Discovery of Novel Pyrido-pyridazinone Derivatives as FER Tyrosine Kinase Inhibitors with Antitumor Activity

Article abstract of DOI:10.1021/acsmedchemlett.8b00631

To obtain a new anticancer drug, we focused on FER tyrosine kinase. Starting with high-throughput screening with our in-house chemical library, compound 1, which has a pyridine moiety, was found. Referring to their X-ray crystal structure with FES proto-oncogene tyrosine kinase, as a surrogate of FER followed by chemical modification including scaffold hopping of the pyridine template, we discovered pyrido-pyridazinone derivatives with potent FER kinase inhibitory activity. Here, we disclose the structure-activity relationship on the scaffold and representative compound 21 (DS21360717), which showed in vivo antitumor efficacy in a subcutaneous tumor model.

Full text of DOI:10.1021/acsmedchemlett.8b00631

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Letter
Discovery of Novel Pyrido-pyridazinone Derivatives as
FER Tyrosine Kinase Inhibitors with Anti-tumor Activity
Toru Taniguchi, Hiroaki Inagaki, Daichi Baba, Isao Yasumatsu, Akiko Toyota,
Yasuyuki Kaneta, Masaki Kiga, Shin Iimura, Takashi Odagiri, Yoshihiro Shibata,
Kiyono Ueda, Maki Seo, Hiroki Shimizu, Tomoki Imaoka, and Kiyoshi Nakayama
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00631 • Publication Date (Web): 15 Mar 2019
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Discovery of Novel Pyrido-pyridazinone Derivatives as FER Tyrosine
Kinase Inhibitors with Anti-tumor Activity
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Toru Taniguchi,*† Hiroaki Inagaki,† Daichi Baba,§ Isao Yasumatsu,‡ Akiko Toyota,† Yasuyuki
Kaneta,† Masaki Kiga,† Shin Iimura,† Takashi Odagiri,† Yoshihiro Shibata,† Kiyono Ueda,¶ Maki
Seo,¶ Hiroki Shimizu,¶ Tomoki Imaoka,† and Kiyoshi Nakayama§
†R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
‡Daiichi Sankyo RD Novare Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
§Daiichi Sankyo Co., Ltd., 3-5-1 Nihonbashi-honcho, Chuo-ku, Tokyo 103-8426, Japan
¶Daiichi Sankyo RD Novare Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
KEYWORDS: FER, inhibitor, tyrosine kinase, FES, pyrido-pyridazinone, anti-tumor
ABSTRACT: To obtain a new anticancer drug, we focused on FER tyrosine kinase (FER). Starting with high-throughput
screening with our in house chemical library, compound 1 which has a pyridine moiety was
found. Referring to their X-ray crystal structure with FES proto-oncogene tyrosine kinase,
as a surrogate of FER followed by chemical modification including scaffold hopping of the
pyridine template, we discovered pyrido-pyridazinone
derivatives with potent FER kinase inhibitory activity. Here,
we disclose the structure-activity relationship on the scaffold
and representative compound 21 (DS21360717), which showed
in vivo anti-tumor efficacy in a subcutaneous tumor model.
FER is a non-transmembrane receptor tyrosine kinase in
the FES family. It plays a pivotal role in cell migration
through the tyrosine phosphorylation of cortactin (CTTN)
and direct interaction with phosphatidic acid.1–2 In breast
cancer cell lines, FER kinase controls migration and
metastasis by regulating integrin-dependent adhesion.
High FER expression in human invasive breast cancer is an
independent prognostic factor that correlates with high-
grade basal/triple-negative tumors and worse overall
survival.3 Recently, a fusion gene of mannosidase alpha
class 2A member 1-FER tyrosine kinase gene (MAN2A1-
FER) was discovered in liver tumors, esophageal
adenocarcinoma, glioblastoma multiforme, prostate
tumors, non-small cell lung tumors, and ovarian tumors.
The expression of MAN2A1-FER led to increases of
proliferation and invasiveness of cancer cell lines, and
conferred liver oncogenic activity in mice.4 These reports
suggest that FER has the potential to be a target for anti-
cancer therapy. However, potent FER kinase inhibitors
have not been investigated intensively and few reports on
an FER inhibitor have been published.5 Here, we disclose
and discuss highly potent FER kinase inhibitors discovered
by high-throughput screening (HTS), and subsequent
scaffold hopping and chemical modification based on the
structural
crystallography.
information
obtained
from
X-ray
Through an HTS strategy with an in-house chemical
library, a pyridine derivative 1 with moderate FER
inhibitory activity was identified. In a preliminary study,
we investigated the basic structure-activity relationship
(SAR) of compound 1 by incorporating additional groups
and modifying its side chains.
Table 1. Preliminary SAR for C-5 position in the
Pyridine Scaffolda
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First, incorporation of a substituent at the 5-position of the
pyridine scaffold was investigated, and we found that
hydrogen can be replaced with Cl, phenyl, or nitrile.
Interestingly, the inhibitory activity was enhanced by Cl or
nitrile. As shown in Table 1, compound 4 having nitrile at
this position showed potent activity. We then progressed
to the preliminary derivatization of the side chains on C-2,
C-3, and C-6 of compound 4.
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Next, we determined the profile of compound 4 in a
cellular assay, as shown in Figure 2. Compound 4
possessed potent growth inhibition activity against
constructed FER-driven Ba/F3 cells and Ba/F3-Mock. At
this stage, the compound showed a preferable profile, but
we were concerned about it from a structural perspective,
due to a hydrophilic carbamoyl group.6 To address this
issue, we next focused on the design of new compounds to
reduce the number of hydrogen bond donors. In particular,
we considered performing scaffold hopping to modify the
carbamoyl group.
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The results are shown in Table 2. In almost all of the
9
compounds
produced
by
these
modifications,
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maintenance of the activities of compound 4 was difficult.
Only compound 10 possessed moderate activity, with IC50
of 57 nM. Removal of the phenyl group from the 2-position
(compound 5), replacement of the aniline with a benzyl
moiety (compound 6), removal of the carbamoyl group at
the 3-position (compound 7), or methylation to the
carbamoyl amine (compound 8) resulted in large loss of
activity. As for the 6-position, the results clearly showed
that cis-cyclohexyl diamine was the optimal structure
(compounds 10 and 11). Terminal amine nitrogen and the
hydrogens on it in the cyclohexyl diamine moiety are
necessary for the activity (compounds 9 and 12). We
confirmed that the side chains in compound 4 as a target
for modification already had good effects on FER inhibitory
activities within the primary derivatization.
To obtain further insight into the mode of binding of
compound 4 to the target protein, we used FES as a
surrogate protein for FER because FES has high homology
to FER. As shown in Figure 1, we obtained the X-ray crystal
structure of FES in complex with compound 4.
Table 2. Side Chain SAR for C-2, C-3, and C-6 Positions
in the Pyridine Scaffolda
Figure 1. Crystal Structure of Compound 4 in Complex
with FES (PDB code: 6JMF). FES is depicted as a cartoon,
sticks, and a grid surface in gray. N atoms are in blue, O
atoms are in red, and an S atom is in yellow. Modeled FES
is in light orange. Compound 4 is shown as a ball-and-stick
model in orange. The hydrogen bond network around the
compound is shown as dashed yellow lines. Two water
molecules participating in the network are shown as
spheres in red. The final model and structure factors were
deposited in PDB with accession code 6JMF. The image
was prepared using PyMOL (The PyMOL Molecular
Graphics System, Version 1.8; Schrödinger, LLC).7
In this complex, compound 4 occupies the ATP-binding
site of FES. The 3-, 5-, and 6-position substituent groups of
the pyridine scaffold of compound 4 undergo hydrogen
bonding with FES: (a) The amine of the 3-carbamoyl group
forms a hydrogen bond with the backbone carbonyl
oxygen atom of E637. The oxygen atom of the 3-carbamoyl
group forms inter- and intra-molecular hydrogen bonds
with the backbone nitrogen atom of V639 and the nitrogen
atom of the 2-aniline moiety, respectively. The criticality of
these interactions between the 3-carbamoyl group and the
FES hinge region for the binding of compound 4 to FES was
experimentally supported by the lack of FER inhibitory
activity for compound 7. (b) The nitrogen atom of the 5-
cyano group forms a hydrogen bond with the side-chain
amino group of catalytic lysine (K590). (c) The linker
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amine of the 6-cyclohexyldiamino group forms a hydrogen
structures might prevent those side chains from being
arranged at a suitable angle or direction to maintain their
activities. On the other hand, compound 15 possessing a
pyrido-pyridazino scaffold had remarkable activity , even
though it did not have the substituents necessary for
strong activity in the case of the pyridine scaffold, that is, a
halogen or cyano group at the 5-position. In fact,
compound 15 was 30 times more potent than parent
compound 1. By conversion to a bicyclic structure like
Type A, it could make better use of occupying the ATP-
binding site. As for inhibitory activities for Ba/F3-Mock
cells, which could be thought as an indicator showing the
selectivity for FER, compound 13, 14 and 15 shows not so
strong inhibitory activities compared with FER IC50 value
and Ba/F3-FER IC50 value. Therefore it could be considered
that these bicyclic structure were not non-selective and
worth for derivatization.
1
bond with the side-chain oxygen atom of D701. The
terminal amine of the 6-cyclohexyldiamino group forms
three hydrogen bonds with the backbone carbonyl oxygen
atom of R687, the side-chain oxygen atom of N688 via a
bridged water molecule, and the side-chain oxygen atom
of D701. These interactions between the terminal amine of
the 6-cyclohexyldiamino group and FES explain the more
than 100-fold decrease in FER inhibitory activity observed
in the comparison between compounds 4 and 9. In
addition to the hydrogen bonds, numerous hydrophobic
interactions are also observed as a result of high shape
complementarity between compound 4 and the ATP-
binding site of FES (see Supporting Information). In this
way, the analysis of the crystal structure provided us with
a sufficient explanation of the high potency, where 2-
anilino, 3-carbamoyl, 5-cyano, and 6-cyclohexyldiamino
groups play important roles in the binding to the ATP-
binding site of the protein. These observations are
consistent with the SAR shown in Tables 1 and 2.
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Next, the optimization of pyrido-pyridazinone derivatives
was performed, focusing on the cyclohexane diamine
moiety. The results are displayed in Table 3. We
investigated the best configuration of the two amines in
the cyclohexane portion. The difference between
compounds 15 and 18 clearly showed that (1R, 2S)-2-
aminocyclohexyl isomer had higher activity. Next, we
incorporated a halogen or cyano group into the 8-position
of the pyrido-pyridazino scaffold to obtain more potent
compounds. The results showed that all compounds
exhibited higher biochemical activity, while halogeno
variants did not possess very potent cellular activity.
Among the derivatives thus far synthesized, compound 21
showed the highest inhibitory activity in biochemical and
cellular growth assays, which encouraged us to evaluate its
in vivo anti-tumor activity.
We then focused our attention on enhancing the
membrane permeability by modifying the carbamoyl
moiety. Our strategy for this conversion consists of the
cyclization depicted in Figure 2 to generate a new bicyclic
scaffold. Two types of cyclization, Type A and Type B,
were investigated: Type A cyclization of 3-carbamoyl to the
4-position of the pyridine template and Type B cyclization
of 3-carbamoyl to the 2-position of the pyridine template.
Table 3. Modification of Pyrido-pyridazino Scaffolda
Figure 2. New Scaffolds Identified by Cyclization. IC50
values were calculated from duplicate experiments by the
least squares method and GI50 values were calculated from
quadruplicate experiments along with 95% confidence
intervals (95% CI) in parentheses. The assay details are
shown in the Supporting Information.
Although these two types appeared to fit in the binding site
according to the model, the results that we obtained
differed markedly from what we anticipated. Type B
compounds did not maintain the activities, whilst Type A
compounds showed moderate to strong activities. For
Type B, which includes compounds 16 and 17, their ring
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Table 4. Pharmacokinetic Properties of 4 and 21 (DS21360717) in Mouse
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We characterized the pharmacokinetic profiles of
compound 4 and DS21360717, which are displayed in Table
4. Although there were no significant differences in the
CLtot between the two, BA of DS21360717 was improved in
comparison with that of compound 4, which was
presumably attributable to an improved membrane
permeability coefficient (Pe). The two compounds showed
almost the same moderate total body clearance (CLtot)
metabolic stability, and protein binding (% bound) in
mouse microsomes (% remaining), even though the
solubility of compound 21 shows poor. But the
bioavailability (BA) of DS21360717 was better than that of
compound 4. These results implied that the improvement
of Pe could confer better BA, via reduction in the number
of hydrogen bond donors as the result of scaffold hopping.
We thought that it was worthwhile subjecting DS21360717
to an in vivo test, and thus carried out anti-tumor study
using a Ba/F3-FER subcutaneous tumor model, the results
of which are shown in Figure 3. As envisioned, DS21360717
exhibited tumor growth inhibitory activity in a dose-
dependent manner without significant body weight loss.
Taking into consideration the fact that mean unbound
plasma concentration upon oral dosing at 10 mg/kg was 3.1
nM, exceeding GI50 for Ba/F3-FER, the anti-tumor efficacy
observed at doses of more than 12.5 mg/kg was considered
reasonable.
The docking model of compound 21 with FES is shown in
Figure 4. It suggests that, while Type A cyclization retains
the hydrogen bonds between the inhibitors and FES, the
shape complementarity around the gatekeeper residue
(M636) is clearly improved compared with that of
compound 4. Further, the additional interactions between
the pyridazinone ring and FES were observed by focusing
on the binding mode of compound 21 and FES; CH/π
interactions with A588 Cβ or L690 Cδ1, aliphatic-
CH···aromatic-CH interactions with M636 Cβ or Cε, and
divalent-S···aromatic-CH interactions with M636 Sδ. As
shown in this figure, their typical distances were
considerable to be suitable for the preferred affinity for
FES.8 The above might be the reason why compound 21
shows high FER inhibitory activity (see Supporting
Information).
Figure 3. Anti-tumor Efficacy of DS21360717 in a Ba/F3-
FER Subcutaneous Tumor Model: (A) The tumor volume
of each group (n = 5). **p<0.01 and ***p<0.001 vs. control.
(B) Body weight change from the start of treatment in mice
treated with DS21360717 (see Supporting Information).
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Scheme 1. Synthesis of Pyridine Derivative 4a
1
2
O
O
N
N
O
EtO
EtO
HN
a
b
N
EtO
HN
N
Cl
N
NH
H
N
3
Cl
N
Cl
Boc
4
22
23
24
5
c
6
7
8
9
NH2
NH2
O
N
N
N
O
e
O
d
HO
HN
HN
N
NH
HN
N
NH
N
NH
H
N
H
N
NH2
Boc
Boc
4
26
25
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aReagents: (a) 3-Methylaniline, DIPEA, MeCN, rt, 96%; (b)
tert-butyl [(1S,2R)-2-aminocyclohexyl]carbamate, DIPEA,
MeCN, 70°C, 83%; (c) LiOH, THF, H2O, rt–50°C, quant.; (d)
NH3 aq., WSCI.HCl, HOSu, DMF, rt, 84%; (e) 2N HCl-
EtOH, rt, quant.
Syntheses of pyrido-pyridazinone derivative 21 are shown
in Scheme 2. Compound 21 was prepared from
commercially available 4-methyl ester 27. Converting the
ester part to aldehyde via alcohol gave 29. Then, protection
of the aldehyde group with trimethyl orthoformate
furnished ketal 30. Ester incorporation into the 3-position
of pyridine with lithium diisopropylamide (LDA) and
methyl chloroformate afforded methyl ester 31. Hydrolysis
of the ketal part gave aldehyde 32, and the following
hydrazidation afforded N-Boc hydrazide 33. Ring closure
was induced by deprotection of the Boc group to provide
pyrido-pyridazinone 34.11 Subsequently, consecutive SNAr
reactions of compound 34 with 3-methylaniline and then
Figure 4. Superposition of Modeled Binding Mode of
Compound 21: (A) Compound 21 and the Crystal Structure
of Compound 4 in Complex with FES. The drawing style of
the crystal structure (FES/compound 4) and its colors are
the same as in Figure 1. Compound 21 is shown as a ball-
and-stick model in green. Its molecular surface is also
shown. (B) Additional interactions observed between FES
and cyclized atoms of compound 21 (docked model; green):
cyan dashed lines are the CH/π interaction between A588
Cβ or L690 Cδ1 and the center of the cyclized ring (pale
cyan sphere). Violet dashed lines are interactions between
M636 Cβ, Sδ, or Cε and aromatic CHs. Distances of the
additional interactions are in Angstrom units.
tert-butyl
[(1S,2R)-2-aminocyclohexyl]carbamate
furnished compound 36. 8-Bromo derivative 37 was
synthesized by bromination of compound 36 with N-
bromosuccinimide (NBS).12-13 Finally, incorporation of the
cyano group was carried out by zinc cyanide and
palladium(0) catalyst on compound 37,14 followed by
deprotection of the Boc group to furnish compound 21
(DS21360717).
Scheme 2. Synthesis of Pyrido-pyridazinone
Derivative 21 (DS21360717)a
N
R
O
R
g
d
HN
MeO
O
Cl
N
Cl
Cl
N
Cl
R
N
Cl
To further evaluate DS21360717, screening was conducted
against a panel of 68 kinases (screened at a concentration
of 200 nM). In addition to strong inhibition of FER and FES,
fourteen kinases conferring greater than 90% inhibition
were identified, which were ALK, FLT3, FMS, KIT, PYK2,
ROS, SYK, TRKA, CDK2/CycA2, CHK2, HGK, IRAK4, MLK1,
and TSSK1. The details are shown in Supporting
Information.
31
32
R = C(OMe)2
R = CHO
34
R = Cl
R = CO2Me
R = CH2OH
27
28
e
f
h
a
b
c
35 R = m-anilino
33 R = CONHNHBoc
29 R = CHO
30 R = C(OMe)2
N
N
N
HN
HN
HN
N
R
j
l
i
O
O
O
HN
N
NH
HN
N
NH
HN
N
NH
H
N
H
N
NH2
Boc
Boc
36
21
(DS21360717)
37
R = Br
k
R = CN
38
Syntheses of pyridine derivative 4 are shown in Scheme 1.
Compound 4 was prepared from commercially available
dichloropyridine 22.9–10 SNAr reaction of 22 afforded aniline
23. Then, another SNAr reaction of compound 23 furnished
N-Boc amine 24. Carboxylic acid 25 was obtained by
hydrolysis of the corresponding ethyl ester 24, and the
following condensation reaction afforded carbamoyl
variant 26. N-Boc deprotection of compound 26 gave
pyridine derivative 4.
aReagents: (a) NaBH4, EtOH, reflux, quant.; (b) Dess-
Martin periodinane, CH2Cl2, rt, quant.; (c) trimethyl
orthoformate, p-TsOH-H2O, MeOH, toluene, reflux,
quant.; (d) LDA, chloroformic acid methyl ester, THF,
−78°C, 27%; (e) TFA, CH2Cl2, rt, quant.; (f) carbazic acid
tert-butyl ester, 1,4-dioxane, rt, quant.; (g) TFA, CH2Cl2, rt,
99%. (h) 3-methylaniline, NMP, microwave, 160°C, quant.;
(i) tert-butyl [(1S,2R)-2-aminocyclohexyl]carbamate, NMP,
microwave 170°C, 45%; (j) NBS, DMF, rt, 87%; (k) zinc
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(2) Itoh, T.; Hasegawa, J.; Tsujita, K.; Kanaho, Y.; Takenawa,
cyanide, tetrakis(triphenylphosphine) palladium(0), DMF,
microwave, 120–130°C, 55%. (l) TFA, CH2Cl2, rt, 87%.
1
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T. The Tyrosine Kinase Fer Is a Downstream Target of
the PLD-PA Pathway that Regulates Cell Migration. Sci.
Signal. 2009, 2(87), ra52.
As for other pyridine derivatives 2-12 and bicyclic
derivatives 13-20, their syntheses and characterizations are
described in the Supporting Information.
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J. M.; Saig, F. A.; Vlug, E. J.; van der Wall, E.; van Diest,
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(14) Alterman, M.; Hallberg, A. Fast Microwave-Assisted
Preparation of Aryl and Vinyl Nitriles and the
Corresponding Tetrazoles from Organo-halides. J. Org.
Chem. 2000, 65, 7984–7989.
In summary, initial SAR study on HTS hits, followed by
scaffold hopping and optimization, furnished a potent,
drug-like, and in vivo-active compound, DS21360717.
Further optimization and characterization of the lead
compound will be reported in due course.
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ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and characterization data of all
compounds, and information on additional studies are
freely available online at http://pubs.acs.org.
AUTHOR INFORMATION
*Corresponding Author
Tel.: +81-3-3492-3131
E-mail address: taniguchi.toru.fm@daiichisankyo.co.jp
ABBREVIATIONS
HTS, high-throughput screening; Pe, permeability coefficient;
CLtot, total body clearance; BA, bioavailability; DIPEA, N-
ethyl-N,N-diisopropylamine; MeCN, acetonitrile; THF,
tetrahydrofuran;
dimethylaminopropyl)carbodiimide;
WSCI.HCl,
1-ethyl-3-(3-
HOSu, N-
hydroxysuccinimide; DMF, N,N-dimethylformamide; EtOH,
ethyl alcohol; LDA, lithium diisopropylamide; NBS, N-
bromosuccinimide; p-TsOH, p-toluenesulfonic acid; MeOH,
methyl alcohol; TFA, trifluoroacetic acid; NMP, N-
methylpyrrolidone
REFERENCES
(1) Sangrar, W.; Gao, Y.; Scott, M.; Truesdell, P.; Greer, P. A.
Fer-Mediated Cortactin Phosphorylation Is Associated
with Efficient Fibroblast Migration and Is Dependent on
Reactive Oxygen Species Generation during Integrin-
Mediated Cell Adhesion. Mol. Cell. Biol. 2007, 32, 6140–
6152.
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