(R)-2-Phenylpyrrolidine substituted imidazopyridazines: A new class of potent and selective pan-TRK inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00050

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have be

Full text of DOI:10.1021/acsmedchemlett.5b00050

Letter
pubs.acs.org/acsmedchemlett
(R)‑2-Phenylpyrrolidine Substituted Imidazopyridazines: A New Class
of Potent and Selective Pan-TRK Inhibitors
Ha-Soon Choi, Paul V. Rucker, Zhicheng Wang, Yi Fan, Pamela Albaugh,‡ Greg Chopiuk,
§
§
Francois Gessier,† Fangxian Sun, Francisco Adrian, Guoxun Liu, Tami Hood,⊥ Nanxin Li, Yong Jia,
Jianwei Che,∥ Susan McCormack, Allen Li, Jie Li, Auzon Steffy, AnneMarie Culazzo, Celine Tompkins,
Van Phung, Andreas Kreusch, Min Lu, Bin Hu,# Apurva Chaudhary,# Mahavir Prashad,# Tove Tuntland,
Bo Liu, Jennifer Harris, H. Martin Seidel,⊥ Jon Loren, and Valentina Molteni*
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Deregulated kinase activities of tropomyosin receptor kinase (TRK)
family members have been shown to be associated with tumorigenesis and poor
prognosis in a variety of cancer types. In particular, several chromosomal
rearrangements involving TRKA have been reported in colorectal, papillary thyroid,
glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic
driver in these tumors. By screening the Novartis compound collection, a novel
imidazopyridazine TRK inhibitor was identified that served as a launching point for
drug optimization. Structure guided drug design led to the identification of (R)-2-
phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving
tumor regression in rats bearing KM12 xenografts. From this work the (R)-2-phenylpyrrolidine has emerged as an ideal moiety to
incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs.
KEYWORDS: Neurotrophins, tropomyosin receptor kinase, TRK, (R)-2-phenylpyrrolidine, imidazopyridazines, GNF-8625
he tropomyosin receptor kinase (TRK) receptors, TRKA,
other cancer settings. For example, BDNF levels have been
TTRKB, and TRKC (also known as NTRK1, NTRK2 and associated with unfavorable pathological parameters and
adverse clinical outcome in breast cancer, and TRKB has
been reported as a strong predictor of aggressive tumor growth
in neuroblastoma.20−23 Selective TRK inhibitors, AZ623 and
GNF-4256, have been shown to inhibit the growth of human
neuroblastoma xenograft tumors and, in combination with
chemotherapy, have prolonged inhibition of tumor re-
growth.24,25 Moreover, high and increased expression of TRK
receptors in pancreatic cancer correlates with tumor pro-
gression, perineural invasion, pain, and metastasis.7,26−28
Given the growing evidence for a role of TRK in cancer there
is interest in discovering developable selective TRK inhibitors
for evaluation in the clinic. Here we describe the discovery of
(R)-2-phenylpyrrolidine substituted imidazopyridazines as a
novel class of selective pan-TRK inhibitors with efficacy in a
KM12 rat tumor model.
The novel benzonitrile substituted imidazopyridazine 1 was
identified as an inhibitor of TRKB (IC50 = 83 nM) from
screening using a TRKB biochemical inhibition assay
(Scintillation Proximity Assay). Submicromolar cellular activity
across the three TRK isoforms was shown in Ba/F3 assays
where Ba/F3 cells were rendered TRKA, TRKB, and TRKC
dependent and IL-3 independent by respectively overexpressing
NTRK3) and their respective neurotrophin ligands (NGF,
BDNF, and NT-3) are implicated in proliferation, survival,
differentiation, and functional regulation of different neuronal
populations in the nervous system, especially during embryo-
genesis.1 In the past decade, the dysregulation of neurotrophin
signaling has been linked to the development and progression
of cancer.2−9 Several different mechanisms have been proposed
involving oncogenic activation of TRK receptors, expression of
alternative and constitutively active splice variants under
hypoxia conditions, point mutations, and genomic rearrange-
ments.2 TRKA rearrangement leading to constitutively active
and ligand-independent fused protein TPM3-TRKA was
originally identified in a colorectal carcinoma biopsy10,11 and
was later found to be associated with a subset of papillary
thyroid carcinomas (PTC).12−15 Recently the TPM3-TRKA
fusion has also been described to be harbored in KM12
colorectal cells.16 Other TRKA fusions with TGF and TPR
have been found in colorectal and thyroid cancers,10−15 and
recently, TRKA fusions with MPRIP and CD74 have been
identified in a subset of patients (3.3%) with lung
adenocarcinoma that did not contain other common genetic
alterations.16 TRKC fusion protein Tel-TRKC has been
identified as the causative event in rare cancers such as
secretory breast carcinoma (SBC),17 congenital fibrosarcoma
(CFS),18 and congenital mesoblastic nephroma (CMN).19 In
addition, there may be a role for small molecule inhibitors in
Received: January 30, 2015
Accepted: March 16, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00050
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ACS Medicinal Chemistry Letters
Letter
Table 1. Cellular Activities of Imidazopyridazines
a
Cellular Ba/F3 assays
b
compd
TRKA
TRKB
TRKC
WT
1
0.85
0.27
0.10
7.13
>10
3.92
1.01
1.92
3.00
2.53
5.89
>10
>10
>10
9.23
>10
>10
>10
4.79
5.91
2
0.74
0.20
0.23
3
0.050
0.17
0.021
0.12
0.006
0.075
0.040
0.003
0.004
0.020
0.011
0.019
0.008
0.98
4
5
0.076
0.006
0.027
0.52
0.027
0.007
0.013
0.13
6
7
8
9
0.17
0.050
0.047
0.029
1.97
10
11
12
13
14
15
16
17
0.075
0.068
3.13
0.021
0.003
0.005
0.001
0.004
0.011
0.001
0.004
0.001
0.004
0.003
0.001
0.002
<0.001
0.002
Figure 1. X-ray cocrystal structure of 1 (carbon in yellow) binding to
the active site of TRKC kinase. Hydrogen bonds are depicted as
dashed lines. Nitrogen atoms are shown in blue, oxygen atoms in red,
and a water molecule depicted as a red sphere.
a
b
Proliferation assays with Tel-TRK fusions; IC50, μM. Proliferation
assay using parental Ba/F3 cells.
the constitutively active Tel-TRKA, Tel-TRKB, and Tel-TRKC
fusions (Table 1). In this assay setting, 1 showed differential
cytotoxicity as compared to parental wild-type (WT) Ba/F3
cells grown in the presence of IL-3. The cellular kinase
selectivity profile of 1 was determined in a Ba/F3 panel using
Ba/F3 cells rendered IL-3 independent by stable transduction
with 29 selected kinases activated by fusion with a dimerizing
protein partner (e.g. BCR or Tel).29 In this panel, 1 showed
reasonable selectivity with submicromolar inhibitory activity for
FLT3 and ROS (Scheme 1, Table 3).
The X-ray cocrystal structure of 1 showed binding to the
inactivated (DFG-out) kinase domain of TRKC and forming a
key hydrogen bond between N1 of the imidazopyridazine and
the hinge amide NH of Met620 (Figure 1).30,31 The
benzonitrile ring was wedged between phenyl rings from the
gatekeeper Phe617, Phe698 from the DFG motif, and the
phenyl ring of the benzylamino group of compound 1. This
phenyl ring sits under the glycine-rich loop and faces the
solvent exposed front of the binding pocket, lending itself to
immediate structure−activity relationship (SAR) investigations.
Chemistry optimization was initiated with the goal of
identifying compounds with improved potency and pharmaco-
kinetic properties suitable for in vivo profiling. New compounds
were screened for antiproliferative effects in Ba/F3-Tel-TRKA,
TRKB, and TRKC assays, and cytotoxicity effects were
compared with parental wild-type (WT) Ba/F3 cells. Initial
efforts focused on exploring the SAR around the benzyl amine
moiety (R1, R2, R4) and the phenyl group linked to the
imidazopyrimidine core (R3) (Scheme 1 and Table 1).
From this work, it appeared that nitrile substitutions in the
phenyl group (R3) at the 3 or 4 ring-position were equally
tolerated (1 vs 2). It was also observed that 3-substitution at
the benzylamine phenyl ring (R1) led to a 10-fold increase in
Scheme 1. Imidazopyridazines SAR Progression and Numbering Assignment
B
DOI: 10.1021/acsmedchemlett.5b00050
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ACS Medicinal Chemistry Letters
Letter
potency relative to the unsubstituted 1, especially with EWG
groups such as fluorine (3 vs 4). Shimming the phenyl ring
(R3) with the potency boosting 3-F substitution at R1 furnished
many tolerated functional groups that could either increase
solubility or further drive potency such as hydroxymethyl
derivative 5, amide 6, and sulfonamide 7.
Methylation of the benzylic amine (R4) led to no significant
change in potency (8 vs 1) suggesting that the benzylic amine is
not involved in a critical proton donating interaction.
Docking models using the cocrystal structure of 1 with
TRKC supported that potency could be further increased by
structurally rigidifying the benzyl amine moiety in a cyclic
fashion by reducing conformational entropy. Cyclic piperdine,
morpholine, and pyrrolidine analogues were synthesized as
racemates (9, 10, and 11) and revealed a potency preference
toward the five-membered heterocycle. A cocrystal structure
between TRKA and 10 indicated a preference for the R-
enantiomer and induced protein conformation change from the
DFG-out to the DFG-in state, which allowed the fluorophenyl
to bind near the ribose binding pocket (Figure 2).30 Moreover,
Figure 3. X-ray cocrystal structure of TRKA with the R-enantiomer of
14. The protein adopts the DFG-in conformation. Compound 14
maintains the key hydrogen bond between the hinge NH of Met592
and N1 of imidazopyridazine. Surprisingly, the imidazopyridazine is
now flipped. This allows for the fluorophenyl group to better fit the
ribose binding pocket compared to 10. Color coding according to
Figure 1.
hydrophobic pocket originally occupied by Phe669 and
provides excellent shape complementary, likely to contribute
significantly to the observed potency improvement. Molecular
modeling indicated that two distinct binding modes (i.e. core
flipping) are indeed possible for the DFG-in conformation. The
preferred binding mode probably depends upon the sub-
stitution on the 6-position of the imidazopyridazine. When this
substitution is the (R)-phenylpyrrolidine, the “flipped”
orientation appears to be preferred as the pyrrolidine anchors
the phenyl group in the hydrophobic pocket.
It is worth noting that since our initial disclosure of the (R)-
2-phenylpyrrolidine in 2008,32 this moiety has become a
privileged TRK structure and was incorporated effectively in
closely related scaffolds33−38 and on unrelated bicycle
scaffolds.39
The cocrystal structure of 14 indicated that the pyridine ring
at the 3-position faces the solvent exposed front of the binding
pocket, suggesting that polar groups in this area should be
tolerated. Indeed, decorating the 3-position (R5) of the pyridine
maintained very good potency across the TRK isoforms (15,
16, and 17).
To determine the in vivo PK properties, 15, 16, and 17 were
administered intravenously to Wistar or Sprague−Dawley rats.
High plasma clearance (CL), moderate volume distribution
(Vss), and short half-life (T1/2) were observed (Table 2). The
plasma clearances of these compounds were about 82% (16 and
Figure 2. X-ray cocrystal structure of TRKA with 10. The
fluorophenyl points toward the pocket usually occupied by the ribose
from ATP. The protein adopts a DFG-in conformation to allow for the
fluorophenyl to bind. Although the 10 racemate was used for
crystallization, only the R-enantiomer binds to TRKA. Color coding
according to Figure 1.
the DFG-in conformation prevented a clash between Phe669
and the compound’s fluorophenyl group. Chiral resolution of
the pyrrolidine derivative 11 into its two enantiomers, 12 and
13, indicated the R-enantiomer (13) as the more potent isomer,
which also demonstrated a 2-fold activity improvement relative
to acyclic compound 3.
Table 2. In Vivo Rat PK Properties after Dosing of 3 mg/kg
IV and 10 mg/kg PO
a
b
b
c
rat PK
15
16
17
By maintaining the 3-F-phenylpyrrolidine in 11, a subsequent
round of optimization at R3 was performed. This effort showed
that replacing the 3-CN-phenyl moiety with a 2-pyridine (14)
led to an 8−20-fold potency improvement across all three TRK
isoforms.
The cocrystal structure of 14 with TRKA revealed an
unanticipated flip of the compound around the imidazopyr-
idazine core while maintaining the same key hinge interaction
(Figure 3).30 Binding could only be observed for the R-
enantiomer where the 3-F-phenyl is optimally positioned in the
CL (mL/min/kg)
Vss (L/kg)
56.9
2.3
45.5
2.4
45.2
4.9
T1/2 (h)
0.83
659
110
10
0.9
1.6
AUC PO (h·nM)
Cmax PO (nM)
F(%) PO
1897
250
26
1879
215
27
a
Route: Intravenous (3 mg/kg) and oral administrations (10 mg/kg).
Formulation as a solution in 75% PEG300/25% D5W. Male
Sprague−Dawley rat. Male Wistar rat.
b
c
C
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ACS Medicinal Chemistry Letters
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17) and 100% (15) of rat hepatic blood flow (55 mL/min/kg).
Compound 17, with an additional heterocyclic group between
the pyridine and the solubilizing group, led to a higher volume
of distribution (4.9 vs 2.3 L/kg), which resulted in a longer half-
life (1.6 vs 0.9 h) compared to 15 and 16. When administered
orally by gavage, 15 showed low bioavailability (10%), while 16
and 17 had 2.6-fold higher oral bioavailability (Table 2). In
mouse brain tissue distribution studies, the AUC and Cmax
ratios of brain to plasma were lower for 17 (∼0.2) compared to
16 (∼0.8) (Supporting Information).
TRKA kinase for proliferation and survival, the compounds
were also very potent with IC50 of 0.001 and 0.01 μM,
respectively.
By virtue of its pan-TRK in vitro potency, good selectivity,
relatively low brain exposure, and overall acceptable PK
properties, 17 (GNF-8625) was selected for in vivo efficacy
studies (synthesis in Supporting Information). In a tumor
xenograft model derived from the KM12 cell line,40 17
demonstrated in vivo antitumor efficacy when administered at
ascending doses twice daily (bid) for 14 days in rats (Figure 4).
Compounds 14, 16, and 17 were tested in the Ba/F3 panel
and showed much improved selectivity across all the kinases
when compared to 1 (Table 3; see Table 1 for TRK activity).
Table 3. Ba/F3 Cellular Kinase Panel (IC50, μM)
a
Ba/F3
1
14
16
17
b
ABL
3.8
4.8
3.1
2.5
9.7
3.9
5.9
6.4
3.0
0.72
3.4
4.0
8.6
4.7
4.1
2.2
2.0
7.5
5.4
5.1
2.0
4.9
6.1
0.30
4.6
4.0
5.5
NA
6.4
NA
6.3
2.4
1.2
2.2
6.2
2.7
4.9
5.3
2.8
2.6
3.3
2.5
5.1
5.3
2.7
1.0
0.79
3.0
3.4
4.7
4.3
2.2
3.7
5.0
0.06
2.5
4.4
4.3
5.2
5.2
5.2
3.0
3.9
5.9
4.3
5.0
4.4
5.9
5.3
4.3
5.3
5.4
6.0
5.6
5.0
7.0
5.2
4.9
5.1
5.7
4.6
6.0
4.8
0.21
4.6
7.9
5.5
5.0
5.5
c
ALK
ALK
NA
NA
>10
9.5
BRAF
BMX
FGFR3
FGFR4
FGR
9.2
8.7
FLT1
FLT3
FMS
NA
8.1
>10
NA
8.8
IGF1R
INSR
JAK2
KDR
Figure 4. KM12 efficacy model in female CRL RNU nude rats with
compound 17 (GNF-8625). Animals were transplanted with KM12
tumor tissues, and dosing began 10 days postimplant. Vehicle and 17
were dosed twice a day (bid) for 14 days. Efficacy measured as tumor
volume.
6.7
6.5
KIT
5.5
LCK
NA
6.4
LYN
MER
MET
PDGFRβ
RET
NA
7.7
In this study, 17 induced 20% tumor regression at 50 mg/kg
bid and achieved partial tumor growth inhibition at 6.25, 12.5,
and 25 mg/kg bid, in a dose-dependent manner. A dose
proportional increase in exposure of compound 17 was
observed, and at the highest doses, a slight increase in body
weight was observed (Supporting Information).
NA
8.0
RON
ROS
NA
0.6
SRC
NA
NA
6.5
SYK
In summary, using a TRK/ligand cocrystal structural guided
approach, we have developed a series of potent, selective, and
efficacious (R)-2-phenylpyrrolidine substituted imidazopyrida-
zine TRK inhibitors. This work has unveiled the (R)-2-
phenylpyrrolidine as a privileged moiety for TRK inhibitors due
to its optimal shape complementarity to the TRK protein’s
hydrophobic pocket available in the “DFG-in” conformation.
TIE1
TYRO3
ZAP70
NA
NA
a
Ba/F3 cells rendered IL-3 independent by stable transduction with
the indicated kinase fused with a Tel dimerization partner unless
b
c
otherwise specified. BCR-ABL. NMP-ALK.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, characterization of compounds,
kinase selectivity, ADMET data for compound 17, and details
of in vitro and in vivo assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
The main kinase off-target activity remained ROS but with an
improved selectivity index (SI) (SI < 3 for 1 and SI >50 for 14,
16, and 17). This demonstrated that (R)-2-fluorophenylpyrro-
lidine-containing analogues were able to achieve higher potency
as well as increased selectivity due to their high specificity at the
hydrophobic pocket.
S
In addition, the anti-TRKA activity for compounds 16 and
17 was further demonstrated in two other cellular systems, Ba/
F3 and KM12. In Ba/F3 cells engineered to express both
TRKA and NGF, both compounds demonstrated potent
antiproliferation activity with IC50 of 0.001 and 0.003 μM,
respectively. In KM12 cells, derived from a colon cancer cell
line that harbors the TPM3-TRKA fusion and is dependent on
AUTHOR INFORMATION
Corresponding Author
*Tel: 858-332-4736. E-mail: vmolteni@gnf.org.
■
Present Addresses
†Novartis Pharma AG, Novartis Institutes for Biomed.
Research, Postfach, CH-4002 Basel, Switzerland.
D
DOI: 10.1021/acsmedchemlett.5b00050
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ACS Medicinal Chemistry Letters
Letter
‡Loma Linda University Medical Center, Rehabilitation
Institute, 25333 Barton Road, Loma Linda, California 92354,
United States.
Genes Producing TRK Oncogenes in Papillary Thyroid Carcinomas.
Genomics 1995, 28, 15−24.
(12) Greco, A.; Mariani, C.; Miranda, C.; Pagliardini, S.; Pierotti, M.
A. Characterization of the NTRK1 Genomic Region Involved in
Chromosomal Rearrangements Generating TRK Oncogenes. Genomics
1993, 18, 397−400.
(13) Bounacer, A.; Schlumberger, M.; Wicker, R.; Du-Villard, J. A.;
Caillou, B.; Sarasin, A.; Suarez, H. G. Search for NTRK1 Proto-
Oncogene Rearrangements in Human Thyroid Tumours Originated
After Therapeutic Radiation. Br. J. Cancer 2000, 82, 308−314.
(14) Beimfohr, C.; Klugbauer, S.; Demidchik, E. P.; Lengfelder, E.;
Rabes, H. M. NTRK1 Re-Arrangement In Papillary Thyroid
Carcinomas of Children After the Chernobyl Reactor Accident. Int.
J. Cancer 1999, 80, 842−847.
(15) Alberti, L.; Carniti, C.; Miranda, C.; Roccato, E.; Pierotti, M. A.
RET and NTRK1 Proto-Oncogenes in Human Diseases. J. Cell.
Phyisiol. 2003, 195, 168−186.
(16) Vaishnavi, A.; Capelletti, M.; Le, A. T.; Kako, S.; Butaney, M.;
Ercan, D.; Mahale, S.; Davies, K. D.; Aisner, D. L.; Pilling, A. B.; Berge,
E. B.; Kim, J.; Sasaki, H.; Park, S.; Kryukov, G.; Garraway, L. A.;
Hammerman, P. S.; Haas, J.; Andrews, S. W.; Lipson, D.; Stephens, P.
§Sanofi Oncology, 640 Memorial Drive, Cambridge,
Massachusetts 02139, United States.
∥Parallel Computing Laboratories, Inc., 3525 Del Mar Heights
Road, #288, San Diego, California 92130, United States.
⊥Novartis Institutes for BioMedical Research, Inc. 250
Massachusetts Avenue, Cambridge, Massachusetts 02139,
United States.
#Novartis Pharmaceuticals Corporation, One Health Plaza, East
Hanover, New Jersey 07936, United States.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Daniel Raymond and Thomas H. Marsilje for help in
proof reading.
■
J.; Miller, V. A.; Varella-Garcia, M.; Janne, P. A.; Doebele, R. C.
̈
Oncogenic and Drug-Sensitive NTRK1 Rearrangements in Lung
Cancer. Nat. Med. 2013, 19, 1469−1472.
(17) Tognon, C.; Knezevich, S. R.; Huntsman, D.; Roskelley, C. D.;
Melnyk, N.; Mathers, J. A.; Becker, L.; Carneiro, F.; MacPherson, N.;
Horsman, D.; Poremba, C.; Sorensen, P. H. B. Expression of the
ETV6-NTRK3 Gene Fusion as a Primary Event in Human Secretory
Breast Carcinoma. Cancer Cell 2002, 2, 367−376.
ABBREVIATIONS
TRK, tropomyosin receptor kinase; NGF, nerve growth factor;
BDNF, brain-derived neurotrophic factor
■
(18) Lannon, C. L.; Sorensen, P. H. B. ETV6-NTRK3: a Chimeric
Protein Tyrosine Kinase with Transformation Activity in Multiple Cell
Lineages. Semin. Cancer Biol. 2005, 15, 215−223.
REFERENCES
■
(1) Huang, E. J.; Reichardt, L. F. TRK Receptors: Roles in Neuronal
Signal Transduction. Annu. Rev. Biochem. 2003, 72, 609−642.
(2) Pierotti, M. A.; Greco, A. Oncogenic Rearrangements of the
NTRK1/NGF Receptor. Cancer. Lett. 2006, 232, 90−98.
(3) Lei, L.; Parada, L. F. Transcriptional Regulation of TRK Family
Neurotrophin Receptors. Cell. Mol. Life Sci. 2007, 64, 522−532.
(4) Lagadec, C.; Meignan, S.; Adriaenssens, E.; Foveau, B.; Vanhecke,
E.; Romon, R.; Toillon, R. A.; Oxombre, B.; Hondermarck, H.; Le
Bourhis, X. TRKA Overexpression Enhances Growth and Metastasis
of Breast Cancer Cells. Oncogene 2009, 28, 1960−1970.
(5) Borrello, M. G.; Bongarzone, I.; Pierotti, M. A.; Luksch, R.;
Gasparini, M.; Collini, P.; Pilotti, S.; Rizzetti, M. G.; Mondellini, P.; De
Bernardi, B.; Di Martino, D.; Garaventa, A.; Brisigotti, M.; Tonini, G.
P. TRK and ret Proto-Oncogene Expression in Human Neuroblastoma
Specimens: High Frequency of TRK Expression in Non-Advanced
Stages. Int. J. Cancer 1993, 54, 540−545.
(6) Ma, J.; Jiang, Y.; Jiang, Y.; Sun, Y.; Zhao, X. Expression of Nerve
Growth Factor and Tyrosine Kinase Receptor A and Correlation with
Perineural Invasion in Pancreatic Cancer. J. Gastroenterol. Hepatol.
2008, 23, 1852−1859.
(7) Zhu, Z.; Friess, H.; diMola, F. F.; Zimmermann, A.; Graber, H.
U.; Korc, M.; Buchler, M. W. Nerve Growth Factor Expression
Correlates with Perineural Invasion and Pain in Human Pancreatic
Cancer. J. Clin. Oncol. 1999, 17, 2419−2428.
(8) Festuccia, C.; Muzi, P.; Gravina, G. L.; Millimaggi, D.; Speca, S.;
Dolo, V.; Ricevuto, E.; Vicentini, C.; Bologna, M. Tyrosine Kinase
Inhibitor CEP-701 Blocks the NTRK1/NGF Receptor and Limits the
Invasive Capability of Prostate Cancer Cells in vitro. Int. J. Oncol.
2007, 30, 193−200.
(9) Davidson, B.; Reich, R.; Lazarovici, P.; Nesland, J. M.; Skrede, M.;
Risberg, B.; Trope, C. G.; Florenes, V. A. Expression and Activation of
the Nerve Growth Factor Receptor TRKA in Serous Ovarian
Carcinoma. Clin. Cancer. Res. 2003, 9, 2248−2259.
(10) Martin-Zanca, D.; Hughes, S. H.; Barbacid, M. A Human
Oncogene Formed by the Fusion of Truncated Tropomyosin and
Protein Tyrosine Kinase Sequences. Nature 1986, 319, 743−748.
(11) Butti, M. G.; Bongarzone, I.; Ferraresi, G.; Mondellini, P.;
Borrello, M. G.; Pierotti, M. A. A Sequence Analysis of the Genomic
Regions Involved in the Rearrangements Between TPM3 and NTRK1
(19) Watanabe, N.; Kobayashi, H.; Hirama, T.; Kikuta, A.; Koizumi,
S.; Tsuru, T.; Kaneko, Y. Cryptic t(12;15)(p13;q26) Producing the
ETV6-NTRK3 Fusion Gene and No Loss of IGF2 Imprinting in
Congenital Mesoblastic Nephroma with Trisomy 11 Fluorescence in
situ Hybridization and IGF2 Allelic Expression Analysis. Cancer Genet.
Cytogenet. 2002, 136, 10−16.
(20) Patani, N.; Jiang, W. G.; Mokbel, K. Brain derived Neurotrophic
Factor Expression Predicts Adverse Pathological and Clinical Out-
comes in Human Breast Cancer. Cancer Cell. Int. 2011, 11, 23−30.
(21) Roesler, R.; Brunetto de Farias, C.; Abujamra, A. L.; Brunetto, A.
L.; Schwartsmann, G. BDNF/TrkB Signaling as an Anti-Tumor
Target. Expert Rev. Anticancer Ther. 2011, 11, 1473−1475.
(22) Izbicka, E.; Izbicki, T. Therapeutic Strategies for the Treatment
of Neuroblastoma. Curr. Opin. Investig. Drugs 2005, 6, 1200−1214.
(23) Douma, S.; Van Laar, T.; Zevenhoven, J.; Meuwissen, R.; Van
Garderen, E.; Peeper, D. S. Suppression of Anoikis and Induction of
Metastasis by the Neurotrophic Receptor TRKB. Nature 2004, 430,
1034−1039.
(24) Zage, P. E.; Graham, T. C.; Zeng, L.; Fang, W.; Pien, C.; Thress,
K.; Omer, C.; Brown, J. L.; Zweidler-McKay, P. A. The Selective Trk
Inhibitor AZ623 Inhibits Brain-Derived Neurotrophic Factor-Medi-
ated Neuroblastoma Cell Proliferation and Signaling and is Synergistic
with Topotecan. Cancer 2011, 117, 1321−1329.
(25) Croucher, J. L.; Iyer, R.; Li, N.; Molteni, V.; Loren, J.; Gordon,
W. P.; Tuntland, T.; Liu, B.; Brodeur, G. M. TrkB Inhibition by GNF-
4256 Slows Growth and Enhances Chemotherapeutic Efficacy in
Neuroblastoma Xenografts. Cancer Chemother. Pharmacol. 2015, 75,
131−41.
(26) Miknyoczki, S. J.; Chang, H.; Klein-Szanto, A.; Dionne, C. A.;
Ruggeri, B. A. The TRK Tyrosine Kinase Inhibitor CEP-701 (KT-
5555) Exhibits Significant Antitumor Efficacy in Preclinical Xenograft
Models of Human Pancreatic Ductal Adenocarcinoma. Clin. Cancer
Res. 1999, 5, 2205−2212.
(27) Ketterer, K.; Rao, S.; Friess, H.; Weiss, J.; Buechler, M. W.;
Korc, M. Reverse Transcription-PCR Analysis of Laser-Captured Cells
Points to Potential Paracrine and Autocrine Actions of Neurotrophins
in Pancreatic Cancer. Clin. Cancer Res. 2003, 9, 5127−5136.
E
DOI: 10.1021/acsmedchemlett.5b00050
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(28) Sclabas, G. M.; Fujioka, S.; Schmidt, C.; Li, Z.; Frederick, W. A.
I.; Yang, W.; Yokoi, K.; Evans, D. B.; Abbruzzese, J. L.; Hess, K. R.;
Zhang, W.; Fidler, I. J.; Chiao, P. Overexpression of Tropomysin-
Related Kinase B in Metastatic Human Pancreatic Cancer Cells. Clin.
Cancer Res. 2005, 11, 440−449.
(29) Melnick, J. S.; Janes, J.; Kim, S.; Chang, J. Y.; Sipes, D. G.;
Gunderson, D.; Jarnes, L.; Matzen, J. T.; Garcia, M. E.; Hood, T. L.;
Beigi, R.; Xia, G.; Harig, R. A.; Asatryan, H.; Yan, S. F.; Zhou, Y.; Gu,
X. J.; Saadat, A.; Zhou, V.; King, F. J.; Shaw, C. M.; Su, A. I.; Downs,
R.; Gray, N. S.; Schultz, P. G.; Warmuth, M.; Caldwell, J. S. An
Efficient Rapid System for Profiling the Cellular Activities of Molecular
Libraries. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3153−3158.
(30) Structure coordinates have been deposited into the PDB: 4YMJ;
4YNE; 4YPS.
(31) Zuccotto, F.; Ardini, E.; Casale, E.; Angiolini, M. Through the
“Gatekeeper Door”: Exploiting the Active Kinase Conformation. J.
Med. Chem. 2010, 53, 2681−2694.
(32) Ferrand, S.; Glickman, F.; Leblanc, C.; Ritchie, C.; Shaw, D.;
Stiefl, N. J.; Furet, P.; Imbach, P.; Stauffer, F.; Capraro, H. G.; Gessier,
F.; Gaul, C.; Albaugh, P. Chopiuk, G. Heterocyclic Compounds as
Antiinflammatory Agents. WO/2008/052734; International Applica-
tion No. PCT/EP2007/009382; May 8, 2008.
(33) Andrews, S. W.; Haas, J.; Jiang, Y.; Zhang, G. Substituted
Imidazo[1,2b]pyridazine Compounds as TRK Kinase Inhibitors. WO/
2010/033941; International Application No. PCT/US2009/057729;
March 25, 2010.
(34) Haas, J.; Andrews, S. W.; Jiang, Y.; Zhang, G. Substituted
Pyrazolo[1,5-a]pyrimidine Compounds as TRK Kinase Inhibitors.
WO/2010/048314 A1; International Application No. PCT/US2009/
061519; April 29, 2010.
(35) Allen, S.; Andrews, S. S.; Condroski, K. R.; Haas, J.; Huang, L.;
Jiang, Y.; Kercher, T.; Seo, J. Substituted Pyrazolo[1,5-a]pyrimidine
Compounds as TRK Kinase Inhibitors. WO/2011/006074 A1;
International Application No. PCT/US2010/041538; January 11,
2011.
(36) Andrews, S. W.; Condroski, K. R.; Haas, J.; Jiang, Y.;
Kolakowski, G. R.; Seo, J.; Yang, H. W.; Zhao, Q. Macrocyclic
Compounds as Trk Kinase Inhibitors. WO/2011/146336 A1;
International Application No. PCT/US2011/036452; November 24,
2011.
(37) Sasmal, P. K.; Ahmed, S.; Tehim, A.; Pradkar, V.; Dattatreya, P.
M.; Mavinahalli, N. J. Substituted Pyrazolo[1,5-a]pyridine as
Tropomyosin Receptor Kinase (Trk) Inhibitors. WO/2013/088256
A1; International Application No. PCT/IB2012/003012; June 20,
2013.
(38) Sasmal, P. K.; Ahmed, S.; Prabhu, G.; Tehim, A.; Pradkar, V.;
Dattatreya, M. P.; Mavinahalli, N. J. Substituted Heterocyclic
Compounds as Tropomyosin Receptor Kinase A (TRKA) Inhibitors.
WO/2013/088257 A1; International Application No. PCT/IB2012/
003022; June 20, 2013.
(39) Stachel, S. J.; Sanders, J. M.; Henze, D. A.; Rudd, M. T.; Su, H.
P.; Li, Y.; Nanda, K. K.; Egbertson, M. S.; Manley, P. J.; Jones, K. L. G.;
Brnardic, E. J.; Green, A.; Grobler, J. A.; Hanney, B.; Leitl, M.; Lai, M.
T.; Munshi, V.; Murphy, D.; Rickert, K.; Riley, D.; Krasowska-Zoladek,
A.; Daley, C.; Zuck, P.; Kane, S. A.; Bilodeau, M. T. Maximizing
Diversity from a Kinase Screen: Identification of Novel and Selective
pan-Trk Inhibitors for Chronic Pain. J. Med. Chem. 2014, 57, 5800−
5816.
(40) Unpublished data.
F
DOI: 10.1021/acsmedchemlett.5b00050
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