Identification of 4-Phenoxyquinoline Based Inhibitors for L1196M Mutant of Anaplastic Lymphoma Kinase by Structure-Based Design

Article abstract of DOI:10.1021/acs.jmedchem.7b01039

Dysregulation of anaplastic lymphoma kinase (ALK) has been detected in nonsmall cell lung cancer (NSCLC) in the form of EML4-ALK fusion. Secondary mutations opposing activity of the first-generation ALK inhibitor crizotinib came into existence, requiring mutation-targeting drug discovery for the powerful second-line treatment. In this study, we report 4-phenoxyquinoline-based inhibitors that overcome crizotinib resistance to ALK L1196M, discovered by the fragment-growing strategy. The protonation of 4-aminoquinoline core could interrupt the ability the N atom of quinoline to act as a hydrogen bond acceptor; therefore, the pK_a and calculated ionization pH values of relevant pyridine-based core moieties were carefully analyzed. The replacement of amine linkage with ether resulted in single-digit nanomolar range inhibitors. The inhibitors exhibited significant antiproliferative effects on H2228 CR crizotinib-resistant cells by decreasing PI3K/AKT and MAPK signaling. This work constitutes the first example for systematic investigation of the effect of ionization pH on activity in this system.

Full text of DOI:10.1021/acs.jmedchem.7b01039

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Article
Identification of 4-Phenoxyquinoline Based Inhibitors for L1196M
Mutant of Anaplastic Lymphoma Kinase by Structure-Based Design
shinmee mah, Jung Hee Park, Hoi-Yun Jung, Kukcheol Ahn, Soyeon Choi, Hyun Seop Tae,
Kyung Hee Jung, Jin Kyung Rho, Jae Cheol Lee, Soon-Sun Hong, and Sungwoo Hong
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01039 • Publication Date (Web): 01 Nov 2017
Downloaded from http://pubs.acs.org on November 1, 2017
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Identification of 4-Phenoxyquinoline Based Inhibitors
for L1196M Mutant of Anaplastic Lymphoma Kinase by
Structure-Based Design
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Shinmee Mah,a,b+ Jung Hee Park, Hoi-Yun Jung, Kukcheol Ahn, Soyeon Choi, Hyun Seop Tae,
c+
a,b
a,b
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Kyung Hee Jung, Jin Kyung Rho, Jae Cheol Lee, Soon-Sun Hong* and Sungwoo Hong*
aDepartment of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon
4141, Korea,
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bCenter for Catalytic Hydrocarbon Functionalizations, Institute of Basic Science (IBS), Daejeon 34141,
Korea,
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cDepartment of Biomedical Sciences, College of Medicine, Inha University, Incheon, 22212, Korea.
dDepartment of Asan Institute for Life Sciences and Oncology, Asan Medical Center, University of Ulsan,
College of Medicine, Seoul 05505, Korea
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ABSTRACT
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Dysregulation of anaplastic lymphoma kinase (ALK) has been detected in non-small cell lung cancer
(NSCLC) in the form of EML4-ALK fusion. Secondary mutations opposing activity of the first generation
ALK inhibitor crizotinib came into existence, requiring mutation-targeting drug discovery for the
powerful second-line treatment. In this study, we report 4-phenoxyquinoline-based inhibitors that
overcome crizotinib resistance to ALK L1196M, discovered by the fragment-growing strategy. The
protonation of 4-aminoquinoline core could interrupt the ability the N atom of quinoline to act as a
hydrogen bond acceptor; therefore, the pKa and calculated ionization pH values of relevant pyridine-
based core moieties were carefully analyzed. The replacement of amine linkage with ether resulted in
single-digit nanomolar range inhibitors. The inhibitors exhibited significant antiproliferative effects on
H2228 CR crizotinib-resistant cells by decreasing PI3K/AKT and MAPK signaling. This work constitutes
the first example for systematic investigation of the effect of ionization pH on activity in this system.
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INTRODUCTION
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Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinases (RTKs) and the
insulin receptor superfamily, and it is activated through dimerization upon binding the growth factor
midkine (MK) and pleiotrophin (PTN) in the extracellular region. Activated ALK phosphorylates
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intracellular substrates and thereby switches on downstream cell signaling pathways to facilitate cell
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proliferation and survival signaling through the PI3K/AKT, JAK/STAT3 and MEK/ERK pathways.1,2
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Dysregulation of ALK signaling has been implicated in various human cancers, including non-small cell
lung cancer (NSCLC), inflammatory myofibroblastic tumor, anaplastic large cell lymphoma, squamous
cell carcinoma, and diffuse large B-cell lymphoma.3-7 Therefore, the hyperactivation of ALK kinase has
become a well-validated target for the development of therapeutics to treat several cancers. In addition, it
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has been demonstrated that the impairment of ALK activity with small-molecule inhibitors is effective in
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suppressing the growth of cancer in xenograft models in vivo.8-11
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Accordingly, much effort has been devoted to the discovery of small-molecule ALK inhibitors,
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including crizotinib (Chart 1), the dual inhibitor of ALK and c-MET.9,10, 12, 13 Despite the clear benefits
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of crizotinib, many ALK-dependent cancer cells tend to acquire various mutations that can render the
ALK enzyme constitutively active by impeding the inhibitor binding in the ATP-binding site.8, 14-20 The
emergence of drug resistance has prevented the first-generation anti-NSCLC drug crizotinib from
continued use in clinical treatment, which led to the development of the drug-resistant mutants of ALK.
In particular, the mutation L1196M is most frequently identified in crizotinib-resistant patients. The
emergence of the L1196M mutant has complicated clinical treatment, highlighting the importance of
identifying new ALK inhibitors with high potency against the L1196M mutant. The substitution of Met
for Leu could block the access of inhibitors to the deeper pocket near the gatekeeper residue in the ATP-
binding site due to an increase in steric hindrance, representing a major obstacle for drug design.22,23 In
this regard, the identification of potent inhibitors that target the L1196M mutation would have important
therapeutic implications.
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The accumulation of three dimensional structure data on ALK-inhibitor interactions for various mutants
has provided insight into overriding the drug resistance of ALK-dependent cancers.24-27 Recently, we have
revealed new molecular scaffolds for the development of new inhibitors targeting the L1196M mutant by
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the virtual screening of a large chemical library. Our research was stimulated by the possibility that new
inhibitors of the L1196M mutant could be rationally designed utilizing 3D structural information. This
study was undertaken to identify new 4-quinoline-based inhibitors of the L1196M mutant of ALK through
structure-based design strategies and to demonstrate the biological evaluation of various quinolone
derivatives in terms of their anti-cancer effects on H2228 CR cells, which are crizotinib-resistant cells. A
design strategy for improving their potency using electrostatic features and the effect of ionization pH of
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-quinoline scaffold to analyze the binding mode in detail was also discussed.
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Chart 1. Chemical structures of crizotinib and representative 4-phenoxyquinoline based inhibitor.
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RESULTS AND DISCUSSION
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Identification of 4-Aminoquinoline Moiety. With the goal of developing a new structural class of
potent L1196M mutant inhibitors, we investigated the starting structure for de novo design and the affinity
of various hinge-binding scaffolds to accommodate the increased bulk of the side chain of Met1196 in the
ATP binding site, employing an L1196M crystal structure (PDB ID: 2YFX) as a template. Figure 1a
shows the lowest energy conformation as calculated with the Discovery Studio 4.5 software. Based on
comparing structural features and the interactions formed between various fragments and the L1196M
mutant, the 4-aminoquinoline core appeared to be stabilized through hydrogen bonding with the backbone
amide groups of a residue in the hinge region (Met1196) of the ATP-binding site. Interestingly, a larger
gatekeeper Met1196 residue was found at the interface formed by van der Waals contacts between 4-
aminoquinoline and the L1196M mutant (Figure 1b), indicating that favorable hydrophobic interactions
were formed in the L1196M mutant. The quinoline serves as a hydrogen-bond acceptor with the backbone
amidic nitrogen of Met1199 in the middle of the hinge region, whereas the 6-membered bicyclic aromatic
ring of quinoline seems to be further stabilized through the van der Waals contacts of its aromatic rings
with the side chains of Leu1122, Ala1148 and Leu1256. It is noteworthy that in the L1196M mutant, the
increased steric hindrance of the gatekeeper mutation needs to be most carefully considered for drug
design, and the 4-aminoquinoline scaffold resides in proximity to the side chain of the Met1196
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gatekeeper without causing a steric clash. Because the 4-aminoquinoline scaffold binds tightly at the hinge
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region (Met1199), it is predicted to serve as a good starting point from which potent inhibitors can be
derived. In addition, the 4-aminoquinoline scaffold can be easily diversified via a convergent synthetic
approach for the rapid elucidation of structure-activity relationship (SAR). The terminal 4-amino group
points toward the DFG (Asp1270-Phe1271-Gly1272) motif in the activation loop (A-loop) and a vacant
binding pocket comprising Arg1253-Asn1254-Cys1255-Leu1256-Gly1269-Asp1270 and Asp1203 to
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accommodate a suitable fragment. A significant improvement in potency is thus expected to result from
introducing a suitable chemical moiety at the terminal amino group. Furthermore, the ATP pocket is
connected to a narrow aisle composed of Glu1132, Leu1198 and Ala1200, implying that a fragment on
C7 of quinoline would be stabilized through hydrophobic interactions with the region near the solvent
front. Therefore, we planned to extend the structures by introducing various chemical moieties at these
two positions (Frag1 and Frag 2 in Figure 1b). For these reasons, 4-aminoquinoline was selected as a
putative core from which more potent inhibitors could be derived in our studies.
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Figure 1. (a) Expected configuration of 4-aminoquinoline moiety in ATP binding site of ALK L1196M.
(b) Design of new scaffolds and opportunities for modification in the schematic active site of ALK
L1196M. Each dotted line represents a hydrogen bond.
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Chemical Synthesis. The de novo design process identified a variety of promising (hetero)arene
systems for introduction at the 4- and 7- positions of the quinoline core. The compound library was
established by a convenient route allowing the rapid exploration of the SAR profile and the general
synthetic route for representative quinolone derivatives, as shown in Scheme 1. The C4 position of
quinoline was functionalized with NH- or O-linked aryl groups prior to performing a Suzuki coupling
with the C7 (hetero)arene partners. The quinoline moiety can be easily equipped with a variety of
substituted anilines via addition and elimination processes under acidic conditions to afford the key
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intermediate B. An assortment of the C7 groups of 4-aminoquinolines were then explored through
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palladium-catalyzed Suzuki cross-coupling with corresponding arylboronic acids or esters, affording the
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desired product C. For a focused 4-phenoxy group array, S Ar reactions between 7-bromo-4-
N
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chloroquinoline and substituted phenol were conducted under basic conditions. A variety of aryl groups
were then installed at the 7-position by Pd-catalyzed cross-coupling with the appropriate aryl boronic
acids or esters. An additional deprotection step removing the Boc-protection group of the solubilizing tail
was conducted to produce the final compounds.
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Scheme 1. Synthesis of Quinoline Derivatives
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Reagents and conditions: (a) aniline, 35% HCl, iPrOH, 80 °C, 1.5 h; (b) phenol, K
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CO
-Bpin, Pd(dppf)Cl
O = 2:1, 90 °C, 12 h; after c or d, additional deprotection under TFA, DCM, rt, 1 h
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, DMF, 140 °C,
12 h; (c) R
Cs CO , toluene/H
for 11, 14-30.
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Array at C7. To identify potent inhibitors, we initially focused our design attempts on the 4-
aminoquinolone scaffold. First, we hypothesized that the overall activity of 4-aminoquinolone might be
improved by introducing a suitable moiety into the molecule to establish additional π interactions with
Gly1202 and hydrogen bonding or ionic interactions with polar amino acid residues at the solvent front.
Based on our structural analysis, we planned to install a variety of (hetero)arene appendages at the C7
position to evaluate the substituent space of 4-aminoquinoline while the phenyl group at the C4 position
remained fixed (Table 1). The resulting derivatives were subjected to a subsequent full IC50 value
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determination. To the best of our knowledge, the 4-aminoquinoline scaffold is not present in any of the
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ALK inhibitors reported so far. The (hetero)arene groups at the 7 position seem to be stabilized in the
internal region of the ATP-binding site because the chemical moiety group at the C7 position points to
the center of the N-terminal domain in docking simulations (Figure 2). Of particular significance is the
observation that a profound effect on potency was displayed upon the installation of the 4-(1H-pyrazol-
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-yl)piperidine group. Molecular docking study of compounds 11 consistently indicated that the pyrazolyl
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piperidine moiety of the appendage plays an important role in determining activity by establishing a
hydrogen bonding interaction with Glu1210. This effect is apparently one of the reasons for the better
inhibitory activity of compounds bearing the pyrazolyl piperidine moiety.
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Table 1. Exploration of groups at the C7 position of 4-phenylaminoquinoline derivatives.
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IC50 (μM)
Compd.
R
WT
L1196M
112±33
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H
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65.7±9.9
Br
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2.99±1.62
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2.70±0.69
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4.23±1.17
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3.81±0.38
10.3±4.3
6.08±1.47
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staurosporine
0.0019
0.00078
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Identification of O-Linked Quinoline Scaffold. Although compound 11 is a newly identified
L1196M inhibitor with micromolar potency, we decided to make some modifications to the structural and
electrostatic features of 4-aminoquinoline scaffold to find more potent inhibitors with at least
submicromolar-level inhibitory activity. For the 4-aminoquinoline core, the acidic conditions can stabilize
the quinoline tautomer where the quinoline is readily protonated to give the quinolinium ion. From this
perspective, we speculated that errors in the binding modes and scoring function could occur from
underestimation of the tautomerism and protonation of the 4-aminoquinoline scaffold, which might be
invoked to explain, at least partly, the lower inhibitory activity. To determine the potential effect of the
tautomerism, the binding modes of 4-phenylaminoquinoline at different pH values were thoroughly
investigated with docking simulations. As depicted in Figure 2, we note that the quinoline tends to be
mostly protonated (marked with red dashed circle) in the physiologically relevant pH 6.5-8.5 range,
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preventing the quinoline scaffold from establishing a hydrogen bond at the hinge region. The introduction
of the additional tautomerism and protonation factors seemed to guarantee better prediction accuracy of
the ligand binding affinity. Therefore, it would be reasonable to consider their electrostatic features to
design new inhibitors with increased potency. For this reason, we investigated the relevance of the effect
of ionization pH and inhibitory activity.
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Figure 2. (a) Binding mode of 4-anilinoquinoline obtained through preparing inhibitors at pH above 11.16
followed by normal docking study (PDB: 2YFX). (b) Calculated binding mode of 4-
phenylaminoquinoline at pH 6.5-8.5. Protonated site is marked with red dashed circle (PDB: 2YFX). (c)
Ionization pH of each marked unit and existing form in the blue- and orange-colored pH ranges.
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Figure 2c shows the ionization pH and range of each protonated/deprotonated form of the pyridine and
quinoline derivatives calculated in the Discovery Studio program. By the resonance effect of the 4-
aminopyridine moiety, the lone pair electron at N of the 4-amino group is transferred to the C-N bond at
the C4 of quinoline to form an N-positively charged imine and delocalized to raise the electron density of
the quinoline nitrogen. The ionization pH reaches 9.1 in both pyridine and quinoline, even higher in 4-
phenylamino-substituted scaffolds, to cause the compounds to mostly exist in protonated form at
physiological pH. We speculated that decreasing the resonance stability by replacing the amino linker
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with an ether linkage would lower the ionization pH, and consequently, the deprotonated neutral form of
-phenoxyquinoline scaffolds would be preferred. Indeed, the calculated ionization pH of 4-
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phenoxyquinoline is 6.44, revealing that the majority of this moiety exists in the neutral form so that the
quinoline nitrogen is able to establish hydrogen bonding in the ATP-binding site. In addition, protonation
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and tautomerism can influence the structural length and bond rotatability of the compounds. In this sense,
the structure of the 4-phenoxyquinoline scaffold tends to be more flexible and could serve as a more
effective surrogate for the corresponding 4-aminoquinoline core. To assess this hypothesis, the influence
of protonation and tautomerism was investigated by changing the amino linkers along with studying the
SAR profiles (Table 2).
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To our delight, changing the linking moiety from NH in the 4-aminoquinoline scaffolds to O results in
remarkable increases in the inhibitory activity. For example, the biochemical potency appears to increase
by a factor of 40 in going from 23 (L1196M, IC50 = 129 nM) to 24 (L1196M, IC50 = 3.3 nM) due to the
replacement of the amine linker with ether, the latter of which is identified as the exclusively preferred
linker (Table 2). This result indicates the importance of the neutral form of quinoline moieties,
participating in a hydrogen bond with the hinge to effectively impair the enzymatic activity in the sense
that the 4-alkoxyquinoline scaffold is more capable of acting as a hydrogen bond acceptor than the
corresponding 4-aminoquinoline moiety. Next, various substituents can be introduced on the 4-phenoxy
ring to maximize the binding affinity in the DFG motif in the A-loop and the nearby vacant binding pocket.
As can be inferred from the similar IC50 values of 14, 18 and 22, various patterns of substitution are
acceptable at the 4-phenoxy ring to achieve submicromolar inhibitory activity. The phenol group in 30 is
likely to function as a hydrogen bond acceptor to Lys1150, as reflected by the excellent IC50 value
(L1196M, IC50 = 7 nM). Therefore, it becomes apparent that the biochemical potency of quinolone-based
L1196M mutant inhibitors can be augmented to the low nanomolar level by introducing an ether linkage.
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The inhibitory activity of 14 compared to 13 with a CH -linked moiety also indicates the superiority of
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the 4-phenoxy group as the optimal substructure for binding in mutant ALK (L1196M).
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Table 2. Inhibition effects of quinoline scaffold attached with different linkages at C4.
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IC50 (nM)
Compd.
Ar
X
WT
L1196M
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11
NH
1,803±451
1,179±268
2
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23
13
CH
2
288±43
126±32
242±26
118±18
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O
NH
O
219±41
62±12
35.4±11.3
377±150
155±42
30.7±9.6
504±201
58.8±11.3
1,075±146
41.5±10.5
58±11
NH
O
NH
O
1,483±98
256±86
20
40
41
42
4
4
2
1
NH
O
181±34
22
148±30
26.3±7.4
129±31
45
46
47
48
4
50
51
52
53
5
5
56
57
58
59
2
2
2
2
2
2
3
4
5
6
7
8
NH
O
201±67
50.2±31.5
1,664±420
231±69
3.3±0.8
NH
O
926±241
93.5±31.2
547±201
28.4±11.2
NH
O
1,064±318
33.6±7.5
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1
2
3
4
5
6
7
8
9
29
NH
O
26.4±7.3
50.9±14.5
7.1±2.3
0.67
3
0
5.2±1.4
staurosporine
1.7
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
Binding Mode Analysis of Inhibitor. To address structural and mechanistic aspects relevant to the
potent inhibition by the newly identified inhibitors, we analyzed the interactions that stabilize the binding
mode of inhibitors in the mutant ALK (L1196M) protein structure (PDB ID: 2YFX). The binding modes
of 30 in the ATP-binding sites of the L1196M mutant is presented in Figure 3. The lowest-energy
conformation shows the hydrogen bond formation between the N of quinoline and the backbone of
Met1199, and this quinoline core is stabilized via hydrophobic interaction with the alkyl chain of Leu1122
and Ala1148 on the upper side and Leu1256 at the bottom of the pocket. A small pocket formed by
Val1130, Met1196 and Lys1150 is occupied by a 56.1° tilted phenyl head group, and this hydroxyl group
serves as an anchor by forming additional two hydrogen bonds with the two opposite-charged side chains
of Lys1150 and Asp1270. Interestingly, a larger gatekeeper Met1196 residue was found at the interface
formed by van der Waals contacts between the terminal phenoxy moiety and the ALK mutant (L1196M)
(Figure 3). The piperidine on the inhibitor appeared to point toward the solvent front by forming a salt
bridge with Glu1210 located on the outside of the kinase domain. Further stabilization may be achieved
through the van der Waals contacts of its the pyrazole moiety with the hydrophobic side chains of Leu1122,
Gly1201, Gly1202 and Ala1200. A similar binding mode would be speculated to occur in the cases of
compounds 20, 28 and 30, which form interactions such as hydrogen bonding. The overall structural
features derived from the docking simulations confirmed that the inhibitory activities of compound 30
were attributed to the multiple hydrogen bonds and hydrophobic interactions established simultaneously
in the ATP-binding site of the L1196M mutant.
4
4
45
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5
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1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
Figure 3. Calculated binding mode of 30 at the ATP binding site of ALK L1196M (PDB ID: 2YFX).
Each dashed blue line and orange line indicates a hydrogen bond and salt bridge, respectively. Gatekeeper
was displayed in semitransparent solid surface.
3
3
34
Mechanism of action (MOA) studies. To understand the interaction and binding modes of these
35
36
inhibitors with ALK L1196M, mechanism of action (MOA) studies including ATP competition evaluation
37
3
39
and K determination were performed. As shown in Figure 4, the progress curves are linear regardless of
i
40
41
the amount of inhibitor, suggesting the inhibition by 24 is not time-dependent.
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
Figure 4. Progress Curves for ALK (L1196M) with 24.
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The apparent K
m
is increased when inhibitor concentration is increased in Michaelis-Menten plot (Figure
1
2
3
4
5
6
7
8
9
5
A) and all lines are converged on the Y-axis in the double-reciprocal plot (Figure 5B). As shown in the
Lineweaver-Burk plots (Figure 5B), 24 appears to impair the kinase activity of the mutant in the ATP-
competitive fashion.
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
Figure 5. Michaelis-Menten Plot (A) and Lineweaver-Burk Plot (B) for ALK (L1196M) with 24
53
5
5
56
When % enzymatic activities of slopes relative to that of DMSO control at each ATP concentration are
57
58
plotted against inhibitor concentration and draw IC50 curves, the IC50 values of 24 were increased when
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ATP concentration was increased, as expected for common ATP-competitive inhibitors.
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
Figure 6. IC50 curves for ALK (L1196M) at different ATP concentrations with 24
19
20
2
Next, global fit was performed for 24 and results are listed in the table in Figure 7. The high value of Ki’
2
23
24
(almost infinity) means the compound has very little affinity for enzyme/ATP complex, which turns ATP-
25
26
competitive inhibition of ALK L1196M by 24. The results from MOA studies show that 24 is not time-
27
2
29
dependent and competitive with respect to ATP with the K
i
value of 22 nM and K value of 32.6 M.
m
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
5
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53
5
5
Parameter
Vmax
Value
Std. Error
0.0010
2.1206
1.5171
0.0000
0.0427
K
m
32.5684 μM
21.9872 nM
K
i
i
K ’ value
4.08474e+019 nM
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58
Figure 7. IC50 curves for ALK (L1196M) at different ATP concentrations with 24.
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ALK Mutant and Kinase Selectivity Profiling. As summarized in Table 3, compounds 24, 28, and 30
were tested against a panel of the most common secondary ALK mutants (L1196M, C1156Y, F1174L,
G1269A, L1152R, G1202R).13,26,32 Notably, compound 30 exhibited good potency against the secondary
ALK mutants (L1196M, C1156Y, F1174L, G1269A, L1152R) with IC50 values ranging from 7 to 30 nM,
although it showed an inhibitory activity against the G1202R mutant in the micromolar range.
1
2
3
4
5
6
7
8
9
1
1
12
1
14
Table 3. IC50 values of 24, 28 and 30 against ALK mutants.
15
16
17
18
19
20
2
2
23
24
25
26
27
IC50 (μM)
Compd.
L1196M
0.0033
0.0284
0.0071
0.00073
C1156Y
0.0409
0.0422
0.0115
0.0017
F1174L
0.141
G1269A
0.0351
0.0384
0.0162
0.00074
L1152R
0.159
G1202R
9.43
2
2
3
4
8
0
0.154
0.155
8.91
0.0093
0.0016
0.0296
0.0029
4.45
staurosporine
0.0038
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
To obtain a detailed picture of the selectivity profile of this series, potent inhibitor 24 was further
subjected to kinase selectivity profiling with a panel of 85 cancer related kinases on the kinome tree in a
high-throughput binding assay at a concentration of 1 M (see Table S1 in Supporting Information for
more detail). Overall, 24 showed moderate to good specificity profiles, and kinases against which 24
demonstrated meaningful off-target activity percent of control (POC < 10) were ALK (POC = 2.3),
AURKA (POC = 9.1), FLT3 (POC = 5.2), KIT (POC = 4.4), RET (POC = 3.3), TIE2 (POC = 4.9) and
VEGFR2 (POC = 2.3). Other kinases inhibited by 24 are ACVR1B (POC = 14), LTK (POC = 13), FGFR1
(POC = 12), MEK2 (POC = 17) and MET (POC = 15).
45
46
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50
51
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53
5
5
Effect on Cell Proliferation and Cell-Signaling Downstream. This series of potent inhibitors were
further tested for cellular growth inhibition against cancer cells. We tested cell viability assay with FDA
approved ALK inhibitors (crizotinib, ceritinib and alectinib) along with the representative compound 30
against Ba/F3 EML4-ALK WT (EA WT) and Ba/F3 EML4-ALK L1196M (EA L1196M) cells. As shown
in Figure 8a, crizotinib did not effectively inhibit cell proliferation of Ba/F3 EA L1196M cells while
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crizotinib significantly inhibited proliferation in Ba/F3 EA WT cells. On the other hand, compound 30
effectively inhibited the cell growth and was comparable to ceritinib and alectinib against both wild type
BaF3 EML4-ALK cells and BaF3 EML4-ALK cells with L1196M in a dose-dependent manner. 8,16, 33
Next, we carried out an evaluation of their antiproliferative activity on the H2228 and H2228 CR cell
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
34
lines, the latter of which is crizotinib-resistant. Next, H2228 and H2228 CR cells were treated with
different doses of crizotinib for 72 h, and then MTT assays were performed. As expected, crizotinib
significantly inhibited proliferation in H2228 crizotinib-sensitive cells (IC50 = 2.5 μM) but did not
effectively inhibit cell proliferation of H2228 CR crizotinib-resistant cells (IC50 = 10 μM). In order to
investigate whether 30 could overcome the resistance to crizotinib, H2228 CR cells were treated with
different doses of crizotinib, ceritinib, alectinib and 30 for 72 h. The results from the MTT assay indicated
that H2228 CR cells exhibited high resistance to crizotinib, whereas 30, ceritinib, and alectinib effectively
inhibited the cell growth of H2228 CR cells in a dose-dependent manner (Figure 8b).
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
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5
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1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
Figure 8. Cell viability of crizotinib, ceritinib, alectinib and 30. Cell viability of crizotinib, ceritinib,
alectinib and 30 were measured in (a) Ba/F3 EML4-ALK WT (EA WT), EML4-ALK L1196M (EA
L1196M), H2228 and (b) H2228 CR human lung cancer cells by MTT assay. EA = EML4-ALK
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
To identify the molecular mechanism of 30 in overcoming acquired crizotinib resistance, we examined
the effect of 30 on PI3K/AKT and MAPK signaling activation, which were reported to be not only the
main downstream signaling pathways of ALK, but also the key mechanism for crizotinib resistance. In
H2228 crizotinib-sensitive cells, crizotinib obviously inhibited the expression of p-AKT, downstream of
PI3K/AKT signaling, but not p-ERK, downstream of MAPK signaling. However, 30 inhibited the
expresssion of both p-AKT and p-ERK (Figure 9). More importantly, 30 remarkably inhibited the
expression of both p-AKT and p-ERK in H2228 CR crizotinib-resistant cells, compared with crizotinib
4
45
(Figure 9). Overall, these results showed that 30 successfully overcame crizotinib resistance by decreasing
46
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48
PI3K/AKT and MAPK signaling.
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1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
Figure 9. Compound 30 increased crizotinib sensitivity and overcame crizotinib resistance by decreasing
PI3K/AKT and MAPK signaling in crizotinib-sensitive and -resistant human lung cancer cells. H2228
and H2228 CR human lung cancer cells were treated with the indicated concentrations of 30 and crizotinib
for 3 h, and then the expression levels of p-AKT and p-ERK were measured by western blotting. Data are
presented as means ± S.D. (***P < 0.005).
52
53
5
5
Two leading compounds 24 and 28 were subjected to pharmacokinetic studies. As shown in Table 4a,
56
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rat pharmacokinetic parameters were determined after oral administration and intravenous injection,
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60
which showed fair oral bioavailability (24: 44.8%, 28: 45.5%, 30: 24.8%). Next, we assessed the hERG
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inhibition of representative compounds of this series, and IC50 values for 24, 28, and 30 were determined
as shown in Table 4b. The assay is based on the competition of fluorescently labeled Tracer binding to
the membrane preparation containing hERG. Compounds 24, 28, and 30 exhibited moderate inhibition of
1
2
3
4
5
6
7
8
9
the hERG channel, with activities ranging from 2.46 to 3.12 μM.
1
1
12
Table 4. (a) Rat pharmacokinetic properties of compound 24 and 28 and (b) IC50 values of compounds
13
14
2
4, 28, and 30 against hERG.
15
16
(a)
17
18
19
20
2
2
23
2
AUClast
Compd.
Dose (mg/kg) T1/2 (hr)
T
max (hr)
C
max (ng/mL)
F (%)
(hr*ng/mL)
IV
1.0
3.3
0.1
4.0
0.1
4.0
0.1
6.0
94.1
130.7
48.9
83.9
83.5
78.4
166.0
744.6
95.9
2
2
3
4
8
0
PO
IV
10.0
1.0
44.8
45.5
24.8
10.7
3.5
PO
IV
10.0
1.0
436.7
155.1
384.0
25
26
27
PO
10.0
28
29
30
31
(b)
3
3
3
35
24
.12
28
2.46
30
E-4031
0.0012
hERG
IC50 (μM)
3
2.76
36
37
38
39
4
41
CONCLUSION
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
By fragment based drug design and modification followed by analysis of chemical properties, we
identified 4-phenoxyquinoline derivatives as ALK and ALK L1196M inhibitors. Starting from the 4-
aminoquinoline moiety, a fragment-growing strategy was adopted to promote hydrophobic interaction,
solvation effect and advanced interactions. The electrostatic properties of molecular structures in
physiological conditions were carefully considered for the structural design of inhibitors. In the range of
physiological pH conditions, electron delocalization from the 4-aminogroup to the quinoline nitrogen is
likely to lead to protonation of the 4-aminoquinoline moiety that blocks the quinoline nitrogen from
59
6
binding the hinge amino acid backbone. The scaffold was redesigned to eliminate this problematic
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Journal of Medicinal Chemistry
character, altering the linker from NH to O. The enzymatic inhibition of ALK L1196M dramatically
increased, especially in the case of 4-phenoxyquinoline bearing p-substitutuent, whose binding stability
is demonstrated in the docking model. The new inhibitors exhibited significant antiproliferative effects
on H2228 CR crizotinib-resistant cells by inhibiting the expression of both p-AKT and p-ERK. These
results show the advantages of the 4-phenoxyquinoline moiety and have considerable implications for the
drug design. Further studies to increase cellular potency by modifying cellular permeability and water-
solubility are currently in progress.
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
EXPERIMENTAL SECTION
19
20
2
2
23
24
25
26
27
28
General Methods and Materials. Unless stated otherwise, reactions were performed in a flame-dried
glassware under positive pressure of nitrogen using freshly distilled solvents. Analytical thin layer
chromatography (TLC) was performed on precoated silica gel 60 F254 plates, and visualization on TLC
was achieved via UV light (254 and 354nm). μW irritation was conducted by Anton Paar Microwave 300
29
30
31
single-mode microwave synthesis reactor. Flash column chromatography was utilized on silica gel (400-
3
3
1
6
30 mesh). H NMR was recorded on 600MHz, 400MHz or 300 MHz and chemical shifts were quoted
34
35
36
37
38
39
40
41
4
in parts per million (ppm) referenced to the appropriate solvent peak or 0.0 ppm for tetramethylsilane.
The following abbreviations were used to describe the peak splitting patterns when appropriate: br = broad,
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet. Coupling constants,
13
J, were reported in hertz unit (Hz). C NMR was recorded on 100 MHz or 150MHz and was fully
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
decoupled by broad band proton decoupling. Chemical shifts were reported in ppm referenced to the
center line of a triplet at 77.0 ppm of chloroform-d. Mass spectral data were obtained from the KAIST
Basic Science Institute by Bruker Daltonik HR-MS using the EI method or Agilent LR-MS. High-
performance liquid chromatography analyses for checking mass spectral data (using EI method) of
synthesized compounds were performed on a Agilent HPLC equipped with an Agilent Poroshell 120 EC-
C18 reverse phase column (4.6 × 50 mm, 2.7 Micron) and by quadrupole LC/MS. The mobile phase was
58
59
a mixture of MeOH (0.1% TFA) and H O (0.1% TFA). Compound purity was determined by integrating
2
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the peak areas of liquid chromatogram, monitored at 254 nm. Flow rate (0.5 mL/min). Gradient system:
from MeOH (0.1 % TFA) 10% and H O (0.1% TFA) 90% (0 min) to MeOH (0.1% TFA) 90% and H
(0.1% TFA) 10% (18 min), the solvent ratio was maintained for 23 min; the solvent ratio was changed to
MeOH (0.1% TFA) 10% and H O (0.1% TFA) 90% (25 min). All final compounds were found to have
95% purity. Commercial grade reagents and solvents were used without further purification except as
1
2
3
4
5
6
7
8
9
2
2
O
2
>
1
1
12
13
14
15
indicated below. Unless stated otherwise, all commercial grade reagents and solvents were used without
additional purification.
16
17
18
Preparation of Compounds
19
20
2
2
Preparation of Boron Coupling Partners for Suzuki Coupling.
23
24
25
R
2
-Bpin (4,4,5,5-tetramethyl-1,3,2-dioxaborolane substituted compounds) reagents for Suzuki coupling
26
27
(GP I-2 and II-2) were prepared by synthesis of iodo-substituted intermediates followed by miyaura
28
2
30
borylation in the case of 11-30. To synthesize 3-10, commercially available boronic acid or Bpin-
31
3
substituted reagents were used. Preparation of synthesized boron reagents were proceded through reported
3
34
synthetic procedures in J. Med. Chem. 2011, 54, 6342–6363 (synthesis of 68). In the case of R -Bpin
2
35
3
37
38
39
reagents for 12, 1-(tetrahydro-2H-pyran-4-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole was prepared by following procedure: A solution of 4-iodo-1-(tetrahydro-2H-pyran-4-yl)-1H-
40
41
o
pyrazole (95.5 mg, 0.3434 mmol) in anhydrous THF (1 mL) was cooled to 0 C under an atmosphere of
42
4
4
N
2
. 2 M iPrMgCl solution in THF (0.26 mL, 0.5151 mmol) was added dropwise for 10 min, and the
45
46
o
mixture was stirred at rt for 1 h. To the reaction mixture at 0 C was added 2-methoxy-4,4,5,5-tetramethyl-
47
4
1,3,2-dioxaborolane (87.2 μL, 0.5323 mmol) and the resulting mixture was stirred at rt overnight. The
49
5
51
o
reaction was quenched with saturated NaCl (aq.) at 0 C, and the mixture was extracted with EtOAc (100
52
53
mL x 3). The organic layers were dried over Na
2
SO , filtered, and concentrated under redused pressure.
4
5
5
56
The residue was directly used for Suzuki coupling without further purification.
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General Procedure (GP) I-1 for the Synthesis of Compounds 1, 2 and 3 and 7-bromo-N-
1
2
3
4
5
6
7
8
9
phenylquinolin-4-amine Intermediates.
In a round bottom flask, 7-bromo-4-chloroquinoline (0.092 mmol) and aniline (0.10 mmol) were
dissolved in 1 mL of iPrOH. A catalytic amount (1 drop) of 35% hydrochloric acid was added to this
1
1
o
mixture at room temperature and the final mixture was stirred for 2 h at 80 C. The reaction mixture was
12
1
14
15
16
17
18
19
20
2
2
23
24
cooled to room temperature, and the precipitate was filtered with iPrOH when the precipitate is observed
in the crude mixture. When the whole materials are solved in the solvent after reaction, the solvent was
evaporated and small portion of diethyl ether was added to precipitate product from mixture. The
precipitate was filtered with diethyl ether, and the solid was dried under reduced pressure obtaining
products or intermediates that was submitted to the next reaction without further purification. (J. Med.
Chem. 2014, 57, 701−713)
25
26
27
28
29
General Procedure I-2 for Suzuki Coupling of 4-anilinoquinoline Substrates.
30
31
3
The mixture of 7-bromo-N-phenylquinolin-4-amine substrate 2 (0.0334 mmol), proper boronic ester or
3
34
35
3
37
38
39
40
41
42
4
4
boronic acid reagent (0.100 mmol), Pd(PPh
3
)
4
(0.0033 mmol) and K
2
CO
3
(0.100 mmol) in dioxane:H
O were added to the mixture
. Filtered
solution was concentrated under reduced pressure and purified on flash column chromatography
dichloromethane/methanol = 20:1) to give desired product. If inseparable impurity is observed, further
2
O
o
(=1:1, 2 mL) was stirred for 1 h at 120 C under μW irradiation. EtOAc and H
2
and the organic materials were extracted with EtOAc (50 mL x 3), followed by dring with MgSO
4
(
45
46
purification was proceded by flash column chromatography (EtOAc/diethyl ether = 20:1). If necessary,
47
4
o
boc deprotection was proceded in the DCM (0.5 mL) solution of product. At 0 C, trifluoroacetic acid
49
50
51
(0.3 mL) was added dropwise and the resulted mixture was stirred at room temperature for 1 h. The solvent
52
53
was evaporated under vacuo, and washed with diethyl ether (1 mL x 3) to give pure product as the TFA
5
5
56
salt.
57
58
59
General Procedure II-1 for the Synthesis of 7-bromo-4-phenoxyquinoline Intermediates.
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7
-bromo-4-phenoxyquinoline. 7-bromo-4-chloroquinoline (15.0 mg, 0.0623 mmol), phenol (5.9 mg,
.062 mmol), and K CO (21.5 mg, 0.156 mmol) were placed in a round bottom flask. The reagent mixture
atmosphere and the reaction mixture was stirred
overnight at 140 ℃. After cooling to room temperature, the mixture was diluted with EtOAc and H O,
and the organic materials were extracted with EtOAc (50 mL x 3). The combined organic phases were
dried over anhydrous MgSO and filtered. The organic layer was concentrated under reduced pressure
1
2
3
4
5
6
7
8
9
1
1
12
13
0
2
3
was dissolved in N, N-Dimethylformamide under N
2
2
4
14
15
and purified on flash column chromatography (dichloromethane/methanol = 40:1) to give desired product
16
1
18
1
as a pale yellow solid (15.0 mg, 80%). H NMR (400 MHz, methanol-d
4
) δ 8.60 (d, J = 5.3 Hz, 1H), 8.29
19
20
(d, J = 8.9 Hz, 1H), 8.17 (s, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.56 – 7.51 (m, 2H), 7.40 – 7.32 (m, 1H), 7.29
2
2
–
7.23 (m, 2H), 6.61 (d, J = 5.4 Hz, 1H). (Bioorg. Med. Chem. 2005, 13, 1069–1081)
23
24
2
26
General Procedure II-2 for Suzuki Coupling of 4-phenoxyquinoline Substrates.
27
2
29
30
31
The mixture of 7-bromo-4-phenoxyquinoline substrate (0.0500 mmol), tert-butyl 4-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate as a boronic ester
3
3
34
35
36
37
38
reagent (0.060 mmol), Pd(dppf)Cl
=2:1, 1.5 mL) was stirred overnight at 90 C. H
EtOAc (50 mL x 3). The combined organic phases were dried over anhydrous MgSO
2
.CH
2
Cl
2
(0.0025 mmol) and Cs
2
CO
O were added and the extraction was conducted with
, filtered and
3
(0.165 mmol) in toluene:H O
2
o
(
2
4
39
4
41
42
4
4
45
concentrated. The residue was purified on flash column chromatography (DCM/MeOH = 40:1) to give
desired product. Inseparable impurity was purified with flash column chromatography (diethyl
ether/EtOAc = 1:20). Additional deprotection of boc group was conducted with the solution of product in
46
4
48
o
DCM (0.5 mL) in a 10 mL round bottom flask. After cooling to 0 C, trifluoroacetic acid (0.3 mL) was
49
50
added dropwise and the resulted mixture was stirred at room temperature for 1 h. The solvent was
51
52
evaporated under vacuo, and washed with diethyl ether (1 mL x 3) to give desired product as the TFA
53
5
5
salt.
56
57
58
N-phenylquinolin-4-amine (1). Compound 1 was prepared (11.7 mg, pale yellow solid, 58% yield)
59
60
according to GP I-1 from 4-chloroquinoline (15.0 mg, 0.092 mmol) and aniline (9.2 μL, 0.10 mmol) as a
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Journal of Medicinal Chemistry
1
starting material. H NMR (400 MHz, methanol-d
4
) δ 8.35 (d, J = 5.8 Hz, 1H), 8.31 (d, J = 7.4 Hz, 1H),
1
2
3
4
5
6
7
8
9
7
.88 (d, J = 8.5 Hz, 1H), 7.72 (dd, J = 8.4, 6.9 Hz, 1H), 7.58 – 7.49 (m, 1H), 7.47 – 7.33 (m, 4H), 7.21 (t,
1
3
J = 7.2 Hz, 1H), 6.92 (d, J = 5.5 Hz, 1H). C NMR (150 MHz, methanol-d
4
) δ 151.3, 150.8, 149.2, 141.4,
+
+
[M+H]+:
1
2
31.0, 130.6, 128.7, 126.3, 126.0, 124.7, 122.7, 121.0, 102.2. HRMS (ESI ) m/z calcd. C15
H
13
N
2
21.1073, found: 221.1076.
1
1
12
1
14
7-bromo-N-phenylquinolin-4-amine (2). Compound 2 was prepared (171.4 mg, yellow solid, 93%)
15
16
according to GP I-1 from 7-bromo-4-chloroquinoline (150 mg, 0.619 mmol) and aniline (62.1 μL, 0.680
17
18
1
mmol) as a starting material. H NMR (400 MHz, methanol-d
4
) δ 8.51 (d, J = 9.1 Hz, 1H), 8.37 (d, J =
19
20
2
2
23
7
6
.1 Hz, 1H), 8.14 (s, 1H), 7.93 (d, J = 9.1 Hz, 1H), 7.60 (td, J = 7.3, 1.7 Hz, 2H), 7.53 – 7.41 (m, 3H),
13
.89 (d, J = 7.1 Hz, 1H). C NMR (150 MHz, methanol-d ) δ 157.6, 144.1, 140.6, 138.1, 131.9, 131.3,
4
24
2
+
12BrN +
[M+H] : 299.0178,
+
129.7, 129.4, 126.7, 126.1, 123.7, 117.6, 101.6. HRMS (ESI ) m/z calcd. C15
H
2
26
27
28
found: 299.0193.
29
30
31
Compounds 3-10 and 12 were prepared by using a same procedure described for the synthesis of 2 as a
3
3
GP I-1, followed by similar procedure to GP I-2.
34
35
3
37
N,7-diphenylquinolin-4-amine (3). Compound 3 was prepared (4.6 mg, pale yellow solid, 2 steps 29%
3
39
1
yield) according to GP I. H NMR (600 MHz, DMSO-d
6
) δ 9.19 (s, 1H), 8.52 (d, J = 8.9 Hz, 1H), 8.49
40
41
(d, J = 5.4 Hz, 1H), 8.14 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.87 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.6 Hz,
42
4
4
45
46
2
H), 7.45 (t, J = 7.6 Hz, 3H), 7.40 (d, J = 7.8 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 6.93 (d, J = 5.4 Hz, 1H).
) δ 150.6, 148.5, 148.1, 141.1, 140.3, 139.2, 129.4, 129.1, 128.1, 127.0,
13C NMR (150 MHz, DMSO-d
6
47
4
+
+
+
125.6, 124.0, 123.7, 123.1, 122.6, 118.7, 101.4. HRMS (ESI ) m/z calcd. C21
H
17
N [M+H] : 297.1386,
2
49
50
51
found: 297.1409.
52
53
5
N-phenyl-7-(pyridin-4-yl)quinolin-4-amine (4). Compound 4 was prepared (3.9 mg, pale yellow solid,
5
56
57
58
1
2
2
steps 24% yield) according to GP I. H NMR (600 MHz, DMSO-d
6
) δ 9.09 (s, 1H), 8.70 (d, J = 5.1 Hz,
H), 8.54 (d, J = 8.8 Hz, 1H), 8.52 (d, J = 5.3 Hz, 1H), 8.30 (s, 1H), 7.98 (d, J = 8.6 Hz, 1H), 7.92 (d, J =
59
60
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Journal of Medicinal Chemistry
Page 28 of 46
4
.9 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 6.96 (d, J = 5.3
1
2
3
4
5
6
7
8
9
1
3
Hz, 1H). C NMR (150 MHz, DMSO-d ) δ 151.5, 150.4, 149.2, 147.7, 146.2, 140.34, 137.7, 129.4, 127.1,
6
+
+
+
123.9, 123.4, 122.9, 122.5, 121.5, 120.0, 101.9. HRMS (ESI ) m/z calcd. C20
H
16
N [M+H] : 298.1339,
3
found: 298.1362.
1
1
7
-(6-aminopyridin-3-yl)-N-phenylquinolin-4-amine (5). Compound 5 was prepared (4.2 mg, ivory
12
1
14
1
solid, 2 steps 25% yield) according to GP I. H NMR (400 MHz, chloroform-d, methanol-d
4
cosolvent) δ
15
16
8
.37 (d, J = 5.5 Hz, 1H), 8.32 (s, 1H), 8.05 (d, J = 8.6 Hz, 1H), 8.00 (s, 1H), 7.81 (d, J = 8.7 Hz, 1H), 7.63
17
18
(d, J = 8.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 2H), 7.28 (s, 2H), 7.14 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 5.5 Hz,
19
20
2
2
23
24
2
1
3
1
1
C
H), 6.60 (d, J = 8.5 Hz, 1H). C NMR (100 MHz, DMSO-d
6
) δ 159.6, 151.0, 149.6, 147.5, 146.3, 140.6,
+
38.8, 135.6, 129.3, 124.1, 123.6, 122.9, 122.6, 122.3, 122.2, 118.2, 108.1, 101.2. HRMS (ESI ) m/z calcd.
+
+
20
H
17
N [M+H] : 313.1448, found: 313.1458.
4
26
27
2
29
7-(furan-2-yl)-N-phenylquinolin-4-amine (6). Compound 6 was prepared (7.7 mg, yellowish brown
3
31
1
solid, 2 steps 50% yield) according to GP I. H NMR (400 MHz, DMSO-d
6
) δ 9.32 (s, 1H), 8.49 (d, J =
3
3
34
35
36
37
38
8
1
1
.9 Hz, 1H), 8.46 (d, J = 5.7 Hz, 1H), 8.14 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.88 (dd, J = 1.8, 0.7 Hz,
H), 7.53 – 7.42 (m, 2H), 7.42 – 7.35 (m, 2H), 7.25 (dd, J = 3.5, 0.8 Hz, 1H), 7.20 (tt, J = 7.2, 1.4 Hz,
13
H), 6.87 (d, J = 5.6 Hz, 1H), 6.69 (dd, J = 3.4, 1.8 Hz, 1H). C NMR (150 MHz, DMSO-d ) δ 152.1,
6
39
4
41
4
149.8, 148.9, 144.0, 139.8, 131.5, 129.5, 124.5, 123.3, 123.0, 121.0, 120.6, 118.2, 112.5, 108.2, 101.2.
+
+
+
HRMS (ESI ) m/z calcd. C19
H
15
N O [M+H] : 287.1179, found: 287.1199.
2
4
4
45
46
N-phenyl-7-(thiophen-2-yl)quinolin-4-amine (7). Compound 7 was prepared (6.3 mg, white solid, 2
47
4
1
steps 39% yield) according to GP I. H NMR (400 MHz, chloroform-d, methanol-d
4
cosolvent) δ 8.35 (d,
49
50
51
52
53
J = 5.5 Hz, 1H), 8.11 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.49 (dd, J = 3.6, 1.1 Hz,
H), 7.38 (dd, J = 8.5, 7.3 Hz, 2H), 7.34 (dd, J = 5.1, 1.1 Hz, 1H), 7.32 – 7.26 (m, 2H), 7.19 – 7.13 (m,
1
5
5
56
1
3
1H), 7.10 (dd, J = 5.1, 3.6 Hz, 1H), 6.89 (d, J = 5.5 Hz, 1H). C NMR (100 MHz, chloroform-d, methanol-
57
58
d
4
cosolvent) δ 150.9, 148.9, 148.8, 143.3, 139.9, 135.8, 129.7, 128.4, 126.0, 124.8, 124.5, 124.5, 123.6,
59
60
+
+
+
1
23.1, 121.6, 119.0, 101.8. HRMS (ESI ) m/z calcd. C19
H
15
N S [M+H] : 303.0950, found: 303.0975.
2
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Journal of Medicinal Chemistry
N-phenyl-7-(1H-pyrazol-4-yl)quinolin-4-amine (8). Compound 8 was prepared (6.6 mg, white solid, 2
1
2
3
4
5
6
7
8
9
1
steps 43% yield) according to GP I. H NMR (400 MHz, DMSO-d
6
) δ 13.06 (s, 1H), 8.93 (s, 1H), 8.42 (d,
J = 5.3 Hz, 1H), 8.36 (d, J = 8.8 Hz, 2H), 8.14 (s, 1H), 8.09 (s, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.47 – 7.30
13
(
m, 4H), 7.14 (tt, J = 7.1, 1.5 Hz, 1H), 6.86 (d, J = 5.3 Hz, 1H). C NMR (150 MHz, DMSO-d
6
) δ 150.9,
1
49.6, 147.5, 140.6, 136.7, 133.8, 129.3, 126.3, 123.6, 123.6, 122.9, 122.6, 122.4, 120.6, 117.9, 101.0.
1
1
12
13
+
+
+
HRMS (ESI ) m/z calcd. C18
H
15
N [M+H] : 287.1291, found: 287.1302.
4
14
15
16
7
-(1-methyl-1H-pyrazol-4-yl)-N-phenylquinolin-4-amine (9). Compound 9 was prepared (12.4 mg,
17
18
1
pale yellow solid, 2 steps 76% yield) according to GP I. H NMR (600 MHz, DMSO-d
6
) δ 9.12 (s, 1H),
19
20
2
8
.42 (d, J = 5.4 Hz, 1H), 8.40 (d, J = 8.8 Hz, 1H), 8.37 (s, 1H), 8.07 (s, 1H), 8.03 (s, 1H), 7.80 (d, J = 8.7
2
23
Hz, 1H), 7.43 (t, J = 7.7 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 6.85 (d, J = 5.5 Hz,
24
2
1
3
1H), 3.91 (s, 3H). C NMR (150 MHz, DMSO-d
6
) δ 150.2, 148.6, 148.1, 140.3, 136.6, 133.9, 129.4,
26
27
28
+
+
1
28.7, 123.9, 122.9, 122.8, 122.6, 122.6, 121.2, 117.7, 100.9, 38.8. HRMS (ESI ) m/z calcd. C19
H
17
N
4
29
30
+
[
M+H] : 301.1448, found: 301.1467.
31
3
3
7
-(1-ethyl-1H-pyrazol-5-yl)-N-phenylquinolin-4-amine (10). Compound 10 was prepared (8.1 mg, pale
34
35
36
1
yellow solid, 2 steps 48% yield) according to GP I. H NMR (400 MHz, DMSO-d
6
) δ 9.09 (s, 1H), 8.57
37
38
–
8.45 (m, 2H), 7.93 (s, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H), 7.44 (dd, J = 8.5, 7.1 Hz,
39
4
41
4
2H), 7.41 – 7.34 (m, 2H), 7.20 – 7.12 (m, 1H), 6.96 (d, J = 5.3 Hz, 1H), 6.54 (d, J = 1.8 Hz, 1H), 4.25 (q,
13
J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSO-d
6
) δ 151.5, 148.8, 147.7, 141.7,
4
4
45
1
40.4, 138.3, 131.1, 129.4, 128.4, 124.8, 123.9, 123.0, 122.5, 119.3, 106.5, 101.9, 44.3, 15.5. HRMS
46
4
48
+
+
+
(ESI ) m/z calcd. C20
H
19
N [M+H] : 315.1604, found: 315.1627.
4
49
50
51
N-phenyl-7-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)quinolin-4-amine (11). Compound 11 was prepared
52
53
1
(
11.1 mg, pale yellow solid, 3 steps 69% yield) according to GP I. H NMR (400 MHz, methanol-d
4
) δ
5
5
56
8.55 (d, J = 8.8 Hz, 1H), 8.43 (s, 1H), 8.32 (d, J = 7.1 Hz, 1H), 8.16 (s, 1H), 8.09 – 7.93 (m, 2H), 7.59
57
58
(dd, J = 8.4, 7.1 Hz, 2H), 7.52 – 7.22 (m, 3H), 6.84 (d, J = 7.0 Hz, 1H), 4.67 (dt, J = 9.8, 4.8 Hz, 1H),
59
60
1
3
3
.88 – 3.52 (m, 2H), 3.28 – 3.16 (m, 2H), 2.47 – 2.18 (m, 4H). C NMR (150 MHz, methanol-d ) δ 155.7,
4
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