/1.
3
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 2.42 (br. s., 3 H) 2.70 (br. s., 4 H) 3.18 – 3.44
6
(m, 4 H) 3.26 (s, 6 H) 3.41 (d, J=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 H) 6.26 (dd, J=8.9, 2.1 Hz, 1 H) 6.94 – 7.03 (m, 3 H) 7.24 (dd, J=8.9, 1.5 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H) 7.41 (d, J=8.7 Hz, 1 H) 7.48 (s, 1 H) 7.79 (d, J=8.9 Hz, 1 H) 8.32 (d, J=8.3 Hz, 1 H)
+
10.06 (s, 1 H) 12.64 (s, 1 H). LCMS (ESI) m/z 579 (M + H) ; HRMS (ESI) calcd for
+
C H F N O + H 579.2890, found 579.2890.
31
36
2
6
3
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-2-(2-fluoro-ethylamino)-4-(4-methyl-
piperazin-1-yl)-benzamide (9)
Eluant for column chromatography: DCM/MeOH = 95/5.
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 2.24 (s, 3 H) 2.43 - 2.48 (m, 4 H) 3.26 - 3.31
6
(m, 4 H) 3.48 (ddt, J =27.7, J =5.4, 4.9, 4.9 Hz, 2 H) 4.04 (s, 2 H) 4.60 (dt, J =47.7,
HF HH HF
JHH=4.9, 4.9 Hz, 2 H) 6.12 (d, J=2.2 Hz, 1 H) 6.27 (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 H) 7.40 (d, J=8.5 Hz, 1 H) 7.50 (s, 1 H) 7.80 (d, J=9.0
Hz, 1 H) 8.36 (t, J=5.4 Hz, 1 H) 10.10 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 523 (M +
(
+
+
H) ; HRMS (ESI) calcd for C H F N O + H 523.2428, found 523.2427.
2
8
29
3
6
2-[(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)amino]-N-[5-(3,5-difluorobenzyl)-1H-
indazol-3-yl]-4-(4-methylpiperazin-1-yl)benzamide (10)
+
ESI(+) MS: m/z 635 (MH ).
N-[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]-2-{[(1-methylazetidin-3-
yl)methyl]amino}-4-(4-methylpiperazin-1-yl)benzamide (12)
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Journal of Medicinal Chemistry
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5
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7
8
9
Eluant for column chromatography: DCM/MeOH/NH 5N in MeOH = 100/10/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 2.25 (s, 3 H) 2.43-2.48 (m, 4 H) 2.62 (br. s., 3
6
H) 2.85 - 2.95 (m, 1 H) 3.31 (m overlapped by water signal, 4 H) 3.37 - 3.42 (m, 2 H)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
.57 (br. s., 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 (dd,
J=8.9, 2.1 Hz, 1 H) 6.93 - 6.99 (m, 2 H) 6.99 - 7.05 (m, 1 H) 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) 7.81 (d, J=8.9 Hz, 1 H) 8.24 (t, J=5.2 Hz, 1 H)
+
1
0.11 (s, 1 H) 12.63 (s, 1 H). LCMS (ESI) m/z 560 (M + H) ; HRMS (ESI) calcd for
+
C H F N O + H 560.2944, found 560.2954.
31
35
2
7
2-Benzylamino-N-[5-(3,5-difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-
yl)-benzamide (13)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 2.21 (s, 3 H) 2.36 – 2.44 (m, 4 H) 3.19 - 3.24
6
(m, 2 H) 4.03 (s, 2 H) 4.38 (d, J=5.6 Hz, 2 H) 6.08 (d, J=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 - 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 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 H)
+
1
0.10 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z 567 (M + H) ; HRMS (ESI) calcd for
+
C H F N O + H 567.2679, found 567.2680.
33
32
2
6
N-[5-(3,5-Difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-piperazin-1-yl)-2-
phenylamino-benzamide (14)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 2.23 (s, 3 H) 2.46 (br. s., 4 H) 3.18 - 3.24 (m, 4
6
H) 4.04 (s, 2 H) 6.52 (dd, J=9.0, 2.2 Hz, 1 H) 6.73 (d, J=2.2 Hz, 1 H) 6.93 - 7.02 (m, 4 H)
7
1
.16 – 7.20 (m, 2 H) 7.24 (dd, J=8.6, 1.5 Hz, 1 H) 7.28 - 7.34 (m, 2 H) 7.41 (d, J=8.6 Hz,
H) 7.54 (s, 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 H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
LCMS (ESI) m/z 553 (M + H) ; HRMS (ESI) calcd for C H F N O + H 553.2522,
3
2
30
2
6
found 553.2499.
2
-Cyclohexylamino-N-[5-(3,5-difluoro-benzyl)-1H-indazol-3-yl]-4-(4-methyl-
piperazin-1-yl)-benzamide (15)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.16 – 1.30 (m, 3 H) 1.33 - 1.45 (m, 2 H) 1.48 -
6
1
3
.58 (m, 1 H) 1.57 - 1.69 (m, 2 H) 1.83 - 1.92 (m, 2 H) 2.24 (s, 3 H) 2.41 – 2.48 (m, 4 H)
.22 - 3.27 (m, 4 H) 3.40 - 3.52 (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 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=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 Hz, 1 H) 10.04 (s, 1 H) 12.61 (s,
+
+
1
5
H). LCMS (ESI) m/z 559 (M + H) ; HRMS (ESI) calcd for C H F N O + H
32 36 2 6
59.2992, found 559.2992.
trans-4-{[2-{[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]carbamoyl}-5-(4-
methylpiperazin-1-yl)phenyl]amino}cyclohexyl benzoate (16)
+
ESI(+) MS: m/z 679 (MH ).
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Journal of Medicinal Chemistry
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cis-4-{[2-{[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]carbamoyl}-5-(4-methylpiperazin-
1
-yl)phenyl]amino}cyclohexyl benzoate (18)
+
ESI(+) MS: m/z 679 (MH ).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N-[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-[(1-
methylpiperidin-4-yl)amino]benzamide (20)
Eluant for column chromatography: DCM/MeOH/NH 5N in MeOH = 100/10/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.35 - 1.48 (m, 2 H) 1.87 – 1.97 (m, 2 H) 2.18
6
(br. s., 3 H) 2.21 (br. s., 2 H) 2.23 (s, 3 H) 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 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 H) 6.94 – 7.03 (m, 3H) 7.24 (dd, J=8.6, 1.5 Hz, 1 H) 7.40 (d, J=8.5 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 H) 10.06 (s, 1 H) 12.62 (s, 1 H).
+
+
LCMS (ESI) m/z 574 (M + H) ; HRMS (ESI) calcd for C H F N O + H 574.3101,
3
2
37
2
7
found 574.3099.
1
4
4
N -[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]-N -[2-(dimethylamino)ethyl]-N -methyl-
2
-(tetrahydro-2H-pyran-4-ylamino)benzene-1,4-dicarboxamide (21)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/10/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.31 - 1.42 (m, 2 H) 1.87 - 1.95 (m, 2 H) 2.04
6
(br. s., 3.25 H, rotamer) 2.24 (br. s., 2.75 H, rotamer) 2.39 (br. s., 1.08 H, rotamer) 2.50
(overlapped by DMSO-d6 signal, 0.92 H, rotamer) 2.91 (br. s., 1.38 H, rotamer) 2.97 (br.
s., 1.63 H, rotamer) 3.31 (overlapped by water signal, 1.08 H), rotamer) 3.45 - 3.52 (m, 2
H) 3.54 (br. s., 0.92 H, rotamer) 3.62 - 3.72 (m, 1 H) 3.79 - 3.85 (m, 2 H) 4.05 (s, 2 H)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
7
1
.55 (d, J=7.9 Hz, 1 H) 6.75 (s, 1 H) 6.94 - 7.04 (m, 3 H) 7.27 (dd, J=8.6, 1.6 Hz, 1 H)
.43 (d, J=8.6 Hz, 1 H) 7.53 (s, 1 H) 7.92 (d, J=7.9 Hz, 1 H) 7.95 (d, J=7.7 Hz, 1 H)
+
0.55 (s, 1 H) 12.74 (s, 1 H). LCMS (ESI) m/z 591 (M + H) ; HRMS (ESI) calcd for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C H F N O + H 591.2890, found 591.2891.
32
36
2
6
3
N-[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]-4-(piperazin-1-yl)-2-(tetrahydro-2H-
pyran-4-ylamino)benzamide (22)
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/10/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.30 - 1.41 (m, 2 H) 1.88 – 1.98 (m, 2 H) 2.80 -
6
2.87 (m, 4 H) 3.17 - 3.22 (m, 4 H) 3.45 - 3.54 (m, 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=2.2 Hz, 1 H) 6.22 (dd, J=9.0, 2.2 Hz, 1 H) 6.94 -
.04 (m, 3 H) 7.25 (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 (d,
7
J=9.0 Hz, 1 H) 8.28 (d, J=7.7 Hz, 1 H) 10.06 (s, 1 H) 12.62 (s, 1 H). LCMS (ESI) m/z
+
+
5
47 (M + H) ; HRMS (ESI) calcd for C H F N O + H 547.2628, found 547.2628.
30 32 2 6 2
1-[4-{[5-(3,5-difluorobenzyl)-1H-indazol-3-yl]carbamoyl}-3-(tetrahydro-2H-pyran-
-ylamino)benzyl]piperidine (23)
4
Eluant for column chromatography: DCM/MeOH/NH 5N in MeOH = 100/5/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.32 - 1.44 (m, 3 H) 1.67 - 1.87 (m, 5 H) 1.95 -
6
2
.02 (m, 2 H) 2.82 - 2.93 (m, 2 H) 3.32 (br. s., 2 H) 3.44 - 3.54 (m, 2 H) 3.63 - 3.74 (m, 1
H) 3.82 - 3.91 (m, 2 H) 4.06 (s, 2 H) 4.23 (d, J=5.37 Hz, 2 H) 6.75 - 6.81 (m, 1 H) 6.94 -
.06 (m, 2 H) 7.13 (s, 1 H) 7.29 (dd, J=8.66, 1.46 Hz, 1 H) 7.45 (d, J=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) 10.14 (br. s., 1 H) 10.54 (s, 1 H) 12.77
7
(
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Page 29 of 68
Journal of Medicinal Chemistry
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9
+
(
br. s., 1 H). ). LCMS (ESI) m/z 560 (M + H) ; HRMS (ESI) calcd for C H F N O +
3
2
35
2
5
2
+
H 560.2832, found 560.2831.
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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/NH 5N in MeOH = 100/10/1.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.30- 1.41 (m, 2 H) 1.62 – 1.75 (m, 2 H) 1.87 –
6
2
.00 (m, 4 H) 2.20 – 2.87 (br. s., 5 H) 2.69 (br. s., 2 H) 3.44 - 3.53 (m, 2 H) 3.63 (m, 1 H)
.78 - 3.85 (m, 2 H) 4.04 (s, 2 H) 4.50 (br. s., 1 H) 6.23 - 6.28 (m, 2 H) 6.95 - 7.04 (m, 3
3
H) 7.26 (dd, J=8.7, 1.6 Hz, 1 H) 7.42 (d, J=8.6 Hz, 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 (s, 1 H) 12.67 (s, 1 H). LCMS (ESI) m/z 576 (M + H) ;
+
HRMS (ESI) calcd for C H F N O + H 576.2781, found 576.2773.
3
2
35
2
5
3
N-{5-[1-(3,5-Difluoro-phenyl)-ethyl]-1H-indazol-3-yl}-4-(4-methyl-piperazin-1-yl)-2-
tetrahydro-pyran-4-ylamino)-benzamide (25)
(
Eluant for column chromatography: DCM/EtOH/NH 5N in MeOH = 100/5/2.
3
1
H NMR (400,5 MHz, DMSO-d ) δ ppm 1.30 - 1.40 (m, 2 H) 1.60 (d, J=7.2 Hz, 3 H)
6
1.89 - 1.98 (m, 2 H) 2.28 (br. s., 3 H) 2.50 (overlapped by DMSO-d6 signal, 4 H) 3.30
(overlapped by water signal, 4 H) 3.46 - 3.54 (m, 2 H) 3.64 - 3.74 (m, 1 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 H) 6.25 (dd, J=9.0, 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 (d, J=8.7 Hz, 1 H) 7.51 (s, 1 H)
.80 (d, J=9.0 Hz, 1 H) 8.31 (d, J=7.7 Hz, 1 H) 10.08 (s, 1 H) 12.62 (s, 1 H). LCMS
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